The balance between excitation and inhibition is critical to the proper function of neural circuits. Aberrant activity is characteristic of numerous neurological disorders, including autism spectrum disorder, Rett syndrome, schizophrenia, and epilepsy. Inhibitory interneurons are embedded in almost all central neuronal networks where they act to influence cell excitability, spike timing, synchrony, and oscillatory activity. GABA, the main inhibitory amino-acid neurotransmitter in mature neurons, is a remarkably multi-functional neurotransmitter: it can bind to either ionotropic GABAA (mediating fast neurotransmission) or metabotropic GABAB receptors (mediating slow neurotransmission) that may be localized extra-, peri-, pre- and postsynaptically. The GABAergic phenotype in vertebrates and invertebrates has been defined classically by the presence of three key players in the presynaptic neurons: (i)glutamic acid decarboxylase (GAD), the enzyme needed to synthetize GABA from glutamate, (ii)the H+-coupled transporter (VGAT) that packages GABA in synaptic vesicles, and (iii)the Na+-coupled transporter (GAT) that recaptures GABA at the nerve terminal after its release in the synaptic cleft. The Caenorhabditis elegans nervous system can be considered as a “microcosm” of the GABA universe as it is much smaller, simpler, and experimentally more accessible than a vertebrate nervous system. For over 20 years, the C. elegans GABAergic nervous system was thought to be composed of only 26 out of the total 302 neurons. However, during my post-doc, I have performed an in-depth revision of the GABAergic nervous system in C. elegans. After optimizing immunohistochemistry techniques in C. elegans for GABA staining and generating several fluorescent reporters, I have significantly given new perspectives on what really define a GABAergic neuron in this model organism. In particular, my work has shown that additional neurons contain GABA but do not always express GAD/unc-25, VGAT/unc-47 and GAT/snf-11, the landmark gene portfolio for classical GABAergic neurons. Indeed, I have identified 22 new GABA-positive cells that do not conform to this classical definition and can be categorized into 4 different types of neurons expressing different combinations of these factors. Two of these types show evidence of alternative modes of GABA transport because they lack expression of known GABA transporters, VGAT/unc-47 and/or GAT/ snf-11, and they do not synthetize GABA. Moreover, in vertebrate dopaminergic neurons, similar observations hint towards the presence of alternative mechanisms for GABA transport too Deciphering these new mechanisms of GABA transport will shed light into the regulation of neural circuits through inhibition. I propose to first take advantage of C. elegans, a powerful genetic model organism, to identify and characterize new presynaptic determinants of the GABAergic neurotransmission, focusing mainly on putative and known transporters. Then, we will test their vertebrate orthologues given that the already known components are very well conserved between mammals and worms. New function for already characterized vertebrate transporters could be uncovered as it happened for the glutamate vesicular transporter 1 (VGLUT1) alias BNPI. To achieve this goal my research project will be organized around two aims: AIM1-Novel actor(s) for GABA packaging (Identification and characterization of alternative GABA transporters for packaging GABA in synaptic vesicles) and AIM2-Novel actor(s) for GABA reuptake (Identfication and characterization of new GABA transporters at the plasma membrane, that provide an alternative supply mechanisms for GABA) Altogether, this project aims to extend our knowledge of the cellular mechanisms underlying inhibitory neurotransmission.

The project proposes a revolutionary method to characterize DNA replication on single molecules. Eukaryotes replicate DNA by activating numerous replication origins that each establishes two divergent replication forks that perform bi-directional DNA synthesis with variable speeds. Despite the advent of DNA micro-array and massive DNA sequencing technologies, origins remain difficult to identify and our knowledge is still incomplete. Solving the nature of origins and analyzing replication progression require high-throughput analyses at the single molecule level because cell-population methods provide only an average picture where inter-cellular variability and rare events are masked in the average. State-of-the-art single molecule methods monitor the incorporation of non-standard nucleotides during replication. However, they provide no sequence information unless they are combined with DNA probes. This is laborious, low resolution and low throughput. High-throughput single molecule genomic profiling of DNA replication remains to be achieved. This challenge can be attained using nanopore sequencing as the monitoring apparatus of non-standard nucleotide incorporation. Sequencing precisely localizes the analyzed molecules on the reference genome and promises high-throughput. Nanopore sequencing is an emerging technology where ionic current through a bioengineered nanopore is recorded during the transit of a single-stranded DNA molecule through the pore. The nucleotide sequence of the molecule is then determined from the resulting current signal variations thanks to dedicated signal processing tools. We carried out a pilot project providing a proof of concept that available nanopore technology sensitivity is sufficient to carry out the project. The main scientific barrier to be lifted is thus to make a nanopore current analysis pipeline discriminating at least one of the non-standard nucleotides compatible with DNA replication studies, in addition to the four canonical nucleotides. We will develop an open-source signal processing pipeline for nanopore current analysis concomitantly with experimental validations and applications. We will follow neural network based approaches that are currently the most efficient methods in the field. The first objective is to detect replication tracks resulting from one short pulse (1-2 minutes) of bromo-desoxyuridine (BrdU), a thymidine analog, allowing to measure replication initiation and progression much more precisely and at higher throughput than the state-of-the-art but also to immediately identify the genomic loci where replication events take place. The second objective is to make the technology also sensitive to replication progression orientation. We will tackle this challenge using two consecutive pulses of BrdU of different concentration and developing the analysis pipeline that can discriminate them. The spatial order of low and high BrdU incorporating regions will provide the replication progression orientation at each pair of BrdU tracks. NanoPoRep project will determine the distributions of single replication fork speed and orientation at all points in the genome of yeast and human cells with a high resolution, and characterize DNA replication stochasticity. NanoPoRep will question the relationship between replication progression and chromatin organization and provide maps of potentially asymmetric replication barriers. The final product delivered by NanoPoRep will both contain software usable by other teams to directly analyze nanopore replication datasets and a programming suite allowing readily adaptation to other experimental settings (different non standard nucleotide or nanopore). NanoPoRep will thus contribute to the emergence of nanopore sequencing as a technological breakthrough for biological assays and diagnostic tools and thus help to respond to challenges in biology and human health.

Transposable elements (TEs) are now widely recognized as major contributors to genome evolution, yet the processes governing their accumulation remain elusive. Mating systems are expected to play a central role, but the net effect of the shift from outcrossing to selfing, which occurs commonly in plants, is not known. Here, we propose to determine the genomic landscape of recent TE insertions in the two outcrossing species Arabidopsis lyrata and A. halleri and to compare it to that of their selfing close relative A. thaliana as well as to that of North American A. lyrata populations that are currently experiencing a shift to selfing. This project is made possible by the advent of long reads sequencing technology, which enables the unambiguous characterization of TEs and other repeat sequences as well as of the complex regions in which they tend to cluster. Specifically, we will produce 100 high-quality genome sequence assemblies for A. lyrata and A. halleri. We will also compare the intensity of natural selection acting on new TE insertions between these three species. Finally, we will analyze the patterns and rate of accumulation of TEs in a key region of the genome, the S-locus, which controls self-incompatibility in outcrossing species. This region shows analogy to sex chromosomes because of a lack of recombination, very low sequence homology among self-incompatibility haplotypes, and high rate of TE accumulation. Our findings should provide major insights into how mating systems impact the accumulation of TEs. This project should also elucidate the role of TEs in driving the evolution of the mating system itself in the Brassicaceae through their rapid accumulation at the S-locus and their potential role in the generation of small RNAs controlling the dominance relationships among S-alleles. The methods and paradigms we will generate should have broad implications for the study of species with high level of heterozygosity and much larger genomes, including humans.

The brain is the most complex organ. Each area of the brain is made up of an intricate network of neurons linked together by synapses, points of contact between neurons. The proper functioning of the brain depends on the correct specific connectivity between subtypes of neurons, usually the axon of a presynaptic neuron and the soma, dendrite, or dendritic spine of a postsynaptic neuron. In the mammalian central nervous system, a typical part of the neuropil contains dozens of synapses per µm3 from many different neurons. The majority of central synapses (~80%) are excitatory, using glutamate as a neurotransmitter, while ~10-20% of synapses are inhibitory (using γ-amino butyric acid (GABA) or glycine as a neurotransmitter). The remaining small minority of synapses are modulatory, using for example dopamine, serotonin or norepinephrine. However, within these neurochemical categories, a great diversity results from differences in the size, efficiency and variability of synaptic transmission, as well as their ability to experience plasticity. Such diversity occurs between neuron types but also within single neurons connecting different types of neurons. Synaptic function depends on a wide repertoire of genes that control adhesion between pre- and post-synaptic elements, the release of neurotransmitters from synaptic vesicles, the type and location of post-synaptic receptors, and the signaling complexes leading to plasticity. Ultimately, knowledge of the molecular composition of synapses would allow us to predict their functional diversity. Moreover, this knowledge is necessary to understand the alterations undergone in genetic diseases affecting the brain or to define therapeutic strategies targeting specific neurons. To further understand the diversity of synaptic function and its alterations in disease, it is essential to combine several state-of-the-art methodologies to identify specific types of neurons, their connectivity with other types of neurons and the morphological, molecular and functional properties of their synapses. We propose to train the next generation of scientists in this field by bringing together a consortium of expert groups, either from leading academic institutions or from thriving biotechnology companies, with expertise in one or more methodological approaches needed to address synaptic diversity: labeling and purification of specific synaptosomes for proteomic analysis (DP/EH, JDW), imaging of living cells at synapse level (CC, DP, SH, ZN), role of adhesion proteins in synapse specification (CC, DKB, JDW), electrophysiology (all groups), in particular pre- and post-synaptic elemental records (SH), electron microscopy and array tomography (ZN). We will combine our expertise to address synaptic diversity in the context of specific synapses such as hippocampal and cerebellar mossy fibers (JDW, SH) and cortical excitatory synapses (JDW, DP, SH, ZN, CC), inhibitory synapses (CC, DKB, ZN), dopaminergic synapses (DP/EH, SH). In addition, the field of synapse function and diversity is at the core of current research on brain disorders, many of which have been recognized as alterations in synapse function or "synaptopathies". Training the next generation of scientists in these rapidly evolving fields will be important in developing innovative strategies for the benefit of every European.

The cerebellum (Cb) is a major brain structure with extensive connections to both caudal brainstem and rostral sub-cortical areas. The Cb is eminently known for its control of motor functions. However recent findings suggest that it could also play a central role in the regulation of emotional behaviors via its interactions with limbic structures like the amygdala. Our hypothesis is that the Cb regulates affective states by computing and adjusting the coherence between external conditioning stimuli experienced and the ensuing emotional responses. Focusing on the processing of fear information, we will examine the affective implications of cerebellar activity by: 1) studying the fear prediction error signals generated by the Cb during emotional learning, 2) establishing the neural pathways that anatomically and functionally link the Cb with the amygdala, 3) examining the synaptic mechanisms that underlie the development of fear memories in cerebello-limbic circuits.