Mingshan Xue - US grants

PROJECT SUMMARY/ABSTRACT Ongoing systematic human genetic studies of neurodevelopmental disorders continue to uncover pathogenic mutations in genes encoding synaptic proteins, demonstrating the importance of these proteins for neurological and neuropsychiatric functions. Although the molecular and cellular functions of many such synaptic proteins have been studied to various extents, the functional roles of these proteins in neural circuits and behaviors are poorly understood because in-depth neurological and behavioral studies in animal models are often lacking. Consequently, the pathological mechanisms underlying these synaptic disorders remain elusive. This knowledge gap can be significantly narrowed by studying a few prioritized genes that are highly penetrant and affect a broad spectrum of neurological and neuropsychiatric features common among neurodevelopmental disorders. The gene encoding syntaxin-binding protein 1 is one such exciting example because it is one of a few genes most frequently mutated in neurodevelopmental disorders. The absence of syntaxin-binding protein 1 abolishes neurotransmitter release. This essential function is well understood at the molecular level, yet it remains unknown how its haploinsufficiency in humans causes a range of neurological impairments including epileptic seizures and intellectual disabilities. Thus, the overall goal of this project is to decipher the synaptic dysfunction of neural circuits in the mouse models of this disorder and understand their relevance to disease pathogenesis at the whole-organism level. The apparently paradoxical effects of syntaxin-binding protein 1 deletion and haploinsufficiency lead to the central hypothesis that its haploinsufficiency preferentially impairs GABAergic inhibitory transmitter release and causes an imbalance between excitation and inhibition, resulting in hyperexcitable neural circuits and neurological deficits, which can be reversed upon restoring protein function in adulthood. We propose to combine genetic manipulations with optogenetic, physiological, and behavioral methods to delineate synapse-specific alterations of neurotransmission in cortical circuits (Aim 1), to determine the contributions of specific cell types to the pathogenesis of the disorder (Aims 2 and 3), and to test the reversibility of the disease phenotypes in adulthood (Aim 3). This project shifts the research focus of syntaxin-binding protein 1 from neurotransmitter release per se to its function at the levels of neural circuitry and behavior. The proposed research will provide mechanistic insights into the pathogenesis of this devastating neurodevelopmental disorder. Beyond this particular disorder, understanding how disruption of excitation-inhibition balance affects neural circuits and behaviors will have ramifications for a growing list of neurodevelopmental disorders caused by mutations that alter synaptic transmission.

The ability of the cerebral cortex to perform incredibly complex functions resides in its intricate neural circuits composed of a vast number of neurons. The synaptic interactions among cortical neurons ultimately manifest as the interplay between excitation and inhibition, two opposing forces that work together to orchestrate the spatiotemporal patterns of neuronal activity. Hence, the relationship between excitation and inhibition (E-I relationship) is fundamental to many functional properties of cortical neurons such as the orientation selectivity and contrast response function of visual cortical neurons. The importance of proper E-I relationship is also underscored by the discovery of altered E-I relationship in many neurodevelopmental and psychiatric disorders. However, the regulation of E-I relationship and the impacts of altering this relationship on the functional response properties of cortical neurons remain poorly understood. Thus, the overall goal of this project is to determine how the activity of individual neurons and homeostatic synaptic plasticity regulate cortical excitation, inhibition, and E-I relationship. To this end, we used the developing mouse visual cortex as a model system and developed molecular approaches to selectively reduce the excitability of a small number of layer 2/3 pyramidal neurons in vivo, such that we can determine the cell-autonomous effect of neuronal activity while minimizing the perturbation to the whole circuit. We found that these neurons counteract the activity perturbation by homeostatic changes at a specific subset of excitatory and inhibitory synapses. These results led to the central hypothesis that homeostatic plasticity differentially modifies distinct synaptic inputs of individual cortical neurons to regulate their E-I relationship, thereby maintaining the activity levels and functional response properties. We propose to combine molecular manipulations with optogenetic, physiological, imaging, and anatomical methods to systematically delineate the homeostatic changes at different synapses originating from distinct presynaptic neuronal types (Aim 1), to identify the underlying synaptic mechanisms of input-specific homeostatic plasticity (Aim 2), and to determine the impact of these synaptic changes on the visual response properties of neurons in vivo (Aim 3). The proposed research connects three levels of investigations from synapse to circuit to system. The successful completion of this project will provide insights into the role of homeostatic synaptic plasticity in regulating E-I relationship and functional response properties of cortical neurons. The outcomes will also have an impact on our understanding of how plasticity mechanisms help the brain cope with perturbations in general.

PROJECT SUMMARY/ABSTRACT Targeted modulation of neural activity is an essential approach in basic and clinical neuroscience research. Optogenetic proteins, such as light-activated ion channels or pumps, enable optical control of neuronal activity with exquisite spatiotemporal precision. Thus, they provide powerful means to interrogate how neural activity contributes to brain functions and alter pathological activity to treat neurological disorders. A variety of excitatory optogenetic tools have been developed to meet different needs of activation paradigms. In contrast, inhibitory tools remain underdeveloped. The most well-developed light-driven ion pumps are still not sufficiently effective in silencing neurons due to their intrinsically low photoefficiency and pumping activity. Newly developed light-gated potassium channels also suffer from their small photocurrents and slow current kinetics. Our discovery of natural light-gated chloride channels, Guillardia theta anion channelrhodopsins 1 and 2 (GtACR1 and GtACR2), led to a new class of inhibitory optogenetic tools that are highly sensitive to light, have outstanding anion selectivity, exhibit time constants of milliseconds, and can generate 10?100-fold larger photocurrents in mammalian cells than previous tools. However, we and others discovered that light activation of light-gated chloride channels in mouse neurons depolarizes the axon and presynaptic terminals to trigger neurotransmitter release even though it inhibits action potentials at the soma. This excitatory action is due to the endogenous high concentrations of chloride in the axon and presynaptic terminals, which create a depolarizing chloride efflux upon channel opening. Thus, axonal excitation impedes the goal of neuronal silencing and complicates the interpretation of experiments using light-gated chloride channels. Another important limitation is that the action spectra of light-gated chloride channels are all within the blue to green- light ranges, limiting their effectiveness in deep brain tissues and flexibility in multiplex optogenetic applications. Therefore, the objective of this project is to overcome these two major limitations of light-gated chloride channels. We will harness protein trafficking machinery, structure-based molecular engineering, high- throughput screening, and protein evolution in nature to eliminate the excitatory effect and expand the action spectra range of natural ACRs. We propose to exploit endogenous protein trafficking mechanisms to restrict ACRs within neuronal somatodendritic domain (Aim 1), perform structure-guided high-throughput mutagenesis screens to create ACR variants with robust outward rectification and photocurrents (Aim 2), and identify spectrally shifted ACR variants through natural ACR homolog screens and high-throughput mutagenesis screens (Aim 3). The proposed research capitalizes on a powerful synergistic collaboration of biophysics, protein engineering, high-throughput screening, neuronal physiology, and system neuroscience. The successful completion of this project will present to the neuroscience community a set of much improved inhibitory optogenetic tools with potent efficacy, minimal side effects, and diverse spectral sensitivities.