Research project #1
Title : Single-neuron transcriptome-to-phenotype mapping using combined patch-clamp
electrophysiology and microfluidic quantitative PCR
Superviser : Jean-Marc Goaillard
Laboratory : Inserm UMR 1072, http://www.unis-neuro.com/
State of the art: Midbrain dopaminergic (DA) neurons play a critical role in the control of motivation and
motricity. In particular, the selective neurodegeneration of substantia nigra pars compacta (SNc) is the
hallmark of Parkinson’s disease (PD) and responsible for the motor symptoms characteristic of this
pathology (tremor, bradykinesia, etc). A growing body of evidence suggests that the vulnerability of SNc
DA neurons to neurodegeneration might stem in the relationship between electrical excitability and
metabolic activity. Voltage-dependent calcium channels (in particular Cav1.3) have been suggested to play
a critical role in linking neuronal ecitability to mitochondrial activity. More generally, many studies suggest
that understanding regulation and dysregulation of excitability in DA neurons may provide important
insights into the pathogenesis of PD. A myriad of ion channels control DA neuron excitability, including
many subtypes of voltage-dependent (including the Kv4.3 and SK3 potassium channels) and –independent
ion channels. We recently performed a transcriptomic analysis on single wild-type SNc DA neurons, which
showed that the levels of expression of ion channels are regulated in a coordinated manner, such that ion
channels are organized in co-regulated modules of expression (Tapia et al., submitted). In parallel, the
electrophysiological characterization of 2 KO mice (for the Kv4.3 and SK3 channels) has revealed that
robustness of SNc DA neurons to genetic perturbation is only partial, with variations in
electrophysiological phenotype specific of each KO. The characterization of the Cav1.3 calcium channel
KO mouse is currently performed. In order to better understand how robustness of electrophysiological
phenotype (or lack thereof) is achieved in SNc DA neurons, we propose now to combine both approaches
and quantify 96 mRNAs (including 53 ion channels or auxiliary subunits) from electrophysiologically
recorded SNc DA neurons in wild-type, Kv4.3, SK3 and Cav1.3 KO mice. This systems-level approach
will provide a unique understanding of the quantitative links between transcriptomic and phenotypic
variations, and help us understand which factors may be involved in excitability dysregulation in DA
neurons. Our preliminary study (Tapia et al., submitted) demonstrated that ion channel and dopaminergic
metabolism genes are organized in modules in SNc DA neurons, suggesting that none of the genes critical
for the function of these neurons are working independently. Therefore, only a systems-level approach that
provides relevant information about the modular structure of gene networks in DA neurons can shed light
on the transcriptomic principles underlying proper function of this particularly vulnerable population of
cells. Objectives: 1. Define the transcriptome-to-phenotype mapping in SNc DA neurons in wild-type
mice. 2. Define the changes in transcriptome-to-phenotype mapping in Kv4.3, SK3 and Cav1.3 KO mice.
3. Define the changes in transcriptome-to-phenotype mapping after pharmacological treatment (in
collaboration with other members of the group).
Methods: Characterization of electrophysiological phenotype. SNc DA neurons will be recorded using
the patch-clamp technique, whole-cell configuration. Spontaneous activity and response of neurons to
various current protocol (steps, white noise) will be recorded. Single-neuron transcriptomics. Following
recording, cytoplasm will be harvested by aspiration. After targeted retro-transcription and preamplification
(with 96 Taqman assays), mRNAs will be quantified using microfluidic qPCR (Fluidigm Biomark HD
platform, Integragen, Evry). Data analysis. Data will be analyzed using mutual information analysis
developed by Pierre Baudot (post-doc in our group). Statistical dependences at all orders between
electrophysiological parameters, between mRNA levels and between these two types of data will be
determined. Expected results : By performing these experiments, we will understand which modules of
ion channels are particularly critical for the stability of SNc DA neuron’s excitability. This will constitute
the first systems-level approach ever performed to achieve an integrated view of DA neuron’s regulation
and dysregulation of activity. Relevance to Clinical Neuroscience : PD is a multifactorial pathology, very
likely determined by the coordinated dysfunction of a multitude of genes, including metabolic and
excitability genes. Only a systems-level approach can provide the integrated view necessary to generate
new promising drugs targeting the many components dysregulated in the disease. This project will
definitely help us to provide such a framework. Feasibility : Our group is routinely using
electrophysiology, single-cell microfluidic qPCR, and mutual information analysis. Our group is currently
funded by an ERC consolidator grant. All necessary means are available to guarantee the success of this
project.
Expected candidate profile: A solid background in molecular biology and/or physiology would
appreciated.
Research project #2
Title : New insights onto the pathophysiology of autism spectrum disorder from targeted
deletion of Tshz3 in mouse brain
Supervisers : Laurent Fasano & Xavier Caubit
Laboratory : IBDM, http://www.ibdm.univ-mrs.fr/fr/institut/presentation/
1. State of the art
Recently, we identified TSHZ3 as a critical gene defining a new syndrome including autistic
features in patients with 19q12 deletion and provided evidence, from studies in mouse models,
for a link between Tshz3 deletion, defects in cortical projection neurons and autism spectrum
disorder (ASD)-like deficits (Caubit et al., Nature Genet. 2016. PMID 27668656).
2. Objectives
The main aim of the project will be to study when, where and how TSHZ3 function is critical
for the proper development and function of the brain.
3. Methods
The successful PhD candidate will address this question by combining molecular,
morphological, behavioral and electrophysiological approaches to characterize a new mouse
model with conditional removal of Tshz3 in cortical projection neurons (CPNs; Where) from
post-natal day (P) 2-3 onward (When). To address the how, the candidate will use large-scale
screening (RNA-seq) to identify differentially expressed genes in the cerebral cortex of wildtype vs. mutant mice. To identify direct targets of TSHZ3 he/she will perform in vivo chromatin
immunoprecipitation (ChIP) experiments. The enriched DNA will be sequenced (ChIP-seq)
and the TSHZ3 target sites will be identified using bioinformatics analysis.
4. Expected results
This project will allow 1) determining the contribution of embryonic vs. post-natal processes to
the ASD-related behavioral deficits and alterations in CPNs function associated with Tshz3
deficiency and 2) identifying for the first time, the direct targets of TSHZ3. Based on our
preliminary results (see “Feasibility”) completion of the project will pave the way to determine
whether genetically restoring Tshz3 expression after birth in specific neuronal populations
improves ASD-like deficits of Tshz3 heterozygous mice and identify novel potential molecular
targets for therapeutic approaches.
5. Feasibility
We have confirmed that Cre activity inactivates Tshz3 in CPNs. Our preliminary data suggest
that postnatal deletion of Tshz3 is sufficient to generate social interaction deficit. We have
strong internal collaboration with Lydia Kerkerian Le Goff Team (electrophysiology) and
Bianca Habermann Team (Bioinformatics).
6. Expected profile of the candidate
For this neuroscience PhD project, we seek one motivated pre-graduate candidate. Training will
be provided in molecular biology, developmental biology, mouse genetics and behavioural
testing. Prior experience with mouse genetics is beneficial but not mandatory. Good knowledge
of English will be appreciated but any good application will be considered.
Research project #3
Title : Characterization of a mouse model for Ohtahara syndrome and pharmacological
interventions.
Superviser : Laurent Villard
Laboratory : Inserm UMR 910, http://www.paca.inserm.fr/
State of the art and objectives
Ohtahara syndrome is the most severe form of intractable epilepsy. During the last few years,
our laboratory has built a cohort of 520 patients with early epileptic encephalopathies including
80 Ohtahara syndrome patients, for whom we collected biological samples and extensive
clinical information. We now have shown that pathogenic variations in the KCNQ2 gene,
encoding the Kv7.2 potassium channel subunit, are the major cause of Ohtahara syndrome.
We have engineered the first Ohtahara syndrome mouse model by knocking-in a variant
present in one of the patient followed locally.
Our preliminary results suggest that we have generated a good model for Ohtahara syndrome.
We now want to shift gears to provide a detailed characterization of the model and use it as a
pre-clinical model to test pharmacological hypotheses.
Upon completion, this project must provide a better understanding of the molecular and cellular
mechanisms causing one of the most severe epilepsy phenotype and will generate new data to
develop therapeutic approaches for this devastating and currently intractable condition.
Methods
We will characterize the motor and cognitive phenotypes of these animals using several tests
routinely used in our laboratory (Phenorack, Barnes Maze, fear conditioning), study brain
morphology (on tissue sections and using 7T MRI) and tissue organization. We will also
perform in vivo electrophysiology (EEG) and study RNA expression using RNA sequencing to
monitor transcriptional changes during the course of the disease. Finally, we will perform
pharmacological interventions in the mouse model to try to prevent seizures.
Feasibility
Our group has been studying mouse models for neurological diseases for the last 15 years,
mostly in the field of intellectual disability. We are familiar with motor and cognitive
phenotyping, autonomic nervous system monitoring and pre-clinical research using
pharmacology or gene therapy. We can perform EEG in rodents. We are also able to purify
brain neurons and analyze RNA-seq data. Additional electrophysiology experiments (brain
slices / isolated cells) are performed in collaboration with other laboratories in Marseille.
Expected profile of the candidate
We are looking for an enthusiastic PhD candidate holding a master degree in neurosciences or
molecular/human genetics. He/she must be willing to work with rodents. He/she must be
interested in epilepsy, genetic diseases and pre-clinical research, and keen to interact with the
other members of the team to perform collaborative work.
Research project #4
Title : Tau protein governs cytoskeletal filament organization in cancer cell – an issue in the
fight against the progression of glioblastoma
Supervisers : Pr. Vincent PEYROT & Dr. Gilles BREUZARD
Laboratory : Inserm S_U911, CRO2, http://www.cro2-timone.fr/CRO2-UMR-INSERM-911Redox-Microenvironment-Cytoskeleton-Colorectal-Tumor-Team-2.php
State of the art: Despite recent advances, the number of patients with glioblastoma (GBM) is
increasing and the main reason for treatment failures remains the ineffectiveness of
conventional treatments for other cancers. Moreover, the dysfunction of cell migration in GBM
is a proven factor of the disease. This process involves strong cell shape modifications and thus
heavily relies on the redistribution and cooperation of cytoskeletal filamentous proteins,
notably actin filaments and microtubules (MT). While the requirement of a crosstalk between
MTs and actin is not questioned, mechanisms underlying the MT-actin organization by crosslinkers remain largely unexplored. In particular, little is known about the functional
contribution of microtubule-associated proteins (MAPs) such as Tau known as a prominent
stabilizer of MTs and promoting in vitro co-organization of MT and actin networks. Thus, a
complete understanding of how cytoskeletal filaments contribute to biological functions in cell
migration and more generally cell shape changes requires a deeper knowledge of how Tau
governs cross-talk of MT and actin cytoskeletons. This project aims at deciphering the role of
Tau in cell migration and the progression of GBM.
Objectives: Our hypothesis of work is that by interacting with both MTs and actin
cytoskeletons, Tau could assist in addressing the (+) end of MTs to focal contacts (a
phenomenon called ‘targeting’) necessary to cell adhesion to extracellular matrix. We propose
(1) to characterize the interaction of Tau with both MTs and actin filaments directly in the cell;
(2) to explore the interaction-activity relationship of Tau on major signaling pathways involved
in cell migration, such as the Rho GTP-ase proteins (Rho, Rac, Cdc42); (3) to determine the
impact of phosphorylation of Tau on three residues candidate as prognostic markers of GBM;
and (4) to determine the impact of anti-cancer agents (taxol, temodal, trichostatin A) on the cointeractions of Tau with MT and actin cytoskeletons.
Methods: The molecular mechanisms of Tau-MT-actin interactions will be investigated using
different GBM cell models (U87 and mutant repressing Tau cells, and primary GBM cell lines
GBM6 and GBM9). The methodologies of this project will involve a combination of optical
instrumentation (FRET, FRAP, STORM imaging) and biological studies.
Expected results: Understanding the molecular mechanisms that regulate the activity of
MAPs, and in particular the tau protein, in the context of tumor progression will improve
fundamental knowledge about these MT regulatory proteins. From a pharmacotherapeutic point
of view, the study of their role in modulating the efficacy of drugs will better predict the
response to chemotherapy and possibly consider these MAPs as new targets in tumor cells.
Finally, the approaches developed for this project will be decisive for the design of drugs
targeting more efficiently tau and microtubules.
Feasibility: The candidate will benefit from the strong expertise of the team in quantitative
imaging tools that combine molecular specificity, high spatial resolution and real-time
measurements of filament organization.
Expected candidate profile: The candidate must have a solid knowledge of the current
techniques of cellular and molecular biology (cell culture, western-blot, transfection…). Skills
in biophotonic approaches would be appreciated. This study is part of the Cancer Plan (SIRIC
label, Site of Integrated Research on Cancer).
Research project #5
Title : Functional characterization of the spontaneous repair process of mammal vestibular
synapses
Superviseur : Dr Christian CHABBERT
Laboratory : LNIA UMR7260 CNRS-AMU , http://lnia.univ-amu.fr/
Summary:
Vertigo is a booming health problem and an unmet medical need.1-4 In France as in the US it is
the third motive for consultation to the general practitioner doctor and 5 % of hospital
emergencies. In over 80 % of cases these conditions result from direct impairment of the
vestibule, the organ of balance located in the inner ear. Synaptic contacts between
mechanoreceptors and vestibular primary neurons that form the vestibular nerve are the most
sensitive area. Acute unilateral vestibular deafferentation (uVD) is believed to be involved in
several vestibular syndromes such as labyrinthitis, vestibular neuritis, vertigo of ischemic
origin, as well as in Menière disease.5 There is currently no targeted and efficient
pharmacological therapy to effectively protect or repair the vestibular synapses under
pathological conditions6. However, an endogenous process of vestibular synapses postlesionnal
self repair occurs in humans as in mammals.7 This process allows under certain conditions to
restore the vestibule function and rescue balance. For several years we have explored the
vestibular synapses insult/repair process, deciphered their different phases and identified
several of the cellular effectors involved.6,8-11 The present project intends to use an original in
vitro model recently developed by our team to characterize further the mechanism of
spontaneous synaptic repair. Cocultures of utricles and Scarpa’s ganglia will be prepared from
C57 mice and actine-GFP mice respectively to authorize on line imaging of the reafferentation
process and analyse of the reafferentation pattern. Direct patch-clamp recording from
reafferented terminals will allow assessing whether reafferented synapses undergo normal
neurotransmission and to study the development of excitability as a function of the pattern of
reafferentation. Through identification of the mechanisms that support functional restoration of
vestibular synapses, this project intend to provide material to significantly impact on the
therapeutic management of these debilitating conditions. Experience in cell culture and/or
patch-clamp welcomed .
References :
1. Chabbert C. Nouvelles avancées pharmacologiques contre les pathologies vestibulaires. Biofutur (2012) 337:
38-39.
2. Chabbert C. New insights into vestibular neuropharmacology. J Vestibular Research (2013) Jan 1; 23(3): 10711.
3. Agrawal Y, Carey JP, Della Santina C, Schubert MC, Minor LB. Disorders of Balance and Vestibular Function
in US Adults: Data from the National Health and Nutrition Examination Survey, 2001-2004. Archives of Internal
Medicine (2009) 169(10): 938-944.
4. Vertiges. Science & Avenir (2014) N°805 Mars pp182-183.
5. Foster CA, Breeze RE. The Meniere attack: An ischemia/reperfusion disorder of inner ear sensory tissues.
Medical Hypotheses 81 (2013) 1108–1115.
6. Dyhrfjeld-Johnsen J, Gaboyard-Niay S, Saleur A, Brugeaud A, Chabbert C. Ondansetron reduces lasting
vestibular deficits in a model of severe peripheral excitotoxic injury. Journal of Vestibular Research (2013) Jan 1;
23(3): 177-86.
7. Ochi K, Ohashi T, Watanabe S. (2003). Vestibular-evoked myogenic potential in patients with unilateral
vestibular neuritis: abnormalVEMPand its recovery. Journal Laryngology Otology, 117(2), 104-108.
8. Brugeaud A, Travo C, Dememes D, Lenoir M, Llorens J, Puel JL, Chabbert C. Control of hair cell excitability
by vestibular primary sensory neurons. Journal of Neuroscience (2007) 27: 3503-3511.
9. Travo C, Gaboyard-Niay S, Chabbert C. Plasticity of Scarpa's Ganglion Neurons as a Possible Basis for
Functional Restoration within Vestibular Endorgans. Frontier in Neurology (2012) 3:91.
10. Desmadryl G, Gaboyard-Niay S, Brugeaud A, Travo C, Dyhrfjeld-Johnsen J, Chabbert C. Histamine H4
receptor antagonists as potent modulator of mammal vestibular function. British Journal of Pharmacology (2012)
167: 905-916.
11. Gaboyard-Niay S, Travo C, Saleur A, Broussy A, Brugeaud A, Chabbert C. Correlation between afferent
rearrangements and behavioral deficits after local excitotoxic insult in the mammal vestibule: an animal model of
vertigo symptoms? Disease Models and Mechanisms (2016) 9: 1181-1192.
Research project #6
Title : Investigate the role of oligodendrocyte progenitors in the adult mouse brain under physiological
and pathological conditions
Superviseur : Myriam Cayre & Pascale Durbec
Laboratory : IBDM, http://www.ibdm.univ-mrs.fr/equipe/stem-cells-and-brain-repair/
State of the art: Oligodendrocyte progenitor cells (OPC) are generated during development; a majority
of them mature during perinatal life to form myelinating oligodendrocytes, thus carrying central nervous
system myelination, which will allow to increase action potential conduction speed of the along axons.
However many OPC remain undifferentiated in the adult brain: these cells represent 5 to 8 % of all brain
cells and are disseminated throughout brain parenchyma. The main known function of this cell
population is to contribute to spontaneous myelin repair after lesion. After acute brain insult and
demyelination, OPC proliferate, migrate to the lesion and differentiate into myelinating
oligodendrocytes. Because of the energy necessary for myelin production, maintenance and repair,
oligodendrocytes are uniquely vulnerable to damage and senescence. Myelin breakdown is detected
very precociously in Alzheimer’s disease (AD) patients and in mouse models, even before amyloid and
Tau pathology. Myelin loss underlies both age-related cognitive decline and dementia in AD patients.
Less is known about OPC function in physiological conditions, although recent studies suggest myelin
remodelling all life long. Few experiments suggest that newly formed myelin may be important for
motor skill learning. In order to better understand the role of OPC in the adult brain, we generated in the
lab a genetically modified mouse line in which the human receptor of diphtheria toxin is specifically
expressed in OPC. This will allow us to specifically kill OPC in a timely controlled manner using
diphtheria toxin injection.
Objectives: The objectives of this PhD project will be:
to characterize this new “Olig2-PDGFRa-DTR” mouse line
to determine the impact of OPC deletion on myelin remodelling (in physiological conditions)
to determine the impact of OPC deletion on motor and cognition fucntions (in physiological conditions)
to determine the impact of OPC deletion on myelin repair (in demyelination conditions)
to test the hypothesis that OPC default might increase the risk to develop Alzheimer’s disease or fasten
its onset
Methods: Adult Olig2-PDGFRa-DTR mice will be examined 4 months and 12 months after diphtheria
toxin injection (or NaCl 0.9% for control group). Oligodendrocyte density will be analyzed (CC1
immunolabeling), and newly born oligodendrocytes will be identified by EdU/CC1 co-labeling in order
to estimate adult oligodendrogenesis. The impact of OPC deletion on myelin properties and axonal
integrity will be examined by electron microscopy (g ratio, % of myelinated axons, ultrastructural
alterations of myelin sheath, axon swelling…) and by immunolabeling (SMI32 and ßAPP for suffering
axons, Nav and Caspr for node/paranode length and spacing analyses). Olig2-PDGFRa-DTR mice will
be extensively characterized at behavioral levels in order to detect any sensory-motor and/or cognitive
dysfunction. Sensory-motor abilities will be assessed using dynamic hot/cold plate, tail flick and the
grip test. Motor coordination and balance will be assessed using the rotarod test. Learning as well as
short and long-term memory will be evaluated using the Morris water maze (spatial memory) and object
recognition task (non-spatial memory). Anxiety will be assessed in the elevated plus maze.To test
whether preventing myelin remodeling and repair by killing OPC is sufficient to trigger the appearance
of signs considered as hallmarks of AD, aging Olig2-PDGFRa-DTR mice (from 12 to 24 month-old)
will be injected with DTA (or NaCl 0.9% for controls). Levels of synaptophysin, PSD95 (pre- and
postsynaptic proteins respectively), phosphorylated Tau and Aß will be examined in hippocampus and
cortex (structures known to be largely affected in AD) both by western-blots and
immunohistochemistry.
Expected results: This work will increase our knowledge on oligodendrogenesis and myelin biology in
physiological and pathological conditions. It will also determine the validity of a novel mechanism
underlying AD pathogenesis.
Feasibility: The mice are already present in our animal facility, our team has the expertise to drive the
experiments described above and our lab is furnished with all the required equipment.
Expected candidate profile: The candidate will need to work with, and manipulate mice, thus should
be prepared to animal experimentation. Since the project addresses questions from molecular to
behavioural levels, the candidate will need to be skilful…First experience in histology / microscopy and
/ or behavioural analyses would be appreciated.
Research project #7
Title : Neurocomputational bases of Causal Learning (CausaL)
Superviseur : Andrea Brovelli & Paul Apicella
Laboratory : INT, http://www.int.univ-amu.fr/
State of the art: Humans have an extraordinary capacity to infer cause-effect relations. In
particular, we excel in forming beliefs about the causal effect of actions. This ability provides
the basis for rational decision-making and allows people to engage in meaningful life and social
interactions. The formation of causal beliefs relies on learning rules determined by the
contingency between actions and outcomes. Mathematically, contingency is operationalized as
the difference between two conditional probabilities: the probability of outcome O given action
A, P(O|A), and the probability of the outcome when the action is withheld, P(O|¬A). In
everyday life, people perceive their actions as causing a given outcome if the contingency is
positive, whereas they perceive them as preventing it if negative; when P(O|A) and P(O|¬A) are
equal, people report no causal effect. Given its probabilistic nature, causal learning is tightly
linked to the notion of uncertainty. High levels of uncertainty in the estimation of probabilities
call for learning. As learning progresses, such estimation uncertainty decreases and transforms
into risk, which reflects the inherent stochasticity of action’s outcomes even when learning has
completed. Both types of uncertainty dictate people’s choices and the degree of confidence in
the decisions.
Objectives: Our goal is to provide an integrative view of how people form causal beliefs by
characterizing neurocomputational bases of Causal Learning (CausaL) by searching for the
neural correlates of causal learning. We will combine computational models of causal learning
and test these hypotheses on neural data recorded using complementary techniques (human
neuroimaging and behavioral neurophysiology in monkeys).
Methods: At the computational level, we will test Reinforcement Learning models previously
developed for goal-directed learning (Viejo et al., Front Behav Neurosci, 2015) to model the
acquisition of conditional probabilities, causal beliefs, uncertainty estimates and decision
confidences. At the neural level, we will search for correlates of the predicted computations at
different levels: i) directional influences between cortical regions using recent tools developed
for the analysis of Granger causality analyses of MEG and intracranial iEEG data (Brovelli et
al., Cerebral Cortex, 2008; Brovelli et al., Neuroimage, 2011); ii) functional dissociations
across fronto-striatal territories in humans by means of functional MRI using model-based
analyses data (Brovelli et al., J Neurosci, 2015; 2017); iii) functional dissociations across
striatal territories through the analysis of intracranial local field potentials (LFPs) recorded in
behaving monkeys during a probabilistic learning task.
Expected results: We expect to provide an integrative view of the neural correlates of causal
learning by linking different levels of analysis (from theory to data and from brain to behavior)
through a multilevel analysis of brain activity, from local (LFPs and iEEG) to large-scale
(MEG and fMRI) spatiotemporal scales.
Feasibility: The time-consuming and risk-taking part of the project related to the acquisition of
neurophysiological data (MEG and iEEG in humans and LFPs in two monkeys) has been
completed. The computational model has already been developed. The PhD student will
perform an fMRI study and devote to the analysis of existing data.
Expected candidate profile: The PhD candidate is expected to have a degree in life sciences,
physical sciences or engineering, with a strong interest in cognitive neuroscience and
interdisciplinary research. Prior experience in neuroimaging and/or neurophysiology,
computational modeling of cognitive processes and good programming skills (Matlab and/or
Python) are an advantage.
References can be downloaded here: https://sites.google.com/site/andreabrovelli/publications
Research project #8
Title : Molecular mechanisms of Circadian rhythmicity: Role of long non-coding RNA Neat1
Superviseur : Dr. A. M. François-Bellan
Laboratory : AMU-CNRS UMR 7286 CRN2M, http:/crn2m.univ-amu.fr
To adapt to the fluctuations of the environment, living organisms have developed an endogenous clock
organized in mammals around a major element located in the suprachiasmatic nuclei of the
hypothalamus which synchronize numerous central and peripheral oscillators whose coordinated
functioning allows to generate rhythms of biological functions which is based in part on rhythmic
expression of mRNA within the cells. It is now known that mRNA rhythms are not mainly based on
rhythmic transcription since only 20% of rhythmic mRNAs are controlled at the transcriptional level.
However, rhythmic mechanisms that occur after transcription are largely unknown. We characterized
one of these post-transcriptional mechanisms. This mechanism involves nuclear bodies called
paraspeckles which consist of a long non-coding RNA, Nuclear-Enriched Abundant Transcript 1
(Neat1) which serves as a scaffold for RNA binding proteins. Paraspeckles are able to bind mRNAs
they retain inside the nucleus. We have shown in pituitary cells, that all components of paraspeckles as
well as the number of these paraspeckles exhibit a circadian expression pattern. Thanks to their
circadian expression, paraspeckles control therefore the circadian nuclear retention of their target
mRNAs allowing their rhythmic cytoplasmic export and therefore their circadian expression.
The aim of the project is to further characterize the role of Neat1, via paraspeckles, in the posttranscriptional mechanisms allowing the circadian gene expression. The project will be developed
according three tasks: 1. We will evaluate the quantitative contribution of paraspeckles to the circadian
gene expression in pituitary cells. To this end, we have developed several Neat1 KO pituitary cell lines
established using the CRISPR / Cas9 technique. We will determine by RNA sequencing, the set of
mRNAs whose circadian expression depends upon Neat1. 2. Our goal is to better understand the
mechanism that underlies the ability of paraspeckles to retain mRNAs in the nucleus. To this end, we
will attempt to identify the molecular determinants that are necessary for the nuclear retention of
mRNAs by these structures. To address this issue, two approaches will be conducted: a screening of
sequences present in the 3'-UTR region of the mRNAs that we have chararcterized as paraspeckle
targets and a more global bioinformatic approach based on the list of paraspeckle mRNA targets we
have established after Neat1 pull down followed by RNA sequencing. 3. We will decipher the
mecanisms involved in circadian Neat1 expression. In particular we will determine whether the
circadian expression of Neat1 is based on a circadian transcription of the gene. To this end, we will use
a transgene construct corresponding to the luciferase reporter gene under the control of Neat1 proximal
promoter and we will develop a GH4C1 cell line expressing this construct in a stable manner. In these
cells, we will record real-time transcriptional oscillations of this construct using a lumicycle apparatus.
We will also examine whether post-transcriptional mechanisms are involved in Neat1 circadian
expression pattern focusing on alternative 3′ end cleavage and polyadenylation. Indeed, two variant
transcripts of NEAT1, NEAT1-1 and NEAT1-2, share the same 5’ end but are processed alternatively at
the 3’ termini. The 3’ ends of NEAT1-1 and NEAT1-2 are formed by two distinct mechanisms:
canonical polyadenylation and RNase P cleavage, respectively. Since alternative 3′ end cleavage and
polyadenylation have been shown to play major role in circadian regulation, we will look for the
possible existence of a circadian rhythm in 3′ end cleavage and polyadenylation of Neat1 affecting the
respective proportions of its two isoforms. In addition to decipher the post-transcriptional mechanisms
that are involved in the circadian regulation of gene expression, a problematic anchored in the field of
chronobiology, this project from a neuroscience point of view will allow to assign a role in the
neurophysiology to the long non-coding RNA Neat1 whose functions remain enigmatic. Since all the
molecular tools necessary for the realization of this project have already been developed, this project is
ready to start. The candidate recruited should be interested in molecular biology approaches as well as
in bioinformatics approaches.
Research project #9
Title : Trajectories in natural images and the sensory processing of contours
Superviseur : Laurent PERRINET
Laboratory : INT, http://www.int.univ-amu.fr/
State of the art: Binding the different features of objects in images is at the core of visual perception. As such, the
visual system needs to detect local edges and to bind them together to form contours at a higher, more global level.
A state-of-the art theory is that of the “association field”: the confidence of an edge depends on the configuration
of neighboring edges. For instance it is facilitated for co-linear or co-circular edges. This process takes advantage
of the statistical regularities of edges that are present in natural images. In particular, we have developed a method
to quantify the association field in different classes of natural images (Perrinet & Bednar, 2015). At the neural
level, modeling the representation of the image, such as that formed in the primary visual cortex of primates (V1),
this heuristics translates to a set of rules that adapts dynamically the activity of isolated neurons representing edges
into the coherent population activity of contours. Yet, we miss an understanding of the link between these
statistics and the probabilistic rules that binds features together and how this information is dynamically
encoded in V1.
Objectives: In this computational neuroscience project, we will exploit our current expertise in computer vision
for the statistical integration of visual of objects to translate them in the form of probabilistic predictive models for
biological vision. Our core hypothesis is that in natural scenes, contours follow coherent trajectories and
that this knowledge is integrated (learned) by the visual system to optimally inform the representation of
the image.
Methods: First, we will learn the different classes of edge co-occurrences that are relevant to natural images.
Using an existing unsupervised learning algorithm, we will learn these as an independent components analysis.
Such an algorithm extends well to a deep-learning convolutional neural network, but importantly, it will be
informed by our expertise of modeling neural networks in low-level visual areas by including horizontal
connectivity. We expect that relevant features will be mainly the predictable arrangements, such as co-linear or cocircular pairs of edges, but also highly surprising ones, such as T-junctions or end-stopping features. Importantly,
we will be able to compare this representation with that present in higher level areas and to refine our knowledge
on the representation of natural-like images. Second, we have previously found that using synthetic textures could
further advance our understanding of neural computations and perception. These random synthetic textures, coined
“Motion Clouds” were initially targeted to quantify the integration properties of visual motion perception (Leon et
al, 2012, Simoncini et al, 2012). Informed by the generative model of edge co-occurrences studied above, an
extension to such stimuli would be to include dependencies between different elements. As such, we will be able
to manipulate the level of dependency between different elements, whether in space, time or feature space
(orientations). A potential outcome will be to use these in neurophysiological and psychophysical experiments
within the team. In particular, the ability to select different classes of dependencies learned above will make it
possible to evaluate the relative contribution of each component to the association field.
Expected results: Finally, those two tasks converge to a long-term goal of understanding the impact of the
spatio-temporal structure of natural images in the neural computations implementing visual processing in
low-level visual areas and perception. Indeed, the regularities observed in static images can be extended to
dynamical scenes by observing that a co-occurrence in time can be implemented by simple geometrical operations
as they are operated during that period. For instance a co-circularity can be described as a smooth rototranslational transformation of an edge along a smooth trajectory. Importantly, such a distinction should allow us
to determine the hierarchy of different features relevant to describe the full statistics of the feature space (that is, of
spatio-temporal edge co-occurrences). We expect to see that the different independent features should decompose
at various scales both in space and in time. This translates into a probabilistic hierarchical model that would
combine dependencies from different cues. In particular, we expect to see the emergence of differential pathways
for form and motion. Feasibility: The project is based on existing expertise and libraries in computer vision and
computational neuroscience. The extension of this expertise to the dynamical domain will be made possible thanks
to an existing collaboration (JM Morel at ENS-Cachan, G Peyré at ENS-Ulm). The groundbreaking nature of the
work takes advantage of the interaction with neurophysiological and psychophysical experiments thanks to the use
of synthetic textures. While we acquired experience in psychophysical experiments, a potential risk lies with
neurophysiological experiments and their inherent difficulty, but we have submitted grant proposal (F Chavane,
INT; Y Fregnac, UNIC) to reduce such risks.
Expected profile of the candidate: The student should be familiar with computer vision and computational
neuroscience. The generic tools used will be probabilities (applied mathematics), machine learning and the python
programming language. The ideal student should be highly curious into the topics of visual perception and
neuroscience in general.
More information & references @ http://invibe.net/LaurentPerrinet/IcnPhdProgram
Research project #10
Title : In silico brain models for personalized medicine –
Supervisor : Viktor JIRSA
Laboratory : INS – TNG group, http://ins.univ-amu.fr/research-teams/theoretical-neurosciences-group/
State of the art: The Virtual Brain (TVB) is a high-fidelity dynamic network model of the human adult
brain, which was developed and consistently refined since 2010 and made available to the community
via an open neuroinformatics source platform. It is currently the only existing modeling approach to link
mathematical models to neuroimaging signals of individual subjects and patients. This is achieved by
the fusion of an individual’s brain structure with computational neuroscience modeling, which allows
creating one model per patient and systematically assessing the modeled parameters that relate to
individual functional differences. The functions of the brain model are governed by realistic
neuroelectric and neurovascular processes and are constrained by subject-specific anatomical
information derived from non-invasive brain Imaging (anatomical MRI, diffusion tensor imaging
(DTI)). TVB comprises a dynamic neuroelectric simulation, refined geometry in 3D physical space,
detailed personalized brain connectivity, large repertoire of mathematical representations of brain region
models, and a complete set of physical forward solutions mimicking used in non-invasive brain
mapping including functional Magnetic Resonance Imaging (fMRI), Magnetoencephalography (MEG)
and Electro-encephalography (EEG). This approach has been successfully applied to the modeling of
brain disorders including epilepsy, stroke and aging. Relevant to this proposal is the publication by
Proix et al (2017), in which TNG demonstrated the predictive power of Virtual Brain modeling with
regards to surgical interventions for a small cohort of 15 epilepsy patients.
Objectives: The goal is to create novel, minimally invasive surgical approaches in a patient-specific
manner and validate these retrospectively against a large patient cohort. Emphasis will be given to the
oscillatory nature of seizure propagation patterns, which will require the development of novel methods
to characterize connectomes of oscillatory networks.
Methods: Backed by a neuroinformatics platform and simulator (Sanz-Leon et al 2013, 2015), TVB is
used to construct individualized virtual brain models from non-invasive neuroimaging data to study
seizure propagation patterns and explore treatment options. Model attributes can be modified or
redefined, including their geometry, connectivity, regional neurotransmitter distributions and signal
transmission properties. Given the large-scale nature of the brain network, time delays via signal
transmission play a critical role and will be included mathematically and computationally following our
latest approaches in Petkoski et al. Data processing of structural data (tractography, cortex
reconstruction) will be performed using our TVB-specific pipeline on Github (Proix et al (2016), and of
functional SEEG data in close collaboration with the clinicians following our modeling strategies
developed in (Proix et al (2017).
Expected results: these will be threefold 1) Theory: Connectome analyses have not taken time delays
via signal transmission into account so far. The latter may however change the nature of excitatory
and/or inhibitory connections. This project will provide a novel mathematical formulation. 2) Clinical:
As a first application to seizure propagation patterns, the project will identify novel strategies to reroute
or extinguish seizure recruitment of non-epileptogenic areas. 3) Neuroinformatics: Using retrospective
multimodal brain electrophysiology and imaging data of epilepsy patients (N=40; SEEG, EEG, fMRI,
MRI, DTI), we will validate the theoretical predictions and demonstrate the degree to which patient
specificity of the model predictions holds. A database comprising non-invasive and invasive, structural
and functional brain imaging data will be built, as well as all computational simulations for each patient,
and thus establish a unique neuroinformatic resource for scientific and clinical discovery.
Feasibility: Having established both local and international collaborations with clinicians and the TVB
development team as well as being equipped by a high-performance cluster and the TVB platform, our
Institute provides the perfect environment for carrying out this project.
Expected candidate profile: Degree in computational neuroscience, engineering or equivalent level of
knowledge with a strong background in computational neuroscience (networks, modeling, dynamic
system theory) and/or data fitting (Bayesian approaches, Dynamical Causal Modeling (DCM), Monte
Carlo techniques). Experience in software development of software or clinical databases would be an
advantage.
Research project #11
Title: Role of actin nanostructures in presynaptic assembly and function
Supervisor: C. Leterrier, NICN
Laboratory: NICN CNRS-AMU UMR 7259
Axonal transport and organization
Axonal transport is crucial for bringing axonal
proteins to their target compartment, notably
presynapses where information is transmitted to
downstream neurons. Perturbation of axonal
transport and presynaptic organization are hallmarks
of neurodegenerative diseases, with a possible
causal role in the associated cognitive deficits. The
role of actin in axonal transport remains seldom
studied, and its role at presynaptic boutons is
controversial (Figure 1).
Unraveling how axonal actin regulates and
builds the presynaptic compartments will deeply
transform our understanding of the axonal
physiology.
Figure 1: Axonal cytoskeleton and transport. The axon contains
microtubules (A) which support vesicular transport. Actin
cytostructures identified so far include actin rings in the axon initial
segment (B) and shaft (C), and actin filaments of unknown
organization in pre-synapses (D). Adapted from Kevenaar &
Hoogenraad 2015.
New actin structures
New techniques of super-resolution microscopy
now allow fluorescent imaging down the molecular scale (20
nm), opening unprecedented opportunities to directly
visualize molecular assemblies in situ1. In neurons, this has
led to the discovery of several new axonal actin structures by
our lab and others: rings, hotpsots and trails (Figure 2). The
function of these structures is still unknown, but hints to a
role for proper presynaptic function2.
Our hypothesis is that actin structures are crucial for the
proper support of presynapse organization, notably by
forming checkpoints that regulate cargo delivery.
Figure 2:
Identification new
actin structures by
STORM
(A) Young neuron
labeled for ßII-spectrin
(magenta) and actin
(green).
(B) 3D-STORM image
of actin for the axon
boxed in (A).
Submembrane actin
rings, actin hot spots
(orange arrowheads)
and trails (blue
arrowheads) are
visible.
(C) Zoom and
transverse section
showing a region
containing an actin
hotspot.
Project: role of actin nanostructures at presynapses
Actin organization inside and around boutons, and exactly
how components are locally delivered to boutons, is still
largely unknown. Our hypothesis is that actin drives
recruitment of transport vesicles to presynaptic boutons. We
will use a correlative combination of live-cell imaging and STORM superresolution microscopy to study the organization and assembly of presynapses,
focusing on the presence of actin structures (Figure 3). We will characterize
the actin-associated nanoscale architecture in and around boutons, including
changes during formation. Finally, we will determine how actin-based
structures drive component delivery and scaffold assembly at pre-synapses.
Candidate
We are looking for a motivated candidate interested in quantitative biology
and advanced microscopy techniques to join the NeuroCyto team
Figure 3: Actin at presynapses
(www.neurocytolab.org) and tackle this ambitious project!
1.
2.
Leterrier, C. et al. Nanoscale Architecture of the Axon Initial Segment Reveals an Organized and Robust
Scaffold. Cell Rep 13, 2781–2793 (2015).
Ganguly, A. et al. A dynamic formin-dependent deep F-actin network in axons. J. Cell Biol. 104, 20576–417
(2015).
Hypothesis: actin (purple) organizes
the presynapses and is able to stop
presynaptic cargoes (red) transported
along microtubules (grey) for delivery.
Research project # 12
Title : Microglia Alteration and Targeting in Epileptic Encephalopathies
Superviseur : SZEPETOWSKI
Laboratory :INMED, INSERM U901, http://www.inmed.fr
State of the art: The crucial role of microglia (the brain resident immune cells) in brain development, functioning
and disorders is increasingly recognized1. Abnormal functioning of the neuronal networks, as seen in various
epileptic conditions, is also associated with microglial alteration2. However, the exact role played by microglia and
by microglia-neuron reciprocal interactions3-5 in this pathological developmental context, is still largely
undetermined. Neuronal glutamate-gated NMDA receptors may be crucially involved in such interactions 4,5. In
the recent years we found that genetic variants of the human GRIN2A gene, which encodes the GluN2A subunit of
NMDA receptors, are a first and major cause of the epilepsy-aphasia spectrum (EAS) of disorders6-8. This
spectrum comprises focal epilepsies and epileptic encephalopathies with speech, behavioral and cognitive
dysfunctions and is characterized by interictal discharges activated during sleep.
Objectives: In the present project, we postulate i/ that an early alteration of microglia due to NMDARs
dysfunctioning, and/or due to the dysfunctioning of the neuronal networks, occurs in Grin2a knock-out mice; ii/
that this in turn influences the phenotypes seen in those mice; and iii/ that the early targeting of microglia should
improve or prevent against the phenotypes. Therefore, our objectives are to look for early developmental
alterations of microglia in Grin2a KO mice and test whether early intervention on microglia would improve the
phenotypes.
Methods: The project involves a multidisciplinary approach and the use of various tools that include Grin2a KO
and other mouse lines (e.g. microglia-labeled Cx3Cr1-GFP), pharmacological and molecular genetic tools to
deplete or modify microglia, state-of-the-art electrophysiological recordings, two-photon time-lapse imaging,
immunohistochemistry and morphology, cellular/molecular biology, flow cytometry and behavioral studies.
Expected results: In this spectrum of disorders, identifying early neuroimmune events, understanding the
pathophysiology, deciphering what underlies the severity and outcome, and designing early therapeutic strategies,
are all crucial issues that must be addressed. Exploring and targeting the brain immune system will modify the
understanding of EAS and of their epileptic, behavioral and cognitive components. Starting from this study, we
anticipate novel insights of broad interest in the expanded context of the severe pediatric epilepsies where
microglial dysfunctioning might be postulated based on the likely alteration of the corresponding molecular and
neuronal networks. The outcome of our project should thus inform the design of novel, non-classical therapeutic
strategies against severe pediatric epilepsies.
Feasibility: The project relies on an interactive and multidisciplinary network between several PIs and
collaborators having strong and complementary expertises in molecular genetics, cell biology biochemistry,
immunology, epileptology, animal models of altered brain development, postgenomic and therapeutic models of
epilepsies, in vivo and ex vivo electrophysiological recordings, and the analysis of microglia in vitro and in vivo.
The present team is composed of three tenured researchers (1 DR1, 2 CR1) and one PhD student. It will soon be
reinforced by its formal association as from January 2018 with another
INMED 'epilepsy' team (headed by N Burnashev) and which comprises 2 more tenured researchers (2 DR2), one
engineer (IE), and one more PhD student. We have the required equipments and core facilities related with the
different tasks that are being planned (e.g. animal core facilities, cell and molecular biology platform, imaging
platform including confocal microscopes, two photon microscopes, electrophysiology in vivo and in vitro set-ups,
etc.). The Grin2a KO mice and the Cx3cr1-GFP mice are established at INMED and at CIPHE. The requested
animal experimentation protocols have been recorded and approved (n°02308.03) at the French Ministry of
Education and Research.
Expected candidate profile: We are looking for a highly motivated and innovative PhD student to explore the
neuroimmune aspects of genetic epilepsies caused by NMDA receptor dysfunctioning in a neurodevelopmental
perspective. The ideal candidate should have a strong background in neuroscience and experience/background in
immunology. Fluency in either of French or English language is required.
References:
1. Prinz & Priller, Nat Rev Neurosci 2014
2. Devinsky et al. Trends Neurosci 2013
3. Li et al. Dev Cell 2012
4. Eyo et al. J Neurosci 2014
5. Dissing-Olessen et al. J Neurosci 2014
6. Carvill et al. Nat Gen 2013
7. Lesca et al. Nat Gen 2013
8. Burnashev & Szepetowski Curr Opin Pharm 2015
Research project # 13
Title : Role of NeuroD2 in the maturation of cortical networks: possible implications in
Neurodevelopmental disorders
Superviseur : Antoine de Chevigny
Laboratory :INMED, INSERM U901, http://www.inmed.fr
State of the art
Neocortical neurogenesis is composed by a sequence of inter-related events: proliferation and specification of stem and progenitor
cells in the ventricular zone, migration of post-mitotic neuroblasts to the cortical plate, positioning and axon pathfinding ultimately
followed by synaptic integration and a survival/death decision. Alterations in one or more of these processes confer malformations
of cortical development (MCD), which are important causes of mental retardation and other neuropsychiatric disorders such as
autism. Antoine de Chevigny studies synaptic integration and plasticity in vivo and its link with neurodevelopmental disorders. A
gene expression screen done in the lab identified the transcription factor NeuroD2 (ND2) as potentially involved in synaptic
integration of new olfactory neurons. Interestingly, ND2 is not confined to the olfactory bulb but also expressed in projection
neurons (PN) of the neocortex. We therefore started to analyze the neocortical phenotype in ND2 KO mice.
First, while global development and PN layering of the KO neocortex were normal, we found supernumerary spines (the postsynaptic elements of excitatory synapses) on the dendrites of neocortical PN. Interestingly, this phenotype has been observed in
autistic patients and in mouse models of autism. Second, we analyzed the behavior of ND2 KO mice and found that they display
stereotypies and anti-social behaviors, which are behavioral hallmarks of autism spectrum disorders. We are currently seeking for
a direct link between ND2 and neuropsychiatric disorders in humans by collaborating with experts in the genetics of these
disorders.
Objectives
Our main goal is now to gain insight into the molecular, cellular and physiological mechanisms underlying the altered synaptic
connectivity observed in ND2 mutant mice.
Methods
To achieve this goal, the PhD student will:
1. Study whether ND2 cell autonomously regulates synaptic integration/plasticity in neocortical PN. To determine the cellautonomous function of ND2 we recently generated ND2 floxed mice. We will perform in utero electroporation of Cre
recombinase to determine the cell autonomous effect of ND2 deletion on new PN migration, positioning, axonal targeting and
dendritic/synaptic morphogenesis. To quantify structural synaptic plasticity, we will use two-photon live imaging of motor cortical
dendrites before and after a motor learning task. This will allow us to measure activity-dependent spine turnover. In parallel, in
collaboration with the Chavis team at INMED we will perform patch clamp recordings of ND2 KO neurons to measure
inhibitory/excitatory inputs, intrinsic excitability as well as functional synaptic plasticity (LTP and LTD).
2. Study how ND2 regulates synaptic integration of neocortical PN. To seek for ND2 target genes important for synaptic
integration, we will FAC-sort fluorescent ND2 KO vs WT neurons from the postnatal cortex after in utero electroporation of Cre
in floxed vs control embryos, and then perform RNAseq-based comparisons. Bioinformatically-isolated best candidate target
genes will be tested functionally using in utero electroporation of shRNAs in WT embryos. Functionally validated candidates will
then be tested for rescue of synaptic deficits in ND2 KO mice using gain-of-function in utero electroporations.
3. Determine the time window after a neuron’s birth where ND2 deficiency induces synaptic defects. We will breed
ND2flox;ROSA-tdtomato mice with nestin-Cre, DCX-Cre and Camk2-CreERT2 mice in order to delete ND2 in early progenitors,
post-mitotic migrating neuroblasts or mature neurons, respectively. Injecting tamoxifen to Camk2-CreERT2 mice at E18, P30 or
P120 will allow us to delete ND2 in neurons of different ages. For all conditions we will measure synaptic integration of
neocortical PN using aforementioned approaches.
Expected results
We expect that both structural and functional synaptic plasticity will be altered in KO PN, that ND2 target genes are related to
synaptic plasticity, and that ND2 is important during development for spine formation and in adults for experience-dependent
spine remodeling.
Feasibility
We already successfully implemented 2-photon live imaging of cortical synapses in the motor cortex and will teach the student
this sophisticated technique. Antoine de Chevigny is also expert in FACS and RNAseq. Pascale Chavis, DR2 INSERM, is a world
expert in cortical electrophysiology and in particular in functional synaptic plasticity (LTP, LTD).
Expected candidate profile
We are looking for a highly motivated individual who will work as member of a multidisciplinary group working at the interface
of mouse genetics, molecular/cellular neurodevelopment and electrophysiology. Good writing and communication skills are
required. The candidate shall be a constructive team player who thrives in collaborative environments.
Research project # 14
Title: Decoding movement goals and muscle activation patterns from superficial and deep motor
cortical layers in non-human primates – determining the suitability for brain machine interfaces.
Supervisors: Bjørg Elisabeth KILAVIK and Demian BATTAGLIA
Laboratories: Institut de Neurosciences de la Timone & Institut de Neurosciences des Systèmes
http://www.int.univ-amu.fr/_KILAVIK-Bjorg_?lang=en
http://ins.univ-amu.fr/research-teams/team-member/d.battaglia/
State of the art: The cerebral cortex is organized into multiple layers comprising largely distinct
distributions of incoming and outgoing anatomical projections. In motor cortex, the superficial layers
receive the majority of feed-forward sensory inputs and mainly project to local and distant cortical
regions. The deep layers project to sub-cortical regions and the periphery (muscles). It is not clear which
of these layers is the better target for Brain Machine Interface (BMI) applications aiming at driving
screen cursors or robotic prostheses via the decoding of motor plans and commands from motor cortical
activity. While one study in rodents proposed to target deep layers (Parikh et al. 2009 Neural Eng
6:026004), preliminary results obtained in non-human primates (NHP) in our and another lab suggest
stronger and earlier movement goal information in superficial layers (Kilavik 2016 AREADNE abstract;
Chandrasekaran et al. 2015 SfN abstract).
Objectives and methods: We will start by determining the availability of information about movement
goal vs. muscle activation patterns in different motor cortical layers, by doing offline decoding of
existing laminar data recorded in NHPs. In parallel, we will build an online BMI setup for further
experiments, coupled with a NHP upper limb exoskeleton already on site. This setup will be combined
with the use of novel implantable laminar probes, which will provide stable long-term recordings from
multiple channels in superficial and deep motor cortical layers, necessary for testing online BMI
protocols. Different behavioural paradigms will be explored, including (i) brain control of the
exoskeleton motors to control passive, goal-directed arm displacements with screen-cursor feedback
during temporal arm-muscle paralysis and (ii) direct brain control of the screen-cursor by bypassing the
exoskeleton motors, to determine suitability of neuronal activity in superficial and deep motor cortical
layers for different BMI protocols.
We will first use information theory approaches to systematically compare the amount of information
relevant for different applications that can be potentially extracted from different laminar depths
(superficial vs. deep) and/or types of signal (spike trains, local field potentials). Based on these offline
analyses, we will then design optimized fast-to-compute features with good generalization properties,
which are suitable for online decoding (e.g. via feed-forward artificial networks, fuzzy decision systems
or decision/regression trees).
Expected results: We will determine the laminar distribution of information concerning movement
goals and muscle activation pattern during visually cued, self-generated reaching movements. We will
compare the respective contributions of neuronal signals in superficial and deep motor cortical layers,
designing optimized features for online decoding and closed-loop control of BMIs, in the absence of
self-generated muscle activation. These results will allow us to determine the optimal motor cortical
depth for implanted invasive electrodes to harvest signals for motor BMI applications.
Feasibility: NHPs are the choice model for better understanding human cortical sensorimotor functions,
due to their superior phylogenetic proximity compared to other animal models, including rodents.
Particularly relevant for the current project is the similar capacity to perform complex sensorimotor
tasks, the similar projection patterns from motor cortex to the spinal cord, and the similar proportional
thicknesses of different cortical layers. The englobing experimental research program is ongoing, and
essential equipment is already in place. When the student arrives, data is already available for analyses,
and recordings will immediately start in a newly trained animal. The two supervisors are highly
complementary, with renowned expertise in experimental (Kilavik) and computational (Battaglia)
approaches, and can therefore provide a solid support for the student to succeed in this highly
demanding project.
Candidate profile: Candidates with a background in biology, neuroscience, physics or engineering are
particularly encouraged to apply. The work will combine experimental and computational approaches,
with adequate supervision from the highly complementary supervisors. This project provides an
excellent opportunity to gain solid multidisciplinary training at the forefront of integrative
neurosciences.
Research project # 15
Title: Rationalized search of biomarkers for neurodegenerative diseases
Supervisor: François Devred
Laboratory: Plateau Microcalorimétrie Timone, CRO2
http://pharmacie.univ-amu.fr/plateau-microcalorimetrie-timone http://www.cro2-timone.fr/
State of the art: Diagnosis and management of diseases of the central nervous system have
always been challenging for clinicians due to difficulty of direct analysis of the affected tissue.
Indeed, while biopsies of brain or spinal cord would be very informative, they are often not
possible due to functional consequences for the patient. For this reason, significant efforts are
currently being directed towards discovering novel non-invasive ways to diagnose and follow
neurological disorders. We have recently shown that differential scanning calorimetry (DSC), a
thermodynamic method that registers protein denaturation, could be used to obtain diseasespecific signatures from blood plasma of patients affected with Glioblastoma, ALS,
Alzheimer’s (AD) or Parkinson’s disease (PD). These signatures correspond to the combination
of denaturation profiles of only a few abundant proteins circulating in plasma. A given diseasespecific pattern could therefore be due to alteration of a particular protein by a mutation,
chemical or post-translational modification, or by ligand binding. Identifying these precise
modifications will lead to the discovery of novel long-sought biomarkers of the disease.
Objectives: Our primary objective is to develop a new methodology for rational fishing of
biomarkers in plasma from patients with neurodegenerative diseases (ALS, AD or PD).
Methods: Biophysics: Differential Scanning Calorimetry (DSC), Differential Scanning
Fluorimetry (DSF), ThermoShift Analysis (TSA), MALDI and Electrospray Mass
Spectrometry, UV/Visible/Fluo Spectrophotometry. Biochemistry: plasma sample preparation
using
protein
depletion
kits,
HPLC.
Expected results: First, we expect to identify the protein(s) which are responsible for the
specific denaturation signature of ALS, Alzheimer’s or Parkinson’s disease. Second, detailed
analysis of these proteins using mass spectrometry will lead to the identification of specific
molecular changes or new partners, which would constitute future biomarkers.
Feasibility: Feasibility of this approach has already been evaluated and validated for
glioblastoma plasma (ARC, Emergence Canceropole, Gefluc). Moreover, thanks to a Region
PACA grant, we have recently installed a new automatic system for plasma analysis. Some
aspects of this project were described in our recent patent application. This bench-to-bedside
project is supported by synergy between biophysists and clinicians. Indeed, our team, which is
equipped with all the necessary instruments for this project, is working in tight collaboration
with the clinical teams of La Timone Hospital that is providing biological samples of patients.
The PhD student will thus benefit from a nourishing and stimulating scientific environment.
Expected candidate profile: Candidate should have a solid background in biophysics and
biochemistry and should be motivated to work in a multi-disciplinary environment.
Relevance to Integrative or Clinical Neuroscience: This project has both fundamental and
clinically-relevant aspects as it will improve our knowledge of neurodegenerative diseases and
hopefully provide new tools for monitoring and/ or diagnostic of these diseases.
Research project # 16
Title: Take an another step forward to regain the ability to walk.
Supervisor: Frédéric Brocard
Laboratory: Institut de Neurosciences de la Timone, UMR 7289
Introduction: There are about 35,000 and 259,000 patients with spinal cord injury (SCI) in
France and USA that primarily affects young adults, respectively. Car accidents are a common
cause of SCI - where the spine breaks exerts pressure on all or a part of the spinal cord;
however, there are a number of other causes. Sports injuries, falls, and gunshot wounds are
other causes of SCI. Unfortunately, the SCI strongly impairs the walking ability because the
motor network responsible for locomotion is located below the spinal cord lesion. We have
recently demonstrated in the spinal cord of rodents a new class of neurons called pacemakers
which are critical in generating the locomotor activity (Brocard et al., Neuron, 2013). These
pacemakers are activated by a flux of sodium ions named the persistent sodium current (INaP).
Our recent work in Nature Medicine pointed out a molecular mechanism responsible for the
dysregulation of the INaP (Brocard et al., Nature Medicine, 2016) but the impact on both
pacemaker cells and into the operation of the spinal locomotor network remains fully unknown.
Aims: The objectives will be to: 1) examine which of the two major sodium channel isoforms
within the spinal cord (Nav1.6 and Nav1.1) is the molecular determinant for INaP which is
critical for both pacemaker and locomotor activities 2) characterize the dysregulation of the
sodium channel expression and the related INaP after SCI 3) restore the locomotion by targeting
INaP and the expression of the sodium channel (identified in aim 1) by means of genetic tools.
Relevance: This project represents an important step towards a mechanistic understanding of
the organization of the neural circuits forming the spinal locomotor in mammals in normal
condition and after SCI. Furthermore the project aims at improving the quality of life of
patients by reducing motor disorders with an original, effective, tolerable and minimally
invasive treatment designed by gene therapy.
Methods: We routinely perform in vitro and in vivo recordings (intracellular patch-clamp,
calcium imaging, electromyograms…), molecular biology and confocal microscopy for the
analysis of immunolabelings. Our laboratory also holds cutting edge technical platforms
including a two photon imagery to perform calcium imaging experiments and a biosafety
containment level for manipulation of genetic tools.
Profile: The student should have a basic knowledge in Neurosciences and Neurophysiology,
should be motivated, enthusiastic, curious, eagerness to learn and to have a strong capacity to
integrate a dynamic team.
Research project # 17
Title: The neural basis of automatic and flexible execution of learned motor sequences.
Supervisor: David Robbe
Laboratory: Institut de Neurobiologie de la Méditerranée. INSERM UMR901
State of the art. A challenge that animals are constantly facing is to perform learned motor sequences
in environments that can change in unpredictable manners. An extreme but illustrative example would
be that of a tennis player, who has learned to execute a certain number of shots (service, forehand,
backhand, ….) with well-defined kinematics, but in practice these kinematics must be adjusted
according to a wide range of conditions (the exact speed and position of the ball, presence of wind,
position of the opponent, ...). In other words, in term of motor performance, animals can not just rely on
automatism but must also incorporate a flexible control on their movements. For experimental reasons,
the understanding of the interplay between automatic and flexible control on behavior has been mainly
approached through the angle of the psychology of decision making (Balleine & O´doherty, 2010), in
which habitual and goal-directed decisions are assumed to compete and depend on different brain
regions, respectively the sensorimotor and associative corticostriatal systems. However, the algorithm
(and their neural implementation) underlying automatic versus flexible motor control are largely
unknown.
Objectives. The goal of this thesis project is to better understand the relation between automatic and
flexible motor control. One general hypothesis will be tested: Do automatic and flexible motor
control rely on two competing independent subsystems operating at the level of the sensorimotor
and associative striatum? This will be done by combining two approaches: First a psychophysical
approach (Objective 1) to describe the motor performance dependency and limits of rats trained and
challenged in a task designed to manipulate the extent to which a motor sequence can be performed
automatically. Second, a neurophysiological approach (Objective 1) in which large-scale recording of
neuronal activity and perturbation will be performed in the dorsomedial and dorsolateral striatum of rats
performing a learned motor sequence in contexts favorable to either automatic or flexible motor control.
Methods. We will take advantage of an orginal motor task in which rats run on a motorized treadmill to
obtain rewards (Rueda-Orozco and Robbe, 2015). The rules for obtaining rewards and the fact that the
treadmill speed is fixed favor the generation of a stereotyped running sequence with well defined
kinematics. We can manipulate the speed of the treadmill (e.g., trial by trial random variation, closeloop change of speed depending on the animals behavior ...) to force the rat to adjust its kinematics (i.e.,
be flexible) to maintain optimal performance and reward delivery. Thus we can control precisely the
degree of motor automatism versus flexibility and performed psychophysical tests of motor
performance. We have also developed precise excitotoxic lesions of striatum subregions and massive
tetrodes recordings of spiking activity. Both techniques are fully compatible with the treadmill task
(e.g., lesions do not impair basic locomotion or motivation). Finally most of the data analysis (behavior
and neural data) is already implemented in an efficient Python pipeline.
Expected results. With this project we aimed at uncovering the algorithm (or strategy) used by animals
to controls their learned movements in changing environments. In addition the neural implementation of
this strategy and the specific contribution of different regions of the striatum will be addressed. This
project if successful can have clinic impact in the field of motor recovery and degenerative diseases.
Feasibility is high because all the methods are already in place in the team. The experiments combine
purely behavioral experiments with a range of neurophysiological methods (lesion are straightforward
while tetrodes recording are very challenging). The diversity of approach allows to guarantee
publications. For instance a purely descriptive but highly informative behavioral paper is expected at the
end of the first year.
Profile. Biology, computer science or physics (engineering and programming skills are a plus).
Research project # 18
Title: Fronto-temporal interactions in cerebral voice processing
Supervisor: Pascal Belin
Laboratory: Institut de Neurosciences de la Timone
o State of the art Neuroimaging studies have revealed the existence of “temporal voice areas”
(TVAs) in the primate cerebral cortex, areas of higher-level auditory cortex with particular
sensitivity to conspecific vocalizations. The TVAs play a central role in cerebral processing of
voice information (identity, emotion, …) yet they are just one part of a wider “vocal brain”, a
distributed network of voice-sensitive areas. Prominent amongst the nodes of this network are
several prefrontal cortical areas, but the functional role and the interaction of these areas with
the TVAs during voice processing remains poorly understood.
o Objectives This PhD project will investigate and characterize the functional neuro-anatomy
of the prefrontal areas involved in cerebral voice processing in humans and non-human
primates, focusing on information transfer between these prefrontal areas and the voicesensitive TVAs of auditory cortex.
o Methods The student will first perform a literature review in the form of a quantitative metaanalysis of relevant neuroimaging studies resulting in a set of testable hypotheses. He/she will
then use advanced effective connectivity measures to characterize fronto-temporal interactions
in large, already existing functional magnetic resonance imaging (fMRI) datasets acquired in
humans and macaques during auditory stimulation with conspecific and heterospecific
vocalizations, and test the hypotheses generated by the meta-analysis. He/she will then perform
a more robust test of these hypotheses by designing, performing and analyzing a combined
fMRI-transcranial magnetic stimulation (TMS) study: key prefrontal areas will be individually
localized via fMRI in human volunteers performing specific voice perception tasks, and TMS
used to transiently interfere with the neuronal activity of these regions in order to causally test
their functional role in voice processing.
o Expected results This PhD project is expected to considerably clarify the functional role and
anatomo-functional organization of prefrontal areas involved in the processing of voice
information. Revealing the anatomical and functional connectivity between these prefrontal
areas will increase our understanding of how voice information is processed and exchanged by
these cortical areas. The comparison of macaque and human data will bring a comparative
perspective to this investigation, testing whether the observed pattern is unique to humans or
more widely shared amongst primates. Three publications with the PhD student as first author
are expected to result from this work, one for each of the three main phases of the planned
work (meta-analysis; mega-analysis; TMS-fMRI experiment).
o Feasibility The La Timone Neuroscience Institute in Marseille offers an excellent
environment ideally suited to perform the proposed project, including access to a lastgeneration 3T MRI scanner (Siemens Prisma) and primate facilities. The student will join the
team “Neural Bases of Communication” composed of 5 permanent researchers and 8-10
students from Master to postdoctoral level. The proposed supervisor, Pascal Belin, has gained
international recognition by his pioneering work on the cerebral bases of voice cognition, and
has solid mentoring experience.
o Expected profile of the candidate The successful candidate will have excellent background
in Biology, Psychology, or Computing Science. Strong programming skills constitute an asset.
Research project #19
Title: In vivo assessment of neuro-axonal degeneration in multiple sclerosis patients using
diffusion MR spectroscopy at 7T
Superviseurs : Jean-Philippe Ranjeva / Wafaa Zaaraoui
Laboratory: CRMBM-CEMEREM, http://crmbm.univ-amu.fr/Systeme-nerveux-central-Homme?lang=en
State of the art: Multiple sclerosis (MS) is a chronic disease of the central nervous system representing the
leading cause of neurological disability in young adults. This disease is characterized by inflammation,
demyelination and neurodegeneration. Among these pathological processes, neuro-axonal loss is the main
substratum responsible for permanent and irreversible disability. To date, non-invasive assessment of neuroaxonal loss is based on atrophy measurement using MRI. While this parameter is widely used, especially in
clinical trials, it reflects final endpoint of irreversible neuro-axonal loss. Furthermore, due to the fact that
there are no standards for brain volume, individual atrophy measurement can only be estimated
longitudinally which takes several years or even decades. Thus, atrophy is not suitable to assess ongoing
neuro-axonal loss, needed to manage therapeutic strategies to prevent neurodegeneration.
Recent data indicate that mitochondrial injury and subsequent energy failure are key factors in the induction
of neuro-axonal loss. N-acetyl aspartate (NAA) is the second most frequent metabolite within the brain.
Simmons et al demonstrated by immunohistology that NAA is located in neurons and axons in brain, and
hence is regarded as a marker of neuro-axonal function [1]. Despite that NAA was identified almost 50 years
ago, its function in the brain is still obscure. Mitochondria in axons and neuronal cell bodies contained higher
levels of NAA compared to the cytosol, compatible with synthesis of NAA in mitochondria. From neurons,
NAA is released to the extracellular space and taken up by oligodendrocytes. In the latter cells NAA is
converted back to aspartate and acetate that generate lipids for maintaining myelination.
Magnetic resonance spectroscopy (MRS) is a powerful non-invasive method to assess in vivo brain
metabolites. The main brain metabolite detected by MRS is NAA. Nevertheless, MRS does not provide
information about brain structure. Diffusion MRI assesses the microscopic Brownian motion of water
molecules influenced by the size, orientation, and structure of the surrounding tissues, providing insights on
the brain architecture. The pathologic specificity of diffusion MRI is limited because the measured signal is
derived from water protons that are influenced by inflammation and edema in MS. Combining MRS and
diffusion MRI allows assessing diffusion of metabolites, in particular NAA, much more specific to neuroaxonal structure and also function. A preliminary study conducted by Wood et al evidenced a reduction of
NAA diffusivity in corpus callosum of MS patients, emphasizing the relevance of this approach [2].
Objectives: To assess neuro-axonal dysfunction and impaired structure integrity using the diffusion
properties of brain metabolites at the early stages of multiple sclerosis and its link to disability.
Methods and expected results: An innovative multi-voxel diffusion MRS technique will be developed on the
clinical 7T Siemens MR scanner equipping the CRMBM, in close collaboration with Pr Itamar Ronen
(Leiden University), one of the pioneer of this technique. In addition, a second challenge will be to assess
more deeply the microstructural complexity of dendrites and axons. For this, diffusion gradients of different
strengths will be applied to separate the diffusion of metabolites in brain tissue into two types of
microstructural environment: intra-neurite and extra-neurite. This technique, adapted form NODDI approach
that uses water diffusion, yields architectural markers like the fraction of tissue that comprises axons or
dendrites and the spatial configuration of the neurite structures based on their orientation dispersion index.
One may suppose that using diffusion properties of metabolites will be much more robust than water
diffusion to provide microstructural information. The development of this new technique will constitute the
main work of the PhD student. MR exams will be applied in vivo at 7T in MS patients (n=30) at the early
stages of the disease. Patients will be included at Timone University hospital by clinicians also members of
the research team (27 persons).
Feasibility: Patients inclusion and clinical evaluation will be performed at Timone hospital by clinicians with
a high expertise in MS and clinical research (more than 150 published papers). To face the physical
challenges, the PhD student will benefit from the recognized expertise in MR physics of her/his supervisors
(JPR, WZ in CRMBM, lab funded 25 years ago and specialized in medical applications of MR techniques).
Expected candidate profile: Highly motivated candidate holding a Master degree in Neurosciences,
Biomedical Engineering or a related discipline with the following skills: Excellent communication skills/
Ability to work in a team environment/ Experience in programming (C++, MATLAB) will be appreciated.
Ref: [1] Simmons et al, Neuroscience 1991 ; [2] Wood et al, J Neurosci 2012.
Research project # 20
Title : On Uncharted Space In Animals (OUSIA)
Superviseur : HOK Vincent & SARGOLINI Francesca
Laboratory : Laboratory of Cognitive Neurosciences (UMR7291), http://lnc.univ-amu.fr/
State of the art: One of the most studied neurophysiological models of spatial cognition is the
hippocampal place cell system. Place cells are pyramidal neurons selectively activated when the
animal is in certain regions of the environment (place fields). The spatial organization of place
fields across a population of place cells is characteristic of a given environment. Additionally,
hippocampal place cells show sequential reactivations during sleep, a phenomenon called “replay”.
During replay events, hippocampal place cells that fired during exposure to an environment are
orderly reactivated so that the initial experience is recapitulated over a very brief period of time, a
process thought to reflect an offline consolidation mechanism. Recent studies investigating the role
of visual areas in spatial cognition showed a clear place selectivity of primary visual cortical (V1)
neurons in freely moving rodents and, more importantly, showed that these neurons displayed also
robust replay events. In line with these previous results, we found numerous place cells in the
dorsal Lateral Geniculate Nucleus (dLGN – thalamic structure located one synapse upstream of the
primary visual area) showing coding properties that were thought until then hallmarks of
hippocampal function. Moreover, the place selectivity of dLGN cells was maintained in the absence
of visual inputs, i.e. when rats were recorded in complete darkness, thus demonstrating that these
spatially localized firing patterns, once learned, are not likely to depend upon local visual cues
within the experimental room. Thus, our results suggest that a topological representation of space is
defined very early in the brain’s processing of sensory information.
Objectives: Based on these results, we hypothesise that the dLGN supports a much more
integrative function that initially thought. The objective of this project is thus to investigate a new
brain functional network (including V1, dLGN and Superior Colliculus) that we believe to be
instrumental in learning and memory processes. In particular, we will examine whether dLGN
neurons show replay events. We will also investigate inter-structural functional interactions by
inactivating V1 or SC and observe these effects on dLGN neuronal activity.
Methods: In order to address these questions, we will characterize more precisely dLGN place cell
specific properties and investigate brain connectivity networks that underlie memory formation
using behavioural, pharmacological and electrophysiological analyses.
Expected results: If replay is observed in dLGN, this would mean that this structure, a key
component in visual processing, would indeed perform computations largely independent of pure
sensory inputs. Our current view is that the dLGN acts as the true mapping structure of the
navigation system, representing space as a passive computation, while the hippocampus supports an
active spatial process involved in route planning.
Feasibility: These experiments require to record single unit and population activities in dLGN and
to inactivate V1 and/or SC with fluorescent muscimol (optogenetics approach will be considered
later on in this project). All these techniques have been used successfully in the laboratory and
should not present any difficulty.
Expected candidate profile: Candidates should have in-depth training in all aspects of the
integrated research, at both the conceptual and technological levels in a range of modern methods
and technologies, including stereotaxy, single cell recording in vivo, local field potentials and
oscillations recording in vivo and behaviour analysis.
Research project # 21
Title: Do adolescents use the same mechanisms to process adults’ and aged-matched’ social signals?
Supervisors: Marie-Hélène GROSBRAS
Laboratory: Laboratory of Cognitive Neurosciences (UMR7291), http://lnc.univ-amu.fr/
State of the art
Adolescence is characterized by important bodily, emotional, social and cognitive changes, alongside
important structural and functional brain maturation. The importance of peers in adolescents’ life is
widely acknowledged and has been studied with regards to the influence they have on individual
behaviours [1, 2]. Moreover, the rapid transformation of the appearance in body, voice and face during
adolescence is unique in the life-span. Given that the processing of social cues is still maturing [3, 4],
how do the mechanisms at play for recognising these changing signals adapt during adolescence? Could
adolescents be experts at non-verbal communication with other adolescents? Understanding this is
crucial to measure peer influence in the realm of social interactions. Yet very few studies provide clues
to these questions. Face recognition does not seem influenced by face-age. Automatic response to
affective expression is however different when teenagers are exposed to teenagers‘ faces compared to
adults’ [5]. Recent data from our group show that contrary to adults, adolescents are not more attracted
to adults’ faces than to other objects or noise patterns (Geringswald et al in prep). It is unclear whether
this would be the case for adolescents faces.
Objectives
The goal of the PhD project is to test whether adolescents process social signals transmitted by other
adolescents differently than signals from adults, and whether these stimuli engage different brain
resources.
Methods
We will implement eye-tracking and reaction-times experiments to assess developmental and stimulusage effects on automatic attraction of attention by faces or bodies. Secondly we will use functional
magnetic resonance imaging (fMRI) and multivariate analyses methods, to test whether the teenage
brain process differently information from adolescents’ or adults’ faces, bodies or voices. We will
correlate these measures with measures of resistance to peer influence [6].
Expected results
Results from these experiments will allow us to identify how age-proximity (as well as gender) affects
the mechanisms of social perception and to qualify the specificity of adolescence in this respect. We
expect to observe an enhanced interaction between age of model and behavioural effects and brain
response in adolescents. Moreover as adolescents grow older we expect this to be sex-specific.
Feasibility
This project is in direct continuation from a research project funded by AMIDEX in the context of its
junior rising star chairs (until end 2017). It is also directly related to an ANR grant (until end 2019).
Projects on adolescent brain development are currently carried out in the lab by two researchers (C.
Assaiante and myself), two post-doctoral fellows (with another application pending) and one PhD
student. Therefore the applicant will be benefit from a very strong material and collegial support.
Expected profile of the candidate
The applicant will hold a Master in cognitive neuroscience or related topic. (S)he will have a strong
interest in developmental studies, excellent interpersonal skills necessary to recruit and interact with
young participants. Experience with fMRI will be an asset.
1.
2.
3.
4.
5.
6.
Chein, J., et al., Peers increase adolescent risk taking by enhancing activity in the brain's
reward circuitry. Dev Sci, 2011. 14(2): p. F1-10.
Steinberg, L. and K.C. Monahan, Age differences in resistance to peer influence. Dev Psychol,
2007. 43(6): p. 1531-43.
Shaw, D.J., et al., Development of functional connectivity during adolescence: a longitudinal
study using an action-observation paradigm. J Cogn Neurosci, 2011. 23(12): p. 3713-24.
Shaw, D.J., et al., Development of the action observation network during early adolescence: a
longitudinal study. Soc Cogn Affect Neurosci, 2012. 7(1): p. 64-80.
Ardizzi, M., et al., When Age Matters: Differences in Facial Mimicry and Autonomic Responses
to Peers' Emotions in Teenagers and Adults. PLOS ONE, 2014. 9(10): p. e110763.
Grosbras, M.H., et al., Neural mechanisms of resistance to peer influence in early adolescence.
J Neurosci, 2007. 27(30): p. 8040-5.
Research project # 22
Title: Membrane-type matrix metalloproteinases are newly identified pathogenic factors in Alzheimer’s
disease: neuroinflammatory mechanisms of action and pre-clinical target validation
Supervisors: Santiago RIVERA and Emmanuel Nivet
Laboratory: Neurobiology of Cell Interactions and Neuropathophysiology (NICN), UMR 7259, AixMarseille University/CNRS
State of the art
Alzheimer’s disease (AD) is the main neurodegenerative disorder that affects 35 million people worldwide. In the
absence of efficient treatments AD may soon reach epidemic proportions. The search for novel targets and
therapeutic strategies is therefore among the most challenging endeavors of modern neuroscience. This implies a
comprehensive approach to study AD based on the interplay between three fundamental processes involved in
neurodegeneration: amyloidogenesis, neuroinflammation and synaptic dysfunction. We have recently discovered
that membrane type matrix metalloproteinases (MT-MMPs) constitute a new class of proteinases at the crossroads
of these processes (PDMI: 28119565, 27349644, 26202697, 25278878). Leveraging on murine models and on human induced
pluripotent stem cells (hiPSCs)-based technologies, the global objective of this proposal is to further investigate
the mechanisms of neuroinflammation driven by MT5-MMP (MT5) and MT1-MMP (MT1), and further
validate their inhibition as a potential therapeutic strategy in AD. The objective relies on the hypothesis that
MT-MMP-mediated neuroinflammation promotes amyloidogenesis and synaptic dysfunction.
Objectives
1. To evaluate the impact of MT1 and MT5 deficiency neuroinflammation and the underlying mechanisms of
action.
2. To identify and validate new substrates of MT-MMPs that could account for their pro-inflammatory actions.
3. To assess the therapeutic properties of MT-MMP modulating factors in AD cell models.
Methods
Objective 1) Upon inflammatory challenge, we will investigate the impact of MT5 and MT1 at different levels of
the signaling cascade for different AD-related cytokines. We will use primary neural cells from AD mice deficient
for MT5 or MT1, as well as hiPSCs-derived neurons/astrocytes made deficient for these MT-MMPs using
CRISPR/Cas9 technology. Of note, hiPSC-derived neurons recapitulate main AD hallmarks: Aβ accumulation and
tau hyperphosphorylation (pTau). Cell phenotypes will be rescued using lentivirus coding for MT1 or MT5. We
will measure the levels of inflammatory agents activated by IL-1β and/or other inflammatory agents,
solube/oligomeric Aβ APP metabolism  pTau, using qPCR, western blot, ELISA and immunofluorescence
approaches. Whole-cell patch-clamp recording combined with 3D imaging analysis (Imaris) of spine morphology
will be used to investigate synaptic dysfunction.
Objective 2) We will use proteomic analysis to identify new MT5 and MT1 substrates. Gain and loss of function
will be used to validate the role of these substrates in the inflammatory process, using the AD cell models
described above.
Objective 3) Ongoing research in our laboratory aims at identifying efficient modulators (i.e, small organic
molecules, nanobodies) of MT1 and MT5 activity. The candidate hits will be tested for their therapeutic/antiinflammatory properties in our murine and human AD cell models described above.
Expected results
We expect to demonstrate that MT-MMP-mediated neuroinflammation is a key factor in AD pathogenesis.
Consequently, MT-MMP inhibition/modulation may set the basis for new promising therapeutic strategies in AD.
Feasibility
All the state of the art techniques/equipment are available: transgenic AD mice, primary neural cell cultures,
genetically modified iPSCs using CRISPR/Cas9, lentiviral constructs, qPCR, 3D confocal/super-resolution
microscope, qPCR, patch-clamp set-up. The proposed project results from the collaboration between two teams
with recognized complementary skills in the fields of AD, proteinases and cell-based reprogramming approaches.
The teams are part of the Network of Centres of Excellence in Neurodegeneration (CoEN). The project is
supported by French and European funding agencies.
Expected candidate profile
We are looking for an enthusiastic individual who can work both independently, as well as in a group setting. The
successful candidate is capable of conceptual thinking, is looking forward to learning new techniques, has
excellent communication skills, and is a hard-working and well-organized person. Knowledge of molecular
biology, CRISPR/Cas9, and cell culture is advantageous. Also beneficial is experience with knockdown/overexpression systems, cell imaging and/or cell trafficking.