Wednesday, January 15, 2025
1/15/2025
PROJECT SUMMARY/ABSTRACT Genetic studies identify glutamatergic synaptic alterations as a core etiological mechanism of schizophrenia (SZ). Consistent with genetic findings, in vivo and postmortem brain studies identify alterations to molecular, structural and physiological markers consistent with deficient cortical glutamate synaptic neurotransmission. However, the nature of synaptic impairments in SZ at individual cortical glutamate synapses, the most basic unit of neural communication, is unknown. Interrogating measures of pre- and postsynaptic functioning at individually- resolved synapses can provide unprecedented insight into the basis of synaptic dysfunction in SZ. Analysis at this level of resolution requires an electron microscopic approach to directly visualize synaptic structures and quantify ultrastructural correlates of pre- and postsynaptic functions. Despite the potent interpretative power provided by ultrastructural analyses of individual synapses, multiple technical and logistical barriers have prevented the large-scale application to study brain tissue in SZ. We have now developed a workflow permitting three-dimensional (3D) quantitative and nanometer resolution imaging of glutamate synaptic structures via focused ion beam-scanning electron microscopy (FIB-SEM) in postmortem human brain. Thus, in Aim 1, we determine the effect of SZ on ultrastructural correlates of core pre- and postsynaptic processes in individual glutamate synapses located in layer 3 (L3) of dorsolateral prefrontal cortex (DLPFC), a locus of in vivo functional, structural and molecular abnormalities in SZ. Because synaptic signaling requires substantial energy produced by mitochondria localized to synaptic structures, and reduced glutamatergic signaling lowers ATP demand and synthesis at the synapse, ultrastructural analysis of mitochondria localized to glutamate axon terminals informs the relative level of activity at individual synapses. Indeed, mitochondrial abundance, size and morphology change in direct response to altered energetic demand at synapses. Thus, in Aim 2, we use our FIB-SEM approach to quantify ultrastructural correlates of mitochondrial activity in each glutamate synaptic structure identified in Aim 1. Finally, synaptic structure and function are directly regulated by synaptic protein levels. As such, the molecular basis of the synaptic and mitochondrial ultrastructural alterations identified in Aims 1-2 can be informed by proteomic analyses of DLPFC L3 synaptosomes. We have optimized a high throughput workflow to quantify thousands of proteins in synaptosome preparations from DLPFC L3. Thus, in Aim 3, alterations in the DLPFC L3 synaptic proteome in SZ are identified, quantified and mapped to ultrastructural alterations identified in Aims 1 and 2. Our proposed studies will directly inform the structural and molecular bases of cortical glutamate synaptic dysfunction in SZ at an unparalleled level of resolution to inform future functional pathway and molecular therapeutic targets for cortical and cognitive dysfunction in SZ.
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