Network Posters - Dementias Platform UK

Imaging Network:
Image Acquisition and Analysis
Novel measurements of micro-vessel structure and
physiology using MRI
Principle Investigator: Laura M Parkes
Co-Investigators: Ben Dickie, Jose Ulloa, Hamied Haroon, Steve Williams, Julian Matthews, Herve Boutin,
Marie-Claude Asselin, Karl Herholz, Geoff JM Parker
Water Exchange Measurements
Cerebral Blood Flow and arterial
arrival time
We are developing measurements of
water exchange across the vessel wall
as a potential marker of subtle bloodbrain barrier damage. The graph shows
how the Arterial Spin Labelled signal
post contrast agent injection is sensitive
to the extraction fraction of water.
We will add slice multiplexing to our
current Look- Locker Arterial Spin
Labelling sequence to maximise the
time
efficiency
of
blood
flow
measurements.
Beaumont H. et al, Proceedings of the
ISMRM 2016
400 averages 100 averages 25 averages
Cerebral Blood Flow
difference signal (a.u.)
The Imaging Network aims to integrate technical expertise and resources across centres . We aim to
develop a standard dementia imaging protocol for use in multi-centre clinical studies. The network is also
an important platform for rapid uptake and translation of novel imaging methods such as the work
described here. In Manchester we are undertaking EPSRC-funded work into the development of novel MRI
measurements of vascular physiology which may be altered in dementia.
2
Extraction fraction
1
0.5
0
1.5
1
0.5
0
0
1000
2000
3000
post labelling delay time (ms)
Micro-vessel structure
0
Diffusion-weighted
images
can
be
sensitised to capillary
segment length by
altering the duration
and separation time of
the magnetic field
gradients.
This measure will be sensitive to vessel
tortuosity which may be increased in
dementia. In addition, Contrast Agents
can sensitise the MR images to vessel
diameter.
200 ml/min/100ml
Arterial Arrival Time
0
2000 ms
http://juracare.co.za/inside-brain-alzheimers-brain-tour/
Oxygen can be used as a
contrast agent to determine
the availability and uptake
of oxygen in the brain which
may be an important link
between altered blood flow
and neural function.
2
BOLD response (%)
Oxygen Uptake
Small Vessel Disease
Old
Young
1.5
1
0.5
0
0
-0.5
100
200
300
Time (s)
400
500
Loughnan R. et al, Proceedings of the
ISMRM 2016.
-1
From Pre-clinical to Clinical
Measurements will be validated pre-clinically through comparison of MR measurements to histology measurements in
the rat. Longitudinal imaging in a rat model of Alzheimer’s disease will validate the sensitivity of the measurements to
disease.
We will implement the same imaging techniques on the PET-MR clinical scanner.
Importantly, this will allow use of the same imaging biomarkers in pre-clinical and clinical testing of novel treatments.
April 2016
This work is supported by the EPSRC Sensing and Imaging for Diagnosis of Dementias grant (EP/M005909/1).
Imaging Network:
18
Imaging Aβ and Tau in Early Stage AD with [ F]AV45
18
and [ F]AV1451
(Pilot Phase of the MRC Deep & Frequent Phenotyping Study)
Principal Investigator: Azadeh Firouzian,
Co-Investigators: Alex Whittington, Graham Searle,
Ivan Koychev, Simon Lovestone, Roger Gunn
Pilot Deep and Frequent Phenotyping study paves the way towards the full study showing the
possibility of performing a short static PET scan in place of long dynamic one.
Introduction
Results
The MRC Deep and Frequent Phenotyping pilot
study is a multi-centre, non-interventional study
utilising the DPUK platform infrastructure aiming
to identify markers of change in early stage AD
subjects
using
clinical
and
non-clinical
assessments such as PET imaging which is the
focus of this poster.
• All subjects successfully completed at least 60
18
min of dynamic PET imaging for both [ F]AV45
18
and [ F]AV1451.
• Time stability analysis of BPND and SUVr for
18
[ F]AV45 showed that it becomes stable after
18
~30 min and for [ F]AV1451 after ~80 min.
• The relationship between the two measure
started to plateau for scan windows of ~40-60min
18
18
for [ F]AV45 and ~90-110 min for [ F]AV1451
(Figure 2).
• Strong correlation was found between Aβ signal
in thalamus and tau signal in thalamus,
hippocampus and striatum.
Martials and Methods
In the pilot phase,15 subjects (men and women,
aged 50 to 85 years) with early stage AD (MMSE
score of 20-29) underwent PET imaging at
Imanova:
• Dynamic PET scan with
(060min, 150 ± 24MBq) to measure Aβ
18
• Dynamic PET scan with [ F]AV1451 (0120min, 163 ± 10MBq) to measure tau
18
[ F]AV45
Regional binding potentials (BPND) were
estimated from the dynamic PET scans using the
SRTM model with cerebellum grey as reference
region and compared to regional SUVr values
(Figure1) obtained from 20 min static scan
windows.
Tau
Figure2: Coefficient of determination between regional
BPND and SUVr values.
Conclusions
Aβ
Figure1: Tau and Aβ uptake (SUV) in 3 example subjects
with different MMSE scores.
April 2016
• Analysis results using short static scans at
appropriate time windows are similar to ones
obtained from long dynamic scans.
• In early AD population Aβ levels in thalamus were
highly correlated with tau uptake in thalamus,
hippocampus and striatum.
Imaging Network:
Enabling the use of PET Radiotracers for Dementia Research
Principal Investigator: Franklin Aigbirhio and Jan Passchier
Embedded within the Dementia Platform UK (DP-UK) Network are centres with facilities to
conduct patient studies with positron emission tomography (PET). Recent MRC funding has
allowed further enhancement of imaging capabilities through the acquisition of five state-of
the-art combined PET-MRI scanners. To capitalise on this unique infrastructure, UK PET
radiochemistry groups are working together to ensure each of the centres has access to a
broad portfolio of the required short-lived radiotracers for dementia research.
DP-UK Imaging Network – Radiochemistry
Working Group (WG2) Members
Chair: Franklin Aigbirhio (Cambridge),
Co-Chair: Jan Passchier (Imanova).
Members: Erik Arstad (UCL), Mike Carroll
(Newcastle), Tony Gee (KCL), Nick Long
(Imperial), Christophe Lucatelli (Edinburgh),
Chris Marshall (Cardiff), Adam McMahon
(Manchester), Phil Miller (Imperial).
DP-UK PET Imaging and Radiochemistry
Infrastructure

PET/MRI
PET/CT
 Cyclotron
 Mini-cyclotron
 GMP Radiochemistry
 Edinburgh

Newcastle
 Manchester
Remit and Aims
 Cambridge
Enable the DP-UK PET network to access the
required range of radiotracers for dementia
research
 Aim to maximise availability of tracers across
participating centres.
 Facilitate knowledge transfer between centers
i.e. radiotracer methods, standard operating
procedures and IMPD.
 In consultation with other groups, in particular
the Clinical and Data analysis groups guide
the imaging network in a structured and
informed choice of radiotracers for studies.
 In partnership with rest of DP-UK IN be a
portal for interface with industry
 Engage with UK regulatory agencies, to
address issues with EU GMP and clinical trials
regulations,
their
interpretations
and
applications in the use of PET radiotracers.
April 2016
 
Cardiff

Imperial,
 Kings

UCL
PET Radiotracers
The initial focus of the group is to establish
throughout the network radiotracers for
imaging the following pathology.
Pathology
PET Radiotracers
beta-amyloid plaque
18
[ F]flutemetamol
18
[ F]florbetapir
11
[ C]PIB
Tau neurofibrillary
tangles
18
[ F]AV1451,
18
[ F]THK5351
11
[ C]PBB3
Neuroinflammation
(TSPO)
11
[ C]PK11195
[11C]PBR28
18
[ F]DPA714
Informatics Network:
Wearables and Connected Devices
Principal Investigator: John Ainsworth
Co-Investigators: James Cunningham & Matthew Machin
1. Wearables and Connected Devices has produced a
secure hardware environment, suite of software tools and
pool of wearable devices for dementias research
The recent explosion in the production and availability of
‘wearable devices’ such as smart watches and fitness trackers
means that research approaches to fine-grained longitudinal
monitoring of subjects that even a decade ago would have
required specialist bespoke (and costly) hardware can now be
done with commodity of-the-shelf devices
We successfully met our aims of producing an integrated
platform and pool of devices that can be accessed as a readymade wearables component to “plug in” to research projects
giving access to the devices, expertise and computational
infrastructure that will enable the collection of large volumes of
longitudinal data from wearable devices.
2. A series of interactive workshops were held with
researchers, patient groups and members of the
public to direct and inform the makeup of the
platform
In order to assess the requirements of the wearables
platform from a research perspective, the utility,
usability and reception of the devices being purchased
and public perception towards data capture, retention
and use from those devices, a series of interactive
workshops were held.
Giving participants a chance to use a selection of
devices – both on the day and at home over the course
of a week – these gave crucial feedback on attitudes
towards such devices and informed and directed the
make-up of the pool devices purchased and the design
of the accompanying DPUK Sensing Platform.
3rd Party
Integration
Data
seamlessly
transmitted
Setup
Study
3. The DPUK Sensing Platform gives research projects a
programmatic interface and web-based portal allowing
data to be gathered, uploaded and processed in a secure
environment
The sensing platform consists of:
• Secure Data Warehouse – providing central repository for
storing data associated with large number of studies
• Trusted Research Environment – allowing users to process and
analyze data in a certified secure environment
• Web Portal – facilitating study creation and format-agnostic data
upload facilities
UoM
DPUK
Server
Integrated data
Data upload
April 2016
Informatics Network:
Imaging Informatics
Principal Investigators: Clare Mackay & Sebastien Ourselin*
Neuroimaging modalities (MRI, MEG, PET) are a window into the living brain and provide some of the most
powerful tools available to clinical neuroscience. Specialist infrastructure and expertise is required to collect,
store and process imaging data, and technological limitations mean that it is only now that we are able to
develop this on a national scale. The DPUK imaging informatics infrastructure will facilitate data sharing for
existing cohorts, improve robustness and transparency in neuroimaging research, and provide a ready-made
solution for future experimental medicine studies.
We set out to develop:
1.
2.
3.
4.
A catalogue of existing DPUK imaging data (MRI, PET & MEG) with a search and data request capability.
A Data Management System designed and implemented at multiple sites using a generic data model.
A central hub with a capability to receive data from local sites for central data analysis.
Centralised analysis capability (+ designs for future development of automated pipelines)
The solution:
Central Hub
UK Biobank
Oxford
Cohorts
UCL
Edinburgh
Cambs
M’chester
Kings
Imperial
Newcastle
Cardiff
Powered by
https://info.dpuk.org/
Federated infrastructure based on XNAT technology (www.xnat.org),
with nodes at each of the 9 DPUK imaging centres, a central hub (Farr
Inst, Swansea) and a specialist node to facilitate use of the UK Biobank
imaging dataset. The model is easily expandable to additional sites.
Progress update, April 2016:
All hardware deployed (9 sites, the UK Biobank and Farr Institute). Software delivered with phase 1 testing
ongoing at 4 sites & hub. Integration with DPUK portal for authentication and governance in progress. Phase
2 testing (all sites) imminent, followed by training and working with WP1 (cohort engagement).
DPUK imaging informatics already committed to future studies and networks:
MRC/NIHR Deep &
Frequent Phenotyping
MRC UK 7T
network
MRC DPUK PET-MR
Partnership
*On behalf of the Imaging Informatics Team: Lars Engstrom, Matt South, Dave Cash &
Dan Marcus with essential contributions from colleagues at each site.
April 2016
DPUK imaging
network
Informatics Network:
Brain Banking
Principal Investigator: Seth Love,
Co-Investigator: Richard Cain
DPUK and the MRC UK Brain Banking Network are working together to increase capacity in
preparation for receiving highly characterised brains from DPUK cohort participants. As part of
this programme DPUK has funded an RFID technology network-wide solution (RFTrackIT) for
tissue location, auditing storage availability, and returning results.
RFID labelling
Radio-frequency
identification
(RFID)
usesd
elecgtromagnetic fields to identify tags attached to objects
and is suitable for use in extreme environments such as
low temperature storage.
RFID labels support a variety of uses and containers,
including storage in liquid nitrogen, embedding in paraffin
wax in cassettes and labelling of plastic bags.
Low temperature-compatible labels have an adhesive that
supports storage in -80°C, -120°C and -150°C freezers
and liquid nitrogen. These labels can be applied to existing
frost covered vials whilst still frozen.
An entire box or bag of containers
can be scanned in a single read.
The ‘RFTrackIT™’ software
The RFTrackIT system makes the registration of new and existing donations straightforward by
creating sample tree templates. These allow biobanks to map their individual sampling/
dissection protocols and then use the preconfigured sample tree to register all of the samples in
their protocol in a single step. The software also ensures that all discrete samples are uniquely
identified and linked with the primary donation, and labels appropriate to the container size and
storage conditions are produced.
Csols RFTrackIT™
software can precisely
identify well characterised
samples and map their
location
Progress
RFTrackIT has been installed in Bristol (South West Brain Bank) and Oxford for testing and staff
training. Roll-out to the remaining brain banks in the network will follow. Once deployment is
complete existing brain tissue samples throughout the network will be labelled retrospectively.
April 2016
Informatics Network: UK-CRIS Programme
Principal Investigator: Prof Simon Lovestone
Co-Investigators: Mike Denis, Andrew Bucknor, David Newton,
Lars Engstrom, Jeremy Leigh, Nicky Morris, Tina Milkovic
What is CRIS?
Objectives
The Clinical Record Interactive Search solution
provides a technology and governance model to
safely transform clinical data held in a Mental Health
NHS Trust electronic medical record into a rich
pseudonymised data resource for research.
Research Collaboration
The potential benefits of research performed jointly across
collaborating organisations and research performed across
significantly larger datasets is already well known but enabling
that research to take place has historically presented significant
governance issues and risk.
Dementias Platform UK > Informatics >
UK - CRIS Programme > Objectives :
1
Extend deployment of the CRIS solution to a
further 10 Mental Health NHS Trusts.
Using its masking algorithm, CRIS can de-identify the
entire medical record and enable researchers to
access not only structured data but also the 80% of
information that exists as free text notes and
documents.
2
Add scale through the development and
implementation of ‘Federated Querying” giving
access to over 2 million health records through
UK-CRIS.
.
Rapidly refine your search queries in real time
- quickly identify rare events, diseases, states and
their clinical context and hard to find cohorts for
clinical trials or research studies.
A UK-CRIS research portal will provide the opportunity to share
additional relevant research materials (either by publishing
links to published papers, reference material or other sources,
or by simply uploading documents to the portal).
The larger number of Trusts joining UK-CRIS is more likely to
have a greater number of clinicians and academics looking to
collaborate.
3
Establish a record linkage with UK-Biobank to
augment potential data sets through the addition
of physical health data held in UK-Biobank.
Pseudonymised EPR data is loaded into compatible UK-CRIS
databases, enabling collaboration; rather than individual
Principal Investigators or research teams sharing data in an
uncontrolled external environment.
Linkage
with
UK-BioBank
Data
Access Over
Add
10
Clinical Record Interactive Search
With the introduction of ‘federated querying’ through UK-CRIS,
collaborative clinical and academic research becomes an
achievable reality with dramatically easier research becoming
possible within a secure environment under a robust
governance model.
2m Health
Records
Trust Sites
Deployments in this phase
Progress to March 2016
10 Mental Health NHS Trusts have given a full commitment to
UK-CRIS and to implementing an instance of the CRIS solution (See
map of Deployments in this phase)
Core technology components have been analysed and updated to
allow scaling, improvements in performance, enhancements to
functionality and reduction in costs.
1.
Avon & Wiltshire Partnership
2.
Berkshire Healthcare
3.
Devon Partnership
4.
5.
Kent and Medway
Mersey Care
6.
Northumberland Tyne and Wear
7.
North East London
8.
Nottinghamshire Healthcare
9.
Southern Health
10.
South West London
Looking Ahead
The programme will complete deployment of the CRIS system to
all 10 Trust sites by October.
Have in place a simple record linkage system with UK-Biobank.
Provide an online community to support academic collaboration
and the use of CRIS.
Enhancements to system functionality include: introduction of new
search engine technology, rationalisation of database and web
server components to optimise operational service delivery,
introduction of some open source components, improved text
retrieval from scanned or attached documents, enhanced audit
function, improvements to the masking algorithm and fixes to
support the processing of large records.
Utilise the potential
components.
6
A full public tender conducted to procure an Infrastructure as a
Service (IaaS) partner to host the UK-CRIS platform on an
enterprise grade, N3 connected, highly resilient and scalable cloud
environment.
for
other
data
linkages
with
DPUK
Have in place a fully managed solution, with a single point of
contact and dedicated service support.
5
Federated Research
8
Further enhanced the proven Information Governance Model and
developed proposed governance model for Federated Querying
(Including Specialist review and Privacy Impact Assessment of IG
and Federation.)
2
7
1
10
Established engagement with UK-Biobank and developed linkage
model.
4
9
3
Developed an additional extract utility for the Carenotes electronic
medical record system to add to the existing utility for RiO.
UK - CRIS High-Level
Conceptual Model
DECIDE TO COLLABORATE
DEFINED
RESEARCH
PROJECTS
UK Biobank Linkage
SQL / WEB
CLIENT
T1
PROJECT
AUTHORISED
Web / SQLClient (T1)
T1 Study
Result Store
5
3
9
SUBJECT
+
PAYLOAD
4
Federated
Search
STUDY
PUBLICATION
RESULT
TN
6
UKBDATA
REQUEST
Proj.
Specific
LinkIDs
ProcessSteps
UKB
DATASET
1.
2.
3.
4.
5.
Extrack data from Trust EHR
Validate if UKB participant
Search against UKCRIS dataset
Submission of UKB application
Load UKCRISdata to analysis
workspace
6. Request
UKB
data
on
approved application
7. UKB prepares dataset
8. PI download UKB dataset
9. PI merges UKCRIS and UKB dataset
10. Analysis and subsequent publication
ELASTIC SEARCH ENGINE
(WEBCLIENT)
Federation
DB For Trust 1
FEDERATION
FEDERATION
DB T2
FEDERATION
DB Tn
T1
FEDERATION
T2
FEDERATION
OMOP
DB
T1
Tn
FEDERATION
Federation
DB For Trust n
Federation
DB For Trust 2
OMOP
DB
T2
OMOP
DB
Tn
8
T1
T2
CRIS
Trust 1
Tn
M AP DB
CRIS
CRIS
PROJECT
DECLINED
PROJECT
DECLINED
PROJECT
DECLINED
PROJECT
AUTHORISED
PROJECT
AUTHORISED
CRIS
Trust 2
M AP DB
CRIS
Trust n
M AP DB
CRIS
IMPORT
IMPORT
IMPORT
Legend :
UK-BioBank
SECUREDATA IMPORT PIPELINE
SECURE DATA IMPORT PIPELINE
Generate
MD5 IDs
LEGEND :
Feature / Function
PID
2
ACCESS CONTROLS - USER AND
PROJECT BASED, TIME LIMITED
ACCESS CONTROLS
INDIVIDUALLY STORED AND
SECURED PSEUDONYMISED
TRUST DATASETS
DATA UPLOAD FROM TRUST
EPR SOLUTIONS
RECEIPT, VALIDATION, MASKING, ANONYMISATION AND TRUNCATION
Extract / Load
Extract / Load
Extract / Load
Extract / Load
Extract / Load
Person identifiable
Data
PID-Extract
WEB
SERVICES
Extract / Load
PID-Pseudomap
7
Pseudonymised
Anonymised
PID
Pseudonymised
1
Anonymised
EHR
EHR
EHR
UK-Biobank
April 2016
PROJECT
AUTHORISED
LOCAL OVERSIGHT COMMITTEES
- EACH CONTROLLING ACCESS TO
THEIR DATA
Function Feature
Extract / Load
PROJECT
AUTHORISED
10
FEDERATED
SEARCH
FEDERATION
DB T1
PROJECT
AUTHORISED
Contact : [email protected]
[email protected]
www.ukcris.net
SECURE N3 CONNECTION
PROJECT
AUTHORISED
Informatics Network:
Data Portal
Principle Investigator: Ronan Lyons
Co-Investigator: Simon Thompson
Researchers: Justin Biddle, Chris Orton, Karen Tingay
Overview
• Facilitating UK wide record linkage and understanding data capability
• Establishing DPUK UK Secure eResearch Platform (UKSeRP) instance and
Governance structure
• Visualisation of UKSeRP and project application process
• Integrating cohort research data and metadata
Cohort Finder
Cohort Matrix
Expression of Interest
Data Processing
Completed tasks
• Data Portal Cohort Finder, Matrix and User
Manual complete
• DPUK UK Secure eResearch Platform
(UKSeRP) live
• Project Application process built and in last
stages of development
April 2016
Ongoing and upcoming work
• Completion of Data Deposit and Access
Agreements
• Liaison between Swansea University and
UK wide research cohorts for data sharing
to the Data Portal
• Upload and primary testing of cohort data
on Data Portal
Informatics Network: Genomics
Genetic data analysis platform
Principal Investigator: Julie Williams
Research Associate: Elisa Majounie
Overview
The aim of the WP is to create a novel informatics platform
to facilitate specialist or customized analyses of genetic
and genomic datasets from DPUK and their international
collaborators.
Objectives
• Deliver a dedicated informatics platform for dementia
genomics, facilitating timely analyses by DPUK cohorts.
• Provide a front-end genetic results data registry to capture
new analyses results and facilitate meta-analyses.
• Allow analysis pipelines to be created and executed within
the platform and on high performance computing resources,
providing automation of a variety of routine genomic
analyses.
Progress
Completed tasks (March 2016)
• Platform deployed at Cardiff University (ARCCA).
• Genetic data explorer and cohort selection tool available.
• Results registry software complete and tested.
• Workflow managers (Galaxy/RoseCylc) integration.
Cohort selection and Genetic Data Explorer tools
Future work
• Complete integration / deployment of result store with Main
Informatics Portal (Swansea).
• Import cohort data into the platform within secure
environment (Swansea).
• Improve platform functionality based on feedback from
researchers.
Exemplar of collaborative project:
We anticipate cohorts would the DPUK platform for
streamlined analysis. Each cohort could receive their results
and combine these with those from other cohorts through
meta-analysis, thus generating the most powerful GWAS
datasets in dementia research.
Dataset and Results Registry: keyword-enabled searches of repository data
Data and results browser: exploring genes and variants from user-generated
results and publicly-available databases.
April 2016
Technical Infrastructure
Stem Cells Network:
Electrophysiological assessment
of oligodendrocyte precursor cells and oligodendrocytes derived
from human pluripotent stem cells
Principal Investigator: Livesey MR,
Co-Investigators: Magnani D, Selvaraj BT, Hardingham GE, Wyllie DJA, Chandran S
The ability to generate human pluripotent stem cell (hPSC)-derived
oligodendrocyte-lineage cells in vitro provides a novel opportunity for the investigation of human
oligodendrocyte physiology and disease. We have developed a protocol that generates an
enriched population of oligodendrocytes (OLs) from hPSCs (Methods/Figure 1). To date, little
evidence exists to describe the membrane properties of hPSC-derived oligodendrocyte-lineage
cells and, as such, a need to examine the capability of such cells to exhibit multiple physiological
properties that reflect their native counterparts. In this regard, we have performed an
electrophysiological and
pharmacological assessment of the intrinsic properties of
oligodendrocyte progenitor cells (OPCs) and OLs, and also characterised the AMPA receptors
expressed upon these cells. We demonstrate a maturation of the (electro)physiological properties
from OPCs to OLs and use this as a basis for an assessment of the maturation of OLs derived
from Amytrophic Lateral Sclerosis (ALS) patients carrying mutations to the C9ORF72 gene, the
most common genetic form of ALS.
1. Generation of oligodendrocytes from hPSCs.
Rosettes of neural precursors
(NPCs) are generated from SHEF4embryonic stem cells (ESCs) or
induced pluripotent
stem cells
(iPSCs; two lines; Cntrl1, Cntrl2).
Caudalised NPCs are both nestin
and OLIG2 positive. Morphogens
are employed to generate a
predominant population of OPCs
(OLIG2+, PDGFRα+) from which
OLs are efficiently differentiated.
Enriched populations of OLs (>70
%) are achieved within one week of
OPC plate down.
ESC
INTRODUCTION.
A
METHODS. The whole-cell patch configuration
iPSC Cntrl1
was used to record macroscopic currents
from hPSC-derived OPCs and OLs. Patch electrodes were typically filled with a K-gluconatebased solution. Coverslips containing cultured cortical neurones were superfused with a NaClbased extracellular solution. Fluctuation analysis of whole-cell currents evoked by AMPA (10 μM)
in the presence of cyclothiazide (10 μM) at a holding potential of -84 mV were used to estimate
the mean single-channel conductance of the AMPA receptor population within the cell. Data are
presented as mean ± S.E.M. Statistical analysis for was conducted using appropriate tests. p
values: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
iPSC Cntrl2
OLs
demonstrated
extensive
staining for immature OL marker O4
and mature OL marker myelin basic
protein (MBP). Importantly, the level
of co-stain for O4+-cells with
PDGFRα is low (<10%), though
with MBP is high (>80%).
A small percentage (<20 % of all
cells) of astrocytes are present. No
neurones are present within the
culture. Scale bar; 50 μm.
Schematic illustrating the production of OPCs and OLs from hIPSCs
140
3. Developmental changes in AMPA receptor composition.
25
25 s
0
25 s
-200 -150 -100 -50
-500
Mean normalised current-voltage relationships constructed from the sustained membrane
current amplitude (175 ms after voltage-step initiation) for PDGFRα+-OPCs and O4+-OLs
derived from an ESC line (C) and two control (Cntrl 1/Cntrl 2) patient hIPSC lines (D). Data
00
50
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was normalised to the -64 mV current
amplitude.
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-125 -100 -75 -50 -25 0
-25
Holding potential (mV)
25
B 120
A
25
***
*
75

**

50
25
40
20
Week 1 1 3
iPSC91
1 1 3
iPSC92
0
1000
25 ms
500 pA 500 pA
1500
250 pA
500 pA
1000
1000
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750
25 ms
25 ms
500
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250
0
0
0
0
-250
-60
-500
-500
-1000
200 ms
16 mV
-1000
16 mV
-750
-80
-2000
-84 mV
0
50
100
-2000
-84 mV
150
0
50
100
150
E
***
***
40
30
20
10
0
iPSC91
1750
1250
+ TTX
--
--
r
29 l 2
8
G S1
29
8
M S
33 2
7
M V
33 1
7V
2
CONTROL
2000
25 ms
2000
-100
nt
50
35
30
25
20
15
10
5
0
G
C
C
nt
rl
1
C
0
Week 1 3
1 3
iPSC91 iPSC92
CONCLUSIONS
0
H
30
***
2000
20
10
0.8
I
1500
1.0
1.2
1.4
*
***
1000
0
+
+
PDGFR
O4
Week 1 Week 1
+
O4
Week 3
PDGFR+ O4+
O4+
Week 1 Week 1 Week 3
200
0
50
100
150
**
-50
PDGFR+ O4+
Week 1 Week 1
O4+
Week 3
***
20
15
10
5
0
Week 1 3
1 3
iPSC91 iPSC92
200
2. hPSC-derived OPCs and OLS possess intrinsic ion channel properties that are directly comparable to those
described in rodent populations.
-40
-60
150
1. Our data demonstrate the ability to successfully generate OPCs and OLs from hPSCs.
500
0
100
***
ACKNOWLEDGEMENTS
1.6
-30
2500
RIN (M)
***
0.6
RMP (mV)
G
0.4
50
25
A, Immunostaining showing RNA foci (G4C2), a
pathological hallmark of ALS, in Week 3 OLs derived
from 2 patients (iPSC91, iPSC92). Scale bar; 10 μm. B,
Quantification of observed %age of cells showing RNA
foci. C, Cell death assay (caspase-3) showing C9ORF72
mutations are not associated with cell death in Week 3
O4+-OLs. D, No apparent morphological changes in
patient-derived cells. E, Maturation of AMPAR singlechannel conductance is maintained in mutants. F,
Example current-voltage step recordings from Week 3
Control and C9ORF72 OLs. Rectification indices in
C9ORF72 mutants did not differ from controls (iPSC91,
9.0 ± 3.6%,, p>0.30; iPSC92, 16.3 ± 3.3%, p>0.27.
Differentiation to OLs is associated with an increase in whole-cell capacitance (G), a decrease
in input resistance (RIN, H) and a hyperpolarisation of the resting membrane potential (RMP, I).
0.2
0
0
60
D
F
-20
= vs cntrl
1
* = vs cntrl 2
-40
**
ES
60
1500
Membrane potential (mV)
% rectification index

80
0
F

100
50Done
25
PDGFRα+-OPCs, but not O4+-OLs,
exhibit TTX (300 nM)-sensitive action
potentials (F).
The rectification index for each Week 3
O4+-OL was expressed as a percentage of
the respective mean PDGFRα+-OPC
rectification index (E).
C
100
-125 -100 -75 -50 -25 0
-25
Holding potential (mV)
50
5
4. Maturation is not impaired in ALS patient (mutant C9ORF72)-derived OLs.
30
0
0
E
75
Capacitance (pF)
Normallised
current response
75
PDGFR+Week 1
O4+ Week 3
Cntrl 1
Cntrl 2
iP
S
iP 1
iP S2
S C9
iP 1
S C9
2
100
PDGFR+ Week 1
O4+ Week 1
O4+ Week 3
10
A, AMPAR can be composed of 4 subunits, GluA1-4. GluA2 is subject to post-transcriptional editing (Q607 ► R). B, Example traces
of NASPM (3 μM) block in OPCs and OLs recorded at -84 mV. C, Mean data showing NASPM (3 μM) block in OPCs and OLs. D,
Example traces showing AC currents (above) and DC currents (below) of AMPAR-mediated whole-cell responses from OPCs and
OLs. E, Current-variance plot of recordings described in D (X axis truncated at -200 pA for clarity). The slopes of the plots give the
estimated single-channel current of AMPAR-mediated events from which single-channel conductance can be calculated. F, Mean
estimated AMPAR single-channel conductance obtained from OPCs and OLs derived from various hPSC lines.
% O4+/Caspase-3+ cells
100
iPSC-derived lines
D
15
Current (pA)
% cells with foci
ESC-derived line
C
***
rl2
-84 mV
-400 -2000
25 s
***
nt
-84
-84 mV
mV
50
***
C
-300
75
20
rl1
16 mV
Week 1 Week 3
PDGFR O4+
nt
-200 -1000
0
C
25 s
200 pA
16
16 mV
mV
Whole cell capacitance (pF)
00
00
-100
Normallised
current response
00
00
0
20
C
0
00
100 pA
100
40
F
OLIGODENDROCYTE
OPC
1000
100 pA
200
E
60
ES
300
00
00
D
25 ms
25 ms
100
500 pA
pA
2000
80
Conductance (pS)
400
25
25 ms
ms
500
500 pA
pA
AMPA
NASPM
AMPA
NASPM
GluA4
200 pA
00
00
GluA3
10 s
***
100
% NASPM block
B
GluA2
10 s
Variance (pA2)
A
50 pA
hPSC-derived OPCs and oligodendrocytes typically exhibit differential membrane current
responses in response to membrane depolarisation. Whole-cell current recordings from OPCs
(A) and OLs (B) in response to a voltage-step protocol. OPCs and OLs within live cultures
were identified using antibodies against PDGFRα and O4, respectively, conjugated with a
secondary fluorophore antibody (see inset).
GluA1
C
OLIGODENDROCYTE
OPC
Conductance (pS)
B
A
120
100 pA
2. Intrinsic developmental properties of oligodendrocytelineage cells.
3. During differentiation of OPCs to OLs the composition of AMPARs switches from a predominantly GluA2(R)lacking to GluA2(R)-containing population.
4. iPSC-derived OLs harbouring C9ORF72 mutations show no apparent differences in their ion channel
maturation profile compared to 'control' OLs, indicating that this aspect of their development is not
responsible
for
disease-related
dysfunction.
Livesey*, Magnani* et al., (2016) Stem Cells. Published online.
This research was ALSO
funded by The Wellcome Trust
(Grant
092742/Z/10/Z
to
D.J.A.W, G.E.H., S.C), MNDA
(SC), Euan MacDonald Centre
(SC),
Indian
Dept
of
Biotechnology (NV)
and
seedcorn funding from the
Patrick
Wild
Centre/RS
Macdonald Trust (D.J.A.W).
April 2016
Stem Cells Network:
Investigating LRRK2 function through
CRISPR Gene Editing of human induced Pluripotent Stem Cells
Principal Investigators: Richard Wade-Martins and Sally Cowley
Co-Investigators: Rowan Flynn and Cecilia Heyne Lee, Richard Wade-Martins and Sally Cowley
Aims/Overview
• Mutations in Leucine Rich Repeat Kinase 2
(LRRK2) cause inherited and sporadic forms
of Parkinson’s disease. However, the function
of this 286 kDa protein is far from fully
understood. The gene cannot easily be cloned
owing to a large gDNA sequence spread over
51 exons, and so genetic manipulation at its
endogenous locus is required to investigate its
function.
Results
LRRK2 KnockOut
A
gRNA21
CCAGGTACAATGCAAAGCTTAATGGGACCCCAGGATGTTGGAAATGATTGG
GGTCCATGTTACGTTTCGAATTACCCTGGGGTCCTACAACCTTTACATACC
gRNA20
WT
TCTGTCCAGGTACAATGCAAAGCTTAATGGGACCCCAGGATGTTGGAAATGATTGGGAAGT
Clone D10
Δ16bp TCTGTCCAGGTACAATGCAAAG----------------GATGTTGGAAATGATTGGGAAGT
+37bp TCTGTCCAGGTACAATGCAAAGCTTAATGGGACCCCAGGATGTTGGAAATGATTGGGAAGT
ACAATGCAAAGCTTAATGGGACCCCAGGATGTTGGAA
C
D
• LRRK2 is highly expressed in macrophages
and microglia, implying a role for the protein in
this lineage.
• LRRK2 expression was ‘knocked out’ through
introduction of biallelic out of frame indels at
exon 3 (Fig1). This serves as an experimental
negative control and has allowed us to:
• identify macrophage phagocytic/endocytic
pathways that LRRK2 is involved in
• Identify proteins interacting with LRRK2 in
macrophages.
40
3
• We can differentiate Parkinson’s Patientderived and control human induced Pluripotent
Stem cells (iPSc) to dopaminergic neurons,
astrocytes, macrophages and microglia.
• To explore the role of LRRK2 in all these
celltypes (and macrophages specifically in this
study), we have used CRISPR/Cas9 gene
editing to create iPSC lines containing the
following edits:
B
G2019S
*
Fig 1. (A) Schematic of LRRK2 showing location of gRNA target sites in exon 3. Sequencing result of biallelic knockout containing a
deletion and an insertion is shown below. (B) Optimised protocol for generating knockouts. (C) Melt curves resulting from High
Resolution Melt analysis of nucleofected clones. Star indicates melt curve of biallelic knockout. (D) Blotting of total protein isolated
from derived macrophages demonstrates complete loss of LRRK2 in KO line.
B
LRRK2 G2019S Repair
Detection of HDR events – PstI
A
• We have repaired the Parkinson’s disease
associated
LRRK2
G2019S
mutation,
providing an isogenic control to investigate the
mechanism through which it causes disease.
Restoration of normal protein half-life
confirmed reversion to wildtype (Fig 2).
C
D
• We have tagged LRRK2 with mCherry
fluorescent protein - this allows accurate
visualisation of subcellular localisation (Fig 3).
Outlook
• These gene edited iPSc lines enable us to
better understand LRRK2 function in
authentic, terminally differentiated cells,
especially neurons, astrocytes, macrophages
and microglia.
• These lines will also help us develop cellular
phenotypic
screening
assays,
and
subsequent screening of compound libraries,
using the MRC DPUK Stem Cell Network
Capital Equipment.
Fig 2. (A) Plasmid donor template containing silently mutated PAM sites and PstI restriction site. Location of gRNA target sites to
be used in combination with nickase Cas9. (B) Restriction analysis of resulting clones and breakdown of recombination events.
(C) Sequence traces comparing repaired and wildtype clones. (D) Decreased turnover of LRRK2 protein in macrophages in which
the G2019S mutation has been repaired. G2019S results in increased LRRK2 kinase domain activity maintaining Ser935
phosphorylation and slowing protein turnover. Repair of the mutation reverts this phenotype. GNE, LRRK2 inhibitor; CHX,
cyclohexamide.
Tagging of LRRK2 with mCherry
C
A
mCherry
Acknowledgements
D
B
WT stained
The research leading to these results has received support from the Innovative Medicines
Initiative Joint Undertaking under grant agreement n° 115439, resources of which are
composed of financial contribution from the European Union's Seventh Framework
Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution. This publication
reflects only the author’s views and neither the IMI JU nor EFPIA nor the European
Commission are liable for any use that may be made of the information contained therein.
April 2016
mCherry-LRRK2 stained
Fig 3. (A) LRRK2-mCherry donor construct composed of homology arms (HA), mCherry, EF1α promoter, puromycin
resistance/thymidine kinase fusion gene and loxP sites for Cre-mediated excision. (B) Location of gRNA target sites relative to
LRRK2 stop codon. (C) Western blot using mCherry antibody shows successful expression of Lrrk2-mCherry (D) Localization of
LRRK2-mCherry within macrophages, showing punctate cytoplasmic staining.
Stem Cells Network:
Early cellular dysfunctions in Parkinson’s
iPSC-derived dopamine neurons
Principal Investigator: Richard Wade-Martins
Co-Investigators: Hugo Fernandes, Elizabeth Hartfield, Helen Christian, Michiko Yamasaki-Mann, Evangelia
Emmanoulidou, Heather Booth, Jane Vowles, Samuel Evetts, Kostas Vekrellis, Michele Hu, William James, Sally Cowley
Overview
• Mutations in the GBA gene represent the highest genetic risk factor for PD - but the mechanisms underlying this association are unknown
Aims
• Generation of human induced pluripotent stem cells (iPSCs) from PD patients carrying GBA mutations and controls;
• Differentiation of iPSC into dopaminergic neurons – development of a physiologically-relevant cell culture model for PD;
• Elucidate early pathological effects of GBA mutations in the early development of PD, focusing on lysosomal and autophagy dysfunctions.
1. GENERATION OF PATIENT-DERIVED iPSCs
2. DIFFERENTIATION INTO FUNCTIONAL DOPAMINERGIC NEURONS
Confirmed pluripotency and genome integrity
Oct
Nanog
Tra-1-60
SSEA-4
Phase
β3-tubulin
DAPI
Merged
GBA-PD
iPS-MK071-1
TH
iPS-MK071-3
Reprogramming of
patient fibroblasts
Neurons produce dopamine
Expression of high levels of neuronal markers
Control
Electrophysiological maturation
of neurons
Weeks:
6
7
8
9
10
iPSC colonies
3. ER RETENTION AND ER STRESS IN GBA NEURONS
4. AUTOPHAGIC PERTURBATIONS IN GBA NEURONS
GBA mutation leads to ER retention of misfolded GBA protein
C1#2
LC3B-I
LC3B-II
Actin
Reduced GBA activity
Impaired lysosomal clearance in dopamine neurons
PD1#1
PD2#1
C1#2
C2#1
PD2#1
PD1#2
C1#2
Bip
PD1#1
GBA-PD
C1#2
C1#2
C2#1
C2#1
PD1#1
PD1#1
PD2#1
PD2#1
GBA mutation leads to activation of ER stress
Merged
TH
Control
C1#2
PD2#1
PD1#1
PD1#1
PD1#2
C2#1
LC3
GBA-PD
GBA
PD2#1
PD2#1
PD2#1
PD2#1
PD1#2
PD1#1
PD1#1
PD1#1
PD-S#1
C2#1
C1#2
C1#2
C1#2
PD2#1
PD2#1
PD2#1
PD1#2
PD1#1
PD1#1
PD1#1
PD-S#1
C2#1
C1#2
C1#2
C1#2
+ EndoH
C1#2
Increased number of autophagosomes in GBA dopamine neurons
p62
Actin
Actin
6. INCREASED EXTRACELLULAR α-synuclein
5. ENLARGED LYSOSOMAL COMPARTMENT
LAMP2A
Actin
Increased extracellular α-syn
C1#2
PD1#1
PD2#1
Ladder
C1#2
PD1#1
PD1#2
PD2#1
C2#1
C1#2
PD2#1
PD1#1
C1#2
PD2#1
PD1#1
C1#2
C2#1
PD2#1
PD1#1
PD1#2
C1#2
PD1#2
PD1#1
Upregulation of lysosomal markers
No change in α-syn
intracellular levels
Increased number and size of lysosomes in
dopamine neurons
SNCA
Actin
Progress
• Efficient differentiation of dopaminergic neurons from iPSC lines generated from 3 PD-GBA patients and 3 control individuals;
• Multiple dysfunctions were identified in PD-GBA iPSC-derived dopaminergic neurons including ER stress and Autophagic impairments;
• These disturbances might impair proteostasis of dopaminergic neurons and result in the observed increased extracellular α-synuclein
April 2016
Stem Cell Network:
Generating stem-cell derived
Astrocytes for in vitro modelling of tauopathy
Principal Investigator: Thomas Warner
Co-Investigators: John Hardy, Selina Wray, Rickie Patani, Andrey Abramov
Researcher: Nuria Seto-Salvia, Noemi Esteras
Overview
Patient with a
genetic cause
of dementia
Patient fibroblast
biopsy, expansion
and banking
Functional characterisation and disease
phenotype screening of patient-derived
cells versus control cells
Isolation and
banking of patientderived iPSCs
Fibroblasts
Cortical glutamatergic neurons
Neural rosettes
iPSCs
TE7 FSP1 DAPI
OCT4 SSEA4 DAPI
OCT4 SSEA4 DAPI
Reprogramming
Okita 2011
TUJ1 SATB2 DAPI TBR1 CTIP2 DAPI
Cortical differentiation
Shi 2012
Astrocytes
GLAST TUJ1 DAPI
There has been increasing focus on the role of astrocytes in the pathogenesis of neurodegenerative disease. To model these
conditions and understand astrocytic dysfunction we are generating induced pluripotential stem cell (iPSC) derived astrocytes,
using a novel protocol. iPSCs allow us to create models of neurodegenerative disorders, containing the patient’s precise
genome in human neurons and glia that are relevant to the disease process.
Introduction
Astrocytes are a subtype of glial cells that are very abundant in the brain. The roles of astrocytes in the healthy
brain include physical and metabolic support for neurons, detoxification, guidance during migration, regulation
of energy metabolism, transmitter uptake and release, electrical insulation (for unmyelinated axons), transport
of blood-borne material to the neuron and reaction to injury.
Astrocytes also play an important role in neurodegenerative diseases. Tauopathies like Corticobasal
degeneration (CBD) or Progressive Suipranuclear Palsy (PSP)demonstrate astrocytic pathology where 4R Tau
protein accumulates in astrocytes in brain in different forms (Fig1). The aetiology of these diseases and the
mechanisms of Tau accumulation is unknown.
Figure 1. Histopathology : CBD and PSP lesions visualized with TAU
immunohistochemistry.
Objectives
Progress to March 16:
Materials and Methods
We are generating astrocytes from 3
control cells lines using modifications
of 2 previously-published protocols.
This new protocol allows us to freeze
the cells down at any point.
To generate astrocytes in the lab
our protocol starts with dual
SMAD inhibition for cortical
neural differentiation (Shi 2012).
After the first neural rosettes
form, we use a modified protocol
to generate cortical astrocyte
precursors (Serio 2012). For the
last step we use a modified
protocol to generate mature
astrocytes (Gupta 2012).
Validation of this protocol will allow us
to generate astrocytes from patients
cell lines with mutations in the MAPT
gene
to
study
fronto-temporal
dementia
and
other
neurodegenerative diseases.
Figure 2. Astrocyte generation protocol.
Progress to March 16: Positive astrocytes markers and functional assays
D
B
A
More than 90% of mature astrocytes were
positive for astrocytic markers SB100,
GLAST and GFAP (Fig 3+4).
E
Functional assays showed that 67% of
the cells in culture behaved as astrocytes.
We used ATP and Glutamate to stimulate
calcium response. FCCP was used with
Rodamine 123 staining to verify the
viability of the cells (Fig 3:D,E).
C
Figure 3. A: Counting control cell lines for astrocytic markers SB100 and GLAST. B+C: Immunocytochemistry (ICC) for astrocytic markers SB100 (B) and
GLAST (C). D: Calcium measurements in control mature astrocytes at day 195. E: Measure of the viability of the cells following the calcium experiments.
Progress to March 16: Mature astrocytes
Conclusions
Mature astrocytes from our control cell lines show the expected functionality and they are
positive for astrocytic markers. We also checked mitochondrial lipid storage using bodiP
C12 (red) and mitochondrial morphology using mitotracker green (green). This will help
with future studies of mitochondria in healthy and mutant astrocytes (Fig4).
iPSC-derived cortical neurons and astrocytes provide a physiologically-relevant cell
model to understand the molecular mechanisms underlying frontotemporal
dementia and other tauopathies. Ongoing work aims to understand the mechanisms
linking mutations in MAPT, C9orf72 and VCP to neuronal death and use these
models to develop a platform for drug screening.
A
GFAP/TUJ1/Dapi
B
GLAST/TUJ1/Dapi
C
bodiP C12/ Mitogreen
References
•
•
•
•
Figure 4. A+B: ICC with control cell lines using astrocytic markers GFAP and GLAST (green).
C: Live cell mitochondrial imaging.
April 2016
Okita K, Matsumura Y, Sato Y, et al., A more efficient method to generate integration-free human iPS
cells. Nat Methods.2011;8(5):409-12.
Shi et al Shi Y, Kirwan P, Smith J, et al., Human cerebral cortex development from pluripotent stem cells
to functional excitatory synapses. Nat Neurosci.2012;15(3):477-86
Serio A, Bilican B, Barmada SJ, et al., Astrocyte pathology and the absence of non-cell autonomy in an
induced pluripotent stem cell model of TDP-43 proteinopathy. Proc Natl Acad Sci U S
A.2013;110(12):4697-702.
Gupta K, Patani R, Baxter P, et al., Human embryonic stem cell derived astrocytes mediate non-cellautonomous neuroprotection through endogenous and drug-induced mechanisms. Cell Death
Differ.2012;19(5):779-87.
Stem Cell Network:
Using stem cells to investigate the effect of modulation of
ADAM10 on f Aβ production and binding
Principal Investigator: Heledd Jarosz-Griffiths,
Co-Investigator: Katherine Kellett, Alys Jones & Nigel Hooper
Introduction
The cellular prion protein (PrPC) is a high-affinity neuronal receptor for Aβ oligomers, and mediates neuronal impairment via the activation of
fyn kinase (Um et al., 2012; Rushworth et al., 2013). The α-secretase, ADAM10 which cleaves the amyloid precursor protein (APP)
precluding the formation of A, also sheds full length PrPC from the cell surface (Taylor et al., 2009).
We hypothesise that attenuating or activating ADAM10 activity will alter cell surface PrPC and consequently A oligomer binding and toxicity.
A. ADAM10 siRNA reduces soluble
APP
130
40
35
25
sPrPC
35
100
70
pADAM10
mADAM10
40
siRNA
actin
-
sAPP
40
Total
PrPC
Protein(% control)
130
siRNA
-
200
+
C
Total PrPC
sPrPC
150
***
100
**
50
150
Knockdown of ADAM10 reduced shedding of sPrPC by 54% (Fig1A, B) and reduced the αsecretase cleavage of APP, as measured by detection of sAPP, by 67% (Fig 1A, C).
*
100
50
-
-
siRNA
+
+
+
E
+
+
-
F
250
PrPC cell surface
staining (% control)
-
+
+
AO binding
(% control)
200
150
**
200
***
***
#
100
50
150
100
50
0
**
-
siRNA
6D11
+
+
-
+
+
-
siRNA
6D11
+
+
-
100
40
sPrPC
35
140
APP
sAPP 
120
250
*
#
#
#
100
80
60
40
20
***
***
0
APP
D
40
35
pADAM10
mADAM10
actin
40
GI
-
-
+
+
-
-
sAPPα/β
(pg/mg protein)
PrPC
100
70
-
Acit
GI
-
120
sAPPα
sAPPβ
100
***
150
# *
100
50
***
***
***
***
Acit
GI
+
+
-
+
-
+
+
+
To establish the specificity of acitretin for ADAM10 activation, cells were treated with/ without
ADAM10 inhibitor and acitretin. The ADAM10 inhibitor significantly reduced sAPPα by 93% and
the shedding of PrPC by 69% relative to control. Co-application of the ADAM10 inhibitor blocked
the acitretin-mediated increase of sAPP and sPrPC (Fig 2A-C). Acitretin caused a reciprocal
decrease in sAPP and A40/42 levels when sAPP was increased relative to control (Fig 2D,
E).
F
*
60
40
*
20
0
+
Acit
-
500
Aβ40
Aβ42
Aβ40 & Aβ42
(pg/mg protein)
sPrPC
200
Acit
+
+
G
400
300
*
200
100
*
0
Acit
-
100
80
60
40
***
20
120
80
60
40
***
20
Acit -
+
Figure 2. (A) SH-SY5Y cells expressing PrPC were incubated -/+ ADAM10 inhibitor, GI254023X (GI) and -/+ acitretin (20M) diluted in
OptiMEM for 48h at 37oC. Samples were immunoblotted for sAPPα and sPrPC in media and APP, PrPC, and ADAM10 and actin in
lysates. Blots quantified and represented as percentage control (B) Total PrPC and sPrPC (C) APP and sAPPα (n=4). were also
measured by MESO scale analysis of (D) sAPP/ and (E) A40/42 (n=3). (F) Cells were incubated with either acitretin (20M) diluted
in OptiMEM or ddH2O control for 48h at 37oC. Following acitretin treatment, cells were incubated with AβOs (500nM) for 25 min at room
temperature, fixed and immunostained for Aβ-biotin and PrPC. (G) PrPC cell surface staining (n=15) (H) AO binding to cells (n=15); #,
not significant, * p<0.05, ** p<0.01, *** p<0.001).
100
0
0
Acit -
+
-
H
120
shedding and reduces Aβ oligomer binding
Acitretin is a synthetic retinoid which induces gene expression of ADAM10 and promotes secretase activity by non-permissive retinoid acid receptor/retinoid X receptor (RAR/RXR)
heterodimers (Tippmann et al., 2009). Acitretin treatment increased sAPP by 17% and the
shedding of sPrPC by 24% (Fig 2A-C). This increase in shedding resulted in a decrease in
surface PrPC which led to a concomitant decrease in the amount of A oligomers bound to the
cells (Fig 2F, G).
***
80
+
E
+
+
-
PrPC
Figure 1. SH-SY5Y cells expressing PrPC were incubated -/+ siRNA targeted against ADAM10 for 48 h. (A) Cells were then incubated
with OptiMEM for 24 h and immunoblotted for sPrPC and sAPP in media; and APP, PrPC, ADAM10 and actin in lysates. Blots
quantified and represented as percentage control (B) PrPC/sPrPC(C) APP/sAPP (n=4). (D) Following siRNA treatment, cells were preincubated with (+ ) or without (-) PrP antibody, 6D11 for 20min at 37oC, then incubated with AβOs (500nM) for 30min at RT, fixed and
immunostainined for Aβ-biotin and PrPC (B) PrPC cell surface staining (n=15) (C) AO binding to cells (n=15); #, not significant, *
p<0.05, ** p<0.01, *** p<0.001).
C
PrP
0
PrPC cell surface
staining (% control)
130
100
Protein (% control)
Protein (% control)
sAPP
+
+
C
B
AO binding
(% control)
A
*
0
B. Activation of ADAM10 increases
Acit
Knockdown of ADAM10 increased cell surface PrPC by 2-fold relative to control (Fig1D, E). This
increase in PrPC at the cell surface, caused a 2.4-fold increase in the amount of A oligomers
bound to the cells. (Fig 1D, F). Pre-incubation with the 6D11 antibody blocked binding of A
oligomers to PrPC (Fig 1D, F).
***
0
siRNA
and increases Aβ oligomer binding
APP
sAPP
0
D
siRNA
6D11
200
Protein (% control)
B
A
C
PrP
+
-
+
100
50
Acit
F
120
100
80
60
***
40
20
0
Acit
-
+
*
80
APP
PrPC
60
***
40
20
-
+
+
-
Acit
+
34
100
70
40
Acit
pADAM10
mADAM10
actin
-
+
Human iPS-derived cortical neurons were cultured to day 48 before incubating with acitretin.
sAPP was increased relative to control and a reciprocal decrease was seen in A40/42 levels
(Fig 3G-I). The effect of acitretin on the binding and toxicity of Aβ oligomers in the iPS-derived
neurons is currently being investigated.
I
H
130
100
40
100
Acit
-
G
0
-
50
Acit
+
120
AO binding
% control)
PrPC cell surface
taining (% control)
E
*
100
0
0
Rat primary hippocampal neurons were cultured to day 14 before incubating with acitretin.
sAPP was increase by 40% and a reciprocal decrease was seen in A40/42 levels (Fig 3A-C).
Acitretin also reduced cell surface PrP levels in MAP2 stained neurons and there was a
concomitant decrease in A binding (Fig 3D-F).
D
Aβ40
Aβ42
30
sAPPα
25
sAPPβ
200
Aβ40 & Aβ42
(pg/mg protein)
Acit
*
150
150
sAPPα/β
(pg/mg protein)
sAPP
100
C
200
Aβ40 & Aβ42
(pg/mg protein)
B
A
sAPP (% control)
C. Activation of ADAM10 reduces Aβ oligomer binding in neurons
20
15
10
5
150
100
50
0
0
Acit
Aβ40
Aβ42
-
+
Acit
-
+
Figure 3 Primary hippocampal rat neurons were incubated with 20M acitretin diluted in OptiMEM media or DMSO control for 48h.
Media was concentrated and immunoblotted for (A) sAPP, (B) sAPP quantification (n=3) (C) Media was analysed by MESOscale for
A40/42 (n=3) (D) Rat hippocampal neurons were incubated with either acitretin (20M) diluted in OptiMEM or ddH2O control for 48h at
37oC. Following acitretin treatment, cells were incubated with AβOs (500nM) for 25 min at room temperature, fixed and immunostained
for Aβ-biotin and PrPC. (E) PrPC cell surface staining (n=15) (F) AO binding to cells (n=15); #, not significant, * p<0.05, ** p<0.01, ***
p<0.001). (G) Human IPS-derived cortical neurons were incubated with 20M acitretin diluted in OptiMEM media or DMSO control for
48h. Lysates were immunoblotted for APP, PrPC, and ADAM10 and actin. Media was analysed by MESOscale for (H) sAPP/ and (I)
A40/42 (n=2).
Conclusions and future perspectives
These data indicate that modulation of ADAM10 activity influences the binding of A oligomers to PrPC on the surface of cells. Increasing
ADAM10 activity using acitretin decreased the amount of PrPC at the cell surface, resulting in reduced A oligomer binding. Developing
agonists of ADAM10 will not only preclude A formation, but also prevent the binding of soluble A oligomers to PrPC and ultimately prevent
the aberrant activation of signaling pathways which lead to neurotoxicity.
April 2016
@Hooperlabmanchester
@Hooperlableeds