Analysis of huntingtin aggregation by fluorescence and FRET

Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
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Analysis of huntingtin aggregation by fluorescence and FRET microscopy
A. Holloschi1, S. Ritz2, I. Schäfer2, and P. Kioschis1
1
2
University of Applied Sciences Mannheim, Institute of Molecular and Cell Biology, Mannheim, Germany
Institute of Molecular Biology (IMB) gGmbH, Core Facility Microscopy, Mainz, Germany.
Huntington's disease is a hereditary movement disorder that is characterized by progressive neuronal cell death mainly in
the cortex and striatum of the brain. It is caused by an unstable CAG repeat extension in the first exon of the IT-15 gene
which encodes a protein called huntingtin (Htt). The trinucleotide expansion translates into an elongated polyglutamine
(polyQ) stretch. A polyQ length of more than 35 glutamine residues is associated with the appearance of huntingtin
aggregates and the development of the disease. The process of aggregation is not fully understood but its inhibition and its
modulation provide an insight into the mechanisms leading to aggregate formation which might be a target for the
treatment of the disease. Using CFP- and YFP-tagged huntingtin exon 1 fragments we established a cellular model that
visualizes the process of huntingtin aggregation and in which the aggregates could be specifically detected by FRET
microscopy (acceptor photobleaching and fluorescence lifetime microscopy). The time course of the aggregation process
was investigated by image analysis.
Keywords: polyglutamine disease; Huntington’s disease; huntingtin; protein aggregation; fluorescence microscopy; FRET
microscopy; FLIM; image analysis
1. Role of huntingtin aggregates in Huntington’s disease
Huntington’s disease (HD) (OMIM 143100) is a autosomal dominant, age-at-onset, progressive neurodegenerative
disorder caused by an expanded (CAG)n repeat in the exon 1 of the huntingtin gene IT-15 located on chromosome 4
[1]. The expansion above the normal range of 6–35 CAG repeats leads to an elongated poly-glutamine (polyQ) tract that
causes misfolding and aberrant protein-protein interactions thereby conferring a multifaceted toxic gain of function to
the widely expressed huntingtin protein. The age of onset is most critically determined by and inversely correlated with
the length of the expanded CAG repeat. HD is characterized by neurodegeneration and formation of neuronal
intranuclear and cytoplasmic accumulation of aggregated mutant huntingtin, particularly in the striatum and cortex but
also extended to other brain regions. The resulting clinical phenotype summarizes progressive movement dysfunction,
cognitive impairments, psychiatric symptoms, and ultimately death. Currently, no cure or therapy for delaying HDassociated symptoms is available. HD belongs to a set of ten, dominantly inherited neurodegenerative disorders, the
polyglutamine (polyQ) diseases, each caused by expanded polyglutamine (polyQ) tracts in otherwise unrelated proteins
[2, 3]. HD pathophysiological processes are multiple, complex and variable including impairments of transcription,
axonal transport, ubiquitin proteasome system and of mitochondrial function. A key feature in HD pathogenesis is the
poly(Q) dependent self-association and aggregation of mutant huntingtin proteins and of N-terminal toxic htt peptides
generated by proteolytic cleavage. Thereby, aggregates in the nucleus (nuclear inclusions) but also in the cytoplasm,
e.g. neuropils, are formed [4, 5]. The mutant huntingtin can undergo different conformations including aberrantly folded
monomeric forms, a wide-range of oligomeric species, fibril states, and larger insoluble aggregates [6]. The role of
mutant huntingtin aggregation in the pathogenesis of HD as well as the toxic impact of different forms of mutant Htt is
intensely discussed. Aggregation-mediated sequestration of proteins with essential cellular functions could be harmful
to the cell, whereas a protective mechanism resulting from sequestration of the toxic Htt moiety or other cellular
proteins which stimulate mutant Htt clearance would be beneficial. Furthermore, different structures of mHtt aggregates
seem to determine the nature of proteins being trapped. Thus, the resulting toxic effects are also driven by whichever
cell-specific proteins are present. Altogether, this may account for selective dysfunction and degeneration in HD. As a
consequence, the modulation of mHtt aggregation could have beneficial effects on overall toxicity or specific cellular
pathways deregulated in HD. This has been successfully shown by the interaction of a specific intrabody with mutant
huntingtin leading to increased ubiquitination and clearance of cytoplasmic mHtt as well as a subsequent prevention of
mHtt accumulation in neuronal processes and a reduced neurotoxicity [7]. Modulation of the mHtt aggregation process
by shifting the equilibrium toward soluble huntingtin was achieved by reducing the level of histone deacetylase
HDAC4. The resulted delay in cytoplasmic mHtt aggregation alleviated disease progression and led to improvement of
neurological phenotypes in an HD mouse model. These data clearly indicate that cytoplasmic aggregation mechanisms
contribute to HD-related neurodegenerative phenotypes [8]. Elucidating the relationship of different forms of mHtt
aggregates to toxicity and to disease progression is thus an important step in the pathway to therapeutic interventions. In
order to study HD pathomechanisms considerable effort has been invested in the development of in vitro and in vivo
model systems [6, 9]. One way to study the fate of proteins in living cells is to use fluorescent protein (FP) fusions.
Proteins tagged with FPs often retain their biochemical properties and allow the functional analysis of proteins in living
cells. In combination with microscopic techniques FP tags are ideally suited to analyse spatio-temporal processes such
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as aggregation in living cells. Here we report on different fluorescence microscopic techniques applied to investigate
aggregation processes of mutant huntingtin fragments in established in vitro cellular models for HD.
Fig. 1 Schematic representation of the full-sized Htt protein illustrating protein features and the huntingtin fragments (HDex and
HD512) that were used in this study. (aa: amino acid, polyQ: poly glutamine domain, PRD: proline rich domain, HEAT: HEAT
domain).
2. Materials and methods
2.1 Constructs
The plasmids encoding huntingtin-FP fusion proteins were generated by PCR amplification of the huntingtin exon1
cDNA fragments using plasmids pHD514Q17 and pHD514Q68 (Erich Wanker, MDC Berlin), respectively, as
template. The obtained PCR products were subsequently cloned into the pECFP-C1, pECFP-N1, pEYFP-C1 and
pEYFP-N1 vectors (Clontech). The sequences of the fusion proteins were verified by DNA sequencing.
2.2 Cell culture and transfection
COS-1 cells were cultured in DMEM medium (PAA, Cölbe) containing 10% fetal bovine serum, CHO-K1 cells were
cultured in F12 medium (PAA, Cölbe) containing 10% fetal bovine serum. Both cell lines were incubated at 37 °C and
5% CO2. Cells were transfected using Fugene 6 (Roche) as transfection reagent according to the manufacturer’s
recommendations.
2.3 Microscopy
GFP fluorescence was detected using an inverted widefield microscope system from Zeiss (Axiovert 200M, controlled
by AxioVision 3.1 software) with a 75 W-Xenon (Hamamatsu) lamp for excitation. Images were taken with a 12 bit
CCD camera (Axiocam MRm, Zeiss), using appropriate filter sets for CFP (ex 436/20 nm, beamsplitter 455 nm, em
480/40 nm), YFP (ex 500/20 nm, beamsplitter 515 nm, em 535/30 nm) or FRET (ex 436/20 nm, beamsplitter 515, em
535/30 nm). The microscope was additionally equipped with an Apotome device (Zeiss) for structured illumination to
improve axial resolution. For FRET measurements by the acceptor bleaching method, the samples were imaged before
and after bleaching the YFP using the YFP filterset for 5 min (bleaching more than 98% of the YFP signal). ImageJ
software was used to calculate the FRET images from the CFP (donor) images:
FRET = 1-
(1)
Fluorescence lifetime images were obtained by using a Zeiss Axiovert 200 microscope with a tunable titanium sapphire
laser (Chameleon, Coherent) that provided ultra short pulses (120 fs) of 800 nm at high repetition rate (80 MHz). The
output of the laser was directed into a TriM Scope multibeam scanning device (LaVision BioTec, Bielefeld), that
allowed us to scan the sample with up to 64 beams simultaneously. For detection a gated camera was used (PicoStar,
LaVision BioTec, Bielefeld). FLIM analysis was performed using ImSpector 4.0 software (LaVision BioTec,
Bielefeld):
FRET = 1-
(2)
with τDA = Fluorescence lifetime of the donor in the presence of the acceptor and τD = fluorescence lifetime of the donor
in the absence of the acceptor.
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3. Results
3.1 Characterization of huntingtin fusion proteins by fluorescence microscopy
In order to establish a cellular model system that allows us to study certain aspects of huntingtin aggregation and its
modulation by small compounds we decided to use fluorescent protein (FP)-tagged huntingtin. It has been shown that
cellular inclusion bodies (IBs) mainly consist of amino terminal fragments of huntingtin (Zitat). Therefore we selected
huntingtin exon 1 (HDex) and huntingtin aa 1-512 fragments (HD512) as a model for HD (Fig. 1). We generated
expression plasmids encoding huntingtin fragments both with a short polyQ domain of 17 glutamines or with a
pathogenic polyQ length of 68 glutamines, thereby reflecting the non-mutated or the mutated state, respectively. To
verify that fluorescent fusions did not influence the IB formation of huntingtin fragments we further established N- and
C-terminal fluorescent labelling (CFP and YFP, respectively). Plasmids carrying the different huntingtin-FP fusion
fragments were expressed individually in COS-1 and CHO-K1 cells and subsequently examined by fluorescence
microscopy (Table 1). Upon expression all huntingtin-FP fusion proteins initially showed a homogenous distribution in
the cytoplasm no matter whether wild type or mutant fragments were used. In addition, HDex was also observed in the
nucleus whereas the larger HD512 fragments were exclusively detected in the cytoplasm (Fig. 6). No aggregate
formations were noted in cells expressing HD512. In contrast, the smaller HDex huntingtin fragments promote
aggregation in a time-dependent manner in some cells (Fig. 3; 4). We did not observe any differences in data achieved
by CFP- or YFP-tagged fragments regardless of the orientation of the fluorescent tag.
Table 1 Localization of huntingtin-fusion proteins HDex and HD512 and their ability to form aggregates. The subcellular
localization of recombinantly expressed HDex and HD512 fusion proteins was analysed by fluorescence microscopy using structured
illumination (ApoTome) to increase axial resolution. Nuclear inclusions were seen only in cells with mt HDex expression.
Localization
fusion protein
CFP-HD512Q17
YFP-HD512Q17
CFP-HD512Q68
YFP-HD512Q68
CFP-HDexQ17
YFP-HDexQ17
CFP-HDexQ68
YFP-HDexQ68
HD512Q17-CFP
HD512Q17-YFP
HD512Q68-CFP
HD512Q68-YFP
HDexQ17-CFP
HDexQ17-YFP
HDexQ68-CFP
HDexQ68-YFP
cytosol
nucleus
aggregates
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In order to validate the effects of fluorescent tags on aggregate formation we analyzed the consequences of N- and Cterminal fusions to mutant Htt fragments. Cloned HDexQ68 constructs were transfected transiently in CHO-K1 cells
and analysed by fluorescence microscopy after 24 h, 48 h and 72 h. We performed total cell counts and assessed cells
containing inclusion bodies (Fig. 2). The proportion of cells containing aggregates increased with time. Furthermore, we
quantified and compared the occurrence of aggregates in cells expressing C -terminally tagged mutant Htt fragments
compared to cells with N-terminally tagged mutant Htt. Formation of aggregates was significantly higher in cells
expressing the C-terminally tagged variants. The maximum value observed was at 72 h of recombinant htt fragment
expression and about twice as high for cells with fluorescent fusion at the C-terminus of htt fragments (Fig. 2). An
amino terminal tag seems to strongly influence the ability of huntingtin exon 1 fragments to form aggregates. Similar
results were obtained regardless of whether CFP or YFP was used as a tag (fig. 2 A +B). Thus, all further experiments
were performed using fluorescent tags at the C-terminus of Htt fragments as they show significant better aggregate
formation.
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Fig. 2 Analysis of positional effects of FP-tag (N- or C-terminal fusion) on the ability of huntingtin exon 1 fragments to form
aggregates. Cells were transfected with the respective HDexQ68 constructs. After 24, 48 and 72 hours the percentage of transfected
cells showing aggregates was determined by fluorescence microscopy. In each case, at least 200 transfected cells were evaluated per
time point and construct. A) CFP-tagged huntingtin exon 1 fragments, B) YFP-tagged huntingtin exon 1 fragments.
We next examined the time course of aggregation processes of HDexQ68-YFP fragments in living cells. 24 hours
after transfection, cells transiently expressing HDexQ68-YFP were transfered into a POC-Mini cell culture chamber
(Zeiss) and observed under the microscope for 10 hours. Images were taken at 10 min intervals and analysed using
ImageJ. The diameter of aggregates was determined and plotted against time (fig. 3).
Fig. 3 Time course of aggregate formation in living cells. COS cells were transfected with pHDexQ68-YFP and seeded on glass
coverslips. After 24 hours, cells were transferred to a POC mini cell culture-chamber and cultured at 37 °C / 5% CO2 on the
microscope stage. YFP fluorescence was recorded over a period of 10 hours at ten-minute intervals. A) Fluorescence microscopy
images of huntingtin exon 1 aggregate formation. B) Analysis of HDexQ68-YFP aggregate formation. The diameter of aggregates
was determined by ImageJ analysis and plotted against time. Since cells formed aggregates at various time points, the time point 0
was defined as the image before the aggregate first became detectable. (* aggregates of cells which formed several aggregates).
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The overall process of protein aggregation is characterized by initial lag time during which no detectable aggregates
are formed in the cells. For the time course analysis we defined the time point 0 as the image right before the aggregate
became detectable in a selected cell. In this way we were able to compare the aggregation process in different cells
though aggregate formation does not start uniformly in a given cell population. Once the aggregation process started the
aggregates rapidly increased in size. Within 65 ± 35 minutes they reached a maximum size and remained stable
afterwards (fig. 3). Most cells formed only one perinuclear aggregate of 1.86 ± 0.34 µm diameter. The aggregate
diameter in cells forming several aggregates was smaller (1.32 ± 0.39 µm). At the final stage of aggregate formation
almost no fluorescence could be detected in the cytosol indicating that all the HDex68-YFP fragments available in the
cells were almost exclusively localised to the aggregates.
3.2 Detection of huntingtin aggregates by FRET
Förster resonance energy transfer (FRET) is a non-radiative process whereby the excitation energy of a donor
fluorophore is transferred to an appropriately positioned acceptor fluorophore. FRET can occur when the emission
spectrum of a donor fluorophore significantly overlaps the absorption spectrum of an acceptor provided that the
distance separating the two molecules is 8 to 10 nm or less. Thus, FRET is well-suited to the investigation of protein
interactions that occur between two molecules positioned within several nanometers of each other. As this is the case
for mutated huntingtin fragments which undergo aggregation, FRET measurements can be utilized effectively to
analyse the aggregation process. Genetically encoded fluorescent proteins represent the best candidates for highresolution imaging of FRET in live cells. Therefore, we co-expressed CFP- and YFP-tagged huntingtin exon 1 in COS1 cells and analysed them by FRET microscopy using the acceptor photobleaching method (Fig. 4). A FRET signal was
only detected in aggregates of cells transfected with CFP- and YFP-tagged mutant HDexQ68 fragments. During the
initial lag phase of the aggregation process HDexQ68 fragments are still soluble and thus, as expected, no FRET was
observed at this stage. Furthermore, no FRET signal was visible in cells expressing wild-type, soluble HDexQ17.
Fig. 4 Detection of huntingtin aggregates by FRET microscopy. The indicated constructs were co-transfected into COS-1 cells.
After 48 hours cells were fixed with PFA and analysed by a FRET-microscopy (acceptor photobleaching). The CFP fluorescence, the
YFP fluorescence and the FRET efficiency images are shown. A specific FRET signal was detected in the aggregates containing
mutated HDexQ68 fragments.
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The results were further verified by fluorescence lifetime imaging microscopy (FLIM). If FRET occurs, the lifetime
of the donor is reduced. The measured fluorescence lifetime ( ) of CFP, HDexQ17-CFP and soluble HDexQ68-CFP
was 2.2 ns, respectively, whereas the fluorescence lifetime of HDexQ68-CFP in aggregates was 1.25 ns (Fig. 5). Based
on measured fluorescence lifetimes a FRET efficiency of 0.43 was calculated. Both methods of detecting FRET
revealed that FRET can be used to specifically detect aggregates.
Fig. 5 Fluorescence lifetime analysis of huntingtin exon 1-CFP and huntingtin exon 1-YFP cotransfections was performed to
detect huntingtin aggregates. On the left side the fluorescence intensity is shown, whereas on the right side the corresponding
fluorescence lifetime of the donor (CFP) is shown. Co-expression of CFP-and YFP-tagged huntingtin exon 1 fragments with a CAG
repeat of 17 glutamines resulted in a fluorescence lifetime of CFP of 2.2 ns. The same fluorescence lifetime for CFP was obtained
when of CFP-and YFP-labeled huntingtin exon 1 with a CAG repeat of 68 glutamines were co-expressed, as long as the proteins
remained soluble. Once aggregates had formed, a shortened CFP fluorescence lifetime of 1.25 ns could be measured. From the
measured fluorescence lifetimes of the donor CFP, a FRET efficiency of 0.43 was calculated in the aggregates.
3.3 Sequestration of wild-type HDexQ17 and HD512 fragments by aggregates of mutant HDexQ68 fragments
Several studies report that the aggregation process driven by polyQs might sequester essential proteins, such as
transcription factors [10-12], components of ubiquitin proteasome system [13] as well as wt huntingtin [14, 15], thereby
affecting neuronal survival. However, what the true role of inclusions is remains still to be determined. For further
studies we tested the ability of mutant HDexQ68-CFP to sequester wild-type huntingtin. For this purpose we coexpressed mutant HDexQ68-CFP with wt HDexQ17 or with the longer fragment wt HD512 in COS-1 cells and
analysed them by FRET microscopy (acceptor photobleaching) (Fig 6). Both wild-type huntingtin fragments showed a
very good co-localisation with mutant HDexQ68-CFP, except that exon 1 fragments localised to the nucleus whereas
the longer HD512-YFP fragments were exclusively located in the cytoplasm. No FRET was detected when HDexQ68CFP fragments were still dissolved at the beginning of the aggregation process. In contrast, an intense FRET signal was
detected in aggregates formed. The results revealed that indeed, the mutant HDexQ68-CFP aggregates sequester wildtype huntingtin fragments indicated by the precise co-localisation data and the achieved specific FRET signal.
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Fig. 6 FRET analysis (acceptor photobleaching) of cells co-expressing indicated mutant and wild-type huntingtin fragments. In
order to analyse sequestration of wt HDexQ17 and HD512 fragments into HDexQ68 aggregates the corresponding plasmids were cotransfected into COS-1 cells. After 48 hours the cells were fixed with paraformaldehyde and subsequently, FRET analysis was
performed according to the acceptor photobleaching method. The CFP fluorescence, the YFP fluorescence and the FRET efficiency
image are shown.
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4. Discussion
Neuronal intracellular inclusion bodies (IBs) consisting mainly of N-terminal fragments (NTFs) spanning exon 1 of the
Htt protein are the pathological hallmarks of HD. Aberrant splicing of the mutant Htt gene as well as proteolytic
cleavage of mutant huntingtin protein lead to increased levels of amino terminal fragments and promote aggregation
[16, 17]. NTFs typically contain an amphipathic 17-residue stretch at the N terminus (N17), followed by a polyQ tract
and a C-terminal 38-residue proline-rich region (C38). It has been suggested that the N17 and C38 sequences minimize
the toxic effects of aggregates, probably driven by the heterogeneities present in polyglutamine aggregation [18].
However, the role of IBs in the pathogenesis of HD is controversially discussed and it remains unclear whether
inclusions are a response to toxicity or are themselves toxic. Formation of IBs might be a beneficial coping response of
the cell, thereby reducing levels of toxic misfolded proteins. Other reports indicate that inclusions are the main cause of
neuronal damage and that reduced aggregation has shown a beneficial effect in different models, including mouse
models of HD [19]. Thus, a comprehensive understanding of the mechanisms and dynamics leading to aggregate
formation as well as of factors modulating this process is essential for the development of therapeutic approaches.
A very convenient way to study the fate of proteins in living cells is to use fluorescent protein (FP) fusions. Proteins
tagged with FPs often retain their biochemical properties and allow the functional analysis of proteins in living cells. In
combination with microscopic methods FP tags are ideally suited to analyse spatio-temporal processes, such as
aggregation. Therefore, we established expression plasmids encoding CFP- and YFP-tagged huntingtin fragments. We
used huntingtin exon1 fragments and huntingtin aa 1 – 512 fragments with a normal polyQ domain of 17 glutamines to
generate cell models for wild-type huntingtin. In contrast, the same fragments but with an expanded polyQ domain of
68 glutamines were used for cell models with pathogenic huntingtin. Expression in CHO-K1 cells showed that all
fusion proteins were found in the cytoplasm of the cells. The additional nuclear localisation of HDex1-FPs is probably
due to phosphorylation of N-terminal htt [20]. As expected, proteins aggregates were formed only in cells that
expressed mutant HDexQ68-FP fusion. The type of FP (CFP or YFP) had no influence on the ability to form
aggregates. In contrast, the position of the tag significantly affected the aggregation process. While HDex1Q68 with
FPs fused to its C-terminus aggregate efficiently, polyQ-huntingtin fragments with N-terminal fusions clearly resulted
in a reduced number of aggregates. Since the N17 stretch of huntingtin has an important role in seeding the aggregation
process, the N-terminal tagged FP may alter this capacity [18].
Time course analysis of the aggregation process of HDexQ68-YFP in living cells revealed that the formation of
aggregates is completed at 65 ± 35 minutes after their first appearance. This is in agreement with the findings of
Apostol et al. (2003) who observed a similar time of 30 min required for aggregate formation in PC12 cells expressing
mutant huntingtin exon 1 with a polyQ length of 103 glutamines [21]. The rapid increase of aggregate size once the
aggregation process has started, is also in concordance with in vitro studies by Scherzinger et al. (1999) and Chen et al.
(2002) who discussed that the rate-limiting step in the aggregation process is the formation of the aggregation nucleus,
followed by the rapid accumulation of further huntingtin fragments [22, 23]. The analysis of aggregate formation in our
cell models using FRET measurements by microscopy with acceptor photobleaching as well as FLIM revealed that
specific FRET signals could be detected in aggregates only. Thus, FRET is well-suited to investigate aggregation
processes.
Furthermore, a specific FRET signal could also be detected in aggregates of cells co-expressing HDexQ68-CFP and
wt HDexQ17-YFP or the longer HD512-YFP fragments, respectively. These results indicate that wt HDQ17 and
HD512 fusions which have no aggregation potential were sequestered by HDexQ68-CFP aggregates. Obviously,
HDexQ68-CFP has maintained the ability to recruit other proteins into aggregates.
In conclusion, the combination of FRET and FLIM microscopy with heterologous expression of FP-tagged
huntingtin fragments represents a very useful strategy to study the aggregation process of mutated huntingtin and
sequestration of other proteins in living cells. The established cell models were further developed as stable cell lines
with inducible expression of huntingtin protein fragments and were consequently adapted to microtitre plate formats for
highly parallel analysis of aggregate formation by FRET. Using this assay we were able screen a multiple compound
library and to identify modifiers of the aggregation process [24], underlining the applicability of the described approach
in studying pathogenesis and finding new putative therapeutic candidates for HD.
Acknowledgements The support by Ministerium für Wissenschaft, Forschung und Kunst - Baden-Württemberg (to PK),
Bundesministerium für Bildung und Forschung (BMBF; grant FKZ 17 07 X 06 to PK) and the Karl-Völker-Foundation, University
of Applied Sciences, Mannheim is gratefully acknowledged. We thank John Clear for proofreading the manuscript.
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