The long term goal of this research is to understand cellular and molecular mechanisms of activity dependent circuit tuning during developmental critical periods. These are times when proper patterns of neural activity and experience are required for brain circuits to form normally, and when abnormal activity or experience can perturb the outcome. The specific hypothesis here is that two specific Major Histocompatibility Class I genes in mouse (MHCI; HLA in human) H2-Db (Db) and H2-Kb (Kb) expressed in neurons and at synapses regulate activity-dependent synaptic pruning and plasticity during developmental critical periods. MHCI genes are famous for their roles in cell-mediated immunity, but here we study a novel role in neurons. Neuronal MHCI expression in CNS neurons was discovered unexpectedly in an unbiased in vivo screen for genes regulated by neural activity in the developing visual system. Research next showed that loss of function of just 2 of the 50+ MHCI genes, Db and/or Kb, prevents synaptic pruning in vivo and unexpectedly enhances visual cortical plasticity and motor learning. Moreover, just by restoring Db selectively to neurons in vivo, synapse pruning is rescued, as well as LTD and AMPA type glutamate receptor subunit composition at retinogeniculate synapses. These observations demonstrated that Db must function in neurons, and showed that there are separate and parallel roles for Db in neurons vs in immune cells.
Three Specific Aims are proposed here: 1) Validate and extend understanding of the relationship between Db, synapse pruning and AMPA type glutamate receptor (GluA1/2) subunit composition observed in vivo using an in vitro system to manipulate systematically Db levels in cortical Layer 2/3 pyramidal neurons. Expression constructs for Db, Kb, or shRNAs, will be used for gain- or loss-of function, or rescue experiments in cortical neuron cultures from WT vs KbDb-/- or Db-/- mice. Synapse pruning and GluA1/2 subunit composition will be assessed biochemically and electrophysiologically. The effects of blocking synaptic transmission will also be studied. 2) Identify molecular interactions between Db (or Kb) and associated molecules using biochemical methods and Mass Spectrometry (MS). Preliminary results from MS suggest that there may be direct interactions with synaptic components as well as proteins associated with membrane trafficking. Such interactions will be validated using co-immunoprecipitation, and can illuminate how Db (or Kb) might directly or indirectly regulate glutamate receptors. 3) Investigate a possible role of the short intracellular ail of Db in regulating pruning, AMPA receptor subunit composition and receptor trafficking by mutational analysis. Together these experiments should help establish a strong link between MHCI molecules such as Db, synapse pruning, neural activity and AMPA type glutamate receptor subunit composition. This knowledge may make it possible someday to correct neurodevelopmental disorders such as Schizophrenia or Autism in which pruning has gone awry, or even to treat neurodegenerative disorders such as Alzheimer's in which excessive synapse pruning destroys the very neural circuits in which memories reside.
The results of these studies of neuronal function of MHC class I proteins should broaden understanding of how use-dependent changes both in development and in adulthood are encoded in the structure of neural circuits. This understanding is crucial for addressing and ultimately curing childhood learning disorders and even Alzheimer's' and other memory disorders of aging that involve pathological synapse pruning. Neuronal MHCI is also known to be modulated by inflammation and is recognized by cells of the immune system, providing an avenue for direct cross talk between nervous and immune cells in both health and disease. Because MHCI/HLA is one of the most highly associated loci in GWAS studies of Schizophrenia, this research may also contribute to a mechanistic understanding of this devastating mental illness.
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