DeGiosio, R. A. | bioRxiv Science

bioRxiv2022

CACNB4 overexpression decreases dendritic spine density in sex-specific manner

The canonical voltage-gated calcium channel (VGCC) subunit complex is comprised of the 1 subunit, the ion permeable channel, plus three auxiliary subunits: {beta}, 2{delta} and {gamma}. {beta} is the most extensively studied auxiliary subunit and is necessary for proper forward trafficking of the 1 subunit to the plasma membrane. 1 subunits mediate voltagedependent movement of calcium ions into the cytoplasm of neurons, including at dendritic sites, where increased intracellular calcium initiates signaling cascades that shape structural and functional plasticity of dendritic spines. Genetic studies strongly implicate calcium signaling dysfunction in the etiology of neurodevelopmental disorders including schizophrenia. Dendritic spine density (DSD) is significantly decreased in schizophrenia in primary auditory cortex where DSD is driven by loss of small spines, and small spine loss is associated with increased peptide levels of ALFDFLK found in the VGCC {beta} subunit {beta}4. Overexpessing CACNB4 to increase {beta}4 levels selectively reduced small spine density in cortical neuron cultures. The studies described herein set out to validate this in vitro observation in an intact mammalian system within a neurodevelopmental context. We overexpressed CACNB4 in neurodevelopment and assessed DSD and morphology in cerebral cortex of male and female mice at an adult timpoint. We then characterized {beta} protein levels and {beta}4 protein-protein interactions in male and female mouse cortex. Overexpression selectively reduced small dendritic spine density but this effect was present only in female mice and did not appear to result from estrous stage. Instead, the sex-dependent effect on DSD corresponded to sex differences in the {beta}4 interactome of male versus female mice: the VGCC {beta} subunit {beta}1b was significantly enriched in the {beta}4 interactome of brain tissue of male mice, and thus may have served to mitigate VGCC overexpression-mediated spine loss in male mice.

neuroscience↗

bioRxiv2019

MAP2 is Hyperphosphorylated in Schizophrenia and Alters its Function

Schizophrenia (Sz) is a highly polygenic disorder, with common, rare, and structural variants each contributing only a small fraction of overall disease risk. Thus, there is a need to identify downstream points of convergence that can be targeted with therapeutics. Reduction of Microtubule-associated Protein 2 (MAP2) immunoreactivity (MAP2-IR) is present in individuals with Sz, despite no change in MAP2 protein levels. MAP2 is phosphorylated downstream of multiple receptors and kinases identified as Sz risk genes, altering its immunoreactivity and function. Using an unbiased phosphoproteomics approach we quantified 18 MAP2 phosphopeptides, 9 of which were significantly altered in Sz subjects. Network analysis grouped MAP2 phosphopeptides into 3 modules, each with a distinct relationship to dendritic spine loss, synaptic protein levels, and clinical function in Sz subjects. We then investigated the most hyperphosphorylated site in Sz, phosphoserine1782 (pS1782). Computational modeling predicted phosphorylation of S1782 reduces binding of MAP2 to microtubules, which was confirmed experimentally. We generated a transgenic mouse containing a phosphomimetic mutation at S1782 (S1782E) and found reductions in basilar dendritic length and complexity along with reduced spine density. Because only a limited number of MAP2 interacting proteins have been previously identified, we combined co-immunoprecipitation with mass spectrometry to characterize the MAP2 interactome in mouse brain. The MAP2 interactome was enriched for proteins involved in protein translation. These associations were shown to be functional as overexpression of wildtype and phosphomimetic MAP2 reduced protein synthesis in vitro. Finally, we found that Sz subjects with low MAP2-IR had reductions in the levels of synaptic proteins relative to nonpsychiatric control (NPC) subjects and to Sz subjects with normal and MAP2-IR, and this same pattern was recapitulated in S1782E mice. These findings suggest a new conceptual framework for Sz - that a large proportion of individuals have a "MAP2opathy" - in which MAP function is altered by phosphorylation, leading to impairments of neuronal structure, synaptic protein synthesis, and function.

neuroscience↗