Trace amine associated receptor 1 (TAAR1)

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Theses and Dissertations
5-1-2015
Trace amine associated receptor 1 (TAAR1), a
novel astrocyte receptor for METH-mediated
neurotoxicity in HIV-1-associated neurocognitive
disorders (HAND)
Irma E. Cisneros
University of North Texas Health Science Center at Fort Worth, [email protected]
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Recommended Citation
Cisneros, I. E. , "Trace amine associated receptor 1 (TAAR1), a novel astrocyte receptor for METH-mediated neurotoxicity in
HIV-1-associated neurocognitive disorders (HAND)" Fort Worth, Tx: University of North Texas Health Science Center; (2015).
http://digitalcommons.hsc.unt.edu/theses/822
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Trace amine associated receptor 1 (TAAR1), a novel astrocyte receptor for METHmediated neurotoxicity: Implications for HIV-1-associated neurocognitive disorders
A DISSERTATION
Presented to the Graduate Council of
The Graduate School of Biomedical Sciences
The University of North Texas Health Science Center
In Partial Fulfillment of the Requirements
For the degree of DOCTOR OF PHILOSOPHY
Biomedical Sciences
By:
Irma E. Cisneros
April 2015
Under the Supervision of Dr. Anuja Ghorpade
UNTHSC
Fort Worth, TX
ACKNOWLEDGEMENTS
I would like to first thank my mentor Dr. Anuja Ghorpade for her endless support,
patience and training while a graduate student. I have grown as an independent and confident
scientist with a critical eye because of her guidance. I would like to express my deepest gratitude
to the mentoring and constructive criticism received by my committee members, the late Dr.
Robert Wordinger, Dr. Michael Forster, Dr. Harlan Jones, Dr. Raghu Krishnamoorthy and Dr.
Ranajit Chakraborty, I greatly appreciate their support.
Ms. Kathleen Borgmann has been essential in my training experience, with her
increasingly complicated questions that always kept me on my toes. She has been supportive,
academically, mentally and personally, and I would have been unable to achieve my successes
without her. Special thanks are due to Ms. Lin Tang for her technical support and caring attitude.
Thank you also to both past and present members of my lab, Dr. Adam Fields, Dr. Manmeet
Kaur, Dr. Neha Vartak, Dr. Richa Panday, Shruthi Nooka, Chaitanya Joshi, Sangeeta Shenoy and
Mayuri Thete for the numerous discussions. I would also like to thank Dr. Gryzynski and Dr.
Rafal Fundala for their assistance in fluorescence techniques, I-Fen for all her help with confocal
microscopy and Larry for his technical help.
I would also like to acknowledge the Department of Cell Biology Immunology, Deanna
Ranker, Jessica Medina, Monica Castillo and Angelica Ramos for the constant administrative
assistance. Finally, I would like to thank my entire family and friends for their constant support
and encouragement. Particularly, I would like to give thanks to my parents, Jaime and Elma
Naranjo for their sacrifices and continuous prayers.
Finally I would like to thank the National Institutes of Health/National Institutes on Drug
Abuse for supporting my work.
iii LIST OF PUBLICATIONS
1. Cisneros IE, Ghorpade A. Methamphetamine and HIV-1-induced neurotoxicity: Role of
trace amine associated receptor 1 cAMP signaling in astrocytes. Neuropharmacology.
2014; Published online 2014 June 17. Doi: 10.1016/j.neuropharm.2014.06.011
2. Fields J, Cisneros IE, Ghorpade A. Extracellular regulated kinase ½ signaling is a
critical
regulator
of
interleukin-1β-mediated
astrocyte
tissue
inhibitor
of
metalloproteinase-1 expression. PLoS One. 2013; 8(2): e56891. Published online 2013
February 14. doi:10.1371/journal.pone.0056891 PMCID: PMC3572966.
3. Cisneros IE, Ghorpade A. HIV-1, Methamphetamine and Astrocyte glutamate
regulation: Combined excitotoxic implications for Neuro-AIDS, Current HIV Research.
2012. Curr HIV Res. 2012 May 11. PMID: 22591363.
4. Cisneros IE, Borgmann K, Ghorpade A. METH-induced, TAAR1-associated CREB
signaling serves as a master regulator for astrocyte EAAT-2. 2015. (In preparation for
submission to Journal of Biological Chemistry).
iv TABLE OF CONTENTS
Page
ABSTRACT……………………………………………………………………………………...ii
TITLE PAGE……………..……………………………………………………………………..iv
ACKNOWLEDGEMENTS…...………………………………………………………………...v
LIST OF PUBLICATIONS……………………………………………………………..……...vi
ABBREVIATIONS…………………………………………………………………………..….xi
LIST OF ILLUSTRATIONS………………………………………………………………….xiii
CHAPTER
I. INTRODUCTION…………………………………….………..…………………………….1
1.1 Methamphetamine abuse……………………………………………………………...4
1.1.1 Distribution and Pharmacokinetics……………………………………………...7
1.2 HIV-1 Epidemiology………………………………………………………………...12
1.3 Coexistence of METH abuse and HIV-1 Infection…………………………………..13
1.4 Emerging role(s) of astrocytes in HAND and METH abuse associated
neurotoxicity...…………………………………………………………………………...15
1.4.1 Astrogliosis/Morphological Changes………………………………………….18
1.4.2 Calcium Signaling/Oxidative Stress…………………………………………...20
1.4.3 Glutamate homeostasis………………………………………………………...20
1.5 Trace amine associated receptor 1 (TAAR1)……...………………………................22
v 1.6 Astrocyte TAAR1 as a novel mechanistic and therapeutic target for METH abuse in
HAND…………………………………………………………………………………....23
1.7 Objectives of the present study.……………………………………………………....24
II. HIV-1, METHAMPHETAMINE AND ASTROCYTE GLUTAMATE REGULATION:
COMBINED EXCITOTOXIC IMPLICATIONS FOR NEURO-AIDS...………………….25
2.1 Abstract…….…………………………………………………………………………26
2.2 Introduction…………………………………………………………………………...27
2.3 Role of astrocytes in the Central Nervous System………………………….………...28
2.4 HIV-1 & Neurotoxicity…………………………………………………………….....30
2.5 METH & Neurotoxicity…………………………………………………………...….34
2.6 Combined effects: METH, HIV-1 & Neurotoxicity………………………………….37
2.6.1 Blood Brain Barrier…………………………………………………………....39
2.6.2 Astrogliosis…………………………………………………………………....40
2.6.3 Astrocyte-Induced excitotoxicity……………………………………………...42
2.7 Role of Glutamate in the CNS………………………………………………………..45
2.8 Transporters & Receptors…………………………………………………………….46
2.8.1 Excitatory amino acid transporters (EAATs)………………………………....46
2.8.2 Vesicular glutamate transporter-1 (VGLUT-1)…………………………….…48
2.8.3 Metabotropic glutamate receptors………………………………………….…49
2.8.4 Ionotropic glutamate receptors………………………………………………..50
2.9 Synthesis and metabolism of glutamate…………………………………………..…..51
vi 2.9.1 Glutaminase………………………………………………………………...…51
2.9.2 Glutamine synthetase………………………………………………………….52
2.10 Concluding Remarks……………………………………………………………..….54
III. METHAMPHETAMINE AND HIV-1-INDUCED NEUROTOXICITY: ROLE OF
TRACE
AMINE
ASSOCIATED
RECEPTOR
1
CAMP
SIGNALING
IN
ASTROCYTES……………………………………………………………………….….……..55
3.1 Abstract……………………………………………………….………………...……56
3.2 Introduction……………………………………………………………….…………..57
3.3 Materials and Methods………………….…………………………………………....59
3.4 Results.....……………….…………………………………………………………....64
3.5 Discussion and Conclusion.…………………………………..…………………..….68
3.6 Figures……………………………………………………..……………………...….73
3.7 Supplementary Data………………………………………………………………..…81
IV. METH-INDUCED, TAAR1-ASSOCIATED CREB SIGNALING SERVES AS A
MASTER REGULATOR FOR ASTROCYTE EAAT-2….………………………….……...82
4.1 Abstract…………………….………………………………………………………...83
4.2 Introduction……………………….……………………………………………….....84
4.3 Materials and Methods………………………………………………………….........89
vii 4.4 Results..…………………………………….………………………………………...95
4.5 Discussion and Conclusion………………………………………………..………..101
4.6 Figures……………..……………………………………………..…….…………...107
V. SUMMARY AND DISCUSSION……………………………………...………….………123
Future Directions…………………………………………………………………....132
VI. BIBLIOGRAPHY………………………………………………………………...............135
viii ABBREVIATIONS
Ab, Amyloid beta; AIDS, Acquired immunodeficiency syndrome; Akt, Protein kinase B;
AMPA, α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPH, Amphetamine;
ANOVA, Analysis of variance; ART, Anti-retroviral therapy; ATP, Adenosine triphosphate; βPEA, beta-phenylethylamine; BBB, Blood brain barrier; Bcl2, B-cell lymphoma 2; Ca2+,
Calcium; [Ca+2]I, intracellular calcium concentrations; CaMKII, Ca+2/Calmodulin-dependent
protein kinase II; cAMP, cyclic adenosine monophosphate; CCL2; monocyte chemotactic
protein 1; CCR5, C-C chemokine receptor type 5;
CNS, Central nervous system; COX,
Cyclooxygenase; CREB, cAMP response element binding protein; CSF, cerebral spinal fluid;
DA, dopamine; DAPI, 4',6-diamidino-2-phenylindole; DAT, dopamine transporter; EAAT-2,
Excitatory amino acid transporter-2; ECM, Extra cellular matrix; Egr-1, Early growth response
protein 1; EPPTB, N-(3-ethoxyphenyl)-4-pyrrolidin-1-yl-3-trifluoromethylbenzamide; ER,
endoplasmic reticulum; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GFAP, Glial
fibrillary acid protein; GLT-1, Glutamate transporter; GLS, glutaminase; GPCR, G-protein
coupled receptor; GS, Glutamine synthetase; HAART, Highly active ART; HAND, HIV-1associated neurocognitive disorders; HIV-1, Human immunodeficiency virus-1; HIV-1Tat, HIV1 Trans-activator of Transcription; iGluRs, iontropic glutamate receptors; IL, Interleukin; iNOS,
Inducible NO synthase; IP3, 1,4,5-triphosphate; K+, Potassium; LDH, Lactate dehydrogenase;
LPS, lipopolysaccharide; LTP, Long term potentitation; LTR, Long terminal repeats; MAO,
Monoamine oxidase; MAPK, Mitogen activated protein kinases; METH, Methamphetamine;
mGluRs, metabotrobic glutamate receptors; MMP, Matrix metalloproteinases; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Na+, Sodium; NF-κB, Nuclear factor-
ix kappaB; NLS, Nuclear localization sequence; NMDA, N-methyl-D-aspartate; NMDAR, Nmethyl-D-aspartate receptor; NO, Nitric oxide; PBS, Phosphate-buffered saline; PI3K/Akt,
Phosphoinositide 3-kinase; PKA, Protein kinase A; PKC, Protein kinase C; ROS, Reactive
oxygen species; TAAR1, Trace amine associated receptor 1; TH, Tyrosine hydroxylase; TJ,
Tight junction proteins; TNF-α, Tumor necrosis factor-alpha, VGLUTs, Vesicular glutamate
transporters, VMATs, Vesicular monoamine transporters
x LIST OF TABLES
CHAPTER I
Table 1
Prevalence of METH abuse in 2013………………………………………………6
xi LIST OF ILLUSTRATIONS
CHAPTER I
Figure
1.1 Diagnosis of HIV Infection among adults and adolescents by sex and transmission
category, 2011…………………………………………………….................................14
1.2 METH and HIV-1 modulate astrocyte activation…………………………………..….19
Schematic
1.1 Methamphetamine pharmacodynamics in dopaminergic signaling…………...……….10
1.2 Astrocyte function at the blood brain barrier………………………………………...…16
xii CHAPTER II
Figure
2.1 METH, HIV-1 and inflammatory mediators alter EAAT-2 mRNA levels…………….......44
Schematic
2.1 Mechanisms of astrocyte-mediated glutamate regulation……………………………………53
xiii CHAPTER III
Figure
3.1 METH and HIV-1 modulate excitotoxicity and astrocyte viability.………….....73
3.2 Astrocyte TAAR1 expression and localization is regulated by METH and
HIV-1…..…………………………………………………………………….…..74
3.3 Forskolin, METH and β-PEA induce astrocyte intracellular cAMP
signaling………………………………………………………………………....76
3.4 RNA interference downregulates astrocyte TAAR1 protein levels and
intracellular cAMP in response to METH and β-PEA……………………..........77
3.5 METH-induced TAAR1 activation regulates EAAT-2 mRNA levels and
function………………………………………………………………………..…79
Supplementary Figure
3.6 TAAR1 overexpression model in primary human astrocytes……………………..81
xiv CHAPTER IV
Figure
4.1
EPPTB reversed METH-induced decreases in EAAT-2 levels/function and prevented
METH-induced cAMP increases…………………………………………………...…107
4.2
IL-1β regulated astrocyte TAAR1/EAAT-2 levels
and increased METH-mediated intracellular cAMP levels………………………..….109
4.3
NF-κB downregulation significantly increased EAAT-2
levels……………………………………………………………………...…………...111
4.4
METH-induced PKA activation regulated EAAT-2 levels……………………...…..112
4.5
METH increased intracellular calcium fluorescence in
GCaMP6s expressing astrocytes……………………………………………………..114
4.6
METH-induced phosphorylation of CaMKII and PKC………………...…………....116
4.7
METH activated CREB in primary human astrocytes……………………………….117
4.8
PKA knockdown decreased METH-mediated CREB phosphorylation………...…...118
4.9
TAAR1, PKA and NF-κB knockdown resulted in
differential CREB phosphorylation………………………………………………….119
4.10 METH and IL-1β phosphorylated pCREBSer133/142
in the nucleus………………………………………………..……………………….121
xv CHAPTER V
1. Implications of METH-induced TAAR1 activation exacerbating HAND….…………...141
xvi Chapter 1
INTRODUCTION
INTRODUCTION
Methamphetamine (METH) is a neurotoxin and potent psychostimulant with
central nervous system (CNS) effects including strong, long lasting euphoria attributed to
the cascading release of the neurotransmitter, dopamine (1).
More than 12 million
Americans have tried METH at least once in their lifetimes with approximately 1.5% of
them reporting habitual use (2). Furthermore, METH users are 20% more likely to
contract sexually transmitted diseases. METH contributes to direct biological and
physiological effects that increase an individual’s risk of contracting human
immunodeficiency virus (HIV) (2). Accurate statistics documenting the numbers of HIV1 infected individuals who are METH abusers is not available; however, the prevalence
of HIV-1 infections with METH abuse is high. Between 2005-2009 nearly 1 in 4 HIV-1
infected individuals reported treatment for the use of drugs and alcohol with intravenous
drug use accounting for ¼ of HIV/AIDs cases (The NSDUH Report: HIV/AIDS and
Substance Use, 2010, Substance Abuse and Mental Health Services Administration:
Rockville, MD). Thus, mechanisms resulting in combined clinical outcomes of the two
are of significant importance.
HIV-1 results in a progressive failure of the immune system, infecting
macrophages that cross the blood brain barrier (BBB) resulting in neurotoxicity, the
hallmark of HIV-associated dementia (HAD) (3). Additionally, METH addiction and
dependence is correlated with the development of neurocognitive disorders, and
therefore, neurotoxicity is a critical consequence of both METH abuse and HIV-1
infection. Further, studies indicate that METH dependence has an additive effect on
neuropsychological deficits associated with HIV-1 infection and accelerates the severity
2 and onset of HAD and common neurotoxic pathways including excitotoxicity, glial cell
activation and brain hyperthermia (4, 5).
Astrogliosis is an important pathological commonality of METH abuse and HIV1 CNS infection. METH/HIV-1-induced glial cell activation is of specific importance due
to the abundance of astrocytes in the CNS and their contribution to excitotoxicity, a
common mechanism in neurodegenerative and neurotoxic disorders including
METH/HIV-1 combined injury (6, 7). Extracellular levels of glutamate in the CNS
microenvironment
are
tightly
regulated
via glutamate
transporters
expressed
predominantly on astrocytes, and excitotoxicity is a direct result of brain glutamate
dysregulation. During homeostasis, rapid removal of glutamate in the synapse via
glutamate transporters ensures optimal glutamate balance, while in brain injury or
disease, glutamate transporter expression and function may be impaired. Moreover, in
such conditions, transporters can also work in reverse, boosting extracellular glutamate
accumulation in the synapse (8).
Excitatory amino acid transporter-2 (EAAT-2) is responsible for 90% of
glutamate clearance from the synapse and is primarily localized in perisynaptic astrocytes
(8). EAAT-2 dysregulation leads to astrocytes reduced ability to clear glutamate. We
have recently described a novel G-protein coupled receptor (GPCR) in human astrocytes
with promising implications in the regulation of EAAT-2 and glutamate clearance termed
trace amine associated receptor 1 (TAAR1). Downstream signaling of TAAR1 include
increased intracellular cyclic adenosine monophosphate (cAMP), increased intracellular
calcium [Ca+2]i levels and activation of protein kinase A (PKA) and protein kinase C
(PKC), both implicated to regulate EAAT-2 at the transcriptional and post-translational
3 levels. Furthermore, a downstream-phosphorylated target of TAAR1 signaling includes
CREB, an EAAT-2 transcription factor, with co-activating and co-repressor forms.
Additionally, HIV-1 relevant proinflammatory stimuli, IL-1β, contributes to EAAT-2
regulation via signaling through NF-κB. Elucidating the converging and temporal order
of METH- and HIV-1-induced signaling cascades will reveal a better understanding of
the master regulators involved in astrocyte-mediated glutamate clearance, uncovering
therapeutic targets for the attenuation of METH- and HIV-1- induced excitotoxicity.
1.1 METHAMPHETAMINE ABUSE
METH belongs to the phenethylamine and amphetamine class of controlled
substances and is a serious problem in the United States (US) and around the world (10).
Approximately 5% of the US population has tried METH at least once in their lifetimes.
A National Survey on Drug Use and Health: Trends on the Prevalence of METH abuse in
2013 showed that approximately 3.5% of individuals between 12 and 25 years old have
used METH in their lifetime, with approximately half being estimated in the past year
(Table 1.1). Additionally, the consequences of METH abuse on the US economy in 2009
were estimated to be approximately $23.4 billion (RAND Corporation report, 2009).
METH is classified as a Schedule II drug and regulated under the Controlled
Substance Act, available through a prescription (11). It is clinically prescribed to treat
hyperactivity disorders, obesity and narcolepsy, at low doses and for limited periods of
time (11). METH is used recreationally as a “club drug” with effects lasting 6-8 hours
and as long as 24 hours after use (12). METH is taken orally, snorted, smoked or injected
and results in physical effects including increased wakefulness, loss of appetite, increased
4 physical activity, increased respiration, irregular heart beat, increased blood pressure and
hyperthermia (13). Users compulsive drug seeking behavior is a result of intense
euphoria making METH an extremely addictive substance with high abuse potential.
Chronic METH abuse systematically destroys the body and leads to METH-induced
psychosis characterized by a loss of social contact and experiences of delusion, paranoia,
hallucinations and obsessive/compulsive disorders (14).
5 Prevalence of METH abuse in 2013 (in percentage)
DRUG
Methamphetamine
Table 1.1:
TIME
PERIOD
Lifetime
Past Year
Past Month
AGES 12 – 17 AGES 18-25 AGES 26 or OLDER
0.50
3.00
5.50
0.30
0.90
0.40
0.10
0.30
0.20
A National Survey on Drug Use and Health: Trends on the
Prevalence of METH abuse in 2013
Individual age groups were surveyed about their habitual use of METH in their
lifetime, within the past year and within the past month.
Modified Table from A National Survey on Drug Use and Health: Trends on the
Prevalence
of
METH
abuse
in
2013,
www.drugabuse.gov/drugsabuse/methaphetamine
6 1.1.1 Distribution and Pharmacokinetics
METH is distributed throughout the majority of organs in the body (15). Positron
emission tomography (PET) demonstrates that the order of highest METH uptake occurs
in the lungs, liver and brain however the brain remains one of the last organs to clear
METH (15). METH’s bioavailability is ~ 90.3 +/- 10.4% in human subjects administered
METH via smoke inhalation. The elimination half-life was calculated at 11.1 hr for
inhalation of METH compared to 12.2 hr for intravenously administered METH (16).
This data suggest that METH metabolism occurs slowly and distribution throughout the
body may attribute to the physiological and psychological effects that occur with METH
abuse.
Within the CNS METH results in neurotoxicity of the frontal cortex and
hippocampus and causes cell death and damage to the dopaminergic and glutamatergic
nerve terminals in the striatum (Source: NIDA NOTES, Vol. 15, No. 4, September, 2000).
The reward pathways are activated by the neurotransmitter, dopamine that is synthesized
in nerve cells located in the ventral tegmental areas (17). Additionally, METH-mediated
cognitive deficits are related to changes in glutamatergic signaling within the prefrontal
cortex (Parsegian, 2014). Long-term mental health effects such as METH-induced
psychosis are a result of aberrant dopamine and glutamate activity and signaling
communication between the cortex and the striatum (18). Neuroimaging of adult METH
users show enlarged striatal volume with reduced dopamine transporters and receptors.
Furthermore, decreased levels of serotonergic receptors and vesicular monoamine
transporters were observed in striatal subregions. Dysregulated glucose metabolism in
the orbitofrontal/limbic regions was correlated to the development of psychosis (18).
7 METH-induced cell death in the prefrontal cortex, striatum and hippocampus occurs via
molecular mechanisms including mitochondrial damage and endoplasmic reticulum stress
(21), increased caspase 3 and Fas/FasL cell death pathways (22) and Bcl-2 gene
alterations (23). This demonstrates that METH abusers have abnormalities in their brain
chemistry and structures.
METH acts on several neurotransmitter receptors including, dopamine, serotonin
and norepinephrine. Furthermore, it effects dopaminergic and glutamatergic signaling,
specifically associated with the nigrostriatal, mesolimbic and mesocortical pathways (14).
Acute METH use immediately increases dopamine concentrations in the reward areas of
the brain allowing the user to experience pleasure and high levels of motivation,
however, the user quickly saturates dopamine concentrations producing a “binge and
crash” pattern of use in order to repeat that first high (19). These changes are associated
with the direct and indirect effects of dopamine acting as a substrate for neuronal
transporters (20). METH also causes a transient increase in glutamate concentrations
within the striatal areas of the brain.
Interactions between dopaminergic and
glutamatergic signaling can result in damages to the dopaminergic terminals in the
striatum. The intracellular distribution of METH also disrupts vesicular monoamine
transporters (VMATs) allowing increased cytoplasmic dopamine and glutamate
concentrations, reversal in the directionality of dopamine transporters (DAT) and
glutamate transporters in the plasma membrane and an efflux of dopamine and/or
glutamate into the synapse (20). Moreover, vesicular glutamate transporter synthesis and
expression increase with METH abuse, resulting in increased glutamate release into the
synaptic spaces. Molecular mechanisms associated with DAT reversal in membrane
8 orientation has been proposed to be via METH binding to TAAR1 in neurons. METHinduced TAAR1 activation leads to DAT phosphorylation via PKA and PKC, trafficking
the transporter intracellularly and reversing the orientation in the membrane.
Additionally, TAAR1 has been implicated in regulated glutamatergic neurotransmission
via regulation of glutamate receptors and transporters. Increased dopamine in the
extracellular space produces continuous firing of dopamine receptors, saturation of the
signal and dopaminergic terminal damage (Schematic 1.1) (10). Finally, TAAR1 levels
in astrocytes are directly correlated to astrocyte EAAT-2 levels and function. Together,
this suggests METH-mediated regulation of neurotransmitters may be via TAAR1, which
is a promising target for the reduction of METH-mediated deficits in the CNS.
9 Modified from Adderall wikipedia
10 Schematic 1.1: Methamphetamine pharmacodynamics in dopaminergic signaling.
Methamphetamines transport across a presynaptic neuron through the membrane or
DAT. Intracellular localization of METH transports through VMAT2 to synaptic vesicles
stimulating release of dopamine into the cytosol. METH activates PKA/PKC signaling,
resulting in DAT phosphorylation. DAT becomes internalized and dopamine synaptic
concentrations increase. PKC mediates internalization and/or DAT reversal in
orientation.
Modified from Adderall wikipedia
11 1.2 HIV-1 EPIDEMIOLOGY
The HIV pandemic affects an estimated 33 million individuals worldwide with
the predominant form of the virus being, HIV-1. Currently, approximately 1.3 million
people live with HIV-1 infection in the US (24). Management of HIV-1 infection
appeared in 1987 with the release of the first FDA approved antiretroviral medication.
Combination therapy, or highly active anti-retroviral therapy (HAART), became common
practice for treatment and showed a 60%-80% decline in AIDS related deaths (25).
Despite the advent of HAART, HAND afflicts 40-50% of the HIV-infected population.
The severest form of HAND is HAD, described as cognitive, motor and behavioral
disorders and impairment of frontal-subcortical patterns (26). Neuropathological changes
of HAD occur in the subcortical and fronto-striatal areas of the brain, and include the
basal ganglia, deep white matter and the hippocampal regions (27). Neurocognitive
impairments of HIV-1 infected individuals affect functions associated with the
hippocampus including memory and long-term potentiation (LTP) (29). Additionally,
damage to the fronto-striatal circuitry is related to less activity of the prefrontal cortex,
underlying cognitive problems (30). The extended lifespan of HIV-1 infected individuals
allows the damaging effects of the virus to persist in the CNS including the release of
proinflammatory cytokines into the CNS microenvironment and prolonged activation of
glial cells generating long-term neuroinflammation and neurotoxicity (28).
12 1.3 COEXISTENCE OF METH ABUSE AND HIV-1 INFECTION
METH abusers are 20% more likely to acquire HIV-1 infection. Approximately 1.1
million Americans are currently living with HIV/AIDS, with an estimated 50,000 new
infections occurring in the US (31, 32). Accurate statistics documenting the number of
HIV-1 infected individuals who are METH abusers is not available; however, the
prevalence of HIV-1 infections with METH abuse is high. The Center for Disease
Control and Prevention reports that in nearly 40,000 males surveyed, approximately 6%
contracted HIV via injection drug use. The statistics for women were even higher. Out
of approximately 10,500 females surveyed, about 14% contracted HIV from injection
drug use (Fig 1.1). Moreover, nearly 69% of HIV+ patients experience neurocognitive
deficits to some extent, with 8% of acquired immune deficiency syndrome (AIDS)
patients developing HAD (33). METH dependence has an additive effect on
neurophysiological deficits associated with HIV-1 infection and accelerates the severity
and onset of HAND (34-37). In part, the neurological complications in both METH abuse
and HAND are associated with cellular and molecular mechanisms such as glial cell
activation, oxidative stress, neuroinflammation and excitotoxicity (34-38). Coexistence of
HIV/AIDS and METH abuse continues to be a significant societal burden.
13 Diagnoses of HIV Infection among Adults and Adolescents
by Sex and Transmission Category, 2011 (United States and
6 Dependent Areas)
Figure 1.1: Modified from Center for Disease Control and Prevention:
Data Includes persons with HIV infection regardless of stage of disease.
Other includes: hemophilia, blood transfusions and perinatal exposure
14 1.4 EMERGING ROLE(S) OF ASTROCYTES IN HAND AND METH ABUSE
ASSOCIATED NEUROTOXICITY
Despite HIV-1-induced neuropathology, the virus does not actively infect
neurons, rather neuronal dysfunction and toxicity is an indirect consequence of
dysfunctional astrocytes (39). Additionally, astrocytes are a direct target for drugs of
abuse, including METH. Astrocyte activation is an important commonality of METH
abuse and HIV-1 CNS infection. Astrogliosis is multifaceted and ranges from
upregulation of glial fibrillary acidic protein (GFAP), morphological changes, cellular
proliferation, calcium (Ca2+) signaling imbalance, oxidative stress and excitotoxicity
(Scheme 1.2) (40-43).
15 Scheme 1.2: Astrocyte functions at the BBB. Astrocytes are responsible for the integrity
of the BBB by providing ion concentration maintenance, structural support and acting as
a buffer to pathogens and lipophilic compounds that transport across the BBB. HIV-1
infected monocytes cross the BBB and infect the brain microglia that are responsible for
productive HIV-1 infection in the brain. Furthermore, METH is a lipophilic compound
that readily crosses the BBB where it downregulates tight junction proteins decreasing
the BBB integrity and allowing more virus to enter. HIV-1 replication results in
accumulation of toxic compounds and release of proinflammatory cytokines and
chemokines, IL-1β and TNF-α and viral proteins like Gp120, Tat and Vpr, promoting
activation of astrocytes. Moreover, METH results in upregulation of GFAP and
production of ROS and mitochondrial damage. Reactive astrocytes enhance the BBB
16 permeability to promote increased monocyte infiltration into the brain. Simultaneously,
reactive astrocytes increase intracellular Ca+2 and glutamate levels by reducing the
uptake of glutamate, thereby resulting in excitotoxicity.
17 1.4.1 Astrogliosis/Morphological Changes
METH treated astrocytes exhibit morphological changes such as a stellate
appearance, an increase in vacuoles, increased GFAP levels and heterogeneity (44).
METH and HIV-1 Tat combined treatment in astrocytes results in the degradation of
extracellular matrix proteins via increased activity of proteinases that are essential in
cytokine signaling (45). The contribution of astrogliosis to METH neurotoxicity
continues to be an active area of research (50, 51). Gliosis during HIV and METH comorbidities is often measured by correlative changes in GFAP immunoreactivity (50-53).
Astrocytes treated with METH and HIV-1, alone or in combination, demonstrated
reactive astrocyte phenotypes (Fig. 1.2).
Therefore, METH and HIV-1 affected
astrocytes fail to provide metabolic and trophic support for optimal neuronal health and
release neurotoxic molecules and promote inflammation.
18 Figure 1.2: METH and HIV-1 inducee astrocyte activation. Primary human astrocytes
were stimulated with METH (500 µM) ± HIV-1 (p24 10 ng/mL) for 24 h. Astrocytes were
fixed with acetone:methanol (1:1) for 30 mins at 4°C and stained for GFAP and DAPI.
Cell morphology was visualized by immunofluorescence, GFAP (green) and DAPI (blue).
A) Astrocyte activation was observed upon treatment with +/-METH (B), HIV-1 (C) and
METH + HIV-1 (D).
19 1.4.2 Calcium Signaling/Oxidative Stress
In addition to reactive astrogliosis, a common characteristic of METH and HIV-1
infection in the brain is oxidative stress, through both direct and indirect mechanisms.
METH-induced dopamine release into the synapse by neurons, produces a dosedependent increase in [Ca+2]i stimulating transient Ca+2 uptake in mitochondria (54). The
production of aldehydes and hydrogen peroxide occurs as a by product of dopamine
metabolism, inducing lipid peroxidation and activating phospholipase C, and additional
IP3-dependent Ca+2 release from ER and increases in [Ca+2]i by a receptor-independent
mechanism. Astrocytes sensitivity to dopamine- and glutamate-induced [Ca+2]i flux is
responsible for dysregulation of cellular redox homeostasis (55).
1.4.3 Glutamate Homeostasis
METH-induced dopamine signaling activates glutamatergic signaling in the
cortex and increased dopamine concentrations in the prefrontal cortex (57). Further,
synergistic activation of dopamine receptor 1 and 2 increases glutamate signaling in the
striatum (58).
Glutamatergic signaling leads to LTP and learning of activities and
persistent behaviors (59). Moreover, excessive glutamate release increases [Ca+2]i and
downstream activation of proteolytic enzymes, reactive oxygen species generation and
apoptotic pathway activation, ultimately resulting in cellular damage (Bruno et al.,
1993 and Nicholls, 2004).
Glutamate is the major signal transduction neurotransmitter and mediator of
excitatory signals in the mammalian CNS. Glutamate mediates a large amount of
information that regulates brain development and is involved in normal brain function
20 including cognition, memory and learning (60). Glutamate, although essential for brain
function, must be maintained in optimal extracellular concentrations and is therefore,
primarily localized intracellularly (60). The chemical nature of glutamate is dependent
on binding and activating glutamate receptors on the exterior of the cell, in the synaptic
space. The resting concentration of glutamate is a few micromolar in the extracellular
environment. In contrast, intracellular glutamate concentrations are approximately 1-10
millimolar (60).
Moreover, concentrated glutamate in synaptic vesicles of nerve
terminals reach as high as 100 millimolar (60). Mechanisms contributing to excitotoxicity
include downregulation and dysfunction of glutamate transporters, glutamate receptors,
and glutamate synthesizing and metabolizing enzymes in astrocytes; however, the
mechanisms that regulate these functions are unknown (64).
METH increases rat striatal extracellular glutamate concentrations via activation of
the striatonigral pathway (61, 62). Inhibiting glutamate release was neuroprotective
against METH damage to the dopamine terminals in the striatum (62). Increased
oxidative stress through ROS/NOS is suggested to regulate glutamate concentrations via
EAAT-2, mGluRs, VGLUTs and glutamate metabolic enzymes (37, 63). Analysis of the
EAAT-2 promoter showed that NF-κB and CREB are important regulators of EAAT-2
transcription in astrocytes (65) and the rate of NF-κB translocation into the nucleus is
regulated by the activation of PKA, downstream of cAMP (66).
The canonical cAMP pathway involves PKA activation, however, PKC has been
reported downstream of METH-induced TAAR1 activation and increases [Ca+2]i levels in
neurons (67, 68). These pathways regulate genes involved in cell cycle, cell survival,
21 cytokine secretion (69) and CREB responsive genes. Therefore, astrocytes present
themselves as therapeutic targets for the attenuation of METH-induced excitotoxicity.
1.5 TRACE AMINE ASSOCIATED RECEPTOR 1 (TAAR1)
METH’s downstream effects on astrocyte gene expression and function suggest a
molecular mechanism for METH signaling. TAAR1 is a stimulatory GPCR identified to
bind trace amines, such as β-phenylethylamine (β-PEA), tyramine, octopamine, and the
psychostimulant METH, in neurons (70). TAAR1 regulates brain monoamines and
mediates the action of amphetamine stimulants (67). Localization of TAAR1 has been
shown in a subset of dopamine neurons, co-localized with DAT (71). It influences
subcortical monoaminergic transmission and has shown potential antipsychotic,
antidepressant and pro-cognitive effects in vivo models. In cells transfected with flagged
rat TAAR1, there was a widespread intracellular distribution of TAAR1 (72).
Furthermore, TAAR1 never trafficks to the membrane suggesting its activity is restricted
to the intracellular environment. TAAR1 expression in rhesus monkey and mouse brain
coincides with the specific labeling of brain nuclei of the substantia nigra, dorsal raphe
nucleus, ventral tegmental area, hypothalamus, preoptic area, amygdala, solitary tract,
parahippocampal region and the subculum (73). TAAR1 has been identified to have
specific expression patterns in the prefrontal cortex, and therefore may play a role in
cognition (74).
Additionally, TAAR1 agonists and antipsychotic drug, olanzapine
showed the highest TAAR1 activation in the medial prefrontal cortex (75). TAAR1
activation increases intracellular cAMP, PKA and PKC (76, 77). PKA phosphorylates
specific proteins that bind to the promoter regions of DNA, causing modulated
22 expression of specific genes. cAMP also has minor PKA-independent functions, such as,
activation of calcium channels (77). Furthermore, activation of PKC is receptor-driven
and is initiated by TAAR1 (67). TAAR1-mediated DAT internalization by METH was
prevented with a PKC inhibitor but not with a PKA inhibitor, suggesting TAAR1 induced
activation of PKC regulates EAAT-2 post-translational modifications but not intricately
involved in EAAT-2 transcription.
1.6 ASTROCYTE-TAAR1 AS A NOVEL MECHANISTIC AND THERAPEUTIC
TARGET FOR METH ABUSE IN HAND
We recently identified TAAR1 as a novel stimulatory GPCR in human astrocytes
with promising implications in the regulation METH-induced, astrocyte-mediated gliosis
and excitotoxicity. METH is a partial agonist for TAAR1 (78). Recent reports identify
several
therapeutic
drugs
that
decrease
cocaine
seeking
behavior,
dopaminergic/glutamatergic activity, and stress-induced hyperthermia via TAAR1 (75,
79). Moreover, TAAR1-knockout mice have dysregulated cortical glutamate transmission
and aberrant behavioral tests (74). TAAR1 has been identified to contribute to the
pathophysiology of schizophrenia due to the effects of increasing cortical glutamatergic
transmission and the involvement on N-methyl-D-aspartate (NMDA) receptor regulation
(74).
Additionally,
TAAR1
antagonist,
N-(3-ethoxyphenyl)-4-pyrrolidin-1-yl-3-
trifluoromethylbenzamide (EPPTB), has been shown to act as a therapeutic compound in
targeting TAAR1 in the brain, with a high bioavailability and lipophilicity (80). Our
studies center on the mechanisms through which astrocyte-TAAR1 mediates METH
comorbidity in HAND. These data will prove highly relevant as they delineate the role of
23 TAAR1 in the context of METH involvement during HAND, both from basic science and
translational perspectives. The unique properties of TAAR1 in modulating dopamine and
glutamate properties of drugs and psychological disorders; warrants this GPCR as a
promising target for therapeutic intervention in the CNS.
1.7 OBJECTIVES OF THE PRESENT STUDY
METH abuse has long been associated with transmission of HIV-1 infection, both
rapidly resulting in cognitive disorders, in an additive and/or synergistic manner.
Pharmacological treatments for METH can dampen the severity of HIV-1 neurocognitive
disorders. Therefore, two key areas of impact from our studies include, identifying
TAAR1 as a target to attenuate METH-mediated effects in astrocytes; identifying
common pathways that exacerbate excitotoxicity following METH and HIV-1
cotreatment.
The proposed studies are innovative in multiple aspects:
First, we will uncover novel signaling pathways that link astrocyte-TAAR1 to
important astrocyte-mediated regulatory mechanisms in health and HIV, particularly in
the context of substance abuse.
Second, novel regulatory circuits will be explored involving our newly described
METH receptor on human astrocytes to uncover intricate signaling between cAMP,
calcium and excitotoxicity. These will have broader implications for substance abuse
related neurodegeneration.
24 Chapter 2
ROLE OF GLUTAMATE IN THE CNS AND REGULATION DURING METH- &
HIV-1-INDUCED NEUROTOXICITY
25 2.1 ABSTRACT
Glutamate, the most abundant excitatory transmitter in the brain can lead to
neurotoxicity when not properly regulated. Excitotoxicity is a direct result of abnormal
regulation of glutamate concentrations in the synapse, and is a common neurotoxic
mediator associated with neurodegenerative disorders. It is well accepted that METH, a
potent central nervous stimulant with high abuse potential, and HIV-1 are implicated in
the progression of neurocognitive malfunction. Both have been shown to induce common
neurodegenerative effects such as astrogliosis, compromised blood brain barrier integrity,
and excitotoxicity in the brain. Reduced glutamate uptake from neuronal synapses likely
leads to the accumulation of glutamate in the extracellular spaces. Astrocytes express the
glutamate transporters responsible for majority of the glutamate uptake from the synapse,
as well as for vesicular glutamate release.
However, the cellular and molecular
mechanisms of astrocyte-mediated excitotoxicity in the context of METH and HIV-1 are
undefined. Topics reviewed include dysregulation of the glutamate transporters,
specifically EAAT-2, metabotropic glutamate receptor(s) expression and the release of
glutamate by vesicular exocytosis. We also discuss glutamate concentration dysregulation
through astrocytic expression of enzymes for glutamate synthesis and metabolism. Lastly,
we discuss recent evidence of various astrocyte and neuron crosstalk mechanisms
implicated in glutamate regulation.
Astrocytes play an essential role in the
neuropathologies associated with METH/HIV-1-induced excitotoxicity. We hope to shed
light on common cellular and molecular pathways astrocytes share in glutamate
regulation during drug abuse and HIV-1 infection.
26 2.2 INTRODUCTION
METH is a psychostimulant that results in strong, long-lasting, euphoric effects.
METH heightens the libido and impairs judgment, which can lead to risky sexual
behavior, increasing an individual’s risk for acquiring HIV-1. Neurotoxicity is a
consequence of both METH abuse and HIV-1 infection. HIV-1 results in cognitive
deficits collectively referred to as HAND and the most severe form, HAD. METH/HIV-1
combined neurotoxicity share similar results including excitotoxicity, oxidative stress,
glial cell activation, inflammation and hyperthermia (81, 82). Clinical studies indicate
that METH dependence has an additive effect on neuropsychological deficits associated
with HIV-1 infection (34); however, the direct mechanism of action for METH/HIV-1induced neurotoxicity remains unclear. Astrocytes, the most abundant cells in CNS, are
affected by METH and HIV-1, eventually leading to neuronal dysfunction via both direct
and indirect mechanisms. Excitotoxicity, a direct result of glutamate dysregulation, is a
common mechanism of many neurodegenerative and neurotoxic disorders. Transporters
located predominantly on astrocytes tightly regulate glutamate, the most common
neurotransmitter in the brain, by rapidly removing glutamate from the extracellular space
(64). During brain injury or disease, glutamate transporters work in reverse, permitting
an excessive glutamate accumulation outside the cells further increasing synaptic
glutamate concentrations (83). Glutamate transporters also regulate the expression of
glutamate receptors in neurons and their own metabotropic glutamate receptors (mGluRs)
(84). Furthermore, astrocytes possess glutamate synthesis and degradation enzymes (85,
86). Other neurotoxic mechanisms induced by METH and HIV-1; such as astrogliosis,
BBB impairment, excitotoxicity, and inflammation, are likely implicated in intracellular
27 signaling events by which astrocyte modulate glutamate uptake, synthesis and
metabolism.
2.3. ROLE OF ASTROCYTES IN THE CENTRAL NERVOUS SYSTEM
Astrocytes are the most abundant cell type of the central nervous system
responsible for monitoring the integrity of nervous tissue (7). Astrocytes are highly
sensitive and responsive to injury, resulting in extension of their processes, excessive
proliferation and upregulation of GFAP, implicated in transient neuroprotection. In vitro
studies have provided evidence that astrocytes take up or release glutamate in a calciumdependent manner (87) and bind glutamate to produce a functional response through
metabotropic and ionotropic glutamate receptors (iGluRs) (84). Astrocytes also release
glutamate, D-serine, and ATP in a calcium regulated manner (88). These molecules are
responsible for neurotransmission and excitotoxicity, implicating astrocytes in the
regulation of synaptic transmission via crosstalk with neurons (89, 90).
Primary insult to astrocytes occurs along the BBB where they are responsible for
endothelial cell support. The relationship between neurons, astrocytes and blood vessels
makes astrocytes a vital mediator modulating neuronal activity and cerebral blood flow.
Neuropathologies are often associated with the ability of neurotoxic mediators to cross
the BBB.
Inflammatory mediators released from astrocytes compromise the BBB
integrity allowing the passage of otherwise restricted substances into the brain.
Proinflammatory cytokines tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, and IL-6
disrupt endothelial cell function via astrocyte activation (91). These cytokines allow for
increased BBB permeability via microfilament reorganization, NF-κB activation and
28 differential expression of tight junction (TJ) proteins (92, 93). Proinflammatory cytokinemediated release of matrix metalloproteinases (MMP)-9 and -13 further affects BBB
permeability through remodeling and degradation of extracellular matrix proteins (ECM)
(94, 95). Astrocytes are also the main producers of the tissue inhibitors of MMPs
(TIMPs) in the brain (96, 97). Dysregulation of astrocyte TIMP-1 further contributes to
ECM remodeling and degradation (96, 98, 99).
Activated astrocytes in the HIV-1-infected CNS directly affect the BBB via gap
junctions (100). Endothelial cell apoptosis is a result of dysregulation of lipoxygenase
and cyclooxygenase (COX), large/big (Ca+2)-activated (K+) channels (BK channels), and
ATP receptor activation within astrocytes (100). Gap junctions are also responsible for
transmitting various small signaling molecules such as calcium and chemokines.
Glutamate receptor activation propagate [Cai+2] waves in astrocytes (101). [Cai+2] and
inositol phosphate (IP3) increases are also associated with diffusion through gap junctions
from adjacent cells (102). James et al. compared [Cai+2] propagation upon activation
mGluRs and iGluRs (101). While increases in [Cai+2] levels were not significantly
different upon activation of mGluRs and iGluRs by ATP, glutamate and histamine, the
resultant gliotransmitters released were distinct.
Upregulation of GFAP is common characteristic feature observed in
neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and HAD
(103). The increased expression of GFAP is an effect exhibited during astrogliosis along
with cellular migration and extension of astrocyte processes. Ganesh et al. show
inhibition of reactive gliosis decreases protease levels, prevents apoptotic death, and
provides substantial neuroprotection (104). Neuroprotection is mediated by defending
29 neurons from oxidative stress, aiding in synaptic plasticity, and the release of
gliotransmitters such as glutamate and D-serine, CCL2 and IL-1β and neuronal growth
factors (105). Astrogliosis can also provoke injury; for example, astrocytes surrounding
damaged cells secrete ECM proteins, modifying the ECM and inhibiting neuronal
regeneration (106). Reactive astrocytes form a glial scar that can ultimately impair nerve
degeneration (105). The nuclear factor of activated T-cells (NFAT) is a transcription
factor family implicated in the activation of astrocytes responding to inflammatory
mediators IL-1β, TNF-α, ATP and glutamate (105, 107, 108) leading to increased
expression of COX-2 and TNF-α affecting neuronal cells in close juxtapositions (109).
NFAT activation requires translocation from the cytoplasm to the nucleus, triggered by a
[Cai+2]/calmodulin-dependent phosphatase, linking calcium signaling and NFATdependent gene transcription in astrocytes (110).
2.4. HIV-1 AND NEUROTOXICITY
HIV-1 primarily infects the cells of the immune system and progressive
immunodeficiency marks disease progression. HIV-1 primarily infects CD4+ T cells and
cells of the monocyte-macrophage lineage (111). During acute HIV-1 infection virus
replicates rapidly, leading to an abundance of virus in the peripheral blood. Traditionally,
the brain is considered to be an immune privileged organ. In parallel, antiretroviral
therapy (ART) drugs are also excluded from the brain creating a reservoir for virus until
replication is reactivated within the CNS. Thus, even HIV-1 infected patients whose
medications successfully control HIV infection in the periphery may eventually suffer
30 from HAND and information regarding extended, low level HIV-1 infection in the CNS
is limited.
HIV-1 has been linked to a number of neurological symptoms, even in the
absence of confounding diseases. HIV-1 penetrates the BBB, enters the CNS and causes
varying degrees of neurocognitive impairment or HAND, all characterized by cognitive,
motor and behavioral disorders (111). While HAD, a clinical correlate of HIV-associated
encephalitis, often occurs during active, chronic HIV-1 infection of the brain.
Approximately 15% of individuals infected with HIV-1 develop HAD, and
approximately 30% to 60% of individuals develop a less severe form minor cognitive
motor disorders (MCMD). During the pre-ART era, up to 25% of HIV-1-infected patients
developed HAD as the first AIDS-defining illness and early evidence of HIV-1
neuropathogenicity included HIV-1 in cerebrospinal fluid (CSF), abnormal neuroimaging,
and the presence of HIV-1 in tissue obtained by brain biopsies (112). CNS manifestations
observed during HIV-1 viremia associated with seroconversion, include meningitis,
encephalitis, facial palsy, and peripheral nerve disorders proposing HIV neuroinvasion
occurs early during infection.
Microglia, the residual macrophage in the CNS, play a crucial role in
neuroinflammatory disorders [reviewed by (113)]. During the disease process of AIDS,
HIV enters the CNS early in the course of infection, and the virus productively infects
and resides primarily in microglia and macrophages, [reviewed by (114)] although the
non-productive or latent infection of astrocytes has also been reported in the early
decades of HIV/AIDS (115). HIV-infected or immune-stimulated macrophages/microglia
produce neurotoxins, and inflammatory mediators (116-118). Macrophage- and
31 microglia-derived inflammatory cytokines such as IL-1β, TNF-α, arachidonate and its
metabolites such as platelet-activating factor (PAF), free radicals, chemokines, and viral
proteins have been investigated [reviewed by (114)].
While the BBB is important for the prevention of toxic mediators into the brain, it
is targeted to allow entry of HIV-1 infected monocytes into the brain as described by the
classical Trojan Horse hypothesis.
HIV proteins, Tat and gp120, and host
proinflammatory cytokines and chemokines compromise the BBB allowing free HIV-1
and transmigration of HIV-1-infected cells into the CNS (111). Astrocytes, localized in
this microenvironment, interact with endothelial cells and help regulate the BBB
physiology, protecting its integrity (100). Recently, Eugenin et al. describe a novel
mechanism for bystander BBB toxicity, which is mediated by low numbers of HIVinfected astrocytes and amplified by gap junctions, and induces apoptosis of non-infected
BBB cells (100). Furthermore, Ju et al. demonstrate upregulation of both TNF-α and
MMP-9 in response to HIV-1 Tat (119), a protein expressed by infected astrocytes, which
can in turn affect other astrocytes.
Some individuals report early neurological disorders suggested to be associated
with an immunological response to virus. TNF-α, IL-1β, and IL-6 regulatory effects
suppress acute HIV-1 infection in microglia, in contrast to the observed effects seen in
chronically infected monocytic cell lines (120). Although acute HIV-1 replication is
attenuated in the brain, HIV-1 mRNA expression in the CSF has been observed 8 days
post HIV-1 transmission (121). The brain serves as a reservoir for HIV-1, activated by
chronic proinflammatory cytokines. Dementia reflects neuronal loss and progressively
deteriorating CNS function. The mechanisms underlying neurological and behavioral
32 characteristics present in the first months after HIV-1 neuroinvasion are associated with
the HIV-1 Tat (122). HIV-1 Tat is a key mediator of neurotoxicity promoting potentiation
of glutamate and NMDA-triggered calcium, excitotoxic neuron apoptosis (123) and
activation of GFAP expression in astrocytes (124).
Thus, reactive astrogliosis and
glutamate excitotoxicity are both implicated in HIV-1 CNS effects.
HAND persists despite the development of ART (125). ART successfully
suppresses peripheral infection and has lessened the incidence, but not the prevalence of
HAD (126) as few common antiretroviral drugs penetrate the CNS. Furthermore, the
effects of ART on non-infected neural cells such as astrocytes are poorly studied. One
recent study reports that antiretroviral medications increase β-amyloid (Ab) expression
by neurons and accumulation of Aβ plaques is a common feature in HIV-1 infection
(127). Understanding the molecular and cellular mechanisms involved in response to low
level, chronic viral expression in the brain along with the effects of ART on neural cells
may further our understanding of HAND.
While the abundance of astrocytes in the brain would make them an ideal target
for HIV-1 infection astrocytes exposed to HIV-1 lack the ability to actively replicate the
virus and express viral regulatory genes (128). Hao et al. report HIV-1 enters astrocytes
through a receptor mediated endocytic pathway via HIV-1 gp120 in a concentration
dependent manner (129). Electron microscopy revealed HIV virions in clathrin-coated
pits in cytoplasmic vacuoles. Perhaps this is one of the mechanisms that lead to a delayed
or restrictive HIV-1 infection of astrocytes and only ~2% of the brain astrocytes are
likely infected by HIV-1.
33 2.5. METH AND NEUROTOXICITY
METH addiction is a growing public health concern.
METH users exhibit
physical and psychological deficits, differing with acute versus chronic use.
Acute
METH administration depresses appetite, increases wakefulness and alertness resulting in
more physical activity.
Rapid, irregular heartbeat increases blood pressure and
hyperthermia during acute METH exposure.
Acute psychological effects include
euphoria, increased energy levels, heightened libido and feelings of self-esteem and selfconfidence. Chronic abuse of METH contributes to anxiety, depression, aggressiveness,
social isolation, psychosis, mood disturbances, and psychomotor dysfunction.
Neuropsychological studies of chronic METH users detected deficits in attention span,
working memory, and decision-making.
These neuropsychiatric complications are
related to drug-induced neurotoxic effects including damage to dopaminergic and
serotonergic terminals [reviewed by (10)].
The high lipophilic nature of METH, the attached methyl group, allows it to cross
the BBB and cellular membranes increases the potentiation of neuropsychological
effects. Furthermore, BBB compromise has been shown in the hippocampus, 24 h post
METH injection, as measured by decreased TJ proteins, zonula occludens (ZO)-1,
claudin-5 and occludin, and increased activity and immunoreactivity of MMP-9 (130).
Increased BBB permeability potentiates METH distribution to the brain and dysregulates
dopamine (DA) concentrations. DA, a catecholamine, activates the psychological reward
system, preventing normal functioning of the DAT.
DAT dysregulation increases
synaptic DA by 12-fold, triggering the reward response. Psychological effects do not last
long, as tolerance increases the euphoric feelings decrease.
34 Chronic METH abuse
eventually depletes the DA storage, attenuating the users euphoria. DA metabolism
generates reactive oxygen species (ROS) and long-term increases in synaptic DA levels
and subsequent oxidative stress mediates its neurotoxicity. Consistent with this
observation reduction in DA production attenuates METH toxic effects (131).
Withdrawal symptoms occur with the abrupt disruption of METH abuse in
approximately 90% of cases. Withdrawal enhances irritability, fatigue and intense
craving for the drug linked to the recurrence of relapse.
Zorick et al. investigated
symptoms that appear during the first several weeks of METH abstinence, and how
cravings for METH evolve into psychiatric symptoms persisting beyond a month of
abstinence (132). They found that while depressive and psychotic symptoms largely
resolved within a week of abstinence, cravings did not decrease until the second week,
and then continued at a reduced level to the fifth week. Low straital DA functioning is
associated with relapse in METH abstinent individuals and is the large contributing factor
in relapse (132).
Importantly, protracted abstinence leads to a reversal of DAT
dysregulation in METH abusers and a clearer understanding of these mechanisms may
lead to therapeutic prevention in relapse (132). The direct mechanism inducing METH
neurotoxicity is vague. TAAR1, is a stimulatory GPCR localized on membranes of
dopaminergic neurons (133). Activated TAAR1 results in DAT internalization, reversal
in the membrane and efflux of DA into the synapse. TAAR1 is a potential therapeutic
target to effectively treating METH addiction.
Astrocytes have receptors located on their cell membranes for neurotransmitters
and peptides, such as glutamate and DA making them susceptible to drug toxicity (134).
METH sensitizes astrocytes, making them more vulnerable to their environment inducing
35 activation and increasing GFAP expression (82). METH-activated astrocytes modulate
glutamatergic transmission, and downregulate glutamate transporter and receptor
expression and activity. Astrocytes release neurotransmitter intermediates including:
glutamate, ATP, D-serine, adenosine, and cytokines, and can modulate neuronal activity.
Acute METH intoxication activates a large percentage of astrocytes, which may diminish
with chronic METH exposure (7).
Astrocytes exhibit a form of excitability and communication through changes in
[Cai+2].
The activation of glutamate receptors are coupled to various intracellular
signaling cascades, including activation of PKC, Akt, cAMP/PKA/ cAMP responsive
element binding protein (CREB), and mitogen-activated protein kinase (MAPK) and
janus kinase (JAK) pathways (135). There is limited information concerning the
mechanisms that underlie METH-induced astrocytic responses but METH has been
implicated in the long-lasting PKC-dependent behavioral sensitization of astrocytes (136).
Activation of astrocyte mGluRs in astrocytes results in reduced intracellular cAMP. A
reduction of intracellular cAMP and activation of Akt pathway are required for mGluR to
exhibit cytoprotective characteristics, but cAMP levels are mediating the activation of the
PI3 kinase (PI3K)/Akt pathway (137). Chronic METH use is also associated with
decreases in the expression of CREB, which modulates immediate early genes (IEG)
expression via activation of the cAMP/PKA/CREB signal transduction pathway (138).
The METH-induced renormalization of the expression of several IEGs in rats chronically
exposed to METH introduces a cellular mechanism that may be responsible for relapse in
METH abstinent individuals. Nitric oxide (NO) insult in astrocytes also affects signaling
pathways that are associated with EAAT-2 regulation. NO insult causes dimerization of
36 p65-c-rel, NF-κB subunits, and the reduced production of cAMP and the activation of the
PI3K/Akt pathway reduce the dimerization of NF-κB subunits, allowing for recuperation
of NF-κB transcriptional activity responsible for regulation of EAAT-2 transcription
(137).
Astrogliosis is a common pathological feature exhibited during METH abuse.
The JAK/signal transducers and activators of transcription (STAT) pathway modulate
astrogliosis and are exacerbated during METH exposure (135). Extracellular stimuli,
such as epidermal growth factor and other proinflammatory cytokines induce nuclear
import of transcription factors and gene expression regulating the rewarding effects of
METH (139).
Herbert et al. sought out to identify whether the MAPK and JAK are
responsible for the glial response following injury. They found that GFAP upregulation
occurred as early as 6 hours and reached a 3-fold induction 48 hours following METH
exposure (140).
METH downregulates extracellular regulated kinase (ERK) and
upregulates p38 MAPK. In the brain of HIV-infected METH users, there is induction of
interferon (IFN)-inducible genes, suggesting a dysregulation of the innate immune
responses (141).
2.6. COMBINED EFFECTS: METH, HIV-1 AND NEUROTOXICITY
Drugs of abuse exacerbate neurocognitive deficits associated with HIV-1,
suppressing immunity and increasing viral replication in the CNS (81). METH increases
the expression of the HIV-1 coreceptors CXCR4 and CCR5 in monocyte-derived
dendritic cells and dysregulate inflammatory cytokine production. HIV-infected brains
show a METH-induced expression of IFN-inducible genes involved in the innate immune
37 responses (141). METH-induced immunomodulatory mechanisms include secretion of
pro- and anti-inflammatory cytokines and chemokines that are also regulated during the
course of HIV-1 infection suggesting commonality of immune regulation (86). Oxidative
stress, a result of METH exposure, interferes with T-cell ability to control HIV-1
infection. Oxidative stress, as a result of [Cai+2] increases, leads to loss of mitochondrial
function indicated by decrease in mitochondrial membrane potential, increased
mitochondrial mass, enhanced protein nitrosylation and diminished protein levels of
complexes I, III, and IV that was paralleled with reduced IL-2 secretion (35).
Antioxidants prevented METH damage by preserving the protein levels of functional
mitochondrial complexes I, III, and IV (35).
Although astrocytes do not actively
contribute to the replication of HIV-1, studies show that cocaine results in astrocyte HIV1 replication both at the proviral DNA level measured by HIV-1 long terminal repeat
(LTR)-R/U5 amplification, and secretion of HIV-1 p24 antigen (142, 143).
Infected macrophages and microglia are responsible for productive replication of
HIV-1 in the brain resulting in the release of viral proteins such as HIV-1 gp120 and Tat.
These viral proteins bind to DAT and impair its function initiating a feedback loop of
increasing synaptic DA concentrations further increasing HIV-1 replication and
inflammatory mediators production (144). METH also causes an increase of DA
concentration in the synapse modulating HIV-1 neurotoxicity. A mutant strain of mice
(VMAT2 LO) was utilized to show exacerbated loss of DAT and tyrosine hydroxylase
along with enhanced astrogliosis following METH insult to the striatum (131). The direct
neurotoxic effects of METH thus aggravate HIV-1-associated neuronal injury, increasing
the combined effects of proinflammatory mediators, ROS, and excitotoxicity.
38 2.6.1. Blood Brain Barrier
The BBB function is to separate circulating blood from brain extracellular fluid,
preventing materials present in the blood from entering the brain. As discussed earlier,
HIV-1 neuroinvasion occurs as a result of HIV-1 infected monocytes crossing the BBB
and infecting resident microglia, each secreting proinflammatory mediators and ROS.
METH also elicits inflammation that contributes further to the diminished integrity of the
BBB. METH and HIV-1 infection comorbidity leads to a greater risk of rapid HIV-1
neuroinvasion, therefore, increased risk of developing HAND.
TJ proteins are an integral part of this microenvironment.
Rho kinase
phosphorylates TJ proteins, claudin 5 and occludin in brain tissues of patients diagnosed
with HIV encephalitis leading to diminished BBB integrity (145). As discussed
previously, HIV-1 Tat has been shown to disrupt TJs associated with astrocytes along the
barrier, increasing the permeability of the BBB. Astrocytes treated with HIV-1 Tat
showed increased MMP-9 mRNA and protein levels, as meditated by NF-κB and MAPK
(119). METH also decreases occuldin protein expression in a dose-dependent manner
(146). Furthermore, HIV-1 gp120 in conjunction with METH altered BBB permeability
by modulating TJ protein expression; as ZO-1, junctional adhesion molecule (JAM)-2,
occludin, claudin-3 and claudin-5 impairments were involved Rho-A activation (147).
Both METH and HIV-1 gp120 independently and concurrently modulate TJ protein
expression. Furthermore, synergistic effects of METH and HIV-1 increase the activity of
MMPs (148), by increasing the release of MMP-1 and MMP activator from human
neuron and human astrocyte cultures (148).
39 2.6.2. Astrogliosis
GFAP is a type III intermediate filament protein exclusively expressed in
astrocytes and widely used as an astrocytic marker and the molecular mechanisms of
increased GFAP expression are important for the study of neuropathologies (124). METH
induces long lasting astrocyte activation through PKC (141) (55). Kiamura et al. showed
that chronic METH use results in an increase in astrocytes desensitized to METH effects
(7, 149). Acute METH use increases brain hyperthermia, much stronger, and more
rapidly than body hyperthermia (150), causing astrocyte activation (151) and
exacerbation of neuroinflammatory processes in the brain (152). Hyperthermia also
induces heat shock proteins in astrocytes in response to METH (153). The underlying
mechanisms for GFAP upregulation is not well understood.
In the striatum of chronic METH users who died of drug intoxication, both GFAP
and S-100β were visualized by immunohistochemistry. The number of GFAP-positive
astrocytes was not significantly higher as compared to S-100β-positive cell density,
demonstrating the absence of reactive gliosis in the striatum of chronic METH users i.e.
those who did not abstain for prolonged periods from METH use. Acute METH use
markedly increases activated astrocytes resulting in PKC phosphorylation, therefore
developing METH-induced desensitization (154). [Cai+2] in astrocytes are responsible for
excitability and communication stimulated by neuronal synaptic activity. Astrocytes
sensitivity to DA- and glutamate-induced [Cai+2] flux is responsible for deficiency in
cellular redox homeostasis (55). Nuclear factor-erythroid 2-related factor 2 (Nrf2) is an
important regulator of redox homeostasis. METH-induced astrogliosis is initiated during
40 Nrf2 deficiency, indicated by increased striatal expression of GFAP (52). Nrf2 inhibits
Tat-induced HIV-1 LTR transactivation in MAGI cells (155) and prevents increases of
TNF-a mRNA and IL-15 protein expression in GFAP positive cells. HIV-1 Tat-enhanced
cellular expression of Nrf2 at transcriptional and translational levels activating the
expression of antioxidants, indicating that Nrf2 has a key role in primary
neuroprotectivity.
IEG delays or initiates the expression of other genes (156). Several IEG members
are associated with METH/HIV-1-induced astrogliosis. HIV-1 Tat upregulates GFAP at
the transcriptional level through cis-acting elements of the early growth response-1 (Egr1) promoter (124). HIV-1 Tat exposure of human astrocytes results in elevated platelet
derived growth factor (PDGF) expression levels, regulated by the downstream
transcription factor Egr-1, leading to increased proliferation and release of CCL2 and IL1β (157). METH-induced DA fluxes lead to differential expression of IEGs. Acute
METH injections caused delayed induction of the Egr family of transcription factors in a
concentration-dependent fashion (156). Chronic METH treatment reduces expression of
AP1, Erg1–3, and Nr4a1 transcription factors below control levels and acute injection of
METH increased c-fos, fosB, fra2, junB and Egr1–3 mRNA levels in the striatum (21,
138).
Egr-1 patterns during acute and chronic METH abuse suggest a correlation
between astrocyte activation and Egr-1 expression.
The Wnt/β-catenin pathway is downregulated in astrocytes exposed to METH and
HIV-1 Tat, a signaling pathway associated with regulation of gene expression (38).
Estrogen recovers individual METH and HIV-1 Tat effects on β-catenin signaling, but
not their combined effects. Increased Myo-inositol, an in vivo marker used to study
41 astrocyte activation measured by neuroimaging is indicative of astrocyte activation (158).
Data from HIV-1+/METH+ subjects describe an increase in the levels of myo-inositol
compared to HIV-1+/METH- and HIV-1-/METH+ (158, 159).
2.6.3. Astrocyte-induced excitotoxicity
DA increases upon exposure to METH regulate the expression of EAAT-2 in
developing striatal astrocytes, suggesting that DA controls the availability of glutamate
(160). HIV-1 downregulates EAAT-2 in astrocytes, increasing extracellular glutamate
concentrations (5), inhibiting glutamate uptake (161) and reversing the transporter
orientation, further increasing extracellular glutamate concentrations (83). While EAAT2 downregulation in primary human astrocytes in response to HIV-1 infection, METH
abuse, and in the presence of inflammatory mediators is widely accepted, synergy in
these pathways is unclear. Our results show EAAT-2 mRNA attenuation during shortterm exposure to a combination of stimuli (Figure 2.1). METH, HIV-1 and IL-1β, a
prototypical proinflammatory cytokine led to significant decreases in EAAT-2 mRNA
levels demonstrating combined effects on glutamate regulation. The mechanism by which
EAAT-2 transcription decreases is unclear, but may be important in developing
therapeutic agents preventing the loss of EAAT-2 expression and function.
EAAT-2 is transcriptionally and translationally regulated, but the mechanisms by
which HIV-1 and METH downregulate EAAT-2 expression are unknown. NF-κB is an
important transcriptional regulator of the EAAT-2 promoter and under specific conditions
may act as a transcriptional repressor (162). Increased ROS/NOS is a common result of
METH and HIV-1 exposure in the brain and is suggested to regulate EAAT-2, mGluRs,
42 VGLUTs and glutamate metabolic enzymes. NO insult dimerizes the p65-c-rel NF-κB
subunits suppressing EAAT-2 transcription. In astrocytes recuperation of NF-κB
transcriptional activity is initiated by activation of the PI3K/Akt pathway, mediated by
cAMP levels (137), increasing EAAT-2 (163). Fragments of Ntab, partial sequence of
non-coding RNA, located in the 3’ UTR of EAAT-2 gene act as enhancers for EAAT-2
transcription (164), but Ntab regulation during METH/HIV-1-induced neurotoxicity has
not been investigated. Translational regulation of EAAT-2 is influenced by
phosphorylation, methylation (165) and sulfhydryl oxidation (162). Methylation results in
silencing of EAAT-2 in astrocytes (165).
Enzymes responsible for glutamate metabolism are dysregulated during
METH/HIV-1-induced neurotoxicity. GS was shown to be upregulated during an
inflammatory CNS state (166) or when exposed to inflammatory cytokines (167).
Studies addressing the molecular mechanisms associated with astrogliosis during METH
abuse (44) demonstrate rapid depletion of GS in METH-treated astrocytes. METHinduced oxidative stress led to the significant decrease in GS and increased levels of
intracellular glutamate (44). HIV-1 gp120 reduced glutamine concentrations and GS
expression with N-acetylcysteine effectively counteracting the effects of HIV-1 gp120
(168) GS is also decreased in HIV-1 infected macrophages (169). In vitro studies with
HIV-1 infected macrophages demonstrate that GLS regulation in infected macrophages
may be the mechanism of glutamate regulation during infection (170, 171).
GLS
dysregulation in microglia is proposed to enhance neurotoxicity, suggesting that HIV-1 in
the CNS increases glutamate concentrations via dysregulation of enzymes, transporters
and receptors (172).
43 Figure 2.1: METH, HIV-1, and inflammatory mediators alter EAAT-2 mRNA
expression. Primary human astrocytes were treated with METH (500 µM), HIV-1ADA,
and IL-1β (20 ng/mL) for 24 hours. RT-PCR was performed to determine the mRNA
expression patterns of EAAT-2. METH, HIV-1 and IL-1β independently led to significant
decreases in EAAT-2 mRNA levels. A combination of stimuli further attenuated EAAT-2
mRNA levels indicating additive/synergistic effects.
44 2.7. ROLE OF GLUTAMATE IN THE CNS
Glutamate is an essential neurotransmitter vital for cognition, learning and
memory and is critical for LTP and synaptic plasticity (64). Astrocytes regulate
extracellular glutamate concentration via glutamate uptake and conversion to glutamine.
i.e. the glutamate-glutamine shuttle (173). Glutamate transporters are responsible for up
to 90% of glutamate uptake, and are necessary for the termination of excitatory signals.
Thus, they prevent glutamate receptor overstimulation, which is potentially detrimental to
neuronal functioning (6). Besides glutamate transporters and glutamate receptors, the
enzymes responsible for glutamate synthesis and metabolism are also vital components of
the shuttle (85, 86, 174).
Regulation of glutamate concentrations is crucial for optimal glutamatergic
neurotransmission as approximately 40% of all synapses are glutamatergic (175). Since
glutamate receptors can only be activated extracellularly, only cells expressing plasma
membrane glutamate receptors are sensitive to glutamate signaling (64, 176). In addition,
approximately 99.99% of glutamate is stored intracellularly where it remains inactive
preventing deleterious neurotoxic effects (176). Thus, accumulation of excess
extracellular glutamate and subsequent overstimulation of glutamatergic receptors
disrupts neuronal [Ca+2]i homeostasis leading to increased reactive oxygen/nitrogen
species (ROS/NOS) (54). Oxidative stress damages neurons by increasing levels of
cytotoxic transcription factors and free radicals (177, 178). Dysfunctional glutamate
transporters and resultant increases in glutamate levels are implicated in epilepsy, stroke
and other neurodegenerative diseases, as well as in neuroinflammation (179).
45 2.8. TRANSPORTERS AND RECEPTORS
Two distinct classes of glutamate transporters are involved in the regulation work
to transport glutamate across cell membranes, (EAATs and VGLUT). EAATs are
dependent on the electrochemical gradients of sodium and potassium ions (6). VGLUTs
pack glutamate into vesicles to be released back into the synapse as neurotransmitters
independent of the electrochemical gradient (180). Glutamate receptors are responsible
for the glutamate-mediated excitation of neural cells, and are essential for neural
communication and LTP. To date two types of glutamate receptors have been identified,
each with high affinity for glutamate (64). iGluRs form an ion channel pore, which
activates upon glutamate binding. mGluRs activate cationic ion channels through GPCR
and intracellular signaling. Schematic 2.1 illustrates the glutamate transporters,
receptors, and enzymes as localized on astrocytes. How each is implicated in glutamate
regulation is discussed below.
2.8.1. Excitatory Amino Acid Transporters
Extracellular glutamate concentration is controlled by EAATs that include five
cloned subtypes (181, 182). EAAT-1 and EAAT-2 are primarily detected in astrocytes
(183). The astrocytic transporters EAAT-1 and -2 are essential for protection against
excitotoxicity as was shown in EAAT gene knockout experiments in mice (184).
Astrocytes protect from excitotoxicity via clearance of extracellular glutamate, and
glutamate metabolism within glutamate-scavenging cells is vital to prevent excitotoxicity.
This is accomplished by astrocytic glutamine synthase (GS) rapidly converting glutamate
into glutamine (173).
46 The majority of glutamate is removed by astrocyte EAAT-2. During normal
functioning of EAAT-2, glutamate and sodium (Na+) ions bind extracellularly and couple
the transporter, lead to a conformational change and then releases glutamate into the
cytosol. Intracellular potassium (K+) then binds inducing a second conformational
change, which releases the K+ extracellularly and resets the transporter (6). The Na+ and
K+ electrochemical gradients are used as a driving force by EAAT to take up extracellular
glutamate against a several thousand-fold concentration gradient (185) and are required
for proper EAAT-2 function.
EAAT-2 function is sensitive to ATP levels, phosphorylation status, membrane
translocation, and interactions with other ions (6). Lowering ATP levels affect glutamate
uptake, independent of the EAAT-2 expression.
Reduced glutamate uptake in
hyperthermia is due to depletion of ATP (186). Changes in ATP regulation in
hyperthermia results in reduced cotransport of ions via EAAT-2, thus diminishing its
functional activity (83).
EAAT-2 transcriptional regulation was studied by cloning of the EAAT-2
promoter, revealing potential regulatory transcription factor binding elements including
NFAT and NF-κB (187). NFAT recruitment inhibits expression of EAAT-2 (188) and
accumulation of extracellular glutamate activates astrocytic NFAT, suggesting an autoregulatory loop (189). Nuclear translocation of NFAT has been associated with forms of
dementia (188). Positive and negative regulators of EAAT-2 promoter activity include
cAMP and TNF-α, respectively (187). Furthermore, underlying basal and enhanced
EAAT-2 mRNA expression is regulated by a NF-κB dependent mechanism (187).
EAAT-2 transcripts contain 5’ untranslated regions (UTR) larger then 300 nucleotides,
47 indicating translational regulation (190). Translation of EAAT-2 mRNA was determined
to be regulated by corticosterone, β-lactam antibiotics and retinol (190). Astrocyte gap
junctions also modulate glutamate homeostasis. Connexin43 double knockout mice
exhibit changes in EAAT-2 mRNA and protein expression in cerebrum (191).
Connexin43 is a major gap junction protein of astrocytes that functions in buffering of
potassium and propagation of [Ca+2]i waves (192). EAAT-2 protein expression is
significantly downregulated in chronic neurological disorders, reducing glutamate uptake,
and increasing extracellular glutamate. During acute injury or insult, these resultant
conditions induce upregulation of EAAT-2 expression by astrocytes to compensate for
decreased glutamate transport (193). Glutamate has been shown to modulate mRNA and
protein levels, and the kinetic properties of glutamate transporters (194). The complexity
of EAAT-2 transcriptional and translational regulation facilitates the fine balance of
glutamate levels and synaptic homeostasis.
2.8.2. Vesicular Glutamate transporter-1
Recent investigations into gliotransmission, or astrocytic involvement in
neurotransmission, have shown that astrocytes can exocytose neurotransmitters (180,
195). Spontaneous [Ca+2]i oscillations observed in astrocytes induce glutamate release
through VGLUT-1 localized on astrocyte processes (196-198). The mechanisms of
glutamate release from astrocytes remains partially undefined, but inhibiting exocytosis
related proteins such as synaptobrevin-2, prevents calcium dependent release of
glutamate demonstrating glutamate release via exocytosis (197).
Bezzi et al. show
VGLUT1/2 and the vesicular soluble N-ethylmaleimide-sensitive factor attachment
48 protein receptor (SNARE) protein, cellubrevin, are colocalized to vesicular organelles
adjacent to glutamate receptors (199). Upon activation of mGluRs, vesicles undergo
calcium and SNARE exocytic fusion accompanied by glutamate release (199). Further
studies using VGLUT-1 overexpression constructs in astrocytes indicate astrocytes
require both cytosolic glutamate and VGLUT-1 to release glutamate by [Ca+2]i induction
(200). These investigations highlight the importance of [Ca+2]i signaling in modulating
glutamatergic transmission.
While astrocyte vesicular release of glutamate aids in
neurotransmission and synaptic plasticity (196), increased expression of VGLUT-1 has
been shown to cause excitotoxic neurodegeneration (201). Thus, despite the fact that
VGLUT-1 is not primarily responsible for extracellular glutamate clearance or
metabolism, it is an essential component of glutamate modulation and facilitates the role
of astrocytes in synaptic homeostasis via spontaneous glutamate release (196).
2.8.3. Metabotropic glutamate receptors
mGluRs are members of the group C family of inhibitory GPCRs activating
[Ca+2]i increases regulating protein expression. Expression of functional group I mGluRs
are involved in the increase of [Ca+2]i in astrocytes (84).
Haak et al. investigated
modulation of glutamate-induced [Ca+2]i increases in astrocytes. Their data shows that
glutamate acts on astrocytic mGluR subtypes, which regulate astrocyte activity through
neurotransmitter stimulation and contributes to the modulation of neuronal activity (202).
Activation of mGluRs has also been shown to initiate neuroprotection by the release of
nerve growth factor (NGF) and S-100β from astrocytes promoting neuronal
differentiation and survival (203). Vice versa, astrocyte mGluRs and EAAT-2 are also
49 regulated by neuronal activity (89). Activation of mGluRs following neuronal glutamate
release activates an IP3 pathway, resulting in an intracellular release of [Ca+2]i and ATP
(204).
Neurons are traditionally viewed as the predominant cell type associated with
neurotransmission and synaptic plasticity.
Recently, astrocytes involvement in
neurotransmission, termed, gliotransmission, has also implicated their importance in
synaptic plasticity. Neuronal activity also participates in dynamics of the astrocyte
glutamate transporter reducing the localization of EAAT-2 and enhancing neuronal
activity (89). Neurons induce the expression of EAAT-2 via NF-κB subunits, p65 and
p50, binding and enhancing the activity of the EAAT-2 promoter and that dominantnegative inhibitors of NF-κB signaling completely blocked neuron-dependent activation
of a NF-κB reporter construct (205). Exogenous expression of the NF-κB subunits p65
and/or p50 induced EAAT-2 expression and EAAT-2-mediated transport activity (205).
Astrocytes physically adhere to neurons via integrin receptors initiating activation of
PKC (206). Astrocytic contact induces excitatory synaptogenesis in the neuron upon
METH exposure causing phosphorylation of PKC in both cell types and astrocyte
activation (206).
Data suggest METH-induced, PKC-dependent activation of
glutamatergic transmission may contribute to psychological dependence (139). Thus,
neuron-astrocyte cross-talk plays a critical role in regulation of astrocyte mGluRs.
2.8.4. Ionotropic glutamate receptors
Most fast excitatory synaptic transmission is mediated through glutamate acting
on iGluRs.
Two types have been reported, the a-amino-3-hydroxy-5-methyl-4-
50 isoxazolepropionate (AMPA)/kainate (non-NMDA) and the NMDA receptors both
directly gate ion channels. Recently, NMDA receptors (NMDAR) mRNA and protein
expression was demonstrated in primary human astrocytes.
Immunocytochemistry
confirmed the localization of NMDR protein with GFAP on the surface suggesting the
likelihood that the receptors are functional. Glutamate activated the NMDAR increasing
[Ca+2]i in astrocytes that was blocked with the addition of specific NMDAR antagonists
(207). NMDAR activation via glutamate opens cation channels ubiquitously. Neuronal
NMDAR are of particular importance for their contribution to the modulation of
excitotoxicity important in METH and HIV-1 as discussed later.
2.9. SYNTHESIS AND METABOLISM OF GLUTAMATE
Two enzymes regulate glutamate metabolism and synthesis: glutaminase (GLS)
and glutamine synthetase (GS), respectively. While GLS expression is low in astrocytes,
GS is predominantly expressed in astrocytes. Functional homeostasis of these enzymes is
critical for neurotransmission and prevention of excitotoxicity.
2.9.1. Glutaminase
GLS catalyzes the conversion of glutamine to glutamate and while it is not
prominently localized to astrocytes, it is highly expressed in neurons of the CNS. GLS is
regulated by ADP levels and is thus dependent on mitochondrial integrity. Elevated
metabolism, decreased intracellular ROS and overall DNA oxidation are linked to GLS
activity in both normal and stressed cells. Through GLS modulation of ROS levels, p53
expression is also regulated, thus inhibiting genomic oxidative damage. Although scarce
51 in astrocytes, GLS localizes predominantly between the mitochondrial membranes (86,
166) and active, phosphorylated GLS localizes specifically to the outer face of the inner
mitochondrial membrane in astrocytes (85).
2.9.2. Glutamine synthetase
GS metabolizes nitrogen in an ATP-dependent reaction catalyzing the
condensation of glutamate and ammonia to form glutamine and is predominately
expressed in glia and microglia. The glutamate amide group serves as a nitrogen source
for the synthesis of glutamine pathway metabolites. Highly expressed in astrocytes
during CNS stress, GS colocalizes with GFAP (174). Recently Mongin et al. proposed a
simplified, quantitative protocol to determine GS and GLS activity by measuring the
mitochondrial conversion of glutamate to the tricarboxylic cycle intermediate αketoglutarate in the oxidative deamination and transamination reactions. Using conditions
optimized for astrocytes and appropriate pharmacological controls Mongin et al.
conclude astrocytes express measureable amounts of GS and GLS indicating their
contributions to synaptic plasticity and neurotransmission (86).
52 Schematic 2.1: Mechanisms of astrocyte-mediated glutamate regualtion. Astrocytes
are intricately involved in glutamate regulation through several mechanisms. EAAT-2
(1) is responsible for uptake of extracellular glutamate into the cell, where intracellular
glutamine synthetase (2) catalyzes the reaction forming glutamine from glutamate.
Glutaminase (3) is the enzyme that catalyzes the reaction to convert glutamine to
glutamate where astrocytes can package it into vesicular glutamate transporters (4). The
expression of functional metabotropic glutmate receptors (5) allows glutamate signaling
leading to increased intracellular calcium.
53 Concluding remarks
In this review, we have examined the current information on the influences of
METH and HIV-1 infection in the context of glutamate regulation in the brain.
Excitotoxicity is a common feature of multiple neurological and neurodegenerative
disorders and is typically associated with a dysregulation of astrocytic glutamate
transporters. Astrocytes regulate extracellular glutamate concentrations through EAAT-2
activity and, although there is no direct infection and replication of HIV-1 in astrocytes or
clear understanding of the mechanism of METH action in astrocytes, METH and HIV-1
independently
downregulate
EAAT-2
expression.
METH/HIV-1-induced
BBB
impairment and astrogliosis are also evidence of METH/HIV-1-induced effects in
astrocytes. Thus, several lines of evidence demonstrate common outcomes in METH and
HIV-1 in the CNS; yet, the combined mechanisms of injury and mechanistic synergy
remain to be elucidated. In the post-ART era, HIV-1 patients are living longer and METH
abuse is on the rise. Thus, further investigation into the comorbidity pathways of METH
and HIV-1 in the CNS are warranted. Furthermore, given the importance of astrocytes in
CNS homeostasis and their critical role in maintaining CNS integrity, understanding their
dysfunction during METH abuse and HIV-1 infection, particularly in the context of
excitotoxicity, is critical for developing future therapeutic strategies for neuro-AIDS, in
the post-ART era.
54 Chapter 3
METHAMPHETAMINE AND HIV-1-INDUCED NEUROTOXICITY: ROLE OF
TRACE AMINE ASSOCIATED RECEPTOR 1 CAMP SIGNALING IN
ASTROCYTES
55 3.1 ABSTRACT
METH is abused by 4.3% of the US population with approximately 10-15% of
HIV-1 patients reporting its use. METH abuse accelerates the onset and severity of
HAND and astrocyte-induced neurotoxicity. METH activates G-protein coupled
receptors such as TAAR1 increasing intracellular cAMP levels in presynaptic cells of
monoaminergic systems. To our knowledge METH-mediated activation of astrocyte
TAAR1 has not been reported. In the present study, we investigated the combined effects
of METH and HIV-1ADA on primary human astrocyte TAAR1 expression and function,
chemokine levels, and excitotoxicity. Our results demonstrate combined conditions
increased TAAR1 mRNA levels four-fold and significantly increased intracellular cAMP
levels.
METH and beta-phenylethylamine (β-PEA), known TAAR1 agonists, also
increased intracellular cAMP levels in astrocytes.
Further, TAAR1 knockdown
significantly reduced intracellular cAMP levels in response to METH and β-PEA in
astrocytes, indicating signaling through astrocyte TAAR1. Interleukin (IL) -6 and
CXCL8 mRNA and protein levels increased upon treatment with HIV-1ADA alone and in
combination with METH. METH + HIV-1ADA decreased EAAT-2 mRNA and
significantly decreased glutamate clearance in parallel experiments. Thus, our data shows
that METH exposure activated TAAR1 leading to intracellular cAMP responses,
decreased glutamate clearance abilities and in combination with HIV-1ADA increased
astrocyte mediated inflammation. To our knowledge this is the first report implicating
astrocyte TAAR1 as a novel receptor for METH during combined injury in the context of
HAND.
56 3.2. INTRODUCTION
METH is an addictive pharmacological psychostimulant of the CNS. Ten percent
of METH becomes biologically available within ten min of smoke inhalation, due to its
high lipophilic nature (15). METH generates an imbalance in the release and reuptake of
dopamine, norepinephrine and epinephrine producing intense euphoria followed by hours
of stimulation, excitation and alertness (208). Risky behavior accompanies strong
neurological impulses associated with METH abuse resulting in the high prevalence of
METH users who acquire HIV-1 infection (4, 37). Further, clinical research describes
that individuals infected with HIV-1 actively participate in METH abuse (209-211).
HIV-1 infection often results in cognitive impairments, collectively termed
HAND (126). Neurotoxic outcomes of METH abuse and HIV-1 CNS infection include,
but are not limited to: brain hyperthermia, release of inflammatory mediators and ROS,
excitotoxicity, and astrogliosis (34, 37, 212); however, the molecular basis for these
effects remains elusive. Additionally, as the most abundant cells of the CNS, astrocytes
are a significant cell type affected by peripheral stimuli, such as METH and HIV-1.
Astrocytes function to support brain homeostasis and maintenance of the blood
brain barrier (213). Astrocyte responses to external stimuli encompass increased
intracellular cAMP signaling and excitotoxicity (7, 134). Astrocytes are responsible for
clearing approximately 90% of extracellular glutamate from the synaptic cleft via EAAT2 (6). Additionally, cytokines, chemokines, biogenic and trace amines, neurotransmitters
and pharmacological agents induce intracellular signaling cascades in astrocytes through
activation of membrane and cytosolic receptors (214-218).
57 METH binds to cell membrane and intracellular receptors initiating rapid
signaling events in astrocytes (75, 219-222). TAAR1 binds METH and regulates
dopamine transporter trafficking in neurons (67).
TAAR1 is a stimulatory GPCR
activated by the metabolites of biogenic amines (222). Biological agonists for TAAR1
are trace amines, such as β-PEA, octopamine and tryptamine (222). Activation results in
intracellular cAMP signaling events, activation of PKA and phosphorylation of
downstream targets (75). METH exposure of astrocytes led to decreased EAAT-2 levels
and function. Astrocyte cAMP signaling may regulate transcriptional, translational and
post-translational modifications of EAAT-2. To our knowledge, expression and function
of TAAR1 in astrocytes is not yet documented.
In this report, we investigated astrocyte TAAR1 in the presence of METH and
HIV-1. We show METH +/- HIV-1 regulated EAAT-2 and TAAR1 expression and
function. Further, METH and β-PEA exposure led to cAMP induction in astrocytes,
which was prevented by TAAR1 RNA interference (RNAi). Finally, we demonstrate that
a reduction in TAAR1 levels significantly increased, whereas, TAAR1 overexpression
significantly decreased astrocyte glutamate clearance. Taken together, we propose that
astrocyte responses to METH are mediated via TAAR1 activation and combined toxicity
of METH and HIV-1 result in synergistic effects on astrocyte cAMP signaling and
glutamate clearance abilities. We report for the first time a novel astrocyte receptor for
METH-induced intracellular cAMP signaling.
58 3.3 MATERIALS AND METHODS
3.3.1 Isolation, cultivation and activation of primary human astrocytes
Human astrocytes were isolated from first and early second trimester elected aborted
specimens as previously described (97). Briefly, tissues ranging from 82 to 127 days
were procured in full compliance with the ethical guidelines of the National Institutes of
Health, University of Washington and North Texas Health Science Center. Cell
suspensions were centrifuged, washed, suspended in media, and plated at a density of
20×106 cells/150 cm2. The adherent astrocytes were treated with trypsin and cultured
under similar conditions to enhance the purity of replicating astroglial cells. These
astrocyte preparations were routinely >99% pure as measured by immunocytochemistry
staining for GFAP. Astrocytes were treated with METH (500 µM, Sigma-Aldrich Inc.,
St. Louis, MO) and HIV-1 (p24, 10 ng/mL) alone and in combination at 37°C and 5%
CO2. The HIV-1ADA isolate used was originally isolated from a patient with initials ADA
and propagated in vitro as previously described (223). Dose- and time-kinetics revealed
optimal concentrations for METH and HIV-1 were not toxic to astrocytes (data not
shown).
3.3.2 RNA extraction and gene expression analyses
Astrocyte RNA was isolated as described in (224) 8 hr post-treatment and mRNA levels
were assayed by real-time polymerase chain reaction (PCR). Taqman 5’ nuclease realtime PCR was performed using StepOnePlus detection system and inventoried gene
expression assays (Life Technologies Inc., Carlsbad, CA). Commercially available
TaqMan® Gene Expression Assays were used to measure EAAT-2 (cat no.
59 Hs00188189_m1), TAAR1 (cat no. Hs00373229_s1) and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) (cat no. 4310884E) mRNA levels. GAPDH, a ubiquitously
expressed housekeeping gene, was used as an internal normalizing control. The 25 ml
reactions were carried out at 48ºC for 30 min, 95ºC for 10 min, followed by 40 cycles of
95ºC for 15 s and 60ºC for 1 min in 96-well optical, real-time PCR plates. Transcripts
were quantified by the comparative ΔΔCT method, and represented as fold-change of
control.
3.3.3 Glutamate clearance assay
Primary human astrocytes were plated in 48-well tissue culture plates at a density of 0.15
x 106 cells/well and allowed to recover for 24 hr prior to treatment as described in section
2.1. Following 24 hr treatment, glutamate (400 µM) dissolved in phenol-free astrocyte
medium was added into each well and clearance was assayed at 4 and 10 hr. The assay
was performed and analyzed according to manufacturer’s guidelines (Amplex Red
Glutamic Acid/Glutamate Oxidase Assay Kit, Life Technologies).
3.3.4 Neurotoxicity and viability assays
Primary human astrocytes were plated in 48-well tissue culture plates at a density of 0.15
x 106 cells/well and allowed to recover for 24 hr prior to treatment as described in section
2.1. Following 24 hr treatment metabolic activity, lactate dehydrogenase release and
apoptosis was measured. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay, a colorimetric assay for measurement of metabolic activity, performed for
60 cell viability (225). The MTT assay was performed at appropriate time points, briefly five
percent MTT reagent in astrocyte medium was added to astrocytes and incubated for 2045 min at 37°C. The MTT solution was removed and crystals were dissolved in DMSO
for 15 min with gentle agitation. The absorbance of the DMSO/crystal solution was
assayed at 490 nm in a Spectromax M5 microplate reader (Molecular Devices,
Sunnyvale, CA). Cytotoxicity by lactate dehydrogenase (LDH) release was quantified
using the cytotoxicity detection kit (Roche Diagnostics, Indianapolis, IN, USA)
according to manufacturer’s instructions. DNA fragmentation and apoptosis was assayed
using the double stranded DNA (dsDNA) ELISA (Roche Diagnostics, Indianapolis, IN,
USA) according to the manufacturer’s directions.
3.3.5 Immunocytochemistry
Astrocytes were cultured as adherent monolayers in 48-well plates at a density of 0.1 x
106 cells per well. Cells were fixed with ice cold acetone:methanol (1:1) for 30 min at 20°C and then blocked in phosphate buffered saline (PBS) with 2% bovine serum
albumin (BSA) containing 0.1% Triton X-100 for 1 hr at room temperature. Cells were
incubated with TAAR1 antibody (1:700, rabbit, Abcam, Cambridge, MA) and glial
fibrillary acidic protein (GFAP) antibody (1:1000, chicken, Covance Inc., Emeryville,
CA) in PBS (2% BSA, 0.1% Triton X) overnight at 4°C. Cultures were then washed and
®
stained with Alexa Fluor secondary antibodies (488 nm, green) and (594 nm, red) for 2
hr at room temperature (1:1000, Life Technologies). Nuclei were visualized with 4',6diamidino-2-phenylindole (DAPI) (1:800, Life Technologies). Micrographs were
61 obtained on an ECLIPSE Ti-300 using the NLS-Elements BR. 3.0 software (Nikon Inc.,
Melville, NY).
3.3.6 Confocal analysis
Human astrocytes were cultured on glass bottom 6-well tissue culture plates (MatTek
Corp., Ashland, MA) at a density of 2x106 cells/well. The cells were carefully fixed with
acetone:methanol (1:1) and stained with antibodies against TAAR1, GFAP, and DAPI at
24 hr post-treatment. Micrographs were obtained on an Olympus IX71 Microscope
(Olympus America Inc., Center Valley, PA). Confocal colocalization analysis and twodimensional histogram was performed using BioimageXD software (Free Software
Foundation Inc., Boston, MA).
3.3.7 cAMP assay
Intracellular cAMP levels in astrocytes were measured using a commercially available
homogenous, bioluminescent assay, cAMP-Glo™ Assay, (Promega Corp., Madison,
WI). Adherent monolayers of astrocytes cultured in 96-well plates (50,000 cells/well)
were exposed to forskolin, METH and β-PEA (all from Sigma-Aldrich). Cells were
directly activated and lysed in the tissue culture plate. Lysates were diluted to a final cell
concentration of approximately 1,000 cells/µL in lysis buffer [500 µM 3-isobutyl-1methylxanthine (IBMX) and 100 µM Ro 20-1724, cAMP specific phosphodiesterase
inhibitors (both from Sigma-Aldrich)] and transferred to a white opaque flat bottom 96well assay plates (Corning Inc. Life Sciences, Tewksbury, MA). Intracellular cAMP
levels were assayed using GloMax 96 Microplate Luminometer with dual injectors
62 (Promega).
Data analysis for the half-maximal effective concentrations (EC50) was
performed with Prism V6.0 (GraphPad Software, La Jolla, CA) using sigmoidal doseresponse (variable slope) curve.
3.3.8 Transfection of astrocytes
®
Cultured human astrocytes were transfected with On-Target plus small interfering RNA
(siRNA) pools specific to TAAR1 (siTAAR1), non-targeting control siRNA pools
(siCON, Thermo Scientific, Waltham, MA, USA) and without siRNA (MOCK) or with
overexpression plasmids CON-GFP or TAAR1-GFP (TrueORF cDNA Clones with Cterm GFP tag, PrecisionShuttle Vector System, Origene Technologies Inc., Rockville,
MD) using the AmaxaTM P3 primary cell 96-well Nucleofector kit and shuttle attachment
(Lonza, Walkersville, MD, USA) according to the manufacturer’s instructions. Briefly,
1.6 million astrocytes were suspended in 20 µl nucleofector solution containing siCON or
siTAAR1 (100 nM) or CON-GFP or TAAR1-GFP (0.25 mg/1.6 million cells) and
transfected using protocol CL133. Transfected cells were supplemented with astrocyte
media and incubated for 30 min at 37°C prior to plating. Cells were allowed to recover
for 48 hr prior to experimental use.
3.3.9 Statistical analyses
Statistical analyses were performed using Prism V6.0 with one-way analysis of variance
(ANOVA) and Newman-Keuls post-test for multiple comparisons. Significance was set
at p ≤ 0.05 and data represents means ± standard error of the mean (SEM). Results are
63 representative of two or more independent donors, denoted as n, with a minimum of three
individual replicates in each experiment.
3.4 RESULTS
3.4.1 METH and HIV-1 decreased astrocyte EAAT-2 levels and glutamate clearance
Glutamate clearance through astrocyte EAAT-2 is regulated by extracellular stimuli,
including inflammatory mediators, pharmacological agents and bacterial/viral particles
(8, 226, 227). We measured astrocyte EAAT-2 mRNA levels and their capacity to clear
glutamate following 24 hr of METH and HIV-1, alone and in combination (Fig 3.1A and
B).
EAAT-2 mRNA levels decreased significantly with METH or HIV-1 alone
(*p<0.05, ***p<0.001, respectively) and by approximately 40% during combined
conditions compared to control (***p<0.001, n=5). Similarly, astrocytes ability to clear
extracellular glutamate 10 hr post-glutamate addition was significantly impaired
subsequent to METH and/or HIV-1 treatments (***p<0.001, Fig 3.1B). GFAP is a
recognized astrocyte marker responsive to injury, therefore we measured GFAP mRNA
levels following METH and/or HIV-1 activation (Fig 3.1C). GFAP mRNA levels were
not significantly different. In order to verify that changes in EAAT-2 were not a result of
METH- and HIV-1-induced cytotoxicity, we measured astrocytes metabolic activity by
MTT (Fig 3.1D), cytotoxicity by LDH release (Fig 3.1E), and apoptosis by dsDNA
fragmentation ELISA (Fig 3.1F) following METH and/or HIV-1 treatment. No
significant changes were observed in any parameters 24 hr post-stimulation (Fig 3.1D-F,
respectively).
64 3.4.2 METH and HIV-1 regulate astrocyte TAAR1
To our knowledge, no previous evidence has been published demonstrating METH
activation of astrocyte TAAR1; so we measured TAAR1 mRNA, protein levels and
function in cultured human astrocytes following METH and/or HIV-1 treatment (Fig
3.2). Astrocyte TAAR1 mRNA levels did not change significantly with METH or HIV-1
alone; however, combined treatments significantly upregulated TAAR1 mRNA levels by
approximately 7-fold (Fig 3.2A, ***p<0.001, n=3). In neuronal cells TAAR1 activation
results in intracellular cAMP increases (228), thus we measured METH- and/or HIV-1induced intracellular cAMP levels at 2 min and 10 min post-activation (Fig 3.2B).
METH significantly increased cAMP levels within 2 min compared to control and
continued to rise up to 10 min (**p<0.01). METH and/or HIV-1 significantly increased
intracellular cAMP at 10 min in comparison to control (**p<0.01). In addition, 24 hr
post-METH and/or HIV-1 treatment, astrocytes were immunostained for TAAR1 (Fig
3.2C, D, E and F). Cytoplasmic and nuclear localization was confirmed by confocal
microscopy (Fig 3.2C1, D1, E1 and F1). Consistent with changes in RNA levels,
combined treatments with METH and HIV-1 increased TAAR1 immunostaining (Fig
3.2A and F). Confocal microscopy (Fig 3.2C1, D1, E1 and F1) showed nuclear
localization of TAAR1 (green) and perinuclear colocalization with GFAP (red). HIV-1
treatment alone or in combination with METH increased cytoplasmic and nuclear
localization (Fig 3.2E1 and F1). Taken together, TAAR1 is expressed in astrocytes and
modulated by combined treatments of METH and HIV-1.
65 3.4.3 Forskolin, METH and β-PEA increase astrocyte intracellular cAMP levels in a
dose-dependent manner
Astrocytes were treated with multiple doses of forskolin, METH and β-PEA, followed by
lysis and quantification of intracellular cAMP levels (Fig 3.3A). The half maximal
effective concentration (EC50) for forskolin, METH and β-PEA were measured in
multiple biological astrocyte donors for intracellular cAMP levels. Representative dose
response curves are shown with an EC50 of 10.96 µM for forskolin (Fig 3.3A), 32.86 µM
for METH (Fig 3.3B) and 11.04 µM for β-PEA (Fig 3.3C). When compared across
multiple astrocyte donors the average EC50 was 12.88 ± 1.98 µM for forskolin, 30.6 ±
5.05 µM for METH and 11.01 ± 2.31 µM for β-PEA (Fig 3.3D). The efficacy for both
METH and β-PEA was 40 nM cAMP; however, the lower EC50 for β-PEA compared to
METH indicates that METH has a lower potency compared to the known TAAR1
agonists, β-PEA.
3.4.4 RNAi for TAAR1 decreased astrocyte TAAR1 protein levels and cAMP responses
Astrocytes were MOCK-, siCON- and siTAAR1-transfected by nucleofection, plated for
48 hr and immunostained for TAAR1 following 24 hr of METH treatment. MOCK- and
siCON-transfected astrocytes expressed TAAR1 protein levels as previously observed
(Fig 3.4A and C). METH treatment did not increase TAAR1 protein levels in MOCK- or
siCON-transfected astrocytes; however, astrocytes appeared reactive with greater
colocalization of TAAR1 with GFAP (Figure 3.4B and D). siTAAR1 transfection
decreased TAAR1 expression with or without METH (Fig 3.4E and F). Forskolin-,
METH- and β-PEA-induced intracellular cAMP levels were quantified in MOCK-,
66 siCON- and siTAAR1-transfected astrocytes and data at 500 µM agonists concentration
for each is shown (Fig 3.4G, n=4). Forskolin induction of cAMP was not significantly
different across all treatments, averaging 44 nM (Fig 3.4G). METH-induced cAMP
levels averaged 32.8 nM in MOCK-transfected astrocytes and 32.3 nM in siCONtransfected astrocytes. However, cAMP levels were approximately 88% lower in
siTAAR1-transfected astrocytes, averaging 3.8 nM (Fig 3.4G, ***p<0.001), indicating
METH signaling via TAAR1. Likewise, β-PEA-mediated cAMP levels in siTAAR1transfected astrocytes significantly decreased approximately 95% to an average of 1.6 nM
when compared to the average cAMP of 29.3 nM in MOCK-transfected and 34 nM in
siCON-transfected astrocytes (Fig 3.4G, ***p<0.001).
3.4.5 TAAR1 RNAi and overexpression alter astrocyte glutamate clearance
Astrocytes were transfected as described in section 2.7. Cumulative data from two
astrocyte donors tested in multiple replicates each is shown. METH significantly
decreased EAAT-2 levels in MOCK- (*p<0.05) and siCON-transfected (**p<0.01)
astrocytes (Fig 3.5A). In order to investigate the role of TAAR1 in astrocyte METH
signaling we next employed TAAR1 RNAi. Downregulation of TAAR1 using siRNAs
prevented METH-induced downregulation of EAAT-2 (Fig 3.5A). In parallel, glutamate
uptake was significantly reduced in MOCK- (***p<0.001) and siCON-transfected
(***p<0.001) astrocytes (Fig 3.5B). TAAR1 downregulation alone significantly
increased glutamate clearance (***p<0.001) that was enhanced following METH
treatment (Fig 3.5B, ***p<0.001). To further study the relationship between TAAR1
and EAAT-2, we utilized a TAAR1-GFP overexpression plasmid. TAAR1 upregulation
67 alone significantly decreased EAAT-2 levels (***p<0.001), which was exacerbated by
METH treatment (Fig 3.5C, ***p<0.001).
Likewise, TAAR1 overexpression alone
significantly impaired glutamate clearance abilities of astrocytes (***p<0.001) with little
change following METH treatment (Fig 3.5D). Taken together, our data suggest TAAR1
mediates METH-induced EAAT-2 downregulation and impaired glutamate uptake by
human astrocytes and may serve as a therapeutic target for the attenuation of
excitotoxicity.
3.5 DISCUSSION
We report for the first time a novel receptor that is activated following METH
treatment in primary human astrocytes. In this study, we demonstrate that METH and
HIV-1 regulated TAAR1 expression and cellular localization. TAAR1 agonists, METH
and β-PEA, led to intracellular cAMP signaling that was interrupted with TAAR1
knockdown. METH and/or HIV-1 exposure decreased EAAT-2 expression and the ability
of astrocytes to clear extracellular glutamate. TAAR1 downregulation prevented a
reduction in EAAT-2 mRNA and increased glutamate clearance, which was enhanced
upon METH treatment. In addition, TAAR1 overexpression alone reduced EAAT-2
levels that significantly decreased with METH and attenuated glutamate uptake in
parallel. The mechanisms by which METH increases brain injury in the context of HIV-1
infection remain unclear; however, our studies reveal the effects of METH and HIV-1 on
astrocyte-mediated excitotoxicity and direct mechanisms of METH-induced intracellular
cAMP levels in astrocytes. Our data suggest TAAR1 mediates METH-induced EAAT-2
68 downregulation and impairs glutamate clearance thereby increasing the severity of HIV1-induced excitotoxicity.
TAAR1 mediating METH-induced effects in astrocytes is a novel finding. TAAR1
is an intracellular GPCR involved in dopaminergic signaling (72, 76, 229). TAAR1 plays
a role in the etiology of various neurological disorders including schizophrenia (230).
The neurobiology of schizophrenia is associated with imbalances in the dopaminergic
and glutamatergic system (231). Further, the antipsychotic potential of TAAR1 agonists
have been shown to be effective for the neurological therapy of both increased dopamine
activity and reduced glutamatergic activity in schizophrenic models (230). We observed
basal TAAR1 protein to be distributed both in the cytoplasm and nucleus of primary
human astrocytes. HIV-1 alone or in combination with METH increased TAAR1
localization both in the cytoplasm and nucleus. Intracellular TAAR1 in eGFP-rhesus
monkey transfected HEK293 cells was activated following agonist diffusion to the
cytoplasm (67). While it is unclear how HIV-1 regulates TAAR1, HIV-1 relevant
cytokines, including IL-1β, increased TAAR1 levels in primary human astrocytes (data
not shown). Moreover, in lymphocytes, TAAR1 levels increased following immune
activation and METH-induced TAAR1 activation upregulated CREB (cAMP responsive
element binding protein) and NFAT (nuclear factor of activated T-cell), transcription
factors commonly associated with immune activation and EAAT-2 transcription (68).
Nuclear localization of TAAR1 has not been previously reported; however, there are an
increasing number of documented GPCRs in the nucleus (232, 233). Astrocyte TAAR1
nuclear activity is yet to be investigated.
69 Activation of TAAR1 leads to intracellular cAMP signaling that results in PKA and
PKC phosphorylation and activation (68, 234). Maximal efficacy for METH- and βPEA-induced intracellular cAMP saturated at 40 nM suggesting they are full agonists for
the target receptor. However, the average EC50 for β-PEA was approximately 3-fold
lower than the average METH EC50 further implying that while both agonists produce
similar efficacy, β-PEA has a higher potency for the receptor. Literature suggests that
METH binds monoaminergic transporters/receptors and sigma receptors in neurons, but
the mechanisms in astrocytes are unknown (75). Interestingly, both METH and HIV-1,
alone and in combination, significantly increased intracellular cAMP levels within 10
min of stimulation. Studies in vitro demonstrated that HIV-1 infection of primary T cells
increased intracellular cAMP levels (35, 235, 236) and lymphocytes from HIV-1-infected
patients had two-fold higher intracellular cAMP levels than lymphocytes from uninfected
individuals (237, 238).
The canonical cAMP pathway involves PKA activation. Activation of both PKA and
PKC have been reported downstream of METH-induced TAAR1 cAMP signaling in
neurons (67, 68). These pathways regulate genes involved in cell cycle, cell survival and
cytokine secretion (69). Downstream activation of PKA and PKC leads to the modulation
of regulatory units on the EAAT-2 promoter, translational and post-translational
modifications and ubiquitination (9, 239-242).
Analysis of the EAAT-2 promoter
showed that NF-κB is an important regulator of EAAT-2 transcription in astrocytes (65)
and the rate of NF-κB translocation into the nucleus is regulated by the activation of
PKA, downstream of cAMP (66). METH may be regulating EAAT-2 transcription via
downstream signaling pathways associated with TAAR1 activation; however, previous
70 reports suggest that intracellular activation of cAMP and PKA lead to increased EAAT-2
transcription in astrocytes (162, 243). Others report that cAMP signaling often fails to
induce EAAT-2 transcription in glioma cell lines, proposing that intrinsic factors, such as
DNA methylation, keep the EAAT-2 gene inactive and unresponsive to external stimuli
(165, 244). Additionally, there are three forms of human EAAT-2 transcripts, two are not
constitutively translated, requiring extracellular factors to induce translation (226).
EAAT-2 expression is regulated at both the transcriptional and translational levels. The
efficiency of EAAT-2 translation suggests that EAAT-2 regulation occurs predominately
at the translational and post-translational levels (245).
In vitro, HIV-1 results in EAAT-2 downregulation and reduced glutamate uptake in
astrocytes (5). While METH is known to induce neuronal excitotoxicity, the direct
mechanisms of METH-induced EAAT-2 dysregulation in astrocytes remain unclear.
EAAT-2 activity is regulated through gene expression, protein targeting and trafficking
and post-translational modifications.
METH may be modulating EAAT-2 via
downstream signaling of TAAR1. An increase in intracellular cAMP levels result in
astrocyte glutamate release (246). Furthermore, signaling pathways involving PKC and
PKA are differentially responsible for early and late phase increases of astrocyte
glutamate uptake, EAAT-2 trafficking and degradation (247). Therefore, the robust
changes observed in astrocyte glutamate uptake following METH treatment maybe
mediated by translation and trafficking of EAAT-2. Further, selective TAAR1 activation
in brain slices from mice blocked activity of NMDA antagonists, which was not observed
in TAAR1 knockout mice, suggesting TAAR1 involvement in glutamatergic signaling
(75). Basal TAAR1 levels and the robust changes in glutamate clearance following
71 TAAR1 knockdown and overexpression suggest it may have broader implications in
astrocyte function.
Astrocytes have not been previously shown to express receptors that are directly
sensitive to METH. We show for the first time METH-mediated activation of astrocyte
TAAR1 and its implication in EAAT-2 regulation. Since EAAT-2 accounts for the
majority of glutamate uptake from the synaptic cleft, METH-mediated changes in EAAT2 transcription and activity may result in additive and/or synergistic effects associated
with HIV-1-induced excitotoxicity. Our results are intriguing and indicate that METHinduced TAAR1 activation in human astrocytes is a novel pathway leading to astrocyte
EAAT-2 regulation in the context of HIV-associated neurocognitive disorders and may
have future therapeutic implications.
72 Figure 3.1: METH and HIV-1 modulate excitotoxicity and astrocyte viability.
Astrocytes were treated with METH (500 µM) and/or HIV-1 (p24 10 ng/mL). RNA was
isolated at 8 hr and assayed for EAAT-2 levels by real-time PCR. EAAT-2 levels were
significantly lower in astrocytes treated with METH (*p<0.05), HIV-1 (***p<0.001) and
METH + HIV-1 (A, ***p<0.001, n=5). Glutamate clearance was measured at 4 hr and
10 hr post-glutamate addition and was significantly decreased with METH, HIV-1 and in
combined conditions at 10 hr (B, ***p<0.001, n=3). No significant differences in GFAP
mRNA levels were observed (C). To determine astrocyte viability, metabolic activity (D),
cytotoxicity (E) and apoptosis (F) were measured following 24 hr activation with METH
and/or HIV-1. Transient treatment of METH and/or HIV-1 did not significantly increase
MTT, LDH activity or apoptosis (D-F, n=3). Multiple astrocyte donors (n) were tested,
each analyzed in a minimum of triplicate determinations.
73 74 Figure 3.2: Astrocyte TAAR1 expression and localization is regulated by METH and
HIV-1.
Human astrocytes were treated with METH (500 µM) and/or HIV-1 (p24 10 ng/mL) for 8
hr and RNA was isolated and assayed for TAAR1.
TAAR1 mRNA levels were
significantly increased with METH + HIV-1 cotreatments (A, ***p<0.001, n=3,
independent astrocyte donors). METH- and/or HIV-1-induced intracellular cAMP was
measured at 2 min and 10 min post-activation (B). METH alone significantly induced
intracellular cAMP at both time points (**p<0.01). HIV-1 alone and in combination
with METH significantly induced intracellular cAMP at 10 min post-activation
(**p<0.01). Astrocytes were fixed and immunostained for GFAP (red) and TAAR1
(green). TAAR1 expression was localized in both cytoplasm and nucleus (C, D, E and F).
To further confirm nuclear localization, confocal microscopy was performed (C1, D1, E1
and F1).
Activation with HIV-1 alone (E1) and in combination with METH (F1)
increased nuclear TAAR1 localization.
75 Figure 3.3:
Forskolin, METH and β -PEA induce astrocyte intracellular cAMP
signaling.
Astrocyte intracellular cAMP signaling was induced with forskolin (A) as a positive
control, METH (B) and β-PEA (C), known TAAR1 agonists. Representative donor EC50
values for forskolin (10.96 µM), METH (32.86 µM), and β-PEA (11.04 µM) are shown.
Intracellular cAMP induction assays were determined for multiple biological donors (n),
and cumulative data is illustrated in panel D.
76 77 Figure 3.4: RNA interference downregulates astrocyte TAAR1 protein levels and
intracellular cAMP in response to METH and β -PEA. Human astrocytes were MOCK-,
siCON- and siTAAR1-transfected and plated for 48 hr followed by 24 hr of METH (500
µM) treatment. Cells were fixed and probed for TAAR1 (green) and GFAP (red).
MOCK- (A) and siCON- (C) transfected astrocytes showed TAAR1 protein expression
with and without METH treatment at 24 hr. METH (500 µM) treated MOCK- and
siCON-transfected astrocytes demonstrated similar levels of TAAR1 protein (B and D).
As expected siTAAR1-transfected astrocytes demonstrated reduced TAAR1 expression (E
and F). Intracellular cAMP levels were quantified in MOCK-, siCON- and siTAAR1transfected astrocytes. siTAAR1-transfected astrocytes showed significantly reduced
METH and b-PEA-induced intracellular cAMP (G, ***p<0.001, n=4, independent
astrocyte donors).
78 Figure 3.5: METH-induced TAAR1 activation regulates EAAT-2 mRNA levels and
function. Primary human astrocytes were MOCK-, siCON-, siTAAR1-, CON-GFP- or
TAAR1-GFP- transfected and treated with METH (500 µM). Two independent astrocyte
donors were tested with a minimum of triplicate determinations in each experiment. Data
was analyzed as fold change to internal control and presented as cumulative data for
both donors. RNA was collected at 8 hr and glutamate clearance was measured at 24 hr
post-treatment. (A) EAAT-2 mRNA levels significantly decreased in MOCK- and siCONtransfected astrocytes treated with METH (*p<0.05, **p<0.01). Changes in METHmediated EAAT-2 levels were blocked by siTAAR1 transfection. (B) Glutamate clearance
79 was performed in parallel. METH treatment significantly decreased glutamate clearance
in MOCK- and siCON-transfected astrocytes while siTAAR1 transfection significantly
increased glutamate clearance compared to METH treated controls (***p<0.001). (C &
D) Both TAAR1-GFP-transfection and METH treatment significantly lowered levels of
EAAT-2 mRNA expression in astrocytes and in parallel resulted in an exacerbated
reduction in glutamate clearance abilities (***p<0.001).
80 3.7 SUPPLEMENTARY FIGURES
Supplementary figure 3.6: TAAR1 overexpression model in primary human astrocytes.
Primary human astrocytes were transfected with Control-GFP or TAAR1-GFP
(Addgene). (A) Changes in TAAR1 mRNA levels were measured by RT2-PCR and were
significantly increased with TAAR1-GFP transfection (***p<0.001). (B & C) Astrocytes
transfected with TAAR1-GFP showed peri-nuclear localization compared to CON-GFP,
which was distributed throughout the cytoplasm. Original magnification 200x.
81 Chapter 4
METH-INDUCED, TAAR1-ASSOCIATED CREB SIGNALING SERVES AS A
MASTER REGULATOR OF ASTROCYTE EAAT-2
82 4.1 ABSTRACT
METH regulates astrocyte EAAT-2 via TAAR1 signaling.
Further,
proinflammatory mediator, IL-1β, increases astrocyte TAAR1 levels and METH-induced
intracellular cAMP levels.
Overexpression and/or knockdown of astrocyte TAAR1
correspond to decrease and/or increase in astrocyte EAAT-2 levels and function,
respectively. Astrocyte EAAT-2 is regulated at the transcriptional level by CREB and
NF-κB (8), transcription factors activated by cAMP, calcium and IL-1β. However,
METH-mediated increases in these second messengers and signal transduction pathways
have not been shown to directly decrease astrocyte EAAT-2 transcription. We propose
CREB activation serves as a master regulator of astrocyte EAAT-2. To investigate the
temporal order of events leading to CREB activation, we utilized genetically encoded
calcium indicators, or GCaMPs, to first visualize and quantify METH-induced calcium
signaling. RNA interference targeting PKA, a cAMP-dependent protein kinase, and NFκB subunit p65, in addition to PKA and NF-κB specific inhibitors, support the
involvement of cAMP/calcium and IL-1β, in astrocyte EAAT-2 regulation. Furthermore,
we investigated CREB phosphorylation at serine 133/142, the co-activator and corepressor forms, respectively, following METH-induced activation. Overall, this work
identifies critical signaling pathways and therapeutic targets for astrocyte EAAT-2
recovery.
83 4.2 INTRODUCTION
METH abuse commonly results in neurocognitive decline and the development of
psychiatric symptoms, a shared characteristic of HAND (2). The extent of cognitive
decline associated with HAND is exacerbated with the use of METH and can partly be
attributed to glutamate dysregulation. As a major excitatory neurotransmitter in the CNS,
glutamate is vital in the proper physiological functions of the brain including acquisition
of learning and memory (248). Improper balance of glutamate concentrations, producing
excessive glutamate in the extracellular environment, results in excitotoxicity.
Glutamate transporters are responsible for the synaptic reuptake of glutamate.
The
family of EAATs consists of five transporters, the human EAATs 1-5. EAAT-1 and -2
are distributed in astrocytes, whereas EAAT-2 is responsible for >90% of total glutamate
uptake in the brain (5). Glutamatergic signaling is coupled with glutamate transport
mechanisms; therefore, dysregulation in astrocyte EAAT-2 and the inability to clear
extracellular glutamate results in pathological features associated with aberrant glutamate
neurotransmission. Thus, it is imperative that EAAT-2 be properly regulated.
We have shown that METH activates astrocyte TAAR1, increasing intracellular
cAMP levels (2).
Furthermore, TAAR1 regulates brain monoamines suggesting
astrocytes play a vital role in drug abuse. METH-induced excitotoxicity is, in part, a
result of astrocyte EAAT-2 downregulation. METH-mediated EAAT-2 downregulation
and decreased glutamate uptake is directly exacerbated through TAAR1 upregulation and
reduced or eliminated by TAAR1 knockdown or inhibition. Previous analysis of the
EAAT-2 promoter showed that CREB and NF-κB are positive regulators of EAAT-2
transcription (249). Experiments with primary human astrocytes stably expressing the
84 firefly luciferase human EAAT-2 promoter, demonstrate that promoter activity was
significantly increased by ceftriaxone, amoxicillin and dibutytyl cyclic AMP (249).
Despite CREB phosphorylation and NF-κB activation, EAAT-2 expression is reduced by
both METH and HIV-1 alone and in combined conditions. While information is available
suggesting that EAAT-2 can be positively or negatively regulated by NF-κB, the negative
regulation of EAAT-2 transcription via CREB-mediated negative regulation remains to
be investigated (249).
METH is well known to stimulate calcium signaling in several cell types (250).
Both cAMP and calcium are well established to activate gene transcription containing
conserved cAMP responsive elements (CRE)s (251, 252), through their ability to
phosphorylate CREB at serine 133 (pCREBSer133) increasing dimerization affinity of the
CREB binding protein (CBP) and inducing transcription (253, 254).
The EAAT-2
promoter contains a CREB element at the -310 site that contributes to transcriptional
regulation of EAAT-2 (Scheme 4.1). However, elevated [Ca+2]I can also activate
Ca+2/calmodulin-dependent protein kinase II (CaMKII) that inhibits transcription by
dominating the activating effects of PKA and preventing the dimerization of CREB with
CBP via phosphorylation of CREB at serine 142 (pCREBSer142)(255).
We propose the expression of a novel GPCR, TAAR1, mediates METH-induced
aberrant glutamate concentrations in the extracellular environment, thereby exacerbating
HIV-1-mediated neurodegeneration. This study evaluates the tripartite signaling
including PKA; NF-κB; and calcium, culminating into CREB activation via differential
regulation mediated by METH-induced TAAR1 activation. We expect to uncover novel
85 insights into the mechanism(s) of how METH-abuse leads to astrocyte-neurotoxicity in
the setting of HAND via TAAR1 signaling.
86 87 Schematic 4.1: Proposed TAAR1 Signaling in astrocytes. The EAAT-2 promoter
contains a CREB element at the -310 site that contribute to transcriptional regulation of
EAAT-2. The contribution(s) and interplay between PKA, PKC, NF-κB and calcium on
EAAT-2 signaling is modulating the phosphorylation of the master regulator CREB on
Serine 133 and 142.
88 4.3 MATERIALS AND METHODS
4.3.1 Isolation, cultivation and activation of primary human astrocytes
Human astrocytes were isolated from first and early second trimester elected aborted
specimens as previously described (97). Briefly, tissues ranging from 82 to 127 days
were procured in full compliance with the ethical guidelines of the National Institutes of
Health, University of Washington and North Texas Health Science Center. Cell
suspensions were centrifuged, washed, suspended in media, and plated at a density of
20×106 cells/150 cm2. The adherent astrocytes were treated with trypsin and cultured
under similar conditions to enhance the purity of replicating astroglial cells. These
astrocyte preparations were routinely >99% pure as measured by immunocytochemistry
staining for GFAP. Astrocytes were treated with METH (500 µM, Sigma-Aldrich Inc.,
St. Louis, MO) IL-1β (20 ng/mL), EPPTB (20 µM), H89 (20 µM) alone and in
combination at 37°C and 5% CO2. Dose- and time-kinetics revealed optimal
concentrations for METH and IL-1 β were not toxic to astrocytes (data not shown).
4.3.2 RNA extraction and gene expression analyses
Astrocyte RNA was isolated as described in (224) 8 hr post-treatment and mRNA levels
were assayed by real-time polymerase chain reaction (PCR). Taqman 5’ nuclease realtime PCR was performed using StepOnePlus detection system and inventoried gene
expression assays (Life Technologies Inc., Carlsbad, CA). Commercially available
TaqMan® Gene Expression Assays were used to measure EAAT-2 (cat no.
Hs00188189_m1), PKA (cat no. Hs00373229_s1) and glyceraldehyde 3-phosphate
89 dehydrogenase (GAPDH) (cat no. 4310884E) mRNA levels. GAPDH, a ubiquitously
expressed housekeeping gene, was used as an internal normalizing control. The 25 ml
reactions were carried out at 48ºC for 30 min, 95ºC for 10 min, followed by 40 cycles of
95ºC for 15 s and 60ºC for 1 min in 96-well optical, real-time PCR plates. Transcripts
were quantified by the comparative ΔΔCT method, and represented as fold-change of
control.
4.3.3 Glutamate clearance assay
Primary human astrocytes were plated in 48-well tissue culture plates at a density of 0.15
x 106 cells/well and allowed to recover for 24 hr prior to treatment as described in section
4.3.1. Following 24 hr treatment, glutamate (400 µM) dissolved in phenol-free astrocyte
medium was added into each well and clearance was assayed at 4 and 8 hr. The assay was
performed and analyzed according to manufacturer’s guidelines (Amplex Red Glutamic
Acid/Glutamate Oxidase Assay Kit, Life Technologies).
4.3.4 Viability assays
Primary human astrocytes were plated in 48-well tissue culture plates at a density of 0.15
x 106 cells/well and allowed to recover for 24 hr prior to treatment as described in section
2.1. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a
colorimetric assay for measurement of metabolic activity, was performed for cell viability
(225). The MTT assay was performed at appropriate time points, briefly five percent
MTT reagent in astrocyte medium was added to astrocytes and incubated for 20-45 min
at 37°C. The MTT solution was removed and crystals were dissolved in DMSO for 15
min with gentle agitation. The absorbance of the DMSO/crystal solution was assayed at
90 490 nm in a Spectromax M5 microplate reader (Molecular Devices, Sunnyvale, CA).
4.3.5 Immunocytochemistry
Astrocytes were cultured as adherent monolayers in 48-well plates at a density of 0.1 x
106 cells per well. Cells were fixed with ice cold acetone:methanol (1:1) for 30 min at 20°C and then blocked in phosphate buffered saline (PBS) with 2% bovine serum
albumin (BSA) containing 0.1% Triton X-100 for 1 hr at room temperature. Cells were
incubated with CREB antibody (1:700, rabbit, Cell Signaling), pCREB antibody (1:700,
rabbit, Cell Signaling) and glial fibrillary acidic protein (GFAP) antibody (1:1000,
chicken, Covance Inc., Emeryville, CA) in PBS (2% BSA, 0.1% Triton X) overnight at
4°C. Cultures were then washed and stained with Alexa Fluor
®
secondary antibodies
(488 nm, green) and (594 nm, red) for 2 hr at room temperature (1:1000, Life
Technologies). Nuclei were visualized with 4',6-diamidino-2-phenylindole (DAPI)
(1:800, Life Technologies). Micrographs were obtained on a ECLIPSE Ti-300 using the
NLS-Elements BR. 3.0 software (Nikon Inc., Melville, NY).
4.3.6 Western Blot analysis
Confluent astrocyte cultures in tissue culture plates were treated as described above. At
appropriate time points, total protein, cytoplasmic and nuclear protein extracts were
isolated using the M-PER or NE-PER nuclear and cytoplasmic extraction kit (Thermo
Fisher Scientific). Total protein, cytoplasmic and nuclear protein samples (30 µg,
91 respectively) were boiled with 4 × NuPAGE LDS loading sample buffer at 100°C for 5 to
10 minutes, resolved by NuPAGE 4% to 12% Bis-Tris gel and subsequently transferred
to a polyvinyldifluoride (PVDF) membrane using i-Blot (Life Technologies). The
membrane was incubated with anti-CREB (1:700, rabbit, Cell Signaling), antipCREBSer133 (1:700, rabbit, Cell Signaling), anti pCREBSer142 (1:500, rabbit, Signalway
Antibodies), anti-PKA (1:700, rabbit, Call Signaling), anti-pPKA (1:700, rabbit, Cell
Signaling), anti-CaMKII (1:500, mouse, Santa Cruz), anti-pCaMKII (1:500, mouse,
Santa Cruz), anti-PKC (1:500, rabbit, Cell Signaling), anti-pPKC antibody (1:500, rabbit,
Santa Cruz) overnight at 4°C, washed and then incubated with anti-rabbit/anti-mouse
goat antibody IgG conjugated to horseradish peroxidase (1:5,000, Bio-Rad, Hercules,
CA, USA) for 2 h at room temperature. The membrane was then developed with
SuperSignal West Femto substrate (Thermo Fisher Scientific) and imaged in a
Fluorochem HD2 Imager (Proteinsimple, Santa Clara, CA, USA). β-Actin (1:4,000,
Sigma Aldrich, St. Louis, MI, USA), GAPDH (1:1,000, Santa Cruz Biotechonology,
Santa Cruz, CA, USA) and Lamin A/C (1:3,000, Cell Signaling Technology) were used
as loading controls for cytoplasmic and nuclear extracts, respectively.
4.3.7 cAMP assay
Intracellular cAMP levels in astrocytes were measured using a commercially available
homogenous, bioluminescent assay, cAMP-Glo™ Assay, (Promega Corp., Madison,
WI). Adherent monolayers of astrocytes cultured in 96-well plates (50,000 cells/well)
were exposed to forskolin or METH (all from Sigma-Aldrich) following pretreatment
92 with EPPTB or IL-1β. Cells were directly activated and lysed in the tissue culture plate.
Lysates were diluted to a final cell concentration of approximately 1,000 cells/µL in lysis
buffer [500 µM 3-isobutyl-1-methylxanthine (IBMX) and 100 µM Ro 20-1724, cAMP
specific phosphodiesterase inhibitors (both from Sigma-Aldrich)] and transferred to a
white opaque flat bottom 96-well assay plates (Corning Inc. Life Sciences, Tewksbury,
MA).
Intracellular cAMP levels were assayed using GloMax 96 Microplate
Luminometer with dual injectors (Promega).
4.3.8 Transfection of astrocytes
®
Cultured human astrocytes were transfected with On-Target plus small interfering RNA
(siRNA) pools specific to PKA (siPKA), p65 (sip65) non-targeting control siRNA pools
(siCON), Thermo Scientific, Waltham, MA, USA) and without siRNA (MOCK), or with
plasmids
CON-GFP
or
GCaMP6s,
PrecisionShuttle
Vector
System,
Origene
Technologies Inc., Rockville, MD) using the AmaxaTM P3 primary cell 96-well
Nucleofector kit and shuttle attachment (Lonza, Walkersville, MD, USA) according to
the manufacturer’s instructions. Briefly, 1.6 million astrocytes were suspended in 20 µl
nucleofector solution containing siCON, siTAAR1, siPKA or sip65 (100 nM) or
GCaMP6s (0.25 µg/1.6 million cells) and transfected using protocol CL133. Transfected
cells were supplemented with astrocyte media and incubated for 30 min at 37°C prior to
plating. Cells were allowed to recover for 48 hr prior to experimental use.
93 4.3.9 Confocal analysis
Human astrocytes were cultured on glass bottom 6-well tissue culture plates (MatTek
Corp., Ashland, MA) at a density of 2x106 cells/well. Astrocytes were stimulated with
ionomycin, as appositive control, forskolin or METH and visualized for fluorescence
activity.
Micrographs were obtained on an Olympus IX71 Microscope (Olympus
America Inc., Center Valley, PA). Confocal colocalization analysis and two-dimensional
histogram was performed using BioimageXD software (Free Software Foundation Inc.,
Boston, MA).
4.3.10 Statistical analyses
Statistical analyses were performed using Prism V6.0 with one-way analysis of variance
(ANOVA) and Newman-Keuls post-test for multiple comparisons. Significance was set
at p ≤ 0.05 and data represents means ± standard error of the mean (SEM). Results are
representative of two or more independent donors, denoted as n, with a minimum of three
individual replicates in each experiment.
94 4.4 RESULTS
4.4.1 TAAR1 antagonist, EPPTB, reversed METH-mediated EAAT-2 downregulation
and cAMP activity. Glutamate clearance through astrocyte EAAT-2 is regulated by
METH (2). METH signals through astrocyte TAAR1, inducing intracellular cAMP,
thereby regulating EAAT-2 mRNA levels and activity. Previously we identified that
TAAR1 overexpression decreases astrocyte EAAT-2 mRNA levels and function while
TAAR1 downregulation increases EAAT-2 function. To further investigate the role of
TAAR1 on EAAT-2 expression we measured EAAT-2 mRNA levels in parallel with
glutamate clearance, following 24 hr treatment with METH in the presence or absence of
EPPTB (Fig 4.1A and B). METH significantly reduced both astrocyte EAAT-2 levels
and astrocytes ability to clear extracellular glutamate (Fig 4.1A and B, ***p<0.001,
*p<0.05, respectively). In the presence of EPPTB, METH-mediated decreases in EAAT2 mRNA levels and glutamate clearance were significantly reversed (***p<0.001). To
determine if EPPTB prevented METH-induced increases in intracellular cAMP,
astrocytes were treated with forskolin, as a positive control, and METH +/- EPPTB (Fig
4.1C). It was confirmed that EPPTB alone did not change basal levels of intracellular
cAMP.
Significant increases in astrocyte cAMP levels were observed following
forskolin (100 µM) and METH (100 µM) (Fig 4.1C, ***p<0.001, **p<0.01,
respectively). As expected, the presence of EPPTB did not affect cAMP induction by
forskolin, but did significantly decrease METH-induced cAMP (***p<0.001) confirming
TAAR1 involvement.
95 4.4.2 IL-1β regulated astrocyte TAAR1, EAAT-2 and METH-induced cAMP levels.
IL-1β, being a proinflammatory cytokine in the CNS microenvironment during HAND,
was utilized to mimic HIV-1-associated neuroinflammation. Our previously published
data demonstrated astrocyte TAAR1 is upregulated during combined treatment of METH
and HIV-1, therefore we investigated whether IL-1β changed astrocyte TAAR1 and
EAAT-2 mRNA levels (Fig 4.2A and B). TAAR1 mRNA levels significantly increased
~ 7-fold following IL-1β treatment of astrocytes (***p<0.001). EAAT-2 mRNA levels
were observed to significantly reduced ~ 50% with IL-1β treatment (***p<0.001). A
significant negative correlation was observed in TAAR1 and EAAT-2 levels (Fig 4.2C,
***p<0.001).
To determine if IL-1β-mediated increases in TAAR1 correlated with
elevated METH-induced intracellular cAMP levels, astrocytes were pre-treated with IL1β and then transiently activated with forskolin as the positive control, and METH (Fig
4.2D). Basal cAMP levels did not change with IL-1β pretreatment. Forkolin and METH
significantly increased intracellular cAMP levels (***p<0.001, **p<0.01, respectively).
However, forskolin-induced cAMP levels did not change with IL-1β pretreatment, while
IL-1β pretreatment significantly increased METH-induced cAMP levels approximately
3-fold (***p<0.001).
4.4.3 NF-κ B downregulation increased EAAT-2 mRNA levels.
IL-1β binds and
activates the IL-1β receptor in astrocytes, thereby activating NF-κB and resulting in
dissociation and translocation of subunit p65 to the nucleus. EAAT-2 promoter analysis
reveals three NF-κB elements, suggesting positive transcriptional regulation of EAAT-2
by IL-1β; however, IL-1β significantly reduced EAAT-2 mRNA levels. To elucidate the
96 effects of IL-1β on EAAT-2 mRNA regulation further, we transfected astrocyte with
siRNA specific for NF-κB subunit p65 and measured EAAT-2 mRNA levels in the
presence and absence of METH (Fig 4.3). Downregulation of p65 significantly increased
EAAT-2 mRNA levels that were unchanged with METH treatment (***p<0.001).
4.4.4 METH-induced PKA activation regulated astrocyte EAAT-2 levels
METH-induced activation of TAAR1 results in increased intracellular cAMP levels. The
canonical cAMP pathway activates PKA, therefore, we investigated if METH induced
PKA phosphoryation and whether PKA downregulation or inhibition regulated EAAT-2
levels. Protein collected following time kinetic activation by METH was assayed for
total PKA, pPKA and GAPDH, as an internal control (Fig 4.4A). METH significantly
induced phosphorylation of PKA as early as 10 min that remained unchanged through 30
min (**p<0.01). Astrocytes were transfected with siRNA specific for PKA catalytic
subunit α, and assayed for PKAcα and EAAT-2 mRNA levels (Fig 4.4B and C).
PKAcα downregulation successfully reduced PKA levels by approximately 75% in the
presence or absence of METH (Fig 4.4B, ***p<0.001).
Utilizing the PKAcα
downregulation model, astrocytes were assayed for EAAT-2. PKAcα downregulation
alone significantly increased EAAT-2 mRNA levels (Fig 4.4C, ***p<0.001). EAAT-2
mRNA levels were significantly increased in METH-treated astrocytes transfected with
siPKA as compared to MOCK and siCON (Fig 4.4C, **p<0.01, ***p<0.001,
respectively). To further investigate PKA action on EAAT-2 levels, we used PKA
inhibitor, H89 (Fig 4.4D). The mechanism of action for H89 is to prevent ATP binding
to PKA catalytic subunit, thus reducing phosphorylation of downstream targets. METH
97 significantly decreased astrocyte EAAT-2 mRNA levels (Fig 4.4D, *p<0.05), while H89
significantly increased EAAT-2 mRNA levels (Fig 4.4D, ***p<0.001). Furthermore,
H89, successfully recovered METH-induced decreases in EAAT-2 mRNA levels (Fig
4.4D, ***p<0.001).
4.4.5 METH transiently increased astrocyte intracellular calcium levels.
Previous reports suggest that TAAR1 activation not only results in activation of PKA, but
also induces [Ca+2]i in neurons. To determine if astrocyte TAAR1 activation results in
transient calcium increases, astrocytes were transfected with GCaMP6s, an intracellular
calcium indicator that allows for real-time calcium waves visualized by GFP activation
(Fig 4.5A-E). Analysis of the data is represented over time as a histogram (Fig 4.5F).
Baseline fluorescence had undetectable [Ca+2]i prior to METH stimulation (Fig 4.5A). A
12s treatment of METH produced a fluorescent signal (Fig 4.5B). A spike in [Ca+2]i is
visualized at 100s, with a peak occurring at 150s (Fig 4.5C and F). METH activation, at
200s and 400s, produced punctate calcium fluorescence within the cytoplasm (Fig 4.5D
and E). The signal begins to quench at 400s (Fig 4.5E).
4.4.6 METH induced phosphorylation of CaMKII and PKC.
The literature suggests that METH increases [Ca+2]i levels via TAAR1, and thereby leads
to activation of both CaMKII and PKC. Thus, we treated astrocytes with METH and
collected protein lysates at 15 and 30 min. Lysates were assayed for total CaMKII and
pCaMKII (Fig 4.6A) in addition to total PKC and pPKC (Fig 4.6B). METH increases
the phosphorylated product of both CaMKII and PKC through 30 min.
98 4.4.7 METH activated CREB phosphorylation.
Since METH induces activation of PKA, we evaluated METH-induced phosphorylation
of CREB. Astrocytes were treated with METH at different time points, fixed and
immunostained for GFAP (red) total CREB (green) and pCREBSer133 (green) (Fig 4.7AE). In parallel, protein lysates were collected following METH stimulation and probed
for pCREBSer133 and normalized to total CREB (Fig 4.7F). GAPDH was analyzed as an
endogenous control for protein loading. Basal levels of total CREB and pCREBSer133
were estimated in control astrocytes (Fig 4.7A and B). Increased pCREBSer133 levels
were visualized at 15 and 20 min (Fig 4.7E-H). METH-mediated phosphorylation of
CREB at serine 133 significantly increased at 10 min and remained phosphorylated out to
30 min (Fig 4.7F, *p<0.05, **p<0.01, respectively).
4.4.8 Downregulation of PKA and TAAR1 reduced METH-mediated CREB
phosphorylation.
It is hypothesized that METH-mediated changes in CREB are via METH-induced
TAAR1 activation and downstream phosphorylation of PKA, therefore using siRNA
specific for PKA we measured pCREBSer133 in MOCK-, siCON- and siPKA-transfected
astrocytes. Transfected astrocytes were treated with METH and immunostained for
GFAP (red), total CREB (green) and pCREBSer133 (green) (Fig 4.8A-C1). Total CREB
levels decreased in siPKA-transfected astrocytes (Fig 4.8C) as compared to MOCK- and
siCON-transfected astrocytes (Fig 4.8A and B). In the absence of METH exposure
amounts of pCREBser133 remained unchanged (Fig 4.8A1-C1).
99 METH treatment
phosphorylated CREB at serine 133, which was reduced in siPKA-transfected astrocytes
(Fig 4.8A2-C2).
4.4.9 TAAR1, PKA and NF-kB modulated pCREBSer133
We proposed that CREB serves as the master regulator of EAAT-2 transcription
downstream of METH-mediated TAAR1 activation and IL-1β treatment.
Thus,
siTAAR1-, siPKA- and sip65-transfected astrocytes were transiently treated with METH
pCREBSer133 to total CREB ratios were measured. As expected, METH increased
pCREBSer133 in MOCK- and siCON-transfected astrocytes (Fig 4.9A-C). Furthermore,
TAAR1 and PKA downregulation reduced METH-mediated activation of pCREBSer133
(Fig 4.9A and B). Respectively, p65 downregulation increased pCREBSer133 in the
presence and/or absence of METH (Fig 4.9C).
4.4.10 METH and IL-1β-induced nuclear pCREBSer133/Ser142
Although we have observed total pCREBSer133 following METH treatment, EAAT-2
continued to be downregulated. METH induced activation of CaMKII and a downstream
target of CaMKII activation is pCREBser142, which acts as a dominant negative regulator
of pCREBSer133-induced transcriptional activation. Thus, we investigated the ability of
METH and IL-1β to induce phosphorylation of pCREBSer142 (Fig 4.10A and B).
Respectively, we show that METH and IL-1β
mediated signaling cascades
phosphorylated total CREB at serine 142 up to 60 min and 15 min, respectively. We
confirmed nuclear localization of pCREB in cytoplasmic and nuclear protein lysates
100 collected following METH and IL-1β stimulation and probed for GAPDH, Lamin, total
CREB, pCREBSer133 and pCREBSer142 (Fig 4.10C). We observe translocation of both
pCREBSer133 and pCREBSer142 following METH and IL-1β treatment.
4.5 DISCUSSIONS and CONCLUSIONS
We previously reported that METH induced activation of astrocyte TAAR1 and
increased intracellular cAMP levels.
Moreover, TAAR1 downregulation and
overexpression resulted in astrocyte EAAT-2 transcriptional and functional change. In
this study we investigated mechanisms involved in the regulatory pathways. We have
demonstrated that astrocyte EAAT-2 is regulated by the transcription factor CREB,
downstream of METH-induced TAAR1 activation. Additionally, we have shown that
astrocyte TAAR1 and METH-induced intracellular cAMP levels increased while EAAT2 levels decreased with IL-1β. Tripartite signaling events associated with METH and IL1β, converge to serve as stimulation for CREB, the master regulator of EAAT-2
transcription in astrocytes. Taken together, these studies establish negative regulation of
CREB activity downstream of TAAR1 activation and NF-κB. This suggests a potential
mechanism for PKA/NF-κB/CREB signaling in modulating METH- and HIV-1-induced
EAAT-2 downregulation.
To further determine the role of TAAR1 on astrocyte EAAT-2 regulation we
show TAAR1 antagonist, EPPTB, recovered METH-mediated decreases in EAAT-2
levels. EPPTB alone did not significantly change EAAT-2 levels or astrocytes ability to
clear extracellular glutamate.
However, quantification of intracellular cAMP in
101 astrocytes pretreated with EPPTB and transiently activated by METH decreased
intracellular cAMP levels below baseline. Previous studies have shown that EPPTB
alone reduced cAMP levels below basal levels suggesting constitutive activity of TAAR1
(80). Binding affinity assays for EPPTB showed higher affinity for mouse TAAR1 (Ki 24 nM) when compared to human TAAR1 (Ki >20 µM) (256), and that EPPTB
successfully antagonized cAMP induction of mouse and human TAAR1 by β-PEA (80).
EPPTB characteristics include high lipophilicity, poor aqueous solubility and high
clearance in human microsomes. EPPTB optimization resulted in the ability to achieve
high bioavailability and was detected and quantified in the brain, indicating it to be
sufficient for in vivo pharmacology experiments (256). Therefore, EPPTB was discovered
to be a potent and full antagonist for mouse TAAR1 with lower affinity in human
TAAR1, but to still exert similar effects. Taken together, EPPTB serves as a target for
astrocyte TAAR1 potentially preventing activation of signaling pathways that lead to
EAAT-2 downregulation. Moreover, the optimized product can be used in future in vivo
experiments.
Inflammatory mediator, IL-1β, increased astrocyte TAAR1 mRNA levels
approximately 8-fold and significantly decreased astrocyte EAAT-2 mRNA levels.
Clinical studies in post-mortem brain tissue of HIV encephalitis (HIVE) patients versus
HIV-1 patients without encephalitis, revealed HIVE patients were more than 50% likely
to have a significant reduction in EAAT-2 expression that was correlated to IL-1β
positive microglia (257). This suggests that the release of IL-1β, activates astrocytes and
results in the transcriptional repression of EAAT-2. Moreover, HIV-1- or gp120-induced
EAAT-2 downregulation in human astrocytes prevents efficient glutamate transport by
102 cells (5). Astrocytes treated with IL-1β showed significantly increased TAAR1 mRNA
levels.
Similar increases were previously observed with TAAR1 overexpression in
primary human astrocytes, therefore IL-1β-mediated increases of TAAR1 may serve as a
physiological model for disease progression. Furthermore, astrocytes pretreated with IL1β had higher METH-induced cAMP levels, further supporting the hypothesis that
TAAR1 regulates METH-induced EAAT-2 downregulation, by mediating intracellular
signaling pathways downstream of TAAR1 activation.
We reported that METH induces cAMP dose response curves in primary human
astrocytes. cAMP binds to the regulatory subunits of PKA, releasing the catalytic
subunits and allowing efficient translocation to the nucleus where it phosphorylates
proteins involved in gene transcription. We determined that transient METH treatments
induced PKA phosphorylation and that PKA downregulation or inhibition regulated
astrocyte EAAT-2 levels and function. Previous studies have demonstrated that chronic
METH use leads to PKA activation (258) and that PKA has been shown to be an
important mediator of EAAT-2 transcription (187). This has been further validated with
the use of H89, a PKA inhibitor that increased EAAT-2 mRNA and protein levels in a
dose-dependent manner (259). Moreover, we have shown that H89 alone increased
EAAT-2 mRNA levels and recovered METH-mediated decreases in EAAT-2. Studying
the temporal order of signaling events shines light on the mechanisms associated with
METH-induced EAAT-2 downregulation.
Additionally, METH-induced activation of TAAR1 has been shown to induce
calcium signaling in neurons (67). Consistent with these studies METH transiently
increased calcium signaling in astrocytes as measured by the fluorescence of GCaMP6s-
103 transfected astrocytes. GCaMPs are genetically modified calcium indicators created from
GFP, calmodulin and myosin light chain kinase (M13) (260). Recent studies demonstrate
that METH and glutamate mediate intracellular calcium signals in neurons that are
transfected and overexpressing GCaMP5 (261). Furthermore, GCaMPs used in vivo,
demonstrate calcium increases following METH stimulation (260). Moreover, adult mice
transduced with GCaMP5E driven by the GFAP promoter demonstrated spontaneous
calcium spikes and oscillations in hippocampal synapses that differed between the
astrocyte somata and astrocytic processes (262). Together, this suggests that GCaMPs
are excellent indicators for METH-mediated calcium signaling and can be detected in
astrocytes in vivo.
The generation of intracellular calcium responses results in activation of PKC and
CaMKII. We observed METH and IL-1β phosphorylated CaMKII. Astrocyte CaMKII
activation regulates cellular excitability, cytoskeletal structure and cellular metabolism
(263). One treatment of METH leads to phosphorylated CaMKII in the prefrontal cortex
and nucleus accumbens in mice (264), (265). While cAMP-dependent PKA, PKC and
CaMKs pCREBSer133, the transcription-activating site (266), early studies demonstrate
that Ca+2-induced activation of CaMKII-mediated pCREBSer142 and prevented CREB
dimerization with CBP, thereby ceasing transcription (255). Additionally, METH and IL1β induced pCREBSer133 and pCREBSer142 was confirmed to be in the nucleus.
Downregulation of NF-κB subunit, p65 significantly increased pCREBSer133, and EAAT2 mRNA levels. Preventing NF-κB translocation to the nucleus increases the amount of
pCREBSer133, the co-activator. There is increasing evidence suggesting that pCREBSer142
attenuates CREB activity, regardless of pCREBSer133. Further studies would confirm that
104 pCREBSer142 prevents pCREBSer133 from dimerizing with the co-activators required for
EAAT-2 transcription, downstream of TAAR1 activation.
Together these data demonstrate that regulation and activation of primary human
astrocyte TAAR1 by METH and IL-1β lead to neurotoxic outcomes such as
excitotoxicity.
Astrocyte EAAT-2 levels and function are regulated through shared
mechanisms during METH and HIV-1 CNS infection including second messengers such
as cAMP, calcium and inflammation. Importantly, our data demonstrate astrocyte EAAT2 is directly regulated via TAAR1 activation and regulation and that extrinsic regulation
of signaling factors downstream of TAAR1 and cAMP induction, not only reduce
activation of subsequent signaling factors, but also reduce or eliminate the METHmediated alterations in astrocyte function. Finally, preliminary studies indicate that
astrocyte-TAAR1 may be a novel receptor for the common comorbidity of METH abuse
in HAND and by targeting downstream signaling of TAAR1 we may be able to increase
astrocyte EAAT-2 levels, thereby recovering METH/HIV-1-induced excitotoxicity.
105 106 107 Figure 4.1 EPPTB reversed METH-induced decreases in EAAT-2 levels/function and
prevented METH-induced cAMP increases. Primary human astrocytes were treated
with TAAR1 selective antagonist (EPPTB 20 µM) for 1 hr followed with METH (500 µM)
treatment. RNA was isolated at 8 hr and assayed for EAAT-2. Astrocyte EAAT-2 levels
significantly decreased with METH, which were blocked by EPPTB (A, ***p<0.001).
Glutamate clearance was measured 24 hr following treatment (B). Astrocytes treated
with METH showed significant decreases in glutamate clearance abilities that were
recovered with EPPTB (B, *p<0.05, ***p<0.001, respectively). Intracellular cAMP was
quantified in astrocytes treated with EPPTB +/- forskolin (7.32 µM and 8.5 µM) and
METH (26.3 µM and 20.9 µM) EC50 for two independent donors and represented as fold
change +/- SEM (C). Forskolin and METH significantly increased intracellular cAMP
levels compared to baseline (C, ***p<0.001, **p<0.01, respectively).
significantly reduced METH induction of cAMP
baseline or forskolin cAMP levels.
108 EPPTB
(***p<0.001), but did not affect
Figure 4.2: IL-1β regulated astrocyte TAAR1/EAAT-2 levels and increased METHmediated intracellular cAMP levels. Multiple biological astrocyte donors were treated
with IL-1β (20 ng/mL). RNA was collected and assayed for TAAR1 (A) and EAAT-2 (B).
IL-1β treatment significantly increased astrocyte TAAR1 levels approximately 10-fold
and significantly decreased astrocyte EAAT-2 levels approximately 50% (***p<0.001,
respectively). Correlative analysis demonstrated a significant negative correlation with
an R2 = 0.4443 (C, ***p<0.001).
To evaluate if IL-1β pretreatment significantly
increases METH-mediated intracellular cAMP levels, astrocytes were treated with IL-1β
for 24 hr, to allow for upregulation of TAAR1 mRNA levels.
109 Forskolin or METH
stimulation alone significantly increased intracellular cAMP levels (***p<0.001,
**p<0.01). IL-1β-alone did not alter astrocyte baseline cAMP levels nor did it change
forskolin-induced cAMP levels. However, IL-1β pretreatment significantly increased
METH-induced cAMP levels by approximately 6-fold (D, ***p<0.001).
110 Figure 4.3: NF-kB downregulation significantly increased EAAT-2 levels. Primary
human astrocytes were MOCK-, siCON- and sip65-transfected. Following recovery,
astrocytes were treated with METH (500 µM). RNA was collected at 8 hr and assayed
for EAAT-2 by RT2-PCR. MOCK- and siCON-transfection significantly reduced EAAT-2
mRNA levels following METH treatment, when compared to their untreated controls
(*p<0.05). p65 knockdown significantly upregulated EAAT-2 levels approximately 5fold, that were unchanged with METH treatment (***p<0.001).
111 Figure 4.4: METH-induced PKA activation regulated EAAT-2 levels. Primary human
astrocytes were treated with METH and protein lysates were collected at different time
points. Lysates were probed for GAPDH, as a loading control, total PKA and pPKA (A).
METH significantly increased pPKA at 10 min that remained unchanged through 30 min
(A, **p<0.01). To evaluate METH-induced PKA phosphorylation on EAAT-2 levels,
astrocytes were MOCK-, siCON- and siPKAcα-transfected, treated with METH and RNA
was collected at 8 hr and assayed for PKAcα (B) and EAAT-2 (C). Transfection with
PKAcα significantly reduced astrocyte PKAcα levels (B, ***p<0.001). METH treatment
in MOCK- and siCON-transfected astrocytes significantly reduced EAAT-2 mRNA levels
112 (*p<0.05, respectively) while siPKAcα transfection + METH significantly increased
EAAT-2 mRNA when compared to METH treated controls (C, **p<0.01, ***p<0.001,
respectively).
To further investigate PKA activation on EAAT-2 mRNA regulation,
astrocytes were treated with PKA inhibitor, H89 (20 µM), and METH, alone and in
combined conditions. RNA was collected at 8 hr and assayed for EAAT-2 (D). H89
alone significantly increased EAAT-2 mRNA levels (***p<0.001). METH significantly
decreased astrocyte EAAT-2 levels that were significantly recovered by H89 (D,
*p<0.05, ***p<0.001, respectively).
113 Figure 4.5: METH increased intracellular calcium fluorescence in GCaMP6s
expressing astrocytes. Primary human astrocytes were transfected with GCaMP6s, an
ultrasensitive protein calcium sensor, and allowed to recover.
MOCK-transfected
astrocytes were maintained as controls. Transfected cells were treated with ionomycin
(10 µM), as a positive control (images not shown) and the fluorescence signal was
visualized by confocal microscopy. Images were taken every 500 ms, selected images are
shown (A-E). Basal fluorescence was measured in the absence of external stimuli and
plotted on the histogram as 0 FLU (A and F). Upon 12 s METH exposure a fluorescence
signal appears, but does not become robust until 100s (B and C). The FLU levels appear
114 to peak at 150 ms (F). While punctate calcium vacuoles are observed intracellularly at
200s and 400s (D and E), the fluorescence remains above baseline.
115 Figure 4.6: METH-induced phosphorylation of CaMKII and PKC. Primary human
astrocytes were treated with METH (500 µM) and protein lysates were collected at 15
and 30 min. Increasing amounts of pCaMKII were observed. METH phosphorylated
CaMKII at 15 min following activation that continued to increase through 30 min (A).
METH increased pPKC at 30 min (B).
116 Figure 4.7: METH activated CREB in primary human astrocytes. Primary human
astrocytes were treated with METH (500 µM) and immunostained for CREB (A), pCREB
(D-G) and GFAP (red) at 0, 5, 15 and 30 min. At 15 min, elevated nuclear pCREB was
observed (F).
METH significantly increased pCREB at 10 min, which remained
unchanged at subsequent time points (F).
117 Figure 4.8: PKA knockdown decreased METH-mediated CREB phosphorylation.
Primary human astrocytes were transfected with reagents (MOCK, A-A2), non-specific
siRNA (siCON, B-B2) or PKA targeting siRNA (siPKA, C-C2). Transfected astrocytes
were treated ± METH (500 µM) for 15 min and fixed. Levels of total CREB (top) and
pCREB (middle) were significantly higher in untreated, MOCK- and siCON-transfected
astrocytes (A, A1, B, B1) as compared to siPKA (C, C1). Further, upon treatment with
METH, pCREB levels in MOCK and siCON (A2 and B2) were greater than siPKA (C2)
indicating PKA-mediated activation of pCREB by METH.
118 119 Figure 4.9: TAAR1, PKA and NF-κ B knockdown resulted in differential CREB
phosphorylation. Primary human astrocytes were MOCK-, siCON-, siTAAR1-, siPKAand/or sip65-transfected and treated with METH (500 µM) for 30 min. Protein was
collected and assayed by western blot for pCREBSer133.
Levels of METH-mediated
pCREBSer133 were decreased in astrocytes transfected with siTAAR1 (A) and siPKA (B)
as compared to METH treated MOCK- and siCON-transfected astrocytes. pCREBSer133
level were robustly increased with sip65-transfection alone, which did not change with
METH treatment (C) indicating NF-κB crosstalk with astrocyte pCREBSer133 activity.
120 121 Figure 4.10: METH and IL-1β phosphorylate pCREBSer133/142 in the nucleus. Primary
human astrocytes were treated with METH (500 µM) or IL-1β (20 ng/mL) and protein
lysates were collected at 10, 15, 20, 30 and 60 mins. Levels of GAPDH, total CREB and
pCREBSer142 were measured by western blot. pCREBSer142 was observed to increase
following METH stimulation at 60 min (A). IL-1β induced pCREBSer142 at 15 min that
remained elevated out to 60 min (B). Levels of GAPDH, Lamin, total CREB, pCREBSer133
and pCREBSer142 were measured in the cytoplasm and nucleus by western blot. METH
and IL-1β mediate both pCREBSer133/142 in the nucleus (C).
122 Chapter 5
SUMMARY AND DISCUSSION
123 This study characterizes astrocyte TAAR1 in mediating METH-induced
intracellular signal transduction pathways associated with EAAT-2 regulation.
Furthermore, it identifies CREB as the master regulator of EAAT-2 transcription. METH
and IL-1β-induced regulation of CREB downstream of TAAR1 activation reveal
mechanisms underlying the loss of EAAT-2 during METH abuse in the context of HIV-1
CNS infection. Loss of astrocyte EAAT-2 has been documented in several CNS
disorders, including HAND, Alzheimer’s disease and Parkinson’s. Moreover, drugs of
abuse lead to neurodegenerative disorders that are correlated with aberrant dopaminergic
and glutamatergic neurotransmission.
The effects of METH abuse during HIV-1
infection exacerbate the onset and severity of HAND, therefore characterization of
astrocyte TAAR1 as a METH receptor reveals the potential of TAAR1 being a
therapeutic target to attenuate astrocyte-mediated excitotoxicity.
This study unravels several questions: (1) What are the direct effects of METH on
human astrocytes, (2) How does astrocyte TAAR1 mediate these effects, (3) What is the
disease relevance of TAAR1, (4) How does TAAR1 regulation during METH and HIV-1
infection and inflammation exacerbate excitotoxicity, (5) What is the signaling crosstalk
of TAAR1 activation and HIV-1 relevant stimuli, IL-1β and how do they regulate EAAT2 transcription, (6) Is astrocyte TAAR1 a therapeutic target for METH abuse during
HAND? Distinct patterns of TAAR1 expression in the prefrontal cortex (74), METHmediated connectivity in the prefrontal cortex (267), and the effects of METH-induced
increases of glutamate in the prefrontal cortex (268) of human brains provides the
necessary physiological evidence for exacerbated effects of astrocyte-mediated
excitotoxicity during METH abuse and HAND. Accordingly, data attained from these
124 studies may be highly translatable to human disease.
White matter pathological features are a common characteristic associated with
HIV-1 CNS infection and HAND (269). Autopsies demonstrate predominate
abnormalities in the white matter of HIV-1 infected individuals attributed to a substantial
loss of neurons in the frontal cortex (270). Likewise, METH results in neurochemical
abnormalities in frontal brain structures (271). White matter consists mostly of glial cells,
including astrocytes, and myelinated axons, functioning in the transmission of signals to
other areas of the brain. Furthermore, abnormalities in neuron-astrocyte crosstalk during
METH and HAND reveal abnormalities in the dopaminergic and glutamatergic pathways
associated with the prefrontal cortex (271). Failure of astrocytes to take up excess
synaptic glutamate results in excitotoxicity. Excessive glutamate concentrations in the
prefrontal cortex modulate addictive behaviors and the development of neurocognitive
deficits (271). Downregulation of astrocyte EAAT-2 during METH abuse and HAND
increases glutamate concentrations in the extracellular microenvironment. The direct
effects of METH in astrocytes make this cell types a promising target in identifying
targeted mechanisms for the attenuation of astrocyte-mediated excitotoxicity, by
increasing EAAT-2.
TAAR1 is characteristic of the rhodopsin/β-adrenergic receptors with seven
transmembrane domains (272). The TAAR1 gene is localized to the 109 kb region of
human chromosome 6q23.1, which has been reported to be genetically associated with
schiozophrenia, bipolar disorders and other psychological pathologies (273). Both
TAAR1 mRNA and protein is broadly distributed throughout the brain (272). Basal
levels of TAAR1 were detected in astrocytes, and protein was localized intracellularly, in
125 both the cytoplasm and nucleus (2). TAAR1 localization is vastly reported within the
dopaminergic regions of the brain for specific effects it has on the dopaminergic reward
system (274). As part of the dopaminergic and glutamatergic pathways, the synthesis of
neurotransmitters occurs in the ventral tegmental area and is released in the nucleus
accumbens and the prefrontal cortex (274) (17). Furthermore, TAAR1 is localized in the
prefrontal cortex, in addition to other CNS location, which has a large white matter
volume (275). TAAR1 expression, METH-induced TAAR1 activation, HIV-1 CNS
infection and glutamate regulation in the cognitive areas of the brain offer a strong basis
for functional studies to gauge the importance of TAAR1 in normal and abnormal brain
physiology.
Studies utilizing EPPTB, the TAAR1 selective antagonist, will help delineate the
role of TAAR1 in the CNS. EPPTB was characterized using chimeric human/rat TAAR1
receptor. A cAMP assay identified benzanilides as an antagonist for mouse TAAR1 (80).
The selected hits were functional for the human TAAR1 when compared to the human/rat
chimeric receptor due to the high homology between species. Optimizations were
performed to improve the physiochemical properties, finally producing EPPTB (256).
The optimizations allowed EPPTB to become readily bioavailable, and lipophilic
allowing in vivo studies in the brain.
TAAR1, being a stimulatory GPCR, activates the canonical cAMP pathway. PKA
and PKC have been reported downstream of METH-induced TAAR1 cAMP signaling in
neurons (67, 68). These pathways regulate genes involved in cell cycle, mitochondrial
function, cell survival and cytokine secretion (69) and CREB responsive genes including
EAAT-2. Previous studies have demonstrated that chronic METH use leads to PKA
126 activation, phosphorylation of CaMKII and CREB binding protein (CBP) (258). While
the temporal order of events remains to be determined, both CREB and NF-κB lead to
EAAT-2 transcriptional regulation (162). In conclusion, PKA/CaMKII/CREB-signaling
cascade was involved in METH abuse (258). Targeting these signaling cascades further
sheds light on the temporal order of signaling involved in the tripartite synapse.
Astrocytes are directly involved in the regulation of neurotransmission via the
release of neurotransmitters including glutamate and ATP. Convergent pathways of HIV1 and METH in the CNS involve mechanisms of glutamate balance. As astrogliosis is
recognized as a hallmark of neurodegenerative disorders, their responsiveness to HIV-1
infection and METH abuse in mediating glutamate homeostasis is of high importance. As
discussed, astrocytes possess the machinery necessary for glutamate uptake, glutamate
synthesis and metabolism, glutamate release and the receptors necessary for direct
glutamate-induced intracellular signaling. In the context of METH and HIV-1, several of
these mechanisms have been investigated.
These studies demonstrate that aberrant
glutamate concentrations produce a cascade of events that result in dysregulated
dopamine concentrations, oxidative stress, mitochondrial damage and neuronal apoptosis.
The synaptic release of these neurotransmitters typically results in transient increases in
intracellular calcium levels. Previous studies indicate that pre-treatment with METH for 3
days leads to increased dopamine and glutamate-induced calcium oscillations (276).
Mimicking the METH-induced dopamine release by neurons, exogenous application of
dopamine to astrocytes produces a dose-dependent increase in [Ca+2]i stimulating
transient Ca+2 uptake in mitochondria and mitochondrial dysfunction (54). This was
evident with the transfection of GCaMP6s in astrocytes treated with METH. GCaMP,
127 are organized in a way so that in the presence of calcium, calmodulin undergoes a
conformational change, allowing calcium binding, a rapid deprotonation of the
chromophore and bright fluorescence (260). Following transient METH stimulation
punctate fluorescent vesicles, indicating calcium, were evident in the intracellular
environment. Calcium uptake into the mitochondria results in dysregulated homeostasis
of enzymes involved in oxidative stress. Further, dopamine metabolism produces
aldehydes and hydrogen peroxide, inducing lipid peroxidation and activating
phospholipase C, and additional IP3-dependent Ca+2 release from ER and increases in
[Ca+2]i by a receptor-independent mechanism. Astrocytes sensitivity to dopamine- and
glutamate-induced [Ca+2]i flux is responsible for deficiencies in cellular redox
homeostasis (55).
As discussed, downstream activation of TAAR1 results in increased intracellular
calcium levels.
We show that METH induced phosphorylation of PKA, PKC and
CaMKII. CaMKII is a calcium dependent kinase. CaMKII inhibition in astrocytes results
in increased ATP levels and aberrant calcium signaling (263). Other studies indicate that
astrocytes possess constitutively active CaMKII (277). Several psychostimulants activate
CaMKII and regulate signal transduction pathways. Calcium-mediated activation of
CaMKII induces pCREBSer142, the repressor form of CREB. Phosphorylation of
CREBSer142 acts as a dominant repressor for the pCREBSer133 transcriptional activator.
Additional studies suggest that CaMKII-dependent pCREBSer142 stimulated nuclear
export of CREB into the cytoplasm with approximately 50% total CREB degradation via
the ubiquitination pathway (278). CREB phosphorylated at serine 142 helps explain the
evidence of METH-induced EAAT-2 downregulation regardless of pCREBSer133.
128 We recently reported TAAR1 expression in primary human astrocytes, and while
it is unclear how HIV-1 regulates TAAR1, HIV-1 relevant cytokines, including IL-1β,
increased TAAR1 levels that were accompanied with increased METH-induced
intracellular cAMP levels.
Importantly, we have shown that astrocyte TAAR1
expression regulated astrocyte EAAT-2 levels and function. Moreover, EAAT-2 mRNA
levels were significantly decreased by METH and HIV, alone and in combination with
IL-1β activation. Further, METH and HIV independently reduced astrocyte glutamate
clearance abilities, a measure of alterations in EAAT-2 levels and function. Additionally,
TAAR1 overexpression exacerbated glutamate clearance impairment, while TAAR1
knockdown significantly improved clearance abilities as compared to experimental
controls; indicating TAAR1 as a possible therapeutic target to reduce METH/HIVmediated glutamate excitotoxicity. The significant negative correlation of TAAR1 and
EAAT-2 expression levels suggests that METH-induced TAAR1 signaling alters
activation of transcription factors thereby reducing EAAT-2 expression.
Analysis of the EAAT-2 promoter showed that NF-κB is an important regulator
of EAAT-2 transcription in astrocytes (65). Although the EAAT-2 promoter possesses
several NF-κB elements, IL-1β continues to decrease EAAT-2 mRNA levels.
Intracellular signal transduction of IL-1β in astrocytes stimulates NF-κB activation and
translocation to the nucleus, where it serves as a transcription factor. While NF-κB is
generally recognized as a transcriptional activator, it has been shown in astrocytes to
positively and negatively regulate EAAT-2 transcription (279). Additionally, the rate of
NF-κB translocation into the nucleus is regulated by the activation of PKA, downstream
of cAMP (66). The EAAT-2 promoter also possesses a CREB binding region, suggesting
129 transcriptional regulation (9, 239-242). Both PKC and PKA can phosphorylate NFκB/Rel complexes stimulating translocation to the nucleus. Interestingly, NF-κB
activation of target genes is optimized by interaction of the RelA subunit with CREB
coactivators, CBP and p300 (280), (281). However, the region of pCREBSer133 that
interacts with CBP also interacts with RelA, thereby inhibiting NF-κB activity by
phosphorylated CREB (282). This serves as compelling evidence for CREB and NF-κB
mechanistic crosstalk ultimately influencing how they regulate transcription of target
genes.
Mechanisms
contributing
to
excitotoxicity
include
downregulation
and
dysfunction of glutamate transporters, glutamate receptors, and glutamate synthesizing
and metabolizing enzymes in astrocytes; however, the mechanisms that regulate these
functions are unknown (64). EAAT-2 is regulated at the translational and
posttranslational level more so than the transcriptional level (190). PKA, PKC and
phosphatidylinositol-3-kinase (PI3K) (283) regulate the activities of EAATs.
The
kinases rapid activities are associated with cell surface expression of EAATs (226).
Previous studies indicate that H89 (PKA inhibitor) significantly reduced glutamate
uptake, suggesting regulation at the posttranslational level (283). Possibly, part of the
mechanism downregulating EAAT-2 transcription and glutamate clearance in TAAR1
overexpression model may be via PKC activation and subsequent ubiquitination and
degradation, therefore, the availability of EAAT-2 protein is decreased.
Our goal was to delineate the interplay of cAMP, PKA, cytosolic Ca2+, PKC and
CaMKII in mediating CREB-based EAAT-2 transcriptional regulation. These data
demonstrate that primary human astrocytes express functional TAAR1, which is
130 upregulated during METH and HIV cotreatments. Further, common neurotoxic
mechanisms include excitotoxicity that is at least partially regulated through cAMPdependent mechanisms. Importantly, our data demonstrate astrocyte functions leading to
neurotoxic outcomes such as excitotoxicity can be directly exacerbated through TAAR1
upregulation and reduced or eliminated by TAAR1 knockdown or inhibition.
Additionally, extrinsic regulation of signaling factors downstream of TAAR1 and cAMP
induction, such as PKA, not only reduce activation of subsequent signaling factors, but
also reduce or eliminate the METH-mediated alterations in astrocytes function. Finally,
CREB serves as the master regulator of EAAT-2 transcription in astrocytes, downstream
of METH-induced TAAR1 activation that is exacerbated in the presence of HIV-1
neuroinflammation.
131 FUTURE DIRECTIONS
Astrocyte TAAR1 activation via METH, and regulation in the presence of HIV-1,
suggests that TAAR1 plays a vital role in EAAT-2 transcriptional regulation and
potentially in EAAT-2 translational regulation. We reveal a novel pharmacological target
for the treatment of METH-induced excitotoxicity during HAND. These studies confirm
that METH-induced activation of astrocyte TAAR1 results in signaling pathways that
regulate EAAT-2 transcription. Another possibility for EAAT-2 downregulation is via
METH-induced activation of PKC, which can lead to ubiquitination, internalization,
degradation and/or reversal of EAAT-2 orientation in the membrane. Therefore, further
investigation is warranted for post-translational EAAT-2 regulation. Additionally, the
utilization of a gp120 transgenic mouse model and human brain tissues from HIV-1
infected individuals +/- METH, would assist in delineating the expression patterns of
TAAR1 in the brain during HIV-1 infection. Lastly, proof-of-concept studies would
identify potential agents used to target TAAR1, critical downstream targets of TAAR1
signaling or agents to reverse astrocyte-mediated excitotoxicity. We are highly excited
about the potential of TAAR1 as a therapeutic target for METH abuse during HIV-1 CNS
infection.
132 Proposed mechanisms for METH and HIV-1-mediated excitotoxicity.
HIV-1 enters the brain via infected monocytes. Activation infection present in infected
macrophages, produces viral proteins, inflammatory mediators and reactive oxygen
species. METH, being a lipophilic molecule, crosses the BBB where it interacts with
TAAR1 in astrocytes, induces increases in intracellular cAMP, activation of PKA and
release of catalytic subuntis. PKA translocates to the nucleus and phosphorylates
pCREBSer133, the transcriptional activator. Furthermore, TAAR1 induces intracellular
calcium increases, phosphorylation of CaMKII and translocation to the nucleus,
resulting in phosphorylation of the dominant repressor pCREBSer142. Inflammatory
mediator, IL-1β, activates astrocytes, resulting morphological changes and alterations in
133 gene expression. IL-1β downregulates astrocyte EAAT-2 mRNA levels and function and
increases astrocyte TAAR1 levels and function, therefore exacerbating METH-mediated
effects. IL-1β mediated signal transduction pathways include NF-κB, which is negatively
regulating
EAAT-2
transcription.
Signal
transduction
crosstalk
between
cAMP/PKA/pCREBSer133, calcium/CaMKII/pCREBSer142 and IL-1β/NF-κB/pCREBSer142 .
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