Neuroscience Program

The goal of this proposal is to examine how presynaptic Neurexins (Nrxns) at serotonin (5-HT) synapses impact 5-HT signaling and social behavior. Extensive 5-HT axon terminal innervation throughout the brain corroborates 5-HT’s modulatory role in numerous behaviors including social behaviors, reward, emotion regulation, and learning and memory. Abnormal brain 5-HT levels and function are implicated in Autism Spectrum Disorder (ASD). While 5-HT therapeutics are often used to treat ASD, variable improvements in symptomatology require further investigation of 5-HT-mediated pathology. Many different genes contribute to increased ASD susceptibility and clinical presentation variability. Notably, synaptic dysfunction, specifically dysregulation of synaptic excitation and inhibition, remains a hallmark of ASD pathogenesis. Nrxns are presynaptic cell adhesion molecules that are well characterized in maintaining synapse function for proper neural circuit assembly. The three Nrxn genes transcribed from two promoters (α and β) express six principal Nrxn isoforms (αNrxn1-3, βNrxn 1-3). Additionally, mutations in Nrxn1 and Nrxn2 genes have been reported in ASD. In the current literature, the role of Nrxns at 5-HT synapses has yet to be investigated. Given that aberrant Nrxn and 5-HT function independently contribute to signaling pathology and social behavior impairments, it is critical to understand how Nrxn-mediated 5-HT neurotransmission participates in pathological mechanisms underlying the core deficits of ASD. Here, I will explore how 5-HT signaling mediated through Nrxns regulates social behaviors (Aim 1) and how Nrxns regulate 5-HT circuits relevant to social behaviors (Aim 2). Our group has created a novel mouse model in which the three Nrxn genes are selectively deleted in 5-HT neurons. My preliminary studies indicate that the loss of Nrxns at 5-HT synapses impairs social recognition memory and social reward preference. The hippocampus and nucleus accumbens, respectively, are crucial in these behaviors. In Aim 1, I will determine whether 5-HTergic Nrxns are critical for social behaviors through completion of social (and other complex) behavior studies. In addition, I will explore (i) if and (ii) how 5-HT is necessary for social behaviors using (i) 5-HT therapeutics to augment 5-HT function prior to social behavior studies and (ii) in vivo microdialysis to measure extracellular 5-HT levels during social behavior. In Aim 2, I will perform a mouse breeding and lentiviral rescue approach to determine whether specific Nrxns control social behavior. Furthermore, I will use immunohistochemical and electrophysiological approaches to identity how Nrxn proteins regulate excitatory and inhibitory synapse distribution and physiology. A close examination of Nrxns in 5-HT synaptic function is necessary to shed new light on social behavior disturbances in ASD.

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.

Elaborate mechanisms exist to establish and maintain the appropriate balance of excitation and inhibition (E/I balance) in the brain. Defects in E/I balance are hypothesized to underlie many core clinical symptoms seen in ASD including repetitive behaviors and seizures. Concomitant with E/I imbalance are increased markers of inflammation in the periphery and brain. Central to this inflammation are microglia, a resident macrophage of the central nervous system. Whether microglial inflammatory state drives E/I imbalance in neuropsychiatric disease remains a critical open question. In this proposal, I will leverage my primary mentor’s (Schafer) expertise in using mouse models to study microglia function at synapses with my co- mentor’s (Frazier) expertise as a physician scientist studying neuroinflammatory processes in ASD patients to explore whether microglia-derived cytokine signaling modulates E/I balance. I will use a mouse model with altered inflammatory cytokine signaling to assess how microglial inflammatory cytokine production modulates neuronal excitability (aim1). Next, I will use human ASD functional imaging data and data from patient serum to identify pro-inflammatory cytokines that are dysregulated in ASD patients and assess how these ASD-specific cytokines affect E/I balance in our mouse models (aim2). To start, I already have one candidate TNF􏰀-alpha. The results from these experiments will help to identify novel targets for treating ASD with inflammatory modulation.

The central nervous system is susceptible to many environmental insults and like many organs can be affected by alcohol. Alcohol impacts the brain in a variety of ways including short-term cognitive changes, development of dependence, memory deficits, neuronal loss and initiation of neuroinflammation. An emerging mechanism being studied in the field of central nervous system (CNS) inflammation, extracellular vesicle communication, has not yet been investigated in alcohol-related neuroinflammation and offers the potential for therapeutic intervention. Key components of alcohol-induced neuroinflammation, the cytokines IL-1β and HMGB1, are thought to be released from cells via extracellular vesicles. This study will explore the hypothesis that alcohol alters the release of extracellular vesicles within the CNS and that these vesicles contain content critical to the inflammatory process. Our Preliminary Data reveals that EVs are released by CNS cell types and can be taken up by unstimulated cells. First, we examined the effect of alcohol exposure on microglia and astrocytes in vitro and found that exosomes were stimulated for release at either 50 or 100mM alcohol. These findings were confirmed with western blot against exosome marker CD63 in the supernatant. Next, we used the membrane dye PKH26 to label membranes of microglia which were then stimulated to release EVs by alcohol. Those EVs were transferred to untreated/unlabeled cells and the dye was seen to incorporate in recipient cells, suggesting that those EVs were taken up by the untreated cells. Specific Aim 1 will investigate the effect of alcohol on extracellular vesicle release from primary mouse CNS cells (neurons, microglia or astrocytes) in single cell-type cultures in vitro. Nanoparticle tracking analysis will be used to measure released vesicles size, which will allow for quantification of the two types of released vesicles: exosomes (<150nm diameter) or microvesicles (150nm-1μm). Proinflammatory cytokines IL-1β and HMGB1 will then be measured in vesicles secreted from CNS cell types after alcohol exposure. These experiments will provide important knowledge regarding alcohol's impact on vesicle release as well as vesicle content. As extracellular vesicles are believed to transmit intercellular signals, Specific Aim 2 will explore the effect of transferring alcohol-induced vesicles onto naïve cells. First, extracellular vesicle uptake by primary CNS cell types will be measured. Next brain slices maintained in culture will be exposed to vesicles derived from alcohol-exposed cells and activation of inflammatory pathways will be examined. Finally, IL-1β or HMGB1 will be individually knocked down or overexpressed in CNS cell types and alcohol-induced vesicles will be transferred onto brain slices. These experiments will test the effect that alcohol-induced extracellular vesicles have on other cells, as well as the contribution made by cargo cytokines. Specific Aim 3 will elucidate the impact that alcohol-induced vesicles have on the brain in vivo. First, we will investigate the concentrations of EVs required for intracranial injection and uptake in the brain by using fluorescently-labeled vesicles. Next, vesicles will be stimulated in vitro from primary mouse CNS cells exposed to alcohol. After isolating those vesicles, they will be injected into the brains of naïve mice. Brain tissue will b examined for increases in immune cell activation and upregulation of inflammatory signals. This experiment will provide important information regarding the impact of extracellular vesicles on inflammation in vivo. The first year of this fellowship will be dedicated to quantifying and qualifying the vesicles released by CNS cells after alcohol exposure. Specific Aim 2 will be investigated in years two and three of the fellowship, while Specific Aim 3 will be completed in year three. The final two years of the fellowship will be dedicated to completing the clinical rotations for my MD training as well as any necessary follow up experiments needed for publishing this proposed work.

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.

Adverse health consequences of tobacco use are the leading cause of preventable mortality worldwide, resulting in approximately 6 million deaths per year. The addictive component of tobacco is nicotine, a tertiary alkaloid that binds and activates nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels that are normally activated by the endogenous neurotransmitter acetylcholine (ACh). Neuronal nAChRs are pentamers assembled from various combinations of receptor subunits and different subunit combinations confer different affinities and functionalities to the receptor subtypes. Eleven subunits, ¿2- ¿7, ¿9, ¿10 and ¿2- ¿4, have been identified in mammalian neuronal nAChRs. Interestingly, chronic nicotine or cigarette smoke exposure results in the upregulation of nAChRs in the brain, including structures within the mesocorticolimbic dopaminergic (DAergic) pathway that is implicated in reward and addiction. While not completely understood, nicotine- induced upregulation of nAChRs is thought to contribute to addiction by altering the neural network, possibly resulting in increased tolerance or altered sensitivity to nicotine. While there are many proposed mechanisms for nAChR upregulation, it is largely believed that multiple forms of posttranscriptional regulation is responsible for this phenomenon. Currently, there is not much known about posttranscriptional regulation of mammalian nAChR subunit expression by microRNAs (miRNAs), small single stranded RNA molecules that function as negative regulators of gene expression. However, there is emerging evidence that miRNA expression is decreased in various rodent tissue types in response to nicotine exposure. In addition, recent studies have found that miRNA dysregulation in response to exposure to various drugs of abuse, including cocaine, can influence rewarding properties of the drug and alter addiction-associated behaviors. We have recently generated preliminary data suggesting that a novel regulatory mechanism involving miRNAs may be at work in the nicotine-mediated upregulation of nAChRs. Preliminary experiments from our lab have identified several miRNAs that are predicted to target nAChR subunit mRNA transcripts, in particular miR-494 and miR-542-3p that target ¿4 and ¿2 transcripts, respectively. In Aim 1, I will determine if ¿4 and/or ¿2 are modulated by miR- 494 and/or miR-542-3p in primary midbrain neuronal cultures. In Aim 2, I will determine if miR-494 and/or miR- 542-3p are modulators of nicotine reward-associated behavior in mice. Through these aims, I hope to achieve a better understanding of the role of miR-494 and miR-542-3p in nicotine reward-associated behaviors, possibly revealing new targets for the development of tobacco cessation aids.