Anton Maximov - US grants

DESCRIPTION (provided by applicant): Brain-derived neurotrophic factor (BDNF) is a small secreted protein that plays a fundamental role in nervous system development and in regulating the strength of existing synapses throughout the adult life. Imbalances in BDNF signaling impair several forms of synaptic plasticity and lead to a wide range of cognitive abnormalities. Unlike the classical neurotransmitters, BDNF is secreted by membrane-trafficking vesicular organelles that undergo exocytosis in neuronal processes and postsynaptic spines. The polymorphism in human BDNF gene which selectively abolishes activity-dependent synaptic release of BDNF has been associated with deficits in learning and memory. Remarkably, despite the importance of BDNF in brain development and plasticity, the molecular mechanisms underlying BDNF secretion have not been elucidated. Syt-11 is a member of synaptotagmin family of secretory proteins that are known to regulate exocytosis of various trafficking organelles. Recent genetic studies linked Syt-11 to familiar schizophrenia. The new observations in this proposal implicate Syt-11 to BDNF secretion. Specifically, we show that: i) Syt-11 is exclusively expressed in neurons and is localized on vesicular organelles that undergo activity-dependent exocytosis;ii) Syt-11 co-localizes with BDNF;iii) mouse Syt-11 gene is essential for survival during postnatal development;and iv) genetic deletion of Syt-11 impairs activity- dependent secretion of BDNF and homeostatic synaptic plasticity. Based on these observations we hypothesize that Syt-11 resides on and regulates exocytosis of trafficking vesicles that transport and release BDNF in neurons. This central hypothesis will be tested by several approaches. By using the subcellular fractionations and high-resolution live cell imaging, we will determine whether Syt-11 and BDNF co-traffic in the same secretory vesicles. Importantly, we will identify the sites of vesicle exocytosis and determine how exocytosis correlates with neural activity. As the next step, we will determine the extent to which transport and secretion of BDNF depends on Syt-11, and on interactions of Syt-11 with its effectors. This goal will be accomplished by analyses of subcellular distribution and secretion of BDNF in Syt-11 deficient neurons. Finally, we will perform electrophysiological analyses of cultured neurons and acute slices to test whether genetic deletion of Syt-11 impairs synaptic transmission and BDNF-dependent synaptic plasticity. These studies will provide new significant insights into cellular and molecular mechanisms underlying neurotrophin signaling in brain. Importantly, these studies will elucidate a secretory pathway that when defective causes abnormalities in synaptic and cognitive functions PUBLIC HEALTH RELEVANCE: Secreted brain-derived neurotrophic factor (BDNF) plays a fundamental role in nervous system development and in regulating the strength of existing synapses throughout the adult life. Imbalances in BDNF signaling have been implicated to a wide range of cognitive dysfunctions in humans. In this proposal, we will combine the biochemical, genetic, imaging and electrophysiological approaches to elucidate the mechanisms controlling transport and secretion of BDNF in neurons. These novel studies will provide significant insights into molecular and cellular mechanisms that regulate activity of neural circuitry in brain and link the abnormalities in BDNF secretion to cognitive diseases.

DESCRIPTION (provided by applicant): Brain-derived neurotrophic factor (BDNF) is a small secreted protein that plays a fundamental role in nervous system development and in regulating the strength of existing synapses throughout the adult life. Imbalances in BDNF signaling impair several forms of synaptic plasticity and lead to a wide range of cognitive abnormalities. Unlike the classical neurotransmitters, BDNF is secreted by membrane-trafficking vesicular organelles that undergo exocytosis in neuronal processes and postsynaptic spines. The polymorphism in human BDNF gene which selectively abolishes activity-dependent synaptic release of BDNF has been associated with deficits in learning and memory. Remarkably, despite the importance of BDNF in brain development and plasticity, the molecular mechanisms underlying BDNF secretion have not been elucidated. Syt-11 is a member of synaptotagmin family of secretory proteins that are known to regulate exocytosis of various trafficking organelles. Recent genetic studies linked Syt-11 to familiar schizophrenia. The new observations in this proposal implicate Syt-11 to BDNF secretion. Specifically, we show that: i) Syt-11 is exclusively expressed in neurons and is localized on vesicular organelles that undergo activity-dependent exocytosis; ii) Syt-11 co-localizes with BDNF; iii) mouse Syt-11 gene is essential for survival during postnatal development; and iv) genetic deletion of Syt-11 impairs activity- dependent secretion of BDNF and homeostatic synaptic plasticity. Based on these observations we hypothesize that Syt-11 resides on and regulates exocytosis of trafficking vesicles that transport and release BDNF in neurons. This central hypothesis will be tested by several approaches. By using the subcellular fractionations and high-resolution live cell imaging, we will determine whether Syt-11 and BDNF co-traffic in the same secretory vesicles. Importantly, we will identify the sites of vesicle exocytosis and determine how exocytosis correlates with neural activity. As the next step, we will determine the extent to which transport and secretion of BDNF depends on Syt-11, and on interactions of Syt-11 with its effectors. This goal will be accomplished by analyses of subcellular distribution and secretion of BDNF in Syt-11 deficient neurons. Finally, we will perform electrophysiological analyses of cultured neurons and acute slices to test whether genetic deletion of Syt-11 impairs synaptic transmission and BDNF-dependent synaptic plasticity. These studies will provide new significant insights into cellular and molecular mechanisms underlying neurotrophin signaling in brain. Importantly, these studies will elucidate a secretory pathway that when defective causes abnormalities in synaptic and cognitive functions

DESCRIPTION (provided by applicant): In the mammalian brain, a number of genes essential for circuit development and synaptic plasticity are controlled by neuronal activity. However, the underlying molecular mechanisms are incompletely understood. HDAC4 is a member of the class IIa histone deacetylase family of nuclear repressors that shuttle between the nucleus and cytoplasm and interact with tissue-specific transcription factors (TFs) in a signal-regulated manner. Human genetic studies have shown that mutations in the HDAC4 locus cause mental retardation, but the consequences of HDAC4 deficiency on neural circuits remain elusive. Our recent exciting findings support a hypothesis that HDAC4 is a molecular substrate for NMDA receptor-dependent transcriptional control of synaptic strength. We provide evidence that, in cultured neurons, HDAC4 represses multiple synapse-related genes thereby affecting the structural organization of central synapses and their physiological properties. Furthermore, we show that NMDA receptors prevent the binding of HDAC4 to neuronal chromatin and TFs, and that misregulation of this transcriptional pathway in the mouse forebrain impairs neurotransmission and spatial memory. We have developed a comprehensive genetic toolbox and assembled a team of qualified investigators to rigorously investigate the outcomes of nuclear HDAC4 signaling in mouse models. In SA1, the interplay between synaptic inputs and HDAC4 repressor activity will be tested in mutant animals that are deficient for NMDA receptors, and in a new unique mouse strain that enables acute drug-inducible manipulation of synaptic neurotransmitter release from specific neuronal types in vivo. In SA2, we will use both genetic loss- and gain- of-function approaches to define the role of HDAC4 in regulating basal neurotransmission and experience- dependent forms of synaptic plasticity in the Dentate Gyrus (DG), a brain region that relays cortical information into the hippocampus and promotes several cognitive tasks. To this end, we will interrogate mice that either lack native HDAC4 due to conditional gene silencing, or express a constitutively nuclear repressor mutant in the wildtype background. Finally, imaging experiments directed towards SA3 will test how nuclear HDAC4 signaling impacts the connectivity between excitatory and inhibitory neurons in the DG, and the architectures of their functional synapses. These studies will gain significant new insights into the mechanisms of experience-dependent transcriptional silencing/de-repression in the brain, elucidate the role of HDAC4 in controlling synaptic function, and provide a molecular explanation for a rare human disease.

Abstract. Biomedical research tremendously benefits from approaches that enable pharmacological control of gene expression in live laboratory animals. However, current techniques have limitations that often imped the design of transformative experimental paradigms. These include suboptimal temporal dynamics and undesired side effects of inducing drugs on animal physiology and behavior. Our preliminary studies demonstrate that these obstacles can be overcome with new approaches permitting acute regulation of nuclear protein function with the antibiotic, trimethoprim (TMP). This innovative strategy takes advantage of destabilizing domains (DD) that mediate instant degradation of synthesized proteins of interest (POI). TMP restores the stability of DD-POIs by binding to DD tags with high affinity. When compared to other chemical-genetic methods, TMP-inducible stabilization has several advantages: (i) TMP is an inexpensive commercially available small molecule that efficiently penetrates peripheral tissues and the blood-brain barrier; (ii) TMP does not produce adverse effects in mammals due to lack of endogenous targets; (iii) DDs can be fused to virtually any protein of interest; and (iv) TMP stabilizes translated proteins with rapid time-course that does not depend on rates of mRNA synthesis. We therefore hypothesize that expression of genetically encoded DD-POIs in model organisms will facilitate a broad spectrum of studies that have not been previously feasible for technical reasons. Here we will: 1) Develop a versatile toolbox for TMP-inducible recombination of chromosomal and episomal DNA with destabilized Cre recombinase (DD-Cre). This method can be used with numerous mouse lines carrying loxP- flanked alleles, and to drive tissue-specific expression of genes of interest with recombinant viruses; 2) Leverage TMP-inducible stabilization of site-specific transcriptional repressors (DD-TR) for acute silencing of multiple genes that act in the same pathway, a task that cannot be performed with other methods. Our qualified interdisciplinary team will rely on mouse models to validate these tools in vivo. We will express DD-Cre and DD-TRs in the brain to systematically characterize their sensitivity to TMP, kinetics of stabilization, and activity on substrates. Moreover, we will exploit these novel systems in proof-of-principle neurobehavioral experiments to investigate the mechanisms of learning and memory.

This proposal aims to investigate the molecular basis of structural plasticity of inhibitory GABAergic interneurons (INs) in the mammalian forebrain. Cortical and hippocampal INs play critical roles in perception and memory storage, and their abnormalities have been associated with a broad spectrum of neurological disorders in humans. It is well-established that INs undergo morphological changes and reorganize their networks after sensory experience. This phenomenon is widely believed to be equally important for correct assembly of synaptic connectivity during development and for processing of information in the brain across lifespan, but the underlying molecular mechanisms are poorly understood. By using deep sequencing, in vitro screening and mouse genetics, we have identified an early response gene, Nr4a1, that regulates the architectures of IN networks in the hippocampus. This gene encodes a transcription factor whose function in inhibitory circuits is entirely unknown. Our preliminary studies support the hypothesis that Nr4a1 is essential for appropriate GABAergic inhibition of principal pyramidal neurons and memory storage. We will use innovative approaches to elucidate the role of Nr4a1 signaling in inhibitory circuits in unique mouse models. In Aim1, we will test how ablation of Nr4a1 in specific genetically-defined IN subtypes impacts their morphologies, wiring and physiology. In Aim2, we will examine the consequences of Nr4a1 signaling on sensory processing and memory formation. Finally, experiments in Aim3 will identify Nr4a1 effector genes, and will elucidate the roles of these genes in controlling IN connectivity and function in neuronal cultures. Together, these aims will provide new and significant insights into thus far poorly understood molecular mechanisms of IN plasticity, and will facilitate future studies of experience-dependent transcriptional programs in normal and diseased brains.

The goal of this research is to establish new robust methods for manipulation of specific circuits and genetically defined neuron types in brains of model organisms with small molecules. While several chemical-genetic techniques are already available, these techniques have drawbacks that limit their utility. Our recent work demonstrates that these obstacles can be overcome by using a strategy for acute control of function of proteins of interest (POI) containing destabilizing domains (DD) with the inexpensive commercially available drug, TMP. This compound efficiently crosses the blood-brain barrier, stabilizes DD-POIs with a rapid time course, and does not produce undesired side effects by itself. Since DD tags can be attached to virtually any protein, TMP-inducible stabilization is applicable to a broad spectrum of experimental paradigms. We propose to generate mouse alleles and viral vectors encoding DD-POI fusions suitable for applications ranging from ultra-structural imaging to assessment of complex behaviors. We will develop tools for TMP-dependent recombination of DNA and genome editing (Aim1), acute labeling of behaviorally relevant neuron populations with a reporter compatible with optical imaging and electron microscopy (Aim2), and cell-type-specific control of neurotransmitter secretion (Aim3). Our collaborative team will demonstrate the advantages of these techniques by combining genomics, whole brain imaging, serial electron microscopy, electrophysiology, optogenetics and behavior. New tools will be compared side-by-side against existing technologies, and then distributed to the neuroscience community. We anticipate that these efforts will significantly benefit future studies of the normal brain and mechanisms underlying neurological diseases.

Alzheimer?s disease (AD) is the leading cause of aging-related cognitive decline, affecting more than 5 million Americans over 65 years old, and the number of patients is expected to climb to 13 million as baby boomers age. Our ability to learn and remember declines with age due to progressive changes in synaptic connectivity and function. Synaptic abnormalities also commonly precede neuronal loss during early stages of Alzheimer?s disease (AD) and other neurodegenerative disorders. However, we are only beginning to understand the full spectrum of these abnormalities, their contributions to cognitive decline, and the underlying mechanisms. This challenge is largely attributed to the complexity of the brain and etiologies of aging and neurodegeneration. Hallmarks of advanced Alzheimer?s disease (AD) include accumulations of extracellular amyloid peptides and intracellular hyperphosphorylated tau protein as well as chronic neuroinflammation. While genes for familial AD have been identified, which shed substantial light on the etiology of the disease, the mechanisms behind sporadic onset AD still remain a mystery. While studies of transcriptional dynamics in the brain have been transformative, transcriptional dynamics do not correlate well with protein dynamics because protein synthesis, turnover and subcellular localization are more tightly regulated spatially and temporally than transcription. Studies have suggested that proteostasis declines with age, impairing cells from managing the inevitable misfolding of proteins. Our team will study protein dynamics in animal models based on bio-orthogonal non-canonical amino acid (BONCAT) protein labeling. These methods allow us to measure dynamics in protein synthesis and degradation in specific brain cell types relevant to AD. These measurements will provide new information about the disruption of normal cellular processes. The overreaching goal of our collaborative proposal is to bridge critical gaps in knowledge by leveraging the state-of-the-art methods for bio-orthogonal non-canonical amino acid tagging (BONCAT) and quantitative mass spectrometry (MS) to identify brain cell-type contributions to synaptic and neuronal decline associated with AD.