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Abstract The voltage-gated calcium channel (VGCC) subunit complex is comprised of the α1 subunit, the ion-permeable channel, and three auxiliary subunits: β, α2δ, and γ. β is the most extensively studied auxiliary subunit and is necessary for forward trafficking of the α1 subunit to the plasma membrane. VGCCs mediate voltage-dependent movement of calcium ions into neuronal cytoplasm, including at dendrites, where intracellular calcium spikes initiate signaling cascades that shape the structural plasticity of dendritic spines. Genetic studies strongly implicate calcium signaling dysfunction in the etiology of neurodevelopmental disorders including schizophrenia. Dendritic spine density is significantly decreased in schizophrenia in the primary auditory cortex where it is driven by the loss of small spines, and small spine loss associated with increased peptide levels of ALFDFLK found in the VGCC β subunit β4. Overexpressing the gene that encodes the voltage-gated calcium channel subunit β4, CACNB4, selectively reduced small spine density in vitro. In the current study we extended this observation in an intact mammalian system within a relevant neurodevelopmental context. We overexpressed CACNB4 in early development, assessed spine density and morphology in adult male and female mouse cortex, and characterized β1-4 protein levels and β4 protein-protein interactions. Overexpression reduced small spine density in females. This effect was not dependent on the estrous stage. Instead, it corresponded to sex differences in the murine β4 interactome. The VGCC subunit β1b was significantly enriched in the β4 interactome of male relative to female mice, and thus may have served to mitigate VGCC overexpression-mediated spine loss in male mice.

Acid transport is required for bone synthesis by osteoblasts. The osteoblast basolateral surface extrudes acid by Na+/H+ exchange, but apical proton uptake is undefined. We found high expression of the Cl−/H+ exchanger ClC3 at the bone apical surface. In mammals ClC3 functions in intracellular vesicular chloride transport, but when we found Cl− dependency of H+ transport in osteoblast membranes, we queried whether ClC3 Cl−/H+ exchange functions in bone formation. We used ClC3 knockout animals, and closely-related ClC5 knockout animals: In vitro studies suggested that both ClC3 and ClC5 might support bone formation. Genotypes were confirmed by total exon sequences. Expression of ClC3, and to a lesser extent of ClC5, at osteoblast apical membranes was demonstrated by fluorescent antibody labeling and electron microscopy with nanometer gold labeling. Animals with ClC3 or ClC5 knockouts were viable. In ClC3 or ClC5 knockouts, bone formation decreased ~40 % by calcein and xylenol orange labeling in vivo. In very sensitive micro-computed tomography, ClC5 knockout reduced bone relative to wild type, consistent with effects of ClC3 knockout, but varied with specific histological parameters. Regrettably, ClC5-ClC3 double knockouts are not viable, suggesting that ClC3 or ClC5 activity are essential to life. We conclude that ClC3 has a direct role in bone formation with overlapping but probably slightly smaller effects of ClC5. The mechanism in mineral formation might include ClC H+ uptake, in contrast to ClC3 and ClC5 function in cell vesicles or other organs.

Benzodiazepine (BZ) drugs treat seizures, anxiety, insomnia, and alcohol withdrawal by potentiating γ2 subunit containing GABA type A receptors (GABAARs). BZ clinical use is hampered by tolerance and withdrawal symptoms including heightened seizure susceptibility, panic, and sleep disturbances. Here, we investigated inhibitory GABAergic and excitatory glutamatergic plasticity in mice tolerant to benzodiazepine sedation. Repeated diazepam (DZP) treatment diminished sedative effects and decreased DZP potentiation of GABAAR synaptic currents without impacting overall synaptic inhibition. While DZP did not alter γ2-GABAAR subunit composition, there was a redistribution of extrasynaptic GABAARs to synapses, resulting in higher levels of synaptic BZ-insensitive α4-containing GABAARs and a concomitant reduction in tonic inhibition. Conversely, excitatory glutamatergic synaptic transmission was increased, and NMDAR subunits were upregulated at synaptic and total protein levels. Quantitative proteomics further revealed cortex neuroadaptations of key pro-excitatory mediators and synaptic plasticity pathways highlighted by Ca2+/calmodulin-dependent protein kinase II (CAMKII), MAPK, and PKC signaling. Thus, reduced inhibitory GABAergic tone and elevated glutamatergic neurotransmission contribute to disrupted excitation/inhibition balance and reduced BZ therapeutic power with benzodiazepine tolerance.

Abstract Background Hemolysis occurs in many injury settings and can trigger disease processes. In the kidney, extracellular hemoglobin can induce damage via several mechanisms. These include oxidative stress, mitochondrial dysfunction, and inflammation, which promote fibrosis and chronic kidney disease. Understanding the pathophysiology of these injury pathways offers opportunities to develop new therapeutic strategies. Methods To model hemolysis-induced kidney injury, human kidney organoids were treated with hemin, an iron-containing porphyrin, that generates reactive oxygen species. In addition, we developed an induced pluripotent stem cell line expressing the biosensor, CytochromeC-GFP (CytoC-GFP), which provides a real-time readout of mitochondrial morphology, health, and early apoptotic events. Results We found that hemin-treated kidney organoids show oxidative damage, increased expression of injury markers, impaired functionality of organic anion and cation transport and undergo fibrosis. Injury could be detected in live CytoC-GFP organoids by cytoplasmic localization of fluorescence. Finally, we show that 4-(phenylthio)butanoic acid, an HDAC inhibitor with anti-fibrotic effects in vivo, reduces hemin-induced human kidney organoid fibrosis. Conclusion This work establishes a hemin-induced model of kidney organoid injury. This platform provides a new tool to study the injury and repair response pathways in human kidney tissue and will assist in the development of new therapeutics.

Microtubule-associated protein 2 (MAP2) is the predominant cytoskeletal regulator within neuronal dendrites, abundant and specific enough to serve as a robust somatodendritic marker. It influences microtubule dynamics and microtubule/actin interactions to control neurite outgrowth and synaptic functions, similarly to the closely related MAP Tau. Though pathology of Tau has been well appreciated in the context of neurodegenerative disorders, the consequences of pathologically dysregulated MAP2 have been little explored, despite alterations in its immunoreactivity, expression, splicing and/or stability being observed in a variety of neurodegenerative and neuropsychiatric disorders including Huntington’s disease, prion disease, schizophrenia, autism, major depression and bipolar disorder. Here we review the understood structure and functions of MAP2, including in neurite outgrowth, synaptic plasticity, and regulation of protein folding/transport. We also describe known and potential mechanisms by which MAP2 can be regulated via post-translational modification. Then, we assess existing evidence of its dysregulation in various brain disorders, including from immunohistochemical and (phospho) proteomic data. We propose pathways by which MAP2 pathology could contribute to endophenotypes which characterize these disorders, giving rise to the concept of a “MAP2opathy”—a series of disorders characterized by alterations in MAP2 function.

Alzheimer's disease with psychosis (AD+P) is a heritable phenotypic variant of the disease which is associated with more rapid cognitive deterioration compared to Alzheimer's disease without psychosis (AD–P). Cognitive decline in AD correlates with synapse loss, and our previous studies suggest that those with AD+P have a differentially affected synaptic proteome relative to those with AD–P. In this study, we utilized RNA-sequencing of dorsolateral prefrontal cortex (DLPFC) in a cohort of 80 AD cases to evaluate novel transcriptomic signatures that may confer risk of psychosis in AD. We found that AD+P was associated with a 9% reduction in excitatory neuron proportion compared to AD–P [Mean (SD) AD+P 0.295 (0.061); AD–P 0.324 (0.052), p = 0.026]. mRNA levels contributed only modestly to altered synaptic proteins in AD+P relative to AD–P. Instead, network analysis identified altered expression of gene modules from protein ubiquitination, unfolded protein response, eukaryotic initiation factor 2 (EIF2) signaling and endoplasmic reticulum stress pathways in AD+P. We previously found that neuropathologies account for ~18% of the variance in the occurrence of psychosis in AD. Further inclusion of cell type proportions and differentially expressed modules increased the percent of the variance in psychosis occurrence accounted for in our AD cohort to 67.5%.

Microtubule-associated protein 2 (MAP2) is a crucial regulator of dendritic structure and neuronal function, orchestrating diverse protein interactions within the microtubule network. We have shown MAP2 is hyperphosphorylated at serine 1782 (S1782) in schizophrenia and phosphomimetic mutation of S1782 in mice (MAP2S1782E) is sufficient to impair dendritic architecture. We sought to determine how this hyperphosphorylation affects the MAP2 interactome to provide insights into the disorder's mechanisms. We investigated the MAP2 interactome using co-immunoprecipitation and mass spectrometry in MAP2S1782E and MAP2WT mice. We found that S1782E MAP2 led to a substantial disruption of protein-protein interactions relative to WT MAP2. Reduced interactions with PDZ domain-containing proteins, calmodulin-binding proteins, ribosome proteins, and kinesin proteins may all contribute to dendritic impairments induced by S1782E, and may be linked to schizophrenia pathogenesis. Interestingly, novel gain-of-function interactions with PPM1L and KLHL8 nominated these as regulators of phosphoS1782 MAP2 abundance and potential therapeutic targets in schizophrenia.