BRT/MRC DTA PhD in Clinical Neuroscience
Dr Jan-Willem Taanman (j.taanman@ucl.ac.uk)
1. Generation of induced pluripotent stem cells from MELAS patients carrying the mitochondrial
3243A>G mutation
Background and overall aim:
Mitochondria are small subcellular structures responsible for producing energy in every cell of the human body.
Mitochondria contain their own DNA, which is present in multiple copies. All the genes on this DNA are required
for mitochondrial energy production. Mutations in mitochondrial DNA (mtDNA) are a common cause of human
inherited disorders. There are no specific therapies or cures for mtDNA diseases. Only supportive treatment can
be offered to patients but these are largely ineffective. Advances in the treatment of mtDNA diseases have been
hindered by an incomplete understanding of the disease mechanisms underlying these disorders due to the lack
of genuine disease models.
This project aims to develop patient-specific cell models using induced pluripotent stem (iPS) cell technology, in
which skin fibroblasts from patients are reprogrammed to revert to stem cells [1]. This approach provides a
unique opportunity to produce stem cells derived from an individual patient and differentiate these stem cells into
cell types relevant to the disease.
The specific objectives of the rotation project are:
1. To generate iPS cell lines derived from fibroblast cultures of MELAS patients.
2. To differentiate these patient-specific iPS cell lines into functional neurons for study of neuronal pathogenic
mechanisms.
3. To produce cell lines with different levels of the 3242A>G mtDNA mutation, so that the effect of the mutant
load can be studied.
MELAS fibroblasts with a >75% 3243A>G mutant load and control fibroblasts will be reprogrammed to iPS cells
by transduction with retroviruses transiently expressing pluripotency transcription factors, using our established
protocol. The pluripotent state of the cells will be verified by expression of pluripotency markers. The neuronal
differentiation process will be initiated by a combination of retinoic acid signalling and inhibition of SMAD
signalling to promote formation of cortical neuroepithelial stem and progenitor cells. Subsequent growth factor
withdraw will lead to the formation of terminally differentiated, glutamatergic neurons [4]. The glutamatergic
phenotype will be validated by immunocytochemistry for VGLUT1 and - 2. The clonal 3243A>G mutant load will
be determined by quantitative PCR techniques.
This 3-month project will allow the student to become familiar with iPS cell technology and neuronal cell
differentiation methods. In addition to advanced cell culture and gene transduction techniques, the student will
gain experience with reverse transcriptase quantitative PCR, immunocytochemistry and mtDNA mutation load
analysis
How this project might grow into an exciting 3-year PhD:
Extension of the work into a full 3-year PhD project would allow a thorough investigation of the possible
pathophysiological consequences of the m.3243A>G mutation in neuronal cells, including defects in oxidative
phosphorylation function, mitochondrial membrane potential, oxidative stress, glutamate uptake, and intracellular
+
2+
Na and Ca handling. It is expected that the study of the patient-specific neuronal cell cultures will provide key
insights in the mechanism of clinical expression of the m.3243A>G mutation.
Importance of this study:
This rotation project will open the door to a thorough investigation of the pathophysiological mechanisms of the
3243A>G mtDNA mutation in in vitro models of cell types that are physiologically relevant to the disease. A
better understanding of the pathophysiology will facilitate the rational development of treatments. These patientspecific cell models will provide an ideal test bed for large-scale genetic and drug-based screens. In addition, the
project will serve as a paradigm for the study of pathophysiological pathways of other mtDNA diseases.
References:
[1] Takahashi, K. et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861-872.
[2] Schaefer, A.M. et al. (2008) Prevalence of mitochondrial DNA disease in adults. Ann. Neurol. 63: 35-39.
[3] Elliott, H.R. et al. (2008) Pathogenic mitochondrial DNA mutations are common in the general population. Am. J. Hum. Genet. 83: 254-260.
[4] Shi, Y., et al. (2012) Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nat. Neurosci. 15:477-486.
2. Exploring LRRK2 mutation specific functional phenotypes using human iPS cellular model.
Dr Rina Bandopadhyay and Prof Thomas Warner
Reta Lila Weston Institute, UCL Institute of Neurology
Aims: To develop and characterise LRRK2 mutation specific induced pluripotent stem (iPS) cell lines.
Background:
Missense mutations in the LRRK2 gene are the most common cause of autosomal dominant PD. The G2019S
mutation affects around 2% of Caucasian population but increases to 40% incidence in people from some ethnic
backgrounds. LRRK2 encodes a large multi-domain protein encompassing two enzymatic domains, GTPase and
kinase. Several mutaions in the LRRK2 gene have been reported thus far but only six have been found to be
clearly pathogenic and these are located predominantly in the enzymatic domains (kinase and GTPase). The
G2019S mutation within the kinase domain increases the kinase activity of LRRK2. Variation around the LRRK2
locus also appears to be a risk factor for sporadic PD. For the majority of LRRK2 cases, the clinical signs in
affected carriers are remarkably similar to idiopathic PD. More recently, an important role of LRRK2 in the
immune system has emerged (reviewed in Greggio et al 2012). LRRK2 is abundantly expressed in different
immune cells. In addition to PD, LRRK2 wasfound in genome-wide association studies as a risk factor for
Crohn’s disease, leprosy and inflammatory bowel disease. Recent data accumulated from our lab has shown
that arsenite or H2O2 treatment induces loss of Ser910 and Ser935 phosphorylation in transfected and
endogenous LRRK2. This is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S and
kinase dead D2017A variant, suggesting that in contrast to the effects of acute kinase inhibitors, it is
independent of LRRK2 kinase activity. Furthermore, arsenite stress enhances LRRK2 self-association,
decreases GTP binding in vitro and induces translocation of WT and pathogenic mutant LRRK2 to centrosomes
(Mamais et al 2014). Our data points to oxidative stress being a physiological/pathological regulator of LRRK2
function. We now need to focus our attention on the upstream regulators and downstream consequences of
LRRK2 phosphorylation. We would also like to test if immune cells are similarly affected.
Three-month project:
1) To expand and grow Lrrk2 mutation specific fibroblasts (1 week)
2) Reprogram fibroblasts (3-4 weeks)
3) Purify iPSCs (2-3 weeks)
4) inducing iPSC to neural stem cells (2-3 weeks)
5) maintaining neurons
How this project might grow into exciting 3-year PhD:
After three months, the cells will be ready for further functional characterisation using electrophysiology,
mitochondrial dysfunction, responsiveness to stressors, live cell imaging and outputs of LRRK2 function ie
altered autophagy, neurite shortening etc. We would also like to differentiate the iPSCs into glial cell phenotypes
and study neuron-glia interaction.
Importance of the study:
Induced pluripotent stem cell (iPS cell)-based models hold tremendous potential for the study of human
neurological disease. Patient specific iPSCs that carry all disease relevant genetic alterations could provide
researchers with a unique opportunity to study the cellular and molecular mechanisms of monogenic and
complex diseases in relevant cell types in vitro. These models bridge the gap between studies using animal
models and human clinical research by opening up new avenues for drug development and disease
management. Our project should provide therapeutic pointers for both familial and sporadic PD.
References: Greggio et al J Neuroinflammation, 2014, 11:52. Doi: 10.1186-2094-11-52. Mamais et
BRT/MRC DTA PhD in Clinical Neuroscience
Mr David Choi, Dr Karen Oprych, Dr Wenhui Song
3. Nano-hydrogels for the delivery of drugs or cellular therapies in spinal cord injury repair.
Aims:
The goal of this project is to develop novel injectable nanocomposite hydrogels as vehicles for delivering both
regenerative cell candidates and neuroprotective drugs for the direct treatment of spinal cord injury. A novel and
critical feature of nanocomposite hydrogels is the ability to be independently tuned as both a scaffold to
accommodate the migration of cells into the spinal cord defect formed after spinal cord injury, and as a vehicle
for the controlled release of therapeutic agents. We will engineer growth factor or drug-preloaded polymeric
nanotubes into injectable gelatin and collagen hydrogels with programmed release kinetics. We aim to show that
multifunctional nancomposite hydrogels promote the recruitment of astrocyte and neurites, enhancing neural
tissue repair in a rodent model.
Importance:
Spinal cord injury results in the permanent loss of function, with significant personal, social and economic
impact. Central nervous system (CNS) repair is ineffective due to inhibitory elements of the glial scar that results
after injury. Potential treatments for spinal cord injury include neuroprotective drugs and regenerative cellular
therapies, but these require sustainable and effective delivery systems which can control the release kinetics of
drugs, cells or growth factors for a sufficiently long period to facilitate regeneration and plasticity. Also, a scaffold
is required to bridge the tissue loss and cavitation which commonly occurs after spinal cord injury.
Nanocomposite hydrogels are ideal candidates for both filling the physical CNS defect, and delivering drugs or
cellular therapies to the lesion site.
The 3-month project:
To develop injectable nanocomposite collagen-genipin and Gtn-HPA hydrogels with desired gelation time and
matched modulus, and incorporate drugs (eg methyl prednisolone steroids) and growth factors.
The student will gain direct experience of:
Development of biomaterials, hydrogels, bioengineering of nanotubules
Understanding of the mechanisms of spinal cord injury and repair
Development of the project into a 3-year PhD
To design and produce bioabsorbable polymer nanotubes loaded with growth factors (fibroblast growth factor,
FGF-2 and stromal cell-drive factor-1a, SDF-1a) with controlled size, size distribution, morphology and surface
chemistry as well as controlled release capacity.
To evaluate in vitro, (a) FGF-2-polymer nanotubes with respect to release kinetics, and retained bioactivity and
dosage using an astrocyte migration assay; (b) SDF-1a-polymer nanotubes with respect to release kinetics, and
retained bioactivity and dosage using NPC migration assay; (c) effects of the gel rheological and mechanical
behavior (viz., gel time and viscoelasticity) of nanocomposite collagen gels on release kinetics and astrocyte and
neural progenitor cell migration. And (d) co-cell culture of both astrocyte and neural progenitor cells and cell
therapy candidiates using optimised composite gels from (a) to (c).
Evaluate by hindlimb locomotor behavior and histology of the response to injection of a select formulation of the
nanocomposite collagen-genipin and Gtn-HPA gels in a standardized hemi-resection defect in the rat spinal
cord.
Methods:
Development of biomaterials, hydrogels, bioengineering of nanotubules
Rodent models of spinal cord injury and repair
Tissue processing for histological analysis including dissections and cryosectioning.
Histology and immunofluorescent techniques for assessing axon regeneration.
Epi and confocal fluorescent microscopy.
The student will also be able to observe and gain knowledge of:
In vivo surgical techniques for modelling spinal cord injury.
Principles of laboratory animal management, welfare and post-operative animal care.
Aortic perfusion-fixation techniques.
BRT/MRC DTA PhD in Clinical Neuroscience
Dr Karen Oprych & Mr David Choi
4. Olfactory ensheathing cell influences on spinal motoneuron outgrowth
Aims: Olfactory ensheathing cells (OECs) are a unique type of glial cell found within the olfactory system.
OECs have been heavily investigated for their regenerative properties and as a potential therapy to repair
1–3
injuries to the central nervous system . OEC transplants could be used to enhance recovery after ventral
spinal nerve root injuries, however, we currently know very little about how OECs from different areas of the
olfactory system, and the accompanying cells in OEC cultures (olfactory fibroblasts), affect regeneration and
interact with axons and growth cones of spinal motoneurons. Utilising an organotypic spinal cord slice culture
model, this project aims to investigate the regenerative properties of different OEC cultures (from rat and
humans) and to dissect the dynamic axonal-glial interactions between OEC cultures and spinal motoneuron
outgrowths.
Importance: We have developed protocols for the harvest and culture of human OECs to enable clinical trials in
4
spinal nerve root injured patients at the National Hospital for Neurology and Neurosurgery (NHNN) . The results
from this study will provide crucial information regarding the suitability of different OEC cultures for enhancing
regeneration of spinal motoneurons, and the effect of olfactory fibroblasts within these cultures. This information
will be used to refine the content of our therapeutic OEC transplants for future clinical trials and will contribute to
the success of this potentially life-changing treatment.
Methods: OEC cultures will be generated from rat olfactory mucosa; rat olfactory bulb; or cultured from human
4
mucosal biopsies donated from patients recruited within the NHNN undergoing endoscopic surgery . The
regenerative properties of the different OEC cultures (human, rat, purified and unpurified) will be investigated by
assessing neurite outgrowth from spinal motoneurons in organotypic spinal cord slice cultures. Confocal
fluorescent time-lapse imaging will be employed to dissect the mechanisms by which the different OEC cultures
interact with spinal motoneuron growth cones, and to define the influence of olfactory fibroblasts. Growth cone
dynamics such as migration rate, growth cone elaboration and substrate preferences will be studied. To facilitate
this, protocols for live-cell labelling will be established utilising lipophilic tracers such as DiI and fluorescent
protein transduction.
The 3-month project:
During the 3-month project the student will have the opportunity to experience a range of challenging and
exciting imaging and cell culture techniques that will underpin the main 3-year PhD programme. Specifically the
student will be involved in developing protocols for fluorescent labelling of spinal motoneurons in live spinal cord
slice cultures and transduction of OEC/olfactory fibroblasts with fluorescent proteins via viral vectors.
Subsequently, imaging protocols for time-lapse confocal fluorescent microscopy of labelled co-cultures will be
established.
The student will also gain direct experience of:
1) Generating primary cultures of OECs from human and animal tissue.
2) Cell separation techniques such as immunopanning and magnetic-activated cell sorting.
3) Transduction of cell cultures with fluorescent proteins and live cell labelling techniques.
4) Establishing organotypic spinal cord slice cultures.
5) Epi and confocal fluorescence microscopy.
Development of the project into a 3-year PhD:
After the completion of the 3-month project the student will now have the tools in place to embark on an exciting
three year PhD investigating i) the regenerative properties of different OEC cultures and ii) axonal-glial
interactions between OEC cultures and spinal motoneurons in an in vitro co-culture system. This PhD project
will address the following important questions:
1) How do OECs from the different regions of the olfactory system: the olfactory bulb and olfactory mucosa
influence spinal motoneuron regeneration and affect growth cone behaviour, and how might these
interactions benefit or hinder their potential as a therapeutic treatment for ventral spinal nerve root injuries?
2) What role do olfactory fibroblasts play in mediating the outgrowth promoting properties of OEC cultures?
Answers to these questions will allow us to identify the most suitable location for harvesting OECs for
therapeutic transplants, and, whether olfactory fibroblasts contribute to the reparative effects and
should therefore be included in these transplants.
References: 1) Li Y, Decherchi P, Raisman G. J Neurosci. 2003;23(3):727–31. 2) Ibrahim AG, Kirkwood PA, Raisman G, Li Y. Brain.
2009;132(Pt 5):1268–76. 3) Li Y, Li D, Khaw PT, Raisman G. Neurosci Lett. 2008;440(3):251–4. 4) Kachramanoglou C, Law S, Andrews P,
Li D, Choi D. Neurosurgery. 2013;72(2):170–8; discussion 178–9.
BRT/MRC DTA PhD in Clinical Neuroscience
Mr David Choi & Dr Karen Oprych
5. Transplants of human mucosal olfactory ensheathing cells: safety and efficacy testing for the repair of
the injured spinal cord.
Aims:
During high impact traumas such as motorcycle accidents, spinal nerve roots of the brachial plexus can be torn
away or ‘avulsed’ from the spinal cord resulting in a longitudinal spinal cord injury with debilitating and
permanent paralysis of the shoulder, arm and hand. Despite advances in the surgical treatment of this injury,
recovery of function to the distal arm and hand is rarely achieved and additional treatments are required. We
have developed a surgical technique to avulse cervical spinal nerve roots in rodents as a model to test potential
therapeutic interventions to treat this type of CNS injury. This project aims to refine this model and subsequently
test the safety and efficacy of an exciting potential cellular therapy; olfactory ensheathing cell transplants.
Importance:
Previous studies from our laboratory and others have shown that transplants of olfactory ensheathing cells
1–3
(OECs) are able to support regeneration in animal models of spinal cord injury . We have now developed
protocols for the harvest and culture of human mucosal OECs to enable clinical trials in spinal root injured
4
patients at the National Hospital for Neurology and Neurosurgery (NHNN) . This project will provide crucial
evidence of the safety and efficacy of our human mucosal OEC transplants taking us a step closer to clinical
application of this potentially life-changing therapy.
The 3-month project:
During the 3-month project the student will be required to assist in refining our in vivo model of spinal root injury
and establish important techniques for assessing functional outcomes after therapeutic interventions. This
period will provide a real taster to the main PhD programme as the student will gain experience and/or
knowledge of many of the different techniques required to complete the full 3-year project.
The student will gain direct experience of:
1) Behavioural training of laboratory rodents for the assessment of forepaw function including analysis of grip
strength, gait, and food pellet retrieval.
2) The home office rules and regulations governing the use of animals in research and schedule 1 procedures.
3) Tissue processing for histological analysis including challenging dissections and cryosectioning.
4) Histology and immunofluorescent techniques for assessing axon regeneration.
5) Epi and confocal fluorescent microscopy.
The student will also be able to observe and gain knowledge of:
1) In vivo surgical techniques for modelling spinal cord injury.
2) Principles of laboratory animal management, welfare and post-operative animal care.
3) Aortic perfusion-fixation techniques.
Development of the project into a 3-year PhD
The work conducted in the 3-month project will set the foundations of the 3-year PhD; testing the safety and
efficacy of human OEC transplants for the treatment of spinal nerve root avulsion injuries. The student will use
the protocols developed during the 3-month project to study functional outcomes after human OEC xenografts
into our rodent model of cervical ventral root avulsion. Prior to transplantation, the antigenic characteristics of the
human OEC xenografts will be characterised in detail and screened for tumorigenic cellular impurities. Additional
important questions addressed during this PhD include the effects of immunosuppression and cell dosage, graft
survival, integration, and transplant delivery techniques.
Methods:
OEC transplants: Using previously established protocols human OECs will be cultured from mucosal biopsies
4
donated from patients recruited within the NHNN undergoing endoscopic surgery .
In vivo injury model: Ventral cervical spinal nerve roots of adult Sprague Dawley rats will be surgically avulsed
resulting in the loss of fore-paw function. The nerve roots will be repaired by re-implantation into the ventrolateral
spinal cord. Experimental groups will consist of controls repaired without interventions, those receiving human
OEC therapies and comparison groups receiving fibroblasts or Schwann cell transplants. After a period of
recovery functional outcomes will be assessed using the developed behavioural tests and histological and
immunofluorescent techniques will be employed to assess the extent of axon regeneration into the re-implanted
nerve root; graft survival, integration and migration; and survival of spinal motoneurons.
Other: Human OEC transplants will be characterised by immunofluorescent and confocal techniques.
Tumorigenicity will be tested by in vitro proliferation assays and soft agar colony formation assay.
References:
1) Li Y, Decherchi P, Raisman G. J Neurosci. 2003;23(3):727–31. 2) Ibrahim AG, Kirkwood PA, Raisman G, Li Y. Brain. 2009;132(Pt
5):1268–76. 3) Li Y, Li D, Khaw PT, Raisman G. Neurosci Lett. 2008;440(3):251–4. 4) Kachramanoglou C, Law S, Andrews P, Li D, Choi D.
Neurosurgery. 2013;72(2):170–8; discussion 178–9.
6. BRT/MRC DTA PhD in Clinical Neuroscience
Archy de Berker & Dr. Sven Bestmann
Aims
Stress is a pervasive feature of life for humans and other organisms. We know that one of the functions of stress
is to change the way that organisms learn about the environment (1), but these changes currently lack a precise
computational characterisation. This project aims to use well-tested models of reinforcement learning (2,3) to
describe how learning changes during stress, and relate these changes to the neurobiological impact of stress
upon learning systems (4).
Methods
This project will use physiological measurements (skin conductance, heart rate, breathing) to confirm the
induction of stress via mild electrical shocks to the arm. We will develop a computerized learning task, building
upon tasks that have previously been described as stress-sensitive. Analysis will involve the use reinforcement
learning models to describe which components of learning change during stress.
Three month project
The student will gain experience in the development of a new learning paradigm, and in the coding of that
paradigm using Matlab and Cogent. They will lead recruitment and running of the study, and gain experience in
applying electric shocks and recording physiological data. They will be introduced to the principles and
applications of computational modelling of learning and how these can be used to gain mechanistic insights into
behavioural changes.
How this project might grow into an exciting 3-year PhD
Stress has been found to influence learning and memory in many domains, such as objects, actions, and space.
This project might grow into one identifying the computational commonalities between disparate reports of the
impact of stress upon learning (1). Furthermore, we currently have little insight into how stress interacts with the
brain systems supporting different forms of learning, although there is lots of evidence that chronic stress can
cause atrophy and remodelling of these systems (5). A 3-year PhD could include the use of
magneto/electroencephalography (M/EEG) to characterise the neural processes that relate to behavioural
alterations under stress, and the use of functional Magnetic Resonance Imaging (fMRI) to gain insight into which
brain regions are affected during acute stress. We also have extensive experience in pharmacological
manipulations of learning (6), which could be used to investigate which neurotransmitter systems are responsible
for behavioural changes observed during stress. We are also interested in how stress affects the utilisation of
different learning systems when making decisions, particularly when those systems come into conflict (7).
Bibilography
(1)
(2)
(3)
(4)
(5)
(6)
(7)
Joels et al., Trends Cogn Sci, 2011
Watkins & Dayan, Machine Learning, 1992
Sutton & Barto, Reinforcement Learning: An Introduction, 1998
Schwabe, Hippocampus, 2013
McEwen & Gianaros, Ann N Y Acad. Sci., 2010
Galea et al., Journal of Neuroscience, 2012
Guitart-Masip et al., Trends Cogn Sci, 2014
BRT/MRC DTA PhD in Clinical Neuroscience
Dr Beate Diehl*, Dr Gerold Baier**, Prof Louis Lemieux*
Departments of *Clinical and Experimental Epilepsy and ** Cell and Developmental Biology, UCL
7. Cortical Stimulation to reveal Dynamic Networks in patients with Focal Epilepsy
Introduction:
Epilepsy is the most common serious chronic neurological condition affecting all ages, affecting 450,000 people
in the UK alone. About one third of all people with epilepsy have seizures that do not respond to medical
treatment, leading to cognitive decline, poor quality of life and significantly increased mortality, as well as high
societal costs (1). Epilepsy surgery may be an appropriate option, leading to seizure freedom in up to 80% of
appropriately selected candidates at 1 year post surgery. However, a significant number of patients require
intracranial EEG monitoring to map where seizures start. It remains challenging to establish the extent of
resection to give patients the best chance of seizure freedom. On the intracranial EEG recordings, careful visual
analysis of the seizure is used to guide resection. Interictal (between seizures) spikes are often a good indicator
where seizures may arise from. Recently, the cortical response to single-pulse electrical stimulation has
emerged as a novel marker of epileptogenicity and has also been explored for assessment of connectivity.
Direct electrical cortical stimulation is used in patients with intracranial EEG recordings for two reasons: 1. to map
eloquent cortical function (using 50 Hz stimulation trains lasting up to 5 s); and 2. to delineate epileptogenic tissue
with single-pulse electrical stimulation (SPES). Specifically, SPES allows assessment of connectivity from evoked
responses (cortico-cortical evoked potentials (CCEPs)) (2,3). This is done by averaging over the responses to the
stimulus recorded from all available implanted electrodes. It is a clinical observation that there is variability
between the responses; this has been described (4) but not systematically explored and it is not known what this
variability is due to.
We hypothesize that understanding the shape of the responses and sources of its variability may allow to gain
insights into what renders the epileptogenic network susceptible for epileptiform activity (spikes) to gain strength
and develop into a clinical seizure. To test this we propose to perform a dynamic network analysis of the evoked
responses and to derive model-based networks for normal and abnormal cortical responses. The model network
will be based on coupled neural population (mass) models that capture the dynamics of the background state and
allow for bifurcations into the abnormal seizure rhythm (5).
Aims:
1. To establish the inter-trial variability of single pulse electrical stimulation responses, inside and outside the
seizure onset zone.
2. To investigate the relationship between responses and brain state. In particular, we will investigate the
influence of spiking and, when fortuitously captured, of occurrence of seizures. Further, this will be
characterized inside and outside the seizure onset zone.
3. To characterize the ongoing EEG before and after stimulation (SPES and 50Hz cortical stimulation)
Methods:
All data for the project is already available; furthermore, new SPES and 50 Hz cortical stimulation data will be
obtained in the course of the project as part of clinical care in patients admitted for intracranial EEG
investigations. Over the past 3 years all patients have undergone both high frequency and single pulse
stimulation. EEG segments containing stimulation will be prepared and exported for post-processing and signal
analysis.
Cortical stimulation results for mapping will be reviewed to characterise specific areas of cortex, such as motor
cortex and anterior language areas. The seizure onset zone as identified by clinical analysis will be identified.
We will extract the stimulus-dependent network based on this knowledge and (in addition to absolute amplitudes
and integrals of the response (2)) we will also look at the waveform of the response and compare it to waveforms
obtained in dynamic network models of epileptic rhythms (5).
We will test the hypothesis that the response variability is correlated with (i) the state of slow potential
fluctuations (6) and (ii) the state of modulation of gamma activity (7).
…………….
Three-month sub-project:
This 3-month project will allow the student to be involved in the clinical interpretation and framework of
the intracranial EEG investigations and stimulation on the Sir Jules Thorn telemetry Unit.
In particular, the BRT/MRC student will be involved in generating a pilot dataset:
This 3-month project focuses on exploring one dataset when CCEPS were performed at two timepoints: 1. A
seizure was captured and frequent interictal spikes were present and 2. A second stimulation session was
performed at a time without seizures and fewer spikes.
This dataset will allow exploring responses to single pulse stimulation at rest and the variability of such
responses during this session. It will then explore how responses change in the brain regions in great proximity
to seizure onset during and shortly after a seizure.
How this project might grow into an exciting 3-year PhD:
Over 30 datasets with both single pulse and 50 Hz cortical stimulation are already available for analysis.
This dataset, its analysis and the data-driven model generation will allow to generate hypotheses about specific
stimulation protocols based on the current state of brain activity. Any evidence for state-dependent responses
can be explored in the model network to suggest more specific stimulation protocols. The predictions could then
be tested using real-time monitoring of ongoing dynamics.
……………….
Importance of this study:
This study represents a unique possibility to improve our knowledge of the mechanisms of how seizures start
and spread, and about the dynamic organisation of the human neocortex in general.
……………..
References:
1. Wiebe, S. & Jette, N, Pharmacoresistance and the role of surgery in difficult to treat epilepsy. Nat Rev Neurol 2012; 8: 669-677, doi:
10.1038/nrneurol.2012.181.
2. Keller CJ et al, Corticocortical evoked potential reveal projectors and integrators in human brain networks. J Neurosci 2014; 34: 915263.
3. Matsumoto R et al, Functional connectivity in human cortical motor system: a cortico-cortical evoked potential study. Brain 2007; 130:
181-197.
4. Lesser RP et al, Brain, Short-term variations in response distribution to cortical stimulation. Brain 2008; 131: 1528-39.
5. Goodfellow M et al, Self-organised transients in a neural mass model of epileptogenic tissue dynamics. Neuroimage 2012; 59:
2644-60. Wang Y et al, PLoS Computational Biology 2014, accepted.
6. Wu et al, Role of ictal baseline shifts and ictal high-frequency oscillations in stereo-electroencephalography analysis of mesial
temporal lobe seizures. Epilepsia 2014; 55: 690–698.
7. Alvarado-Rojas C et al, Slow modulations of high-frequency activity (40–140 Hz) discriminate preictal changes in human focal
epilepsy. Scientific Reports 2014; 4: 4545 (1-9).
8. Dr Pietro Fratta, Professor Elizabeth Fisher UCL – Institute of Neurology
PhD project: Investigating the axonal transport and localization of RNA in motor neuron disease models.
The pathogenesis of Motor neuron disease is largely unknown, but two aspects have recently emerged as
important players: a) RNA metabolism alterations and b) axonal transport disruptions.
Furthermore, over the last decade, growing evidence has emerged pointing to the importance of RNA
localization in neurons and their transport in axons.
In order to study the role of RNA transport in motor neuron disease animal models, we will exploit a novel
transgenic mouse, which allows to tag in vivo and isolate RNA from specific cells and cellular sub-compartments.
Rotation Project
The “RNA-tagging” mouse model functions using the Cre/Lox system in order to direct the expression of the
“RNA-tagging” transgene to specific cells. When the mouse is treated with a precursor drug, cells expressing the
enzyme will generate tagged-RNA.
1. The first step will therefore be to validate and characterize the correct expression of the “RNAtagging” transgene. This will require immunohistochemistry and molecular biology techniques.
2. Tagged RNA will be isolated from motor neuron cell bodies and their axons, and tested for purity
and specificity.
Training in mouse spinal cord and nerve dissection, RNA extraction, Q-PCR and western blotting and
immunohistochemistry will be provided.
PhD project
Aim one: identifying the transcriptome of the motor neuron cell bodies and MN axons in vivo.
Year 1
Organise breeding of colony and assign cohorts and perform mouse treatments.
RNA pull-down experiments for cell body and axonal RNA.
RNA sequencing of the cell body and axonal RNA.
Year 2-3
RNA sequencing analysis.
RNA sequencing validation (Fluorescent in situ hybridization; immunohistochemistry and
molecular biology techniques).
Aim two: investigate what changes occur in axonal and synaptic RNA in MND mouse models
Year 2-3
Perform axonal and cell body RNA pull-downs and sequencing in ALS mouse models.
Data analysis and identification of specific changes. Biological validation.
Investigation of selected targets in a wider range of motor neuron disease models.
Reference
Ling, Polymenidou, Cleveland. Converging mechanisms in ALs and FTD: disrupted RNA and protein homeostasis. (2013) Neuron 79: 416
BRT/MRC PhD in Clinical Neuroscience
Dr Janice Holton, Prof Tom Warner, Prof Henry Houlden and Dr Abi Li
Queen Square Brain Bank
9. Developing a cell model to study multiple system atrophy
Background
Multiple system atrophy (MSA) is a progressive neurodegenerative disease of adults affecting approximately 4.4
per 100,000 in the UK for which there are no disease modifying therapies. MSA is a member of the group of the
protein folding neurodegenerative disorders and, similar to Parkinson’s disease (PD), neurodegeneration is
accompanied by intracellular accumulation of abnormally folded insoluble α-synuclein. In MSA the pathological
hallmark is the α-synuclein containing oligodendroglial glial cytoplasmic inclusion (GCI) with additional but
sparse α-synuclein containing neuronal cytoplasmic inclusions, neuronal nuclear inclusions, glial nuclear
inclusions and neuropil threads. In PD α-synuclein predominantly aggregates to form Lewy bodies in neurons.
As fibrillar α-synuclein with similar biochemical properties aggregates in cells in both MSA and PD they are
regarded as members of the group of diseases known as α-synucleinopathies. GCIs are believed to be
important in disease pathogenesis as we previously found that they increase in number with disease duration
and also with neuronal loss (1). At present we have only limited insight into the mechanism of GCI formation and
how this leads to neurodegeneration (2). Despite the long held belief that mature oligodendrocytes do not
express α-synuclein our group has recently demonstrated that they do express α-synuclein mRNA and this may
significantly change our view of the disease pathogenesis (3). Mutations in SNCA, the gene encoding αsynuclein are not associated with MSA, however, we have described a rare point mutation in SNCA (G51D) that
leads to pathological features overlapping between PD and MSA including inclusions resembling GCIs. This may
imply that structural changes in α-synuclein influence the distribution and inclusion type formed in MSA and PD
(4).
Suitable cell models of MSA are lacking and the development of such models would enable us to explore the
cellular mechanisms of disease and provide a model to test therapeutic interventions for MSA.
Aims:
To develop cellular models of MSA by culturing fibroblasts derived from fresh dura donated at the time of brain
donation. Fibroblasts will be cultured to provide one potential cell model for MSA. The project will be further
developed to use these fibroblasts to derive induced neuronal progenitor cells (iNPCs) by direct conversion.
Such cells have tri-lineage potential and can be differentiated to form neurons, oligodendrocytes and astrocytes.
Methods:
Specimens of fresh dura obtained at the time of brain donation will be collected from MSA and Parkinson’s
disease cases, the latter will act as a disease control. To confirm the diagnosis and therefore the suitability of
each case, frozen sections will be cut and stained using immunohistochemistry for α-synuclein at the time of
brain dissection. In suitable cases a sample of dura will be collected and fibroblast cultures will be established.
iNPCs will be generated using the method of Kim et al (5). We also have access to skin fibroblasts from patients
with G51D SNCA mutation providing a further genetically determined model for PD and MSA.
Three-month project:
This 3-month project will allow the student to learn a number of laboratory techniques. The most important of
these will be to culture fibroblasts from fresh dura and to develop the technique of producing tripotent iNPCs.
The student will also become familiar with the methods of immunohistochemistry and learn the diagnostic
features of the α-synucleinopathies MSA and PD.
The student will be working in a laboratory that produces induced pluripotent stem cells and will be able to take
the opportunity to learn about this methodology.
As a member of the Queen Square Brain Bank team the student will have the opportunity to attend brain cut
sessions and to learn about the diagnostic features of a number of different neurodegenerative diseases.
How this project might grow into an exciting 3-year PhD:
This 3-month project paves the way to developing cellular models of MSA using human patient-derived cells.
1. The establishment of patient fibroblast cultures from the two major α-synucleinopathies and from a genetically
determined model of PD/MSA will allow the exploration of these as disease models. This model will be explored
by using microarray analysis of RNA to determine which genes are differentially expressed in PD, MSA and
G51D mutation fibroblasts. Genes identifies will be validated by quantitative PCR and the pathways involved will
be identified. Functional assays will be used to determine the effect of altered mRNA levels on the cellular
processes (6).
2. Recent studies have shown that in sporadic motor neuron disease astrocytes are toxic to neurons in culture
(7-9). We will test the hypothesis that oligodendrocytes are toxic to neurons in MSA. The production of tripotent
iNPCs will be followed by differentiating these cells to form neurons and oligodendrocytes. By co-culturing ineurons and i-oligodendrocytes from MSA and G51D mutation patients we will determine whether ioligodendrocytes are toxic to i-neurons. PD i-neurons and i-oligodendrocytes will act as a disease control.
To explore further the origin of oligodendroglial α-synuclein in MSA we will determine whether MSA derived ioligodendrocytes and i-neurons express α-synuclein mRNA and protein. We will investigate whether these cells
secrete α-synuclein into the culture medium and which cell types are able to take up α-synuclein from the culture
medium.
Importance of this study:
This study will provide a novel cell model to study the α-synucleinopathies in particular MSA. Access to the
G51D SNCA mutation fibroblasts is a unique resource that can be used to inform our understanding of both
MSA and PD and this study will provide the opportunity to develop a unique cell model using these fibroblasts.
References:
1.
Ozawa T, Paviour D, Quinn NP, Josephs KA, Sangha H, Kilford L, et al. The spectrum of pathological involvement of the
striatonigral and olivopontocerebellar systems in multiple system atrophy: clinicopathological correlations. Brain : a journal of neurology.
2004;127(Pt 12):2657-71. Epub 2004/10/29.
2.
Ahmed Z, Asi YT, Sailer A, Lees AJ, Houlden H, Revesz T, et al. The neuropathology, pathophysiology and genetics of multiple
system atrophy. Neuropathology and applied neurobiology. 2012;38(1):4-24. Epub 2011/11/15.
3.
Asi YT, Simpson JE, Heath PR, Wharton SB, Lees AJ, Revesz T, et al. Alpha-synuclein mRNA expression in oligodendrocytes in
MSA. Glia. 2014;62(6):964-70. Epub 2014/03/05.
4.
Kiely AP, Asi YT, Kara E, Limousin P, Ling H, Lewis P, et al. alpha-Synucleinopathy associated with G51D SNCA mutation: a link
between Parkinson's disease and multiple system atrophy? Acta neuropathologica. 2013;125(5):753-69. Epub 2013/02/14.
5.
Kim J, Efe JA, Zhu S, Talantova M, Yuan X, Wang S, et al. Direct reprogramming of mouse fibroblasts to neural progenitors.
Proceedings of the National Academy of Sciences of the United States of America. 2011;108(19):7838-43. Epub 2011/04/28.
6.
Raman R, Allen SP, Goodall EF, Kramer S, Ponger LL, Heath PR, et al. Gene expression signatures in motor neuron disease
fibroblasts reveal dysregulation of metabolism, hypoxia-response and RNA processing functions. Neuropathology and applied neurobiology.
2014. Epub 2014/04/23.
7.
Haidet-Phillips AM, Hester ME, Miranda CJ, Meyer K, Braun L, Frakes A, et al. Astrocytes from familial and sporadic ALS patients
are toxic to motor neurons. Nature biotechnology. 2011;29(9):824-8. Epub 2011/08/13.
8.
Meyer K, Ferraiuolo L, Miranda CJ, Likhite S, McElroy S, Renusch S, et al. Direct conversion of patient fibroblasts demonstrates
non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS. Proceedings of the National Academy of Sciences
of the United States of America. 2014;111(2):829-32. Epub 2014/01/01.
9.
Re DB, Le Verche V, Yu C, Amoroso MW, Politi KA, Phani S, et al. Necroptosis drives motor neuron death in models of both
sporadic and familial ALS. Neuron. 2014;81(5):1001-8. Epub 2014/02/11.
10. Cellular basis of frontotemporal dementia and motor neuron disease caused by C9orf72 repeat
expansion
Supervisor
Adrian Isaacs
Background
Frontotemporal dementia (FTD) is the second most common cause of young-onset dementia after Alzheimer’s
disease and it is becoming increasingly recognised that it has overlapping clinical, neuropathological and genetic
features with motor neuron disease (MND). An expanded intronic repeat in the C9orf72 gene was identified as
the most common genetic cause of both FTD and MND. The mechanism that leads to disease is unknown but
may involve toxic expanded repeat RNA and toxic proteins transcribed from the expanded repeats. We have
developed novel methods for dissecting repeat RNA from protein toxicity, which is a fundamental question for
understanding disease pathogenesis and developing therapies.
Aim of 3-month project
We will investigate repeat RNA and protein pathology in C9orf72 iPS cells and novel neuronal cell models and
assess the effect of repeat RNA and protein on neuronal survival. A range of cellular functions will also be
assessed using immunofluorescence and confocal microscopy, immunoblotting and FACs analysis.
Methods
Western blotting, immunofluorescence staining of cell cultures and human brain sections, fluorescence in situ
hybridisation, confocal microscopy (potentially including live cell imaging), primary neuronal culture, , shRNA
knockdowns.
Scope and outlook
The project will develop into a dissection of the molecular mechanism by which the C9orf72 expanded repeat
causes FTD and MND in a range of novel models using state-of-the-art molecular methods and transcriptomics.
It will also include a therapeutics aim as we are developing small molecules which bind the C9orf72 repeats. We
will test the efficacy of these small molecules in the cellular models that will be developed and characterised as
part of this project.
11.Predictive coding in action selection: A high precision MEG study
Dr. James Bonaiuto & Dr. Sven Bestmann
Aims:
The predictive coding model suggests that the brain updates an internal model of the world to infer the probable
causes of sensory events. Feedback projections to deep cortical layers signal predictions via beta oscillations
and feedforward projections to superficial layers encode prediction errors in gamma oscillations. This project
aims to use high precision techniques for MEG to look at how predictions and prediction errors in action
selection differ in their oscillatory signatures and spatial distribution.
Methods:
Subjects will be scanned in a 3 T MRI unit to acquire structural images which will be used to subject-specific
masks using a 3D printer. This will reduce movement in the MEG allowing small enough coregistration error and
large enough signal-to-noise ratio to allow high-precision mapping to anatomy. Subjects will participate in MEG
studies designed to test predictive coding accounts of action selection. The MRI scans will be used to extract
deep and superficial surfaces. Model evidence for generative models based on each surface will then be
compared to determine which layers the recorded band activity originates from.
Three-month project
This 3-month project will allow the student to become familiar with the basics of brain imaging, subject
recruitment, and data analysis. The student will be involved in:
1) Assisting in recruitment, assessment and scanning of subjects at study entry; this will allow the student to
have a direct contact with subjects and obtain experience in running the MRI scanner;
2) Analyzing MEG data to investigate activity in various frequency bands and their spatial distribution. this will
allow the student to gain experience in the analysis of MEG data using advanced computer programmes
and statistical models.
How this project might grow into an exciting 3-year PhD:
This 3-month project focuses on the experimental techniques which can be used to localize electrophysiological
signals recorded with MEG to anatomical structures with high precision. After that, the student may start a 3-year
PhD which will focus on using these techniques to study various neural correlates of action selection which until
now have only been possible using invasive techniques in non-human primates. This has the potential to be a
very exciting PhD allowing action selection to be studied in humans at a temporal and spatial precision
unachievable until now.
BRT/MRC DTA PhD in Clinical Neuroscience
Prof Huw Morris and Prof John Hardy
Departments of Clinical and Molecular Neuroscience, Institute of Neurology
h.morris@ucl.ac.uk
12. Human knock-out approaches to Parkinson’s disease
Aims:
Study of knock-out animal models have been a classical technique for investigating normal gene function.
Advances in genetic technology means that it is now possible to study loss of function variation on a genome
wide basis in humans. We are investigating genetic variation in early onset Parkinson’s disease (EOPD) and are
studying whole exome data from ~300 individuals, in order to determine new Mendelian genes which cause PD.
In this study the student will identify bi-allelic loss of function (LOF) variation in individuals with EOPD and
determine whether these human “knock-outs” are likely to be relevant to PD. This project will provide training
and experience in current techniques in neurogenetics, with the potential to identify new relevant genes for PD.
The ultimate aim of this work is to develop new tests and rational therapies for these patients.
Methods and Three-Month Project
1) Annotation of whole exome data to identify new bi-allelic (homozygous or compound heterozygous) loss of
function variants (i.e. stop codon, splice site or frame-shift mutations) in this dataset (bio-informatics)
2) Validation of these variants by conventional Sanger sequencing (wet lab work)
3) For validated LOF genes determination of whether these are likely to be relevant to PD based on expression
in relevant tissues, and occurrence of LOF alterations in control subjects unaffected by neurological disease
(bio-informatics and literature review)
4) If time permits, analysis of segregation of these genetic variants within families to provide further information
as to whether they are relevant to PD.
How this project might grow into an exciting 3-year PhD:
Any “hits” will need further investigation:
1) Further analysis of the importance of variation in candidate genes in GWAS, EXOME and new
validation/replication samples
2) Determination of loss/disruption of mRNA/protein with studies of lymphoblastoid cell lines and human
fibroblast cell lines
3) Investigation of the consequences of loss of protein in model systems including fibroblasts, induced
pluripotent cell lines
Importance of this study:
This study has the potential to generate new insights into the pathogenesis of Early Onset PD and the
relationship between known and novel PD genes.
References:
1.
Kilarski LL, Pearson JP, Newsway V, Majounie E, Knipe MDW, Misbahuddin A, Chinnery PF, Burn DJ, Clarke CE, Marion M-H,
Lewthwaite AJ, Nicholl DJ, Wood NW, Morrison KE, Williams-Gray CH, Evans JR, Sawcer SJ, Barker R a, Wickremaratchi MM,
Ben-Shlomo Y, Williams NM, Morris HR. Systematic review and UK-based study of PARK2 (parkin), PINK1, PARK7 (DJ-1) and
LRRK2 in early-onset Parkinson’s disease. Mov Disord. 2012 Oct 6;27(12):1522–9.
2.
Simón-Sánchez J, Kilarski LL, Nalls MA, Martinez M, Schulte C, Holmans P, Gasser T, Hardy J, Singleton AB, Wood NW, Brice A,
Heutink P, Williams N, Morris HR. Cooperative Genome-Wide Analysis Shows Increased Homozygosity in Early Onset Parkinson’s
Disease. Lewin A, editor. PLoS One. 2012 Mar 12;7(3):e28787.
3.
Alsalem AB, Halees AS, Anazi S, Alshamekh S. Autozygome Sequencing Expands the Horizon of Human Knockout Research and
Provides Novel Insights into Human Phenotypic Variation. 2013;9(12).
BRT/MRC DTA PhD in Clinical Neuroscience
ProfsHuw Morris, Dr Jan-Willem Taanman and Prof Tony Schapira, Department of Clinical Neuroscience, UCL
Institute of Neurology
h.morris@ucl.ac.uk
13. Understanding early onset Parkinson’s disease through cell models
Aims:
Parkinson’s disease (PD) is an age related condition, but around 1% of PD patients develop disease before the
age of 40 (Early onset PD – EOPD). In about 10% of these patients we know that there are bi-allelic loss of
function mutations in genes such as parkin and PINK1. We know that these genes are crucially important for
mitochondrial quality control via mitophagy, and that cell and animal models derived from patients with these
diseases show major abnormalities of mitochondrial structure and function. We are engaged in studying
patients with EOPD to identify new pathogenic genes and tp understand disease mechanisms. We have
collected the largest cohort of early onset PD patients in the UK, with > 300 patient samples. In collaboration
with a global consortium, we have access to samples and data related to > 1400 early onset PD cases.
Following exclusion of known genes, our genetic (homozygosity mapping) and clinical (segregation analysis)
work suggests that these patients have an increased rate of autosomal recessive PD, relating to new PD loci.
We are currently engaged in whole exome analysis in an attempt to define new genes. What is currently
unknown is whether the abnormalities of mitochondrial structure and function seen in parkin/PINK1 disease are
also relevant to other patients with EOPD, and whether this will help direct rational therapies. Our aim is to
define the pathogenic mechanisms relating to disease in patients with early onset PD. We are also engaged
with industry in trying to develop new treatment approaches based on these mechanisms.
Methods and Three-Month Project
1) Culture of fibroblasts from EOPD patients with and without mutations in parkin/PINK1 and unaffected controls;
2) Measurement of key markers of mitochondrial structure and function in these patient derived cell lines
including: mitochondrial membrane potential, mitochondrial morphology and ATP production; 3) If time permits,
biochemical characterisation of key proteins in mitophagy in patients with and without mutations in known genes
How this project might grow into an exciting 3-year PhD:
There are numerous potential future research areas:
1) Further investigation of the biochemistry of this process as applied to PD
2) Development of high throughput methods that would allow large-scale biochemical characterisation of PD
patients on the basis of mitochondrial function
3) Characterisation of new candidate genes identified in our accompanying genetic work
4) Generation of neuronal and glial cells using induced pluripotent stem cell technology to further explore these
models
5) Integration of these data with other clinical and biomarker data derived from these patients, to further refine
patient stratification in life
Importance of this study:
The failure of disease modifying treatment trials in PD to date may relate to a lack of stratification of PD patients
based on disease mechanisms.
In the future measures of target engagement and treatment response may relate to the measurement of markers
of mitochondrial function in PD, as identified in this work.
References: 1. Gegg ME, Cooper JM, Chau K-Y, Rojo M, Schapira AH V, Taanman J-W. Mitofusin 1 and mitofusin 2 are ubiquitinated in a
PINK1/parkin-dependent manner upon induction of mitophagy. Hum Mol Genet. 2010 Dec 15;19(24):4861–70. 2. Schapira AH.
Mitochondria in the aetiology and pathogenesis of Parkinson’s disease. Lancet Neurol. 2008 Jan;7(1):97–109. 3. Kilarski LL, Pearson JP,
Newsway V, Majounie E, Knipe MDW, Misbahuddin A, Chinnery PF, Burn DJ, Clarke CE, Marion M-H, Lewthwaite AJ, Nicholl DJ, Wood
NW, Morrison KE, Williams-Gray CH, Evans JR, Sawcer SJ, Barker R a, Wickremaratchi MM, Ben-Shlomo Y, Williams NM, Morris HR.
Systematic review and UK-based study of PARK2 (parkin), PINK1, PARK7 (DJ-1) and LRRK2 in early-onset Parkinson’s disease. Mov
Disord. 2012 Oct 6;27(12):1522–9. 4. Wickremaratchi MM, Knipe MDW, Sastry BSD, Morgan E, Jones A, Salmon R, Weiser R, Moran M,
Davies D, Ebenezer L, Raha S, Robertson NP, Butler CC, Ben-Shlomo Y, Morris HR. The motor phenotype of Parkinson’s disease in
relation to age at Onset. Mov Disord. 2011 Jan;26(3):457–63.
14. Neuronal vulnerability in frontotemporal dementia
Supervisors
Dr Tammaryn Lashley, Dr Selina Wray and Prof John Hardy
Queen Square Brain Bank for neurological diseases
Department of Molecular Neuroscience
Institute of Neurology
Queen Square
T.Lashley@ucl.ac.uk
Background
The von economo neurons (VENs) are bipolar layer V projection neurons that can be found in the anterior
cingulate (ACC) and frontoinsular cortices (FI) (Mesulam MM et al 1982). These neurons have been linked to
frontotemporal dementia, autism and schizophrenia, however little is known about these neurons and their role is
neurodegenerative disease. The VENs form a discrete neuronal network involving the ACC and FI, areas
involved in social-emotional processing, that are affected in behavioural variant frontotemporal dementia
(bvFTD). It has been shown in a small cohort of bvFTD cases there is a 74% reduction in VENs (Seeley WW et
al 2006). These neurons are only found in socially complex mammals and have been shown to represent an
early target in bvFTD (Seeley WW et al 2008). However, little information is known about this specific group of
neurons in the large heterogenous group of frontotemporal dementia’s and why they may be vulnerable to the
pathological processes.
Aims and objectives for 3 month rotation
Utilising our large cohort of frontotemporal dementia cases we wish to identify the VENs through their unique
bipolar morphology and to identify them using in-situ hybridistation methods using recently identified
transcription factors (Cobos I et al 2013).
Aims and objectives for PhD project
Frontotemporal lobar degeneration’s are a large group of heterogeneous neurodegenerative diseases, affected
by three main pathological proteins tau, TDP-43 and FUS. During this project we will assess VENs vulnerability
in all the FTLD subgroups using both morphology and in-situ hybridisation to identify the neurons. Once
identified we will be able to isolate the remaining VENs using laser capture microscopy and detect any proteomic
or expression changes compared to other neuronal populations that render them vulnerable to disease
pathogenesis. The use of pathological tissue will be complimented by differentiating induced pluripotent stem
cells (iPSC) from control and FTD patients into cortical neurons, and using the molecular signatures identified in
tissue to identify VEN precursor cells within our cell cultures.
References
1. Seeley, W.W. Selective functional, regional, and neuronal vulnerability in frontotemporal dementia. Current opinion in neurology
21, 701-7 (2008).
2. Mesulam, M.M. & Mufson, E.J. Insula of the old world monkey. III: Efferent cortical output and comments on function. The Journal
of comparative neurology 212, 38-52 (1982).
3. Seeley, W.W. et al. Early frontotemporal dementia targets neurons unique to apes and humans. Annals of neurology 60, 660-7
(2006).
4. Cobos I et al Human von Economo neurons express transcription factors associated with layer V subcerebral projection neurons.
Cerebral cortex Aug 19 2013.
15.Aggressive Parkinsonisan disorders; Next Generation Gene Discovery.
Supervisors; Henry Houlden and John Hardy
(a) Abstract
Multiple system atrophy (MSA) is a progressive neurodegenerative disorder that manifests in the early sixth decade of
life with parkinsonism, autonomic problems and cerebellar ataxia, the disorder progresses relentlessly with a mean
survival of 6 to 9 years. There is significant clinical and pathological overlap with PSP but the age of onset is diffcient
and there is frequenct Tau deposition in the brain in PSP. MSA and PSP are aggressive forms of Parkinson’s disease
(PD) and they share alpha-synuclein aggregation into inclusion bodies in the brain or Tau protein deposition as their key
pathogenic event (Kara E et al, Curr Opin Neurol. 2013 Aug;26(4):381-94).
In many neurodegenerative disorders such as Alzheimer’s disease and familial and sporadic Parkinson’s disease some
of the greatest advances have come from the discovery of disease genes and genetic risk factors. The insidious clinical
progression of MSA and PSP is similar to other neurodegenerative disorders and there are strong indicators of a genetic
component with the existence of rare small families, siblings and twins with MSA. Like sporadic PD, MSA and PSP are
likely to be made up of a number of risk factor genes that contribute to the development of the disease but reveal a great
deal about the pathogenesis and treatment possibilities (Höglinger GU et al Nat Genet. 2011 Jun 19;43(7):699-705).
(b) Planned work.
We have recently collected MSA brain tissue (370 cases (UK145)) and patient blood samples (640 cases (UK230)) as
the investigating group in an international MSA collaboration. As part of an ongoing PSP collaboration we have over 250
PSP brains and over 500 clinical samples fully characterised. DNA has been extracted in all cases and a genome wide
association study (GWAS) carried out using Illumina 610quad and OmniExpress SNP arrays. We intend to use next
generation sequencing to investigate the implicated genetic regions from the GWAS and to sequence the entire exome
in MSA and PSP cases. We have recently received funding from the Wellcome Trust to purchase an Illumina next
generation sequencer, this core UCL resource will be ideal for this type of project.
Specific aims:
1. Using next generation sequencing we will exome sequence MSA DNA from 300 neuropathologically confirmed
cases and 200 PSP cases. We will analyse this data for risk associated genetic variants and use the 1000
genomes project as control data.
2. In brain tissue from the Queen square brain bank we will analyse the mRNA expression and protein deposition
of the implicated genes in MSA and PSP brain tissue using RT-PCR and immunhistochemistry in comparison to
other parkinsonian disorders.
3. In the MSA and PSP disease genes identified we will characterise them using a combination of overexpression,
knockdown and the generation of iPS cells.
State-of-the-art techniques
This project uses a number of state of the art techniques to investigate the pathogenesis of patients with the
neurodegenerative disorders MSA and PSP. The initial part of the project uses next generation sequencing with
subsequent work on mRNA and then tissue culture characterisation and the development of iPS cells. There will be the
option to attend MSA and neurogenetics clinics for all students. Clinically qualified students will be able to see patients
and be trained in neurogenetics and movement disorders.
Email (h.houlden@ucl.ac.uk) for project details.
16.Genetic and functional characterisation of paroxysmal neurological disorders
Supervisors; Henry Houlden and James Jepson
Ataxia, epilepsy, migraine and related paroxysmal neurological disorders affect over 15% of the population, and account
for an enormous burden to the individual and to society and exhibit strong (~70%) heritability. Understanding the
mechanisms will be an essential step not only towards improved diagnosis, but also towards the development of rational
therapies. A further clue that many forms of epilepsy, migraine, cluster headache and related paroxysmal movement
disorders are synaptopathies comes from the finding that experimental manipulation of neuronal excitability results in
homeostatic compensation of synaptic strength.
This project primarily focusses on identifying disease genes and genetic risk factors in people affected by epilepsy,
migraine and paroxysmal movement disorders and modelling in flies. Specifically this will be by investigating three main
clinical cohorts;
DNA from approximately 100 small families and cases of early onset paroxysmal kinesigenic dyskinesia in whom coding
and exon-intron boundary mutations in the known genes have already been excluded.
Over 800 cases with episodic ataxia, with the known genes excluded. In many instances several affected individuals have
been sampled within families.
Over 1,000 cases of FHM, cluster headache and trigeminal autonomic cephalgias, already screened for mutations in
known FHM genes.
The genetic analysis will focus on exome sequencing in trios to identify genetic defects and to allow;
(i) Prioritisation of genes for further study influenced by a detailed and quantitative understanding of synapse function.
(ii) Systematic exploration of alternative amino acid substitutions, guided by structure-function insights.
(iii) Studying the interaction among several genes, to identify potential genetic modifiers of severity and to test
hypotheses of oligogenic inheritance.
(iv) Use of Drosophila for rapid generation of mutant lines. (The biophysics and molecular components of fast synaptic
transmission are highly conserved, and the reduced redundancy of synaptic genes in Drosophila may offer a greater
sensitivity to the effects of given mutations).
The laboratory environment with be enriched with groups able to take the functional proof of these genetic defects further
with, (i) biophysics of presynaptic vesicle trafficking and exocytosis (James E Rothman) (ii) the interaction of postsynaptic
receptors, ion channels and peri-synaptic neurotransmitter transporters (Rothman/Kullman), (iii) mRNA expression
analysis (Hardy/Houlden), and (iii) vertebrate, invertebrate and human-derived cellular models of synaptic disorders
(Jepson/Kullmann). Together with the collaborators we are optimally positioned to make breakthroughs in disease
causation and apply them to identify therapeutic targets.
Email (h.houlden@ucl.ac.uk) for project details.
BRT/MRC DTA PhD in Clinical Neuroscience
c
17. Investigating the cellular interaction of PrP at regions critical for prion propagation in prion-infected
and non-infected cells
Prof Parmjit Jat and Dr Joo-Hee Waelzlein
Aims:
Prion diseases like Creutzfeld-Jakob disease (CJD) are fatal progressive neurodegenerative maladies that, like
Alzheimer’s and Huntington’s disease, involve accumulation of aberrantly misfolded protein, a hallmark of
Sc
proteinopathies. They are unique in that the misfolded protein, PrP , shares 100% identity with the native
C
cellular form, PrP , in primary but not secondary structure. The cellular pathways involved in prion propagation
c
are unknown. However, PrP expression is crucial for prion propagation and therefore for development of prion
c
disease. We have identified three regions of PrP within the unstructured amino terminus, which are critical for
c
prion propagation. One of these regions corresponds to the high affinity interaction site between PrP and
1
synthetic A 
-oligomers that mimic some toxic properties of AD brain extracts.
We aim to identify the proteins, which bind to these three regions to elucidate the mechanism and signalling
pathways involved in prion propagation and determine their relevance to other protein misfolding disorders.
Methods
c
It has been shown that expression of PrP tagged with the human myc epitope (EQKLISEEDL) at aa230 in
c
0/0
transgenic mice can restore susceptibility to prions in mice devoid of PrP expression (Prnp ) and be converted
Sc
2
to PrP , the pathogenic form . Therefore, mouse (mo)PrP with a human myc tag or a STrEP tag at aa230 will
be constructed and used to stably reconstitute PK1 knockdown cells, a derivative of PK1 cells in which
C
endogenous PrP has been stably silenced. These cells are refractory to prion infection but regain full
susceptibility upon reconstitution with moPrP. Stably reconstituted cells will be challenged with prion-infected
3,4
brain homogenates. If both cell types can propagate prions, the STrEP tag (WSHPQFEK) will be used since it
can be used to purify associated proteins in a single step under physiological conditions, thereby preserving
native complexes.
Further mutations of the tagged moPrP will be prepared in order to create alanine replacements corresponding
to the three critical regions and PK1 knockdown cells will be reconstituted with these mutants. Proteins, which
c
co-purify with PrP will be identified by MALDI-MS mass fingerprinting and LC-MS/MS. This will allow
identification of proteins that bind differentially to the wild type protein compared to the mutants. In addition, it will
be investigated whether the identified interacting proteins are altered upon prion infection and whether they also
c
5,6
bind to PrP in CAD5 and LD9, cell lines that can propagate different prion strains .
Three-month project
This 3-month project will allow the student to become familiar with the basics of cell culture and
molecular biology and also to gain insight into the biology of prions and prion disease.
In particular the student will be involved in:
(1) Making the moPrP-human myc/STREP tag constructs; this will allow the student to experience
molecular cloning and the basics of protein tagging;
(2) Stably transduce this construct into PK1 knockdown cells using retrovirus transduction; this will allow
the student to understand and experience the basics of retrovirus-mediated cell transduction;
(3) Cell culture of PK1-KD, CAD5 and LD9 cells and scrapie cell assay (SCA) to measure prion
propagation; this will allow the student to understand culture requirements and different growth
properties of various cell lines, and to gain experience in sterile cell culture as well as the SCA.
How this project might grow into an exciting 3-year PhD:
This 3-month project will focus on making the moPrP-myc/STrEP tagged constructs and transducing them into
PK1-KD cells. These cells will then be challenged with mouse prions to determine if the moPrP-myc/STREP
Sc
tagged constructs can support prion propagation and be converted into PrP . The student will also undertake
experiments to determine if any cellular proteins co-purify with the tagged mo-PrPc.
During the 3-year PhD, the aim will be to purify and identify the interacting proteins by mass spectrometry. The
student will then undertake functional studies on the identified interacting proteins to determine if these
interactions are required for highly efficient prion infection/propagation in cells. He/she will also generate mice
null for these interacting proteins (which will be done by commercial outsourcing) and challenge them with RML
to determine whether these interactions are required for in vivo prion propagation.
Importance of this study:
c
This study represents a unique possibility to identify novel binding partners to regions of PrP required for prion
propagation, essential for elucidating the underlying mechanism of prion propagation and determine its
relevance to other protein misfolding disorders.
References:
1. Lauren J et al. Nature 2009; 457: 1128.
2. Rutishauser D et al. PLoS ONE 2009; 4: e4446
3. Zafar S et al. J Proteome Res 2011; 10: 3123
4. Schmidt TG et al. Nat Protoc 2007; 2: 1528
5. Qi Y et al. J Neurosci 1997; 17: 1217
6. Mahal SP et al. Proc Natl Acad Sci USA 2007; 104: 20908
BRT/MRC DTA PhD in Clinical Neuroscience
c
18. Investigating the cellular interaction of PrP at regions critical for prion propagation in prion-infected
and non-infected cells
Prof Parmjit Jat and Dr Joo-Hee Waelzlein
Aims:
Prion diseases like Creutzfeld-Jakob disease (CJD) are fatal progressive neurodegenerative maladies that, like
Alzheimer’s and Huntington’s disease, involve accumulation of aberrantly misfolded protein, a hallmark of
Sc
proteinopathies. They are unique in that the misfolded protein, PrP , shares 100% identity with the native
C
cellular form, PrP , in primary but not secondary structure. The cellular pathways involved in prion propagation
c
are unknown. However, PrP expression is crucial for prion propagation and therefore for development of prion
c
disease. We have identified three regions of PrP within the unstructured amino terminus, which are critical for
c
prion propagation. One of these regions corresponds to the high affinity interaction site between PrP and
1
synthetic A 
-oligomers that mimic some toxic properties of AD brain extracts.
We aim to identify the proteins, which bind to these three regions to elucidate the mechanism and signalling
pathways involved in prion propagation and determine their relevance to other protein misfolding disorders.
Methods
c
It has been shown that expression of PrP tagged with the human myc epitope (EQKLISEEDL) at aa230 in
c
0/0
transgenic mice can restore susceptibility to prions in mice devoid of PrP expression (Prnp ) and be converted
Sc
2
to PrP , the pathogenic form . Therefore, mouse (mo)PrP with a human myc tag or a STrEP tag at aa230 will
be constructed and used to stably reconstitute PK1 knockdown cells, a derivative of PK1 cells in which
C
endogenous PrP has been stably silenced. These cells are refractory to prion infection but regain full
susceptibility upon reconstitution with moPrP. Stably reconstituted cells will be challenged with prion-infected
3,4
brain homogenates. If both cell types can propagate prions, the STrEP tag (WSHPQFEK) will be used since it
can be used to purify associated proteins in a single step under physiological conditions, thereby preserving
native complexes.
Further mutations of the tagged moPrP will be prepared in order to create alanine replacements corresponding
to the three critical regions and PK1 knockdown cells will be reconstituted with these mutants. Proteins, which
c
co-purify with PrP will be identified by MALDI-MS mass fingerprinting and LC-MS/MS. This will allow
identification of proteins that bind differentially to the wild type protein compared to the mutants. In addition, it will
be investigated whether the identified interacting proteins are altered upon prion infection and whether they also
c
5,6
bind to PrP in CAD5 and LD9, cell lines that can propagate different prion strains .
Three-month project
This 3-month project will allow the student to become familiar with the basics of cell culture and
molecular biology and also to gain insight into the biology of prions and prion disease.
In particular the student will be involved in:
(4) Making the moPrP-human myc/STREP tag constructs; this will allow the student to experience
molecular cloning and the basics of protein tagging;
(5) Stably transduce this construct into PK1 knockdown cells using retrovirus transduction; this will allow
the student to understand and experience the basics of retrovirus-mediated cell transduction;
(6) Cell culture of PK1-KD, CAD5 and LD9 cells and scrapie cell assay (SCA) to measure prion
propagation; this will allow the student to understand culture requirements and different growth
properties of various cell lines, and to gain experience in sterile cell culture as well as the SCA.
How this project might grow into an exciting 3-year PhD:
This 3-month project will focus on making the moPrP-myc/STrEP tagged constructs and transducing them into
PK1-KD cells. These cells will then be challenged with mouse prions to determine if the moPrP-myc/STREP
Sc
tagged constructs can support prion propagation and be converted into PrP . The student will also undertake
experiments to determine if any cellular proteins co-purify with the tagged mo-PrPc.
During the 3-year PhD, the aim will be to purify and identify the interacting proteins by mass spectrometry. The
student will then undertake functional studies on the identified interacting proteins to determine if these
interactions are required for highly efficient prion infection/propagation in cells. He/she will also generate mice
null for these interacting proteins (which will be done by commercial outsourcing) and challenge them with RML
to determine whether these interactions are required for in vivo prion propagation.
Importance of this study:
c
This study represents a unique possibility to identify novel binding partners to regions of PrP required for prion
propagation, essential for elucidating the underlying mechanism of prion propagation and determine its
relevance to other protein misfolding disorders.
References:
7. Lauren J et al. Nature 2009; 457: 1128.
8. Rutishauser D et al. PLoS ONE 2009; 4: e4446
9. Zafar S et al. J Proteome Res 2011; 10: 3123
10. Schmidt TG et al. Nat Protoc 2007; 2: 1528
11. Qi Y et al. J Neurosci 1997; 17: 1217
12. Mahal SP et al. Proc Natl Acad Sci USA 2007; 104: 20908
4 Yr PhD in Clinical Neuroscience:
Dr Christos Proukakis
19. Confirmation of somatic copy number alterations (CNAs) in DNA from Parkinson’s disease (PD) and
control brain
Aim:
To confirm and fully characterize CNAs detected in brain regions by array CGH
Background:
The traditional view of the genome as invariant in each individual (except in the case of cancer) is rapidly being
eroded. Post-zygotic (somatic) mutations give rise to mosaicism (the presence of cells with different genetic
composition in an organism). Landmark recent papers have discovered single-neuron CNAs, although their
prevalence in different brain regions and role in disease remain unclear. In sporadic diseases like PD, somatic
CNAs could play a role, and might be detectable only in brain-derived DNA. We have obtained data using a
customized aCGH approach from up to 3 regions of PD and control brains. Although some findings have been
validated by digital PCR, additional work is needed to refine them, and confirm others.
Methods for the 3-month project:
Selected CNAs will be analysed. Quantitative PCR will be used to confirm gains and losses. PCR and cloning /
sequencing approaches will be used to further confirm events and define breakpoints.
Scope for PhD:
The investigation of mosaicism in the healthy and diseased human brain is in its infancy. Improving techniques
including microarrays, next-generation sequencing, finer-resolution fluorescent in situ hybridisation and digital
PCR are going to provide increasing information. This pilot project is expected to develop in several ways.
Additional brain regions from cases studied, and additional PD cases, can be analysed. To verify that detected
CNAs arose very early in development, young control and foetal brains will be studied. DNA from differentiating
mesenchymal stem cells will allow determination of when CNAs arise. The student will obtain extensive
experience in a range of cutting-edge techniques, and could make a significant contribution to this expanding
field.
References
1. McConnell et al http://www.ncbi.nlm.nih.gov/pubmed/24179226
2. Gole et al http://www.ncbi.nlm.nih.gov/pubmed/24213699
3. Frank http://www.ncbi.nlm.nih.gov/pubmed/24937287
4. Proukakis et al http://www.ncbi.nlm.nih.gov/pubmed/23674490
20. The identification of genetic modifiers of Huntington’s Disease
Start dates:
3 month project – November 2014, February or May 2015
PhD – September 2015
Supervisors: Prof Sarah Tabrizi, Dr Vincent Plagnol, Prof Simon Mead
Collaborators: Prof Lesley Jones, Prof Peter Holmans, Prof Christian Neri, Prof Douglas Langbehn
Background:
Huntington’s disease (HD) is a devastating and fatal autosomal dominant neurodegenerative condition; the
1, 2
onset of and progression of the disease are variable . HD is caused by a CAG repeat (polyglutamine)
expansion in the 5’ region of HTT on chromosome 4. The gene and its mutation was discovered in 1993 but
despite 20 years of intensive research there is no disease modifying therapy available to patients, we are only
just beginning to understand the downstream pathogenesis caused by the mutation well enough to predict the
best therapeutic targets. While 50-70% of the variability in onset is accounted for by age and CAG repeat length
3, 4
we know that much of the remaining variability is genetically determined . It is the aim of this project to look for
these genetic modifiers. If we identify genes, and the pathways in which they occur, that alter age of onset and
progression in HD then these are primary targets for therapy in this disease as they are factors already proven to
modulate disease progression in man. The genetics can deliver targets known to enhance or delay age of onset
of disease in people, and delaying age of onset is the goal of therapeutic approaches in this disease.
Methods:
This is a clinical genetic project using cutting edge statistical, genetic and bioinformatic techniques, and has the
potential for wet laboratory work to study and validate findings.
In this project the student will be using data collected in two important Huntington’s disease clinical studies,
which the student will have the opportunity to familiarise themselves with:
1. Track-HD, a multicentre prospective observational biomarker study collecting deep phenotypic data
5-8
(imaging, quantitative motor, cognitive, clinical) for 360 subjects annually over 4 years
2. EHDN Registry, a pan-European study with less phenotypic detail than Track-HD, but has the benefit of
9
over 10,000 subjects
The genetic architecture of HD disease modifiers remains unclear so a range of genetic and transcriptomic
techniques are being employed in this project including following up on the results from a recent GWAS study,
whole exome sequencing, transcriptome sequencing (RNAseq) and whole genome sequencing.
Three month project:
This 3-month project will introduce the BRT/MRC student into both the phenotypic and genetic data being used
to examine the influence of non-CAG genetic factors on HD onset and progression, and become familiar with the
complexities of the application of next generation sequencing technologies to probe a clinically relevant
question.
Specifically the student will:
1) Attend study visits for TrackOn-HD and Enroll-HD (a follow-up to Registry http://www.enroll-hd.org), in
order to gain first-hand experience of multi-site clinical research
2) Depending on the timing of the placement, either be involved in the analysis of RNAseq data of subjects
from the Track-HD study, looking for transcripts which are differentially expressed between fast, slow
10
and normally progressing subjects
3) Or, conduct a hypothesis driven analysis of gene hits and single nucleotide polymorphisms (SNPs)
identified as being of interest in a recent HD genome-wide association study (GWAS) to see if they are
11
also significantly associated with onset in the Track-HD cohort
How this project might grow into an interesting and rewarding 3-year PhD:
During a PhD the BRT/MRC student will complete the transcriptome / SNP analysis started during the 3-month
project, and move on to integrating both these datasets with data from whole exome and whole genome
12
sequencing to examine genes, pathways, networks involved in HD pathogenesis and progression . The
student will have the opportunity to work with collaborators at the Institute of Neurology to generate induced
pluripotent stem cells from the some of the same subjects who have been sequenced in the genetic studies. If
a particular gene of interest is identified the student will sequence this gene to validate the finding, and carry out
functional studies in the lab to explore the role of the gene both in vitro, and in ex-vivo patient cultures. Using
data from Track-HD and Registry the student may also want to explore whether there are distinct phenotypic
clusters of HD which associate with different genetic variants.
This BRC/MRC project offers the student the experience of cutting edge clinical research from the individual
patient, through genomics, to the identification of therapeutic targets in a research group with a proven track
record of translational projects, and a set of leading international collaborators.
Selected references
1.
Rosas HD, Reuter M, Doros G, et al. A tale of two factors: what determines the rate of progression in Huntington's disease? A
longitudinal MRI study. Movement disorders : official journal of the Movement Disorder Society 2011;26:1691-1697.
2.
Gayan J, Brocklebank D, Andresen JM, et al. Genomewide linkage scan reveals novel loci modifying age of onset of Huntington's
disease in the Venezuelan HD kindreds. Genetic epidemiology 2008;32:445-453.
3.
Langbehn DR, Brinkman RR, Falush D, Paulsen JS, Hayden MR. A new model for prediction of the age of onset and penetrance
for Huntington's disease based on CAG length. Clinical genetics 2004;65:267-277.
4.
Wexler NS, Lorimer J, Porter J, et al. Venezuelan kindreds reveal that genetic and environmental factors modulate Huntington's
disease age of onset. Proceedings of the National Academy of Sciences of the United States of America 2004;101:3498-3503.
5.
Tabrizi SJ, Langbehn DR, Leavitt BR, et al. Biological and clinical manifestations of Huntington's disease in the longitudinal
TRACK-HD study: cross-sectional analysis of baseline data. Lancet neurology 2009;8:791-801.
6.
Tabrizi SJ, Scahill RI, Durr A, et al. Biological and clinical changes in premanifest and early stage Huntington's disease in the
TRACK-HD study: the 12-month longitudinal analysis. Lancet neurology 2011;10:31-42.
7.
Tabrizi SJ, Reilmann R, Roos RA, et al. Potential endpoints for clinical trials in premanifest and early Huntington's disease in the
TRACK-HD study: analysis of 24 month observational data. Lancet neurology 2012;11:42-53.
8.
Tabrizi SJ, Scahill RI, Owen G, et al. Predictors of phenotypic progression and disease onset in premanifest and early-stage
Huntington's disease in the TRACK-HD study: analysis of 36-month observational data. The Lancet Neurology 2013.
9.
Orth M, Handley OJ, Schwenke C, et al. Observing Huntington's Disease: the European Huntington's Disease Network's
REGISTRY. PLoS currents 2010;2:RRN1184.
10.
Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nature reviews Genetics 2009;10:57-63.
11.
Gandhi S, Wood NW. Genome-wide association studies: the key to unlocking neurodegeneration? Nature neuroscience
2010;13:789-794.
12.
Majewski J, Pastinen T. The study of eQTL variations by RNA-seq: from SNPs to phenotypes. Trends in genetics : TIG
2011;27:72-79.
BRT/MRC DTA PhD in Clinical Neuroscience
Supervisors – Primary - Prof Sarah J Tabrizi
Secondary – Dr Ralph Andre
Collaborators - , Prof Meng Li (Cardiff), Prof Gill Bates (KCL), Prof Tom Warner (UCL)
21. Characterisation of novel genetically related Huntington’s disease patient-derived stem cells.
Aims
Huntington’s disease (HD) is caused by a CAG expansion in the gene encoding the huntingtin (HTT) protein.
Most HD cell models to date are non-human and/or non-neuronal, limiting their use in modelling the human
1,2
They often also rely on
striatal medium spiny neurons (MSNs) that are particularly affected in HD.
overexpression of mutant (m)HTT fragments. Pluripotent stem cells (PSCs) offer new possibilities in generating
3,4,5
Of considerable interest is the association between HTT CAG
disease-relevant human cell populations.
expansion length and disease onset/progression, but genetic heterogeneity can confound group-wise
6
differences in patient-derived PSCs from unrelated individuals. We are addressing this by creating a series of
PSC lines from related HD patients with differing HTT CAG-repeat lengths and unaffected relatives. Fibroblasts
will already have been banked and re-programmed to form induced (i)PSCs. Here, we aim to confirm the
pluripotency of the iPSC lines and initiate differentiation of the cells into MSNs using newly developed protocols.
Methods
Induced PSCs will have been generated from early-passage fibroblasts using integration-free methods.
Sufficient numbers of cells will have been generated for freezing down and banking in liquid nitrogen container.
In this project, undifferentiated iPSCs will be cultured and examined using immunocytochemistry. The cells will
be fixed using 4% paraformaldehyde and primary antibodies to OCT3/4, Nanog, SOX2, TRA-1-81/60 and SSEA7,8
1/3/4 will be used to demonstrate the pluripotent nature of these cells. In addition, RT-PCR will be used to
confirm the expression of pluripotency-associated genes OCT4, Nanog, SOX2, KLF4, Myc, REX1, GDF3 and
3
hTERT.
Following differentiation into embryoid bodies, the potential of cultures to develop into cells from all three germ
9
layers will be assessed using RT-PCR for specific markers of endo-, meso- and ectodermal fate. These
markers comprise GATA4 and AFP for endoderm, RUNX1 and Brachyury for mesoderm, and Nestin and NCAM
3
for ectoderm. In addition, immunofluorescence for GATA4, αSMA and β3-tubulin will be undertaken, again
confirming endo-, meso- and ectodermal lineage respectively.
Finally, the cells will undergo karyotype analysis to ensure chromosomal stability over multiple passages and
8
genomic PCR analysis will be carried out to confirm non-integration of the transforming vectors. CAG repeat
sizing will also be used to confirm the tract length of HTT alleles, which will also ensure that the iPSC lines are
genetically matched to their parental somatic lines.
We will then demonstrate the potential of our iPSCs to differentiate into MSNs using a protocol developed by
Professor Meng Li (Professor of Stem Cell Neurobiology, Cardiff University) in which cultures comprising up to
40% MSNs are differentiated over a six week period (personal communication). This will be undertaken under
the supervision of members of Professor Li’s team. Successful differentiation into MSNs will be demonstrated by
immunocytochemistry staining for neuronal (MAP2), GABA-ergic (GAD65/67) and striatal-specific markers
(DARPP32, CTIP2).
Three-month project
This three-month project will allow the student to familiarise themselves with the latest human stem cell
techniques at the forefront of Huntington’s disease research.
In particular, the student will:
(1) Confirm the pluripotency of the human HD iPSC that have been generated;
(2) Determine the ability of the human HD iPSCs to differentiate into cells of each germ layer;
(3) Establish differentiation of the human HD iPSCs into disease-associated MSNs, in collaboration with
Prof Meng Li at Cardiff University.
How this project might grow into an exciting three year PhD
This three-month project focuses on applying molecular techniques to determine the successful generation of
HD iPSCs and their differentiation into human MSNs. The generation of novel patient-derived stem cell lines will
form the basis of a future three year project concerning mechanisms of pathobiology and the effects of CAG
repeat length on neuronal phenotype. Initially, the cells would be assessed for changes in cell morphology and
viability. Changes in cell morphology throughout the neuronal lifespan would be studied using the highthroughput Cellomics Arrayscan robotic fluorescence microscope, which directly quantifies changes in cell body
morphology, neurite length and branching; cell viability will be assessed using the Cellomics fluorescence based
10
cytotoxity application. This initial phenotyping and characterisation of the cells will form the basis of further
work to dissect the effect of increasing CAG repeat length on the cellular pathogenesis of HD.
Importance of this study
This study represents a unique possibility to further our understanding of the cellular mechanisms of HD, using
novel stem cell based models that can be used to investigate the effects of the disease-causing HTT mutation
on the function of human MSNs.
References (1) Ross CA, Tabrizi SJ. Lancet Neurol. 2011; 10(1):83-98. (2) Cisbani G, Cicchetti F. Cell Death Dis. 2012; 3:e382. (3) Park IH
et al. Cell. 2008;134(5):877-86. (4) An MC et al. Cell Stem Cell. 2012;11(2):253-63. (5) HD iPSC Consortium. Cell Stem Cell.
2012;11(2):264-78. (6) Cahan P, Daley GQ. Nat Rev Mol Cell Biol. 2013;14(6):357-68. (7) Camnasio S, et al. Neurobiol Dis. 2012;46(1):4151. (8) Goh PA et al. PLoS One. 2013; 8(11):e81622. (9) Park IH et al.. Nature. 2008;451(7175):141-6. (10) Wood-Kaczmar A et al. PLoS
One. 2008; 3(6).
22. Adipose Tissue in Huntington’s Disease- dissecting metabolic dysfunction in neurodegenerative
diseases
Primary supervisor: Professor Sarah J Tabrizi
Secondary supervisors: Dr Jennifer Parker, Dr Maria Björkqvist
Background on Huntington’s Disease
Huntington’s disease (HD) is an inherited neurodegenerative disease characterised by the loss of medium spiny
neurons in the striatum. The disease is caused by an expanded CAG repeat sequence within the gene for
huntingtin (HTT). This mutation is translated into an increased polyglutamine repeat length within the HTT
protein which is thought to cause the protein, or certain fragments, to become toxic. HTT is expressed in all
human cells and although its roles have not been clearly defined we know it to be important during development,
particularly of the nervous system (1). HTT also has roles in the functioning of adult cells including in
mitochondrial function, intracellular signalling, regulation of gene expression and in neuronal intracellular
trafficking (2-4). It is possible that the presence of mutant HTT in non-neuronal cells may affect the functioning of
these cells and contribute to the profile of symptoms seen in HD patients. Evidence from studies in immune and
muscle cells suggests this to be the case (5, 6). Thus the benefits of studying HD in non-neuronal cells are
twofold; firstly we can examine the effects of mutant HTT expression in primary human cells, which is not
possible for neurons. Secondly, by learning more about the effects of mutant HTT in peripheral cell types we can
begin to target these cells for therapeutics.
Adipose Tissue in HD
In addition to being a site for triglyceride storage, adipose tissue is an active endocrine organ. Hormones such
as leptin and adiponectin, as well as inflammatory cytokines including interleukin-1β (IL-1 β), IL-6 and tumour
necrosis factor α (TNFα), can be released by adipose tissue (7, 8). Systemic inflammation has been shown to
increase cell death in mouse models of neurodegeneration (9).
Adipose tissue in HD has not been comprehensively studied, but a few observations have been made. In the
R6/2 mouse model of HD the mice are prone to obesity when fed a high fat and sugar diet (10) and even on
normal chow have increased intrabdominal fat mass (11). There is also evidence that there are changes in the
hormones released and genes expressed by fat in R6/2 mice (12). In control human subjects the levels of
adipokines leptin and adiponectin correlate with body mass index (BMI), however in HD gene carriers this
correlation is lost (13) suggesting alterations in human adipose tissue function in HD. There are no published
studies on human adipose tissue itself, but unpublished results from our collaborator Maria Björkqvist suggest
that there are numerous gene expression changes in human HD gene carrier adipose tissue compared with
controls.
There is some evidence for a direct effect of mutant HTT expression in adipocytes. Preadipocytes are the
precursor cells to mature adipocytes. Mutant HTT has been overexpressed in the 3T3-L1 preadipocyte cell line.
When expression of mutant HTT was induced in adipocytes derived from these cells, the expression of some
key adipogenic and mature adipocyte marker genes was reduced, as was the triglyceride storage of these cells
compared to those overexpressing wild type HTT (12). However no studies have been published examining the
effects of mutant HTT in primary human preadipocytes or adipocytes.
Current Work on Adipose Tissue in HD within the Tabrizi Lab
As part of a larger investigation into the effects of mutant HTT expression in peripheral tissues, over the past
year we have collected subcutaneous adipose tissue from 10 control, 10 premanifest HD gene carriers and 10
patients with early stage HD. Our collaborators at the University of Ulm have also collected the same number of
samples. This adipose tissue will be used for RNAseq and proteomics such that we will be able to compare the
gene and protein expression profiles of adipose tissue from premanifest and manifest HD patients with controls
for the first time. Snap frozen adipose tissue from the London participants has been stored for future use, to
follow up on the results of the RNAseq and proteomics. In addition, preadipocyte cultures have been set up from
the majority of the London participants such that the effects of cell intrinsic expression of mutant HTT can be
studied in these cells in vitro. We are also in the process of creating induced pluripotent stem (iPS) cells from HD
patients with varying lengths of CAG repeat. We hope to be able to differentiate these into adipocytes for use in
studies which require greater numbers of cells than we have available as primary preadipocytes.
3 month project outline
The aim of the 3 month project will be to carry out some preliminary experiments on preadipocytes and
adipocytes derived from HD mouse tissue. The student will learn how to obtain and culture these cells as well as
optimising further the differentiation protocol. As the Tabrizi lab has a particular interest in inflammation in HD the
first experiments will centre on measuring the release of inflammatory cytokines from preadipocytes derived from
control and R6/2 mice in response to proinflammatory stimuli. Depending on the results of these preliminary
experiments the student may extend this work into primary human preadipocytes during the 3 month rotation.
The student may also have the opportunity to culture and optimise differentiation protocols for iPS cells.
PhD project outline
The results of the RNAseq and proteomics screens will provide a huge amount of data. The student will be able
to use tissue samples and cells from the same subjects to carry out mechanistic investigations to follow up any
interesting findings from these screens. We anticipate this will involve studying the capacity for differentiation,
release of hormones and cytokines, and response to hormonal and cytokine stimuli in primary HD cells and/or
those derived from HD iPS cells.
Skills and experience gained
During the 3 month rotation: General tissue culture, primary cell culture, iPS cell culture (time allowing),
immunofluorescent staining and confocal microscopy, ELISA or related assays.
During the PhD project (In addition to those above): Western blotting, quantitative PCR, in vitro glucose uptake
assays. Other techniques will depend on the direction in which the student takes their project.
References
1.
Lo Sardo V, Zuccato C, Gaudenzi G, Vitali B, Ramos C, Tartari M, et al. An evolutionary recent neuroepithelial cell adhesion
function of huntingtin implicates ADAM10-Ncadherin. Nat Neurosci. 2012;15(5):713-21.
2.
Ismailoglu I, Chen Q, Popowski M, Yang L, Gross SS, Brivanlou AH. Huntingtin protein is essential for mitochondrial metabolism,
bioenergetics and structure in murine embryonic stem cells. Dev Biol. 2014;391(2):230-40.
3.
Zuccato C, Ciammola A, Rigamonti D, Leavitt BR, Goffredo D, Conti L, et al. Loss of huntingtin-mediated BDNF gene transcription
in Huntington's disease. Science. 2001;293(5529):493-8.
4.
Gauthier LR, Charrin BC, Borrell-Pagès M, Dompierre JP, Rangone H, Cordelières FP, et al. Huntingtin controls neurotrophic
support and survival of neurons by enhancing BDNF vesicular transport along microtubules. Cell. 2004;118(1):127-38.
5.
Träger U, Andre R, Lahiri N, Magnusson-Lind A, Weiss A, Grueninger S, et al. HTT-lowering reverses Huntington's disease
immune dysfunction caused by NFκB pathway dysregulation. Brain. 2014;137(Pt 3):819-33.
6.
Ciammola A, Sassone J, Alberti L, Meola G, Mancinelli E, Russo MA, et al. Increased apoptosis, Huntingtin inclusions and altered
differentiation in muscle cell cultures from Huntington's disease subjects. Cell Death Differ. 2006;13(12):2068-78.
7.
Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked
insulin resistance. Science. 1993;259(5091):87-91.
8.
Mohamed-Ali V, Flower L, Sethi J, Hotamisligil G, Gray R, Humphries SE, et al. beta-adrenergic regulation of IL-6 release from
adipose tissue: In vivo and in vitro studies. Journal of Clinical Endocrinology & Metabolism. 2001;86(12):5864-9.
9.
Cunningham C, Wilcockson DC, Campion S, Lunnon K, Perry VH. Central and systemic endotoxin challenges exacerbate the local
inflammatory response and increase neuronal death during chronic neurodegeneration. J Neurosci. 2005;25(40):9275-84.
10.
Fain JN, Del Mar NA, Meade CA, Reiner A, Goldowitz D. Abnormalities in the functioning of adipocytes from R6/2 mice that are
transgenic for the Huntington's disease mutation. Hum Mol Genet. 2001;10(2):145-52.
11.
Björkqvist M, Petersén A, Bacos K, Isaacs J, Norlén P, Gil J, et al. Progressive alterations in the hypothalamic-pituitary-adrenal
axis in the R6/2 transgenic mouse model of Huntington's disease. Hum Mol Genet. 2006;15(10):1713-21.
12.
Phan J, Hickey MA, Zhang P, Chesselet MF, Reue K. Adipose tissue dysfunction tracks disease progression in two Huntington's
disease mouse models. Hum Mol Genet. 2009;18(6):1006-16.
13.
Wang R, Ross CA, Cai H, Cong WN, Daimon CM, Carlson OD, et al. Metabolic and hormonal signatures in pre-manifest and
manifest Huntington's disease patients. Front Physiol. 2014;5:231.
23.Predicting the behavioral impact of tDCS on perceptual decision making with computational
neurostimulation
Dr. James Bonaiuto & Dr. Sven Bestmann
Aims:
Transcranial direct current stimulation (tDCS) allows for non-invasive and reversible modulation of neural activity
in widespread cortical and subcortical networks. These network changes can influence behavior, depending on
the specific placement of electrodes on the scalp and stimulation parameters. The aim of the project is to test the
role of brain regions involved in various cognitive processes based on predictions made by computational
modeling. This includes regions that accumulate information and formulate a response during perceptual
decision-making (such as parietal and frontal cortex) and regions that support the formation of value
representations during value-based decision-making and reinforcement learning (such as the medial and orbital
frontal cortex). To assess their role, brain stimulation methods will be used to selectively alter the impact of
single brain regions during decision-making.
Methods:
Transcranial direct current stimulation is a non-invasive form of electrical stimulation which has been shown to
modulate underlying brain activity. This causal manipulation of the contribution of selective brain regions will be
crucial in assessing the specific impact on decision making of different brain regions. tDCS electrodes are
placed over frontal and occipital as well as parietal cortices. In addition to using tDCS during behavioural test
sessions, we also plan to use tDCS during functional imaging with EEG (electroencephalogram) or fMRI
(functional magnetic resonance imaging).
Three-month project
This 3-month project will allow the student to become familiar with the basics of behavioural testing,
tDCS, subject recruitment, and data analysis. The student will be involved in:
1) Assisting in recruitment, assessment and stimulation of subjects; this will allow the student to have a direct
contact with subjects and obtain experience in using tDCS;
2) Analyzing behavioural and EEG data to investigate activity the effects of tDCS on accuracy, reaction time and
activity in various frequency bands; this will allow the student to gain experience in the analysis of
behavioural and EEG data using advanced computer programs and statistical models.
How this project might grow into an exciting 3-year PhD: During a PhD, candidates may pursue several
directions. First, a long-term goal of our lab is to continue our development of computational neurostimulation,
i.e. the use of neurobiologically informed computational models that generate predictions about the
consequence of neurostimulation, and to conduct experimental studies in healthy participants to compare the
behavioural consequences of neurostimulation with the predictions generated by these models. Second,
candidates may use oscillatory current stimulation to entrain specific brain networks and to test for their
behavioural consequences. This will provide causal manipulations of oscillatory activity and thereby allow testing
of their mechanistic role in supporting different kinds of behavior.
Dr Frances Wiseman, Professor Elizabeth Fisher, UCL – Institute of Neurology
PhD project
24. Understanding Alzheimer disease in Down syndrome: using a new trisomic animal model
Our recent experimental results demonstrate that a gene or genes on Hsa21 when trisomic significantly
accelerates APP/Aβ plaque pathology, the aim of this project is to understand this process and identify the
causative gene or genes that interacts with APP.
People who have Down syndrome (DS) are at a greatly increased risk of developing Alzheimer disease (AD).
We aim to understand the mechanisms that cause people who have DS to develop AD, as part of the London
Down Syndrome Consortium (http://www.ucl.ac.uk/london-down-syndrome-consortium/about). Our group has
demonstrated that a gene or genes on Hsa21 (other than the Amyloid Precursor Protein (APP) gene) when
trisomic significantly accelerates APP/Aβ plaque pathology. Our more recent experimental data suggests that
trisomy of Hsa21 alters the processing of APP. The aim of this project is to understand this process and identify
the causative gene or genes on Hsa21 that alters how APP is processed.
Rotation Project
APP-CTFs are generated by the cleavage of APP by α-secretase (α-CTF) or β-secretase (β -CTF). Our data
shows that trisomy of chromosome 21 leads to a significant increase in the abundance of both types of APP- Cterminal fragments (APP-CTFs). The project aims to understand which genes on Hsa21 are responsible for this
change. During the 3 month rotation the student will determine the abundance of APP-CTFs in 3 mouse models
of Hsa21 partial trisomy, to map the location of the causative gene. Training in, protein extraction, protein
assays, western blotting and analysis will be provided.
PhD project
Aim one: Does trisomy of Mmu16 modify APP pathology, as Hsa21 does?
Cross Ts1Tyb with J20 tg mouse model of APP
3 cohorts of mice – aged to 14 months, 6 months and 3 months.
Year 1
Apply for home office licence and basic mouse handling training
Organise breeding of colony and assign cohorts
Write experimental request
Year 2-3
Phenotype mice by Y-maze (spatial learning, activity), burrowing (“motivation”)
Quantify APP expression (Q-PCR, western), Aβ (ELISA) in mouse brain
Quantify pathology APP plaque load in mouse brain
Aim two: What is the identity of the gene or genes that modify APP pathology?
Year 1
Learn how to generate cortical neuronal cultures, organise timed matings etc.
Year 2-3
RT-PCR and western blot of candidates in cortical neuronal cultures
Verify APP relevant phenotype in cultured cells (APP expression, Aβ cleavage)
RNAi of candidates that are over-expressed in Mmu16 brain and cortical cells, to determine if
APP phenotype reversed.
Reference
Ruparelia A, Pearn ML, Mobley WC. (2013) Aging and intellectual disability: insights from mouse models of Down syndrome.Dev Disabil Res
Rev. 2013 Aug;18(1):43-50. doi: 10.1002/ddrr.1127.