Neuroscience Program

Huntington’s Disease – a devastating neurodegenerative condition – is caused by a defect in the Huntingtin gene, resulting in the production of alternative forms of Huntingtin mRNA and protein. This proposal will use small RNA drugs to reduce alternative forms of Huntingtin in mouse models of Huntington’s disease and determine the effect on disease features and outcomes. Findings from these studies will provide insight into the mechanisms underlying Huntington’s Disease to inform the development of future therapeutics.

During development of the human brain, neurons are forming a mature neural circuit, which requires major rewiring of synaptic connections. Developmental remodeling helps the brain integrate rewired connections, which when disrupted is linked to neurological disorders such as schizophrenia and autism spectrum disorder. The goal of this project is to identify mechanisms regulating the highly conserved process of synapse remodeling. I will be using the simple model system Caenorhabditis elegans because it undergoes a striking example of remodeling in GABAergic dorsal D-class (DD) motor neurons. Prior work on synapse remodeling has largely focused on the presynaptic side, while my preliminary work has focused on the post-synaptic domain. The Francis lab has identified dve-1 to act as a transcription factor of remodeling, specifically synapse elimination. Through bulk RNA-sequencing (RNA-seq) I have identified 2 downregulated targets of dve-1, the calcineurin-like EF-hand protein CHP1/chpf-1 and the regulatory subunit of CalciNeurin, PPP3R1/cnb-1. Preliminary data has shown a defect of synapse remodeling in both cnb-1 and chpf-1. Additionally, I will determine the functional requirement and site of action for cnb-1 and identify the contribution of calcineurin phosphatase function and the proteasome to the effect of cnb-1 on synapse remodeling. In aim 2, I will further characterize the role of cnb-1 in both synapse elimination and maintenance, while also identifying the effect of calcium binding on synapse remodeling. In aim 3 I will identify potential targets of dve-1 in the remodeling pathway by using neuron-specific RNA-seq. My proposed work will advance the understanding of remodeling and the mechanisms of regulation, potentially providing targets for future exploration.

Regulation of innate immunological self-tolerance, or the ability of cells to discern “self” from “non-self” has long been studied in the periphery in autoimmune disorders, especially in the context of nucleic acids (NA). Understanding of self-tolerance in the central nervous system (CNS), however, has not been thoroughly investigated despite expression of these NA-sensing TLRs by microglia, the primary phagocyte of the CNS. Published data from our lab highlights that microglia retain untranslated RNA transcripts from engulfed myelin for days after phagocytosis in vitro and in human multiple sclerosis patients. Based on these data, I hypothesized that these retained transcripts could aberrantly activate endosomal TLRs. I, thus, induced primary demyelination in UNC93B1 -/- mice, which lack functional NA-sensing TLRs, and observed that these mice remyelinate more efficiently than wildtype. These data suggest that signaling of NA-sensing TLRs suppresses remyelination during demyelinating disease. Several exciting questions have now arisen, which I will tackle in this proposal: 1) Is myelin phagocytosis causing aberrant endosomal TLR signaling? 2) Are microglia the primary cell type driving this response? 3) Does a specific NA-sensing TLR hinder remyelination? I hypothesize that TLR7 is aberrantly signaling in response to engulfed myelin RNAs in microglia and suppressing remyelination. To address these questions, I have acquired powerful in vivo molecular genetic tools to manipulate UNC93B1 and endosomal TLR function. I will first identify molecular pathways that are changed in microglia in vitro in response to chronic myelin phagocytosis and test whether these molecules are UNC93B1-dependent (Aim 1a). I will then determine if the UNC93B1 dependent effects that I observed on remyelination are microglia-specific (Aim 1b). Lastly, I will identify the endosomal TLR underlying these UNC93B1 effects (Aim 2). I am now in a strong position to molecularly dissect the role of NA sensing TLRs in remyelination during demyelinating disease, which has high long-term therapeutic potential.

Disease and injury cause a catastrophic loss of cognitive and motor abilities in the nervous system. Preventing neurodegeneration is critical to maintaining neuronal function. To develop effective treatments, we first need a greater understanding of the genetic and cellular mechanisms that determine whether the nervous system degenerates in response to various insults. Caenorhabditis elegans is a highly tractable model to dissect conserved molecular and cellular mechanisms. The goal of this proposal is to take advantage of the C. elegans model to identify regulatory mechanisms of axon degeneration. To reach this goal, I will apply my developing skills in detailed genetic analyses, laser axotomy, imaging, sequencing, and bioinformatics. The impact of this project is significant. In addition to providing a critical advance in understanding the fundamental mechanisms of neurodegeneration, it will also inform future therapeutic approaches that can be manipulated to protect the nervous system from degenerating.

The goal of this proposal is to dissect the molecular signaling between microglia and neurons that regulates synapse elimination in response to changes in sensory experience. Despite compelling evidence that microglia, the resident brain macrophages, play important roles in eliminating synapses in development and disease, the precise neuron-to-microglia molecular signaling that drives this process is poorly understood. I recently discovered a signaling pathway necessary for microglia-mediated synapse elimination by utilizing the well-described circuitry of the mouse barrel cortex circuit as a model to manipulate sensory experience and dampen neuronal activity. Here I found microglia robustly engulf synapses in the barrel cortex following either whisker lesioning or trimming, and that this engulfment is dependent on the microglial CX3CR1 receptor and its canonical neuronal ligand, CX3CL1, but not complement. Using single-cell RNAseq I also found that neuronal Cx3cl1 was not differentially regulated in the cortex following whisker removal, but the protease Adam10, known to cleave membrane-bound CX3CL1 into a soluble form, is increased following lesioning. Importantly, pharmacological inhibition of ADAM10 resulted in synapse elimination defects that phenocopied CX3CR1 and CX3CL1-deficient mice. These data suggest that post-translational modification of neuronal CX3CL1 by ADAM10 is required to regulate microglial synapse elimination in the cortex following whisker removal. Several exciting new questions have now arisen, which I will tackle in this proposal: 1) What is the cellular source of ADAM10 and is it localized to synapses (Aim 1)? 2) Do other subcortical synapses within the barrel circuit remodel via ADAM10-CX3CL1-CX3CR1 signaling and does this differ between whisker lesioning and trimming (Aim 2)? I hypothesize ADAM10 is derived from layer IV excitatory neurons to regulate microglia- mediated synapse remodeling and that ADAM10 signaling is specific for cortical synapse rewiring after whisker trimming and lesioning, but not for sub-cortical synapse remodeling. To test this hypothesis, I have acquired powerful in vivo molecular genetic tools to manipulate ADAM10 function in specific cells. I have also developed collaborations to learn and perform cutting-edge whole tissue clearing by iDISCO to assess structural remodeling of entire circuits. Finally, I have a strong mentoring team that includes my mentor Dr. Dorothy Schafer with expertise in microglial function within neural circuits, my co-mentor Dr. Andrew Tapper with expertise in structural and functional mapping of brain circuits, and collaborators with expertise in iDISCO. Together, I am in a strong position to molecularly dissect how ADAM10 modulates neuron-microglia signaling necessary for remodeling brain circuits. This could be highly relevant for neurodegenerative disease where microglial dysfunction, synapse loss, and ADAM10 have been implicated. In the process, I will receive training in a variety of microscopy and molecular genetic approaches that will provide a foundation for my future career as an independent principle investigator at an academic institution focused on dissecting functions for glial cells within neural circuits.

Dopamine (DA) neurotransmission is vital for behaviors such as movement and reward, as well as, cognitive functions including mood, learning and memory. Several neuropsychiatric disorders are linked to alterations in DA signaling including Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia, Parkinson's disease, and addiction. The DA transporter (DAT) is imperative for temporal and spatial control of DA signaling. DAT is located at the presynaptic terminal of DAergic neurons and facilitates the termination of DAergic transmission by rapidly clearing released DA. DAT is the primary target of addictive and therapeutic psychostimulants, which compete for DA binding and block uptake through the transporter, preventing DA clearance and leading to the hyper-locomotive and rewarding behaviors associated with drug use. Given that DAergic signaling is highly sensitive to DAT function, understanding the molecular mechanisms that control DAT function and availability is a critical missing piece of the puzzle in understanding DAergic neurotransmission and dysfunction in DA- related disorders. Over two decades of research support that DAT surface expression is acutely regulated by endocytic trafficking. Protein kinase C (PKC) activation with phorbol esters stimulates DAT internalization and thereby decreases DAT surface expression and function. Although considerable progress has been made to define the molecular mechanisms governing basal and PKC-regulated DAT trafficking, there are significant gaps in our understanding of this process in bona fide DAergic terminals. It is not clear how DAT is regulated in response to the endogenous presynaptic receptors that are activated upstream of PKC, such as Gq-coupled receptors, and how the complex signal events stemming from Gq receptor activation integrate to acutely control DAT surface expression. It is additionally unknown whether regulated DAT trafficking is region-specific, or whether altered DAT surface expression impacts DAergic signaling in the striatum. The proposed studies will leverage chemogenetic receptors to test how Gq activation impacts DAT surface levels in a cell- autonomous manner, in both dorsal and ventral striatum. We will capitalize on a novel conditional, inducible, in vivo gene silencing approach to determine the endocytic mechanisms that are required for Gq-mediated DAT trafficking, by both chemogenetic and endogenous presynaptic receptors. We will further employ ex vivo fast- scan cyclic voltammetry to investigate how presynaptic DAT trafficking impacts DA signaling. I anticipate that at the completion of these studies, we will have gained a more in-depth understanding of the complex mechanisms underlying DAT regulation at presynaptic DAergic terminals, and its potential influence on synaptic DA homeostasis.

The goal of this project is fluorescently visualize ATP release and extracellular accumulation at the surface of stimulated microglia. The development of this innovative technology has the potential to enable spatiotemporal imaging of microglial extracellular signaling. For this project, I am exploiting the presence of the cell's glycocalyx to attach ATP-sensitive biosensors at the sites of ATP accumulation. There are two aims to this project: 1) to synthesize a novel, polyhistidine binding moiety that covalently modifies the glycocalyces of living cells and binds recombinant biosensors to measure ion and metabolite efflux and accumulation; 2) to visualize and measure ATP release from pannexin channels in C5a stimulated microglia. The completion of these aims will yield a transformative set of chemical-biological tools and methodologies to investigate the physiology and pathophysiology of pannexin-dependent activity in glia, and potentially in living animals.