Determining cell-type specificity for a nonclassical MHC class I during an activity-dependent cortical critical period.

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7. Project Summary/ Abstract The long-term goal of this research is to understand how neural activity sculpts brain circuits during developmental critical periods. In cerebral cortex, synapses are pruned or stabilized in relation to levels and patterns of activity and normally this process occurs extensively during critical periods and is highly specific. In disease and inflammation, activation of glia can drive excessive pruning and perturb synaptic plasticity. Systematic approaches to treating pruning disorders in disease require understanding cell and molecular mechanisms normally engaged in synapse plasticity and remodeling. In healthy brain, we have discovered that Qa-1, a nonclassical Major Histocompatibility Class I (MHCI) molecule (gene name H2-T23; human HLA-E), is highly expressed in L6 of cerebral cortex and may contribute to activity-dependent plasticity in visual cortex. Qa- 1 has been studied in peripheral immune cells, but until now nothing was known about expression or function in healthy brain. However, Qa-1/HLA-E has been detected in neurons and glia in inflammation and Alzheimer's disease. Experiments proposed here test the hypothesis that Qa-1 expression in neurons restricts sensory- driven plasticity in visual cortex during the critical period. Two specific aims are proposed: 1) Identify cell type expression, activity-regulation and subcellular localization of Qa-1 mRNA in cortex. Cell types expressing Qa-1 mRNA will be identified by RNAScope in situ hybridization in combination with neuronal or glial-specific markers. Visual experience will be manipulated to test if Qa-1 expression is regulated, as occurs for many genes known to mediate activity-dependent plasticity. RiboTag cell type specific gene profiling will be used to detect Qa-1 mRNA isolated specifically from neurons, microglia and synaptosomes. The presence and enrichment of Qa-1 mRNA in L6 cortical neurons and synaptosomes will provide clues about how Qa-1 regulates plasticity during the critical period. 2) Generate a conditional allele of Qa-1 and explore cell-type specificity of Qa-1 function in activity-dependent plasticity. To dissect neuronal vs glial Qa-1 function, the Easi-CRISPR method (Quadros et al. 2017) will be used to insert loxP sites into the H2-T23 gene. Once a stable line is generated, Qa-1fl/fl mice will be crossed to Cre-expressing lines to generate mice lacking Qa-1 in neuronal vs glial cell populations. These lines will then be studied for activity-dependent phenotypes including ocular dominance plasticity and an activity- dependent microglial response. Results should reveal if Qa-1 acts in neurons, in glia or both for intact activity- dependent plasticity in visual cortex. If Qa-1 is required specifically in L6 neurons, results would imply a key role for this MHCI in a major circuit connecting thalamus and cortex. These studies of visual cortex should have broad significance because Qa-1 is expressed throughout neocortex. Moreover, the Qa-1 ortholog HLA-E is expressed in human cerebral cortex, so our studies may illuminate how this MHCI normally functions in human brain, and also contribute to understanding the basis for disorders in which activity-dependent synaptic plasticity and pruning go awry, as are thought to occur in Amblyopia, Autism, Schizophrenia and Alzheimer’s disease.