Adult neural stem cells reside in discrete neurogenic niches in the adult mammalian brain and generate newborn neurons throughout life. Neural stem cells can shuttle between mitotically active or quiescent states. Quiescence may play an essential role in preserving the proliferative capacity of stem cells and limiting the accumulation of genomic mutations by minimizing rounds of DNA synthesis. It is therefore important to understand the mechanisms regulating quiescence of neural stem cells in vivo. Recent studies showed that phosphorylation of the histone variant H2AX, which facilitates the repair of DNA double strand breaks, restricts proliferation of embryonic stem cells and other stem cells. I hypothesize that niche factor-induced active phosphorylated H2AX (γ-H2AX) in nucleosomes maintains the quiescence of adult neural stem cells via an epigenetic mechanism. I will determine the expression of γ-H2AX in different neural stem cell lineages by immunohistology. Clonal analysis will be performed in individual adult neural stem cells with conditional deletion of H2AX to uncover the role of H2AX in controlling the quiescence, self-renewal mode and fate choice of neural stem cells in the adult mouse brain in vivo. I will further examine how GABA, a key niche factor, regulates H2AX activity via optogenetic manipulation of specific interneuron subtype and genetic manipulation of specific GABAA receptor. Moreover, I will investigate the role of H2AX in epigenetic regulation of gene expression with chromatin immunoprecipitation sequencing (Chip-seq) to map the γ-H2AX-occupied DNA sequences and RNA-seq analysis of gene expression in quiescent adult neural stem cells.

This proposal focuses on understanding fundamental properties of actin-like MreB cytoskeletal filaments in prokaryotes. How is the MreB filament remodeled during the cell cycle and differentiation? What are the specific changes of MreB organization and dynamics that allow different growth rate under varying environments? What are the molecular mechanisms and regulators by which the cytoskeleton mediates cell envelope biogenesis and possibly establish other spatial organization within cells? Due to the large number of players involved and functional redundancy in the system, the answers to these basic biological questions remain elusive. We propose to address these questions in the model gram-positive bacterium Bacillus subtilis by employing an interdisciplinary approach combining genetics, genomics, biochemistry and advanced imaging (e.g. single molecule microscopy). The ease with which biochemical and genetic analysis, as well as advanced imaging can be carried out in B. subtilis makes it an ideal system to uncover the molecular mechanism of spatial and temporal regulation of MreB dynamics and organization. We propose to complement these by developing a microfluidic device and computational imaging analysis tools to : 1) Characterize the subcellular patterns of MreB filaments throughout cell cycle with high temporal and spatial resolution in live single cells. 2) Identify components of the regulatory network responsible for modulating MreB assembly. 3) Determine how the complex web of interactions amongst the factors involved in MreB assembly and biogenesis of cell envelope dictate cell elongation and division in different environments.

The neocortex is organized into neural circuits that perform distinct computations, from sensory processing and motor control to memory, learning and language. In these circuits, excitatory and inhibitory neurons are extensively interconnected and the balance between their activities is tightly regulated. Dynamic modulation of cortical states, associated with rapid changes to the excitation/inhibition (E/I) balance, is thought to allow the brain to rapidly respond to changes in behavioral demands. Yet, little is known regarding the mechanisms responsible for rapid modulation of the E/I balance during behavior. Chronic changes to the E/I balance, occurring during early development, were proposed to dramatically alter the structure and function of cortical circuits and give rise to cognitive and behavioral impairment associated with neuropsychiatric disorders. Yet, the impact of such changes on cortical organization has not been directly tested. This research proposal is aimed at elucidating the mechanisms responsible for dynamic modulation of the E/I balance in the prefrontal cortex, a cortical region involved in working memory, attention and goal-directed behavior. Using a novel opto-physiological recording system, we will study the rapid behavioral state-dependent dynamics of prefrontal E/I balance and its regulation by naturally occurring neuromodulation. We will further examine the changes in cortical circuit organization and function resulting from chronic alterations in the E/I balance. These projects address fundamental aspects of cortical function and will be valuable in determining the causal role of E/I balance changes in neurodevelopmental disorders.

The kinetics of neurotransmitter release determines the duration of the postsynaptic response, and therefore has major implications on synaptic strength, circuit development, and cognition. Recent studies also suggest that mutations altering the kinetics of presynaptic release can confer risk for autism spectrum disorders (ASD). Here I propose a comprehensive set of genetic, cell biological, and biophysical experiments to define mechanisms regulating the kinetics or neurotransmitter release at synapses.
Synaptic transmission comprises fast and slow modes of neurotransmitter release, which evoke post-synaptic responses with correspondingly different durations. However, relatively little is known about the molecules and pathways that regulate each form of release. Previous work suggests that the syntaxin-binding protein Tomosyn/TOM-1 might be a selective inhibitor of slow release. Therefore, I propose three Aims for this project, using C. elegans as a model system: 1) I will test several candidate genes for their ability to regulate TOM-1 function; 2) I will analyse how each of these factors regulate TOM-1 activity, and test for four possible mechanisms: altered binding of TOM-1 to SVs, altered dissociation of TOM-1 from SVs, changes in formation of TOM-1 SNARE complexes, and altered TOM-1 expression and synaptic abundance; 3) I will perform a genetic screen to identify novel TOM-1 regulators, and analyse how they inhibit TOM-1 activity as described in Aim 2.
These experiments will provide new insights into the cell biological mechanisms regulating synaptic transmission and plasticity, as well as mechanisms underlying the changes in cognition and development in ASD.