Abstract

The precise exon composition of expressed genes defines fundamental features of neuronal function. Alternative pre mRNA splicing regulates exon composition for >95% of multi-exon genes according to cell-type and influenced by development, cellular activity and disease and this process defines cell-specific functions. However, the mechanisms that regulate cell-specific alternative splicing of physiologically significant events are determined for a relatively small number of genes. Cell specific expression of ion channel splice isoforms influences synapse-specific release probability, neuronal firing frequencies, ion channel sensitivity to G protein coupled receptor modulation, subcellular targeting and ion channel density. I present our studies that describe the cellular mechanism that controls cell-specific splicing of Cacna1b - the gene that encodes the core subunits of all voltage-gated CaV2.2 channels. CaV2.2 channels control transmitter release at numerous synapses in the mammalian nervous system and, in the dorsal horn of the spinal cord, CaV2.2 channel activity regulates the transmission of innocuous and noxious signals from the periphery to central sites. We found striking cell-specific hypomethylation of a Cacna1b exon in noxious heat sensing nociceptors and long-term disruption of this epigenetic modification in an animal model of nerve injury. Our studies offer the most comprehensive description yet, of the mechanisms of cell-specific alternative splicing of a synaptic ion channel gene exon in normal and in disease states. The cellular control mechanisms that determine which isoform is expressed where, when and for how long, are essential for defining protein action within the specific cellular context.

Abstract

The question of how the brain encodes behavior provides an ongoing investigation to many scientists across multiple disciplines. Recently, using a multi-site in vivo recording approach to understand how the brain coordinates behavioral states, we discovered that brain-wide networks of electrical oscillations orchestrate a dynamic emergent network, and that this underlies a complex behavioral brain state, namely that of stress vulnerability. To do this, we used a novel approach including electrical recording electrodes to collect local field potentials (LFPs) across seven brain regions concurrently in freely behaving mice, combined with machine learning algorithms, and behavioral validations in a mouse model of chronic stress, chronic social defeat stress. Using this approach, we identified a robust and specific neurophysiological biomarker of vulnerability to chronic stress. Importantly, vulnerability to chronic stress is assessed prior to stress exposure, as opposed to susceptibility, which is defined post-stress by depressive-like behaviors. This brain network provides, for the first time, a brain-wide electrical signature predicting which animals will develop a depressive phenotype after exposure to chronic social defeat stress. Thus, our stress vulnerability measurement in freely behaving mice enables study of the causes of maladaptive responses to stress as opposed to just correlations. 

Our approach provides a way to identify underlying mechanisms of a maladaptive brain network that may be useful for the development of therapeutics targeting specific brain networks. It also enables us to study a brain state that has largely not been characterized, stress-naïve latent susceptibility to chronic stress. I will discuss the utility of such brain-wide electrical networks in identifying underlying causes of stress-susceptibility across other behavioral phenotypes as well as ongoing work to identify the circuit, cell, and molecular mechanisms driving this brain-wide electrical network activity. 

Abstract

Unipolar brush cells are excitatory interneurons in the granule cell layer of the cerebellum.   They receive glutamatergic contact from mossy fibers onto a large dendritic brush.  Retention of glutamate in the synaptic cleft of this synapse creates a hugely prolonged postsynaptic response.  We have examined how glutamate is cleared from this space, and have identified multiple roles for transporters in setting background glutamate levels, adjusting the dynamics of the synaptic response, and limiting cross-talk between nearby nerve terminals.

Abstract

Striatal neuromodulators have critical roles in motor control, decision-making, reinforcement learning and addiction. These neuromodulators exert their functions through G-protein-coupled receptors (GPCRs) on striatal neurons that activate second messenger intracellular cascades to affect neuronal signaling. Thus, understanding the cellular, subcellular, and synaptic mechanisms underlying neuromodulation is critical to advancing our knowledge of striatal function. I have taken a multifaceted approach to study striatal neuromodulation. The focus of my recent research has been the action of dopamine on different cellular populations of striatal dopamine D2 receptors in the induction of endocannabinoid (eCB)-dependent long-term depression (LTD), the downstream activation/inhibition of the cAMP-PKA signaling pathway, as well as the behavioral implications of this signaling. I identified a dual mechanism for D2R modulation of corticostriatal LTD induction at medium spiny neuron (MSN) synapses involving D2Rs on cholinergic interneurons and indirect pathway MSNs. These findings provide clarity and unify discordant literature within the field. In addition, I can simultaneously measure dopamine release and subsequent downstream GPCR mediated cAMP-PKA signaling in striatal brain slices using a genetically encoded fluorescent cAMP sensor, cAMP Difference Detector in situ (cADDis), and Fast-Scan Cyclic Voltammetry (FSCV). These experiments indicate that during afferent stimulation MSN-cAMP signaling is primarily influenced by dopamine transmission. Behavioral studies performed by our group and others indicate that D2 MSN-cAMP signaling is important for action initiation and invigoration. I will also describe studies that explore neuromodulatory and circuit mechanisms related to Alcohol Use Disorder. Drugs of abuse can interfere with eCB-mediated synaptic plasticity in a synapse-specific, and possibly in an eCB type (i.e AEA and 2-AG) selective manner. Little is known about mechanisms by which ethanol interacts with eCB transmission, including which eCB (AEA or 2-AG) is affected by ethanol. Using a novel, genetically encoded fluorescent eCB GPCR Activation based sensor (GRAB_eCB) to detect eCB levels with high spatiotemporal resolution, I observed that EtOH reduced eCB-CB1 receptor activation at both corticostriatal and striatopallidal synapses. Also, mice with targeted deletion of the 2-AG-synthesizing enzyme diacylglycerol lipase α (DAGLα) in direct pathway MSNs show alterations in ethanol consumption and preference in a sex-dependent manner. Using a multidisciplinary approach, these experiments have helped to further elucidate unique functions of GPCR neuromodulation within the basal ganglia circuitry and the functional relevance of these circuits.

Abstract

Memories thread and unify our sense of being. In recent years, the advent of activity-dependent and inducible optogenetic strategies have enabled us to image, tag, and manipulate defined sets of cells active during discrete periods of time, including learning and recollection. Here, I'll discuss our recent advances in modulating hippocampus and amygdala cells processing specific memories in the context of healthy and psychiatric disease-like states. Our results are twofold: 1) we show that acute activation of hippocampus and amygdala-mediated positive and negative memories can bi-directionally control both reward, social, and aversion-related behaviors; 2) we provide proof of principle data demonstrating brain-wide tagging and molecular characterization of cells active during positive and negative memory formation. Together, we suggest that these defined sets of cells are sufficient to lastingly modulate a battery of healthy and maladaptive behavioral states and provide novel avenues for molecular, circuit, and systems-wide interrogation of memory engrams. 

Abstract

Transposable elements, known colloquially as ‘jumping genes,’ constitute approximately 45% of the human genome. Cells utilize epigenetic defenses to limit transposable element jumping, including formation of silencing heterochromatin and generation of piwi-interacting RNAs (piRNAs), small RNAs that facilitate clearance of transposable element transcripts. We have utilized fruit flies, mice, and postmortem human brain samples to identify transposable element dysregulation as a key mediator of neuronal death in tauopathies, a group of neurodegenerative disorders that are pathologically characterized by deposits of tau protein in the brain. Mechanistically, we find that heterochromatin decondensation and reduction of piwi and piRNAs drive transposable element dysregulation in tauopathy. Based on multiple lines of evidence suggesting that tau-induced transposable element activation is conserved in human Alzheimer's disease and related tauopathies, we have initiated a clinical trial involving antiretroviral therapy to silence transposable element activation in patients with early-stage Alzheimer's disease. Taken together, our studies identify heterochromatin decondensation, piwi and piRNA depletion and consequent transposable element dysregulation as a pharmacologically targetable, mechanistic driver of neurodegeneration in tauopathy.

Abstract

Dr. Do Monte’s talk will focus on the neural circuits and mechanisms responsible for regulating the opposing drives of approaching food and avoiding predator threats. Previous studies have shown that the paraventricular nucleus of the thalamus (PVT) is interconnected with areas of the brain implicated in the control of food seeking and fear responses. He will speculate as to why PVT neurons and its connections with the ventromedial hypothalamus and the ventral striatum may be part of a core neural network that integrates threatening and rewarding information to generate distinct patterns of behavior. Using multidisciplinary tools that include immunohistochemistry, optogenetics, and electrophysiological recordings in freely behaving rats exposed to predator cues, Dr. Do Monte will show recent data suggesting the existence of a hypothalamic-thalamostriatal circuit that suppresses food-seeking behavior under the competing demands of avoiding potential threats.

Abstract

Maintaining genome stability is fundamental for organismal development and survival. Mammalian cells are endowed with a number of tightly regulated pathways providing efficient DNA repair in response to endogenous or exogenous genotoxic agents. However, compromising DNA repair proficiency or increased mutation loads can lead to a broad range of human diseases including those related to immune deficiency, cancer, inflammation, aging, and developmental disorders affecting the nervous system. DNA repair processes are also linked to redox homeostasis. My Ph.D. and postdoctoral studies have focused on elucidating the interplay between redox homoeostasis and two major DNA repair genes, ATM and

H2AX. ATM drives efficient DNA repair in response to DNA double-strand breaks (DSBs) through rapid phosphorylation of the histone variant H2AX. H2AX plays essential roles in DSB repair and genome stability. My research at the NCI has led to the demonstration that mitigation of reactive oxygen species (ROS) by silencing of the NADPH oxidase 4 (NOX4) reduced the severity of features of Ataxia Telangiectasia disease (A-T), such as oxidative DNA lesions, cell death, and lymphoma. During my second postdoctoral training at Johns Hopkins University, I published the first comprehensive studies demonstrating that mice lacking histone H2AX experience increased oxidative stress originating from repressed NRF2-regulated antioxidant genes and mitochondrial dysfunction. As an independent investigator at the University of Texas at Austin, my research is aimed at elucidating the pathways coordinating DNA repair and redox homeostasis in age-related diseases such as cancer and neurological disorders. Our current research addresses the following questions:

1) What is the importance of DNA Damage Response pathways in redox homeostasis?

2) How do deficiencies or mutations in DNA repair genes affect mitochondrial metabolism?

3) What is the relevance of impaired redox homeostasis induced by DNA repair deficiency to age-related neurological disorders?

Abstract

Our ability to detect cold is critical for survival; nevertheless, our understanding of cold processing in the peripheral nervous system is limited. A major outstanding question in sensory physiology is how cold information is transmitted to the central nervous system. Our recent work identified tetrodotoxin-sensitive voltage-gated sodium channels, namely NaV1.1, as critical mediators of action potential firing and excitability in putative cold-sensing sensory neurons. Furthermore, an analysis of native sodium currents found that NaV1.1 channels in this population are largely refractory to entry into slow-inactivated states. Thus, this could be a biophysical mechanism that promotes excitability of peripheral cold-sensing neurons. Finally, we have recently found evidence that NaV1.1 works to transmit cold sensations in vivo. Collectively, these results highlight a new role for NaV1.1 in thermosensation.

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