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The cell-type specific expression of ligand/receptor and cell-adhesion molecules is a fundamental mechanism through which neurons regulate connectivity. Here we determine a functional relevance of the long-established mutually exclusive expression of the receptor tyrosine kinase Kit and the trans-membrane protein Kit Ligand by discrete populations of neurons in the mammalian brain. Kit is enriched in molecular layer interneurons (MLIs) of the cerebellar cortex (i.e., stellate and basket cells), while cerebellar Kit Ligand is selectively expressed by a target of their inhibition, Purkinje cells (PCs). By in vivo genetic manipulation spanning embryonic development through adulthood, we demonstrate that PC Kit Ligand and MLI Kit are required for, and capable of driving changes in, inhibition of PCs. Collectively, these works in mice demonstrate that the Kit Ligand/Kit receptor dyad sustains mammalian central synapse function and suggest a rationale for the affiliation of Kit mutation with neurodevelopmental disorders.

The sodium channel alpha proteins consist of ten homologous human proteins (SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A, SCN11A), many involved in various human physiological pathways and clinical disorders/syndromes. Many human variants connected to these genes remain uncertain for human disease. To better support the understanding of challenging human variants, we have built an ortholog database of the alpha subunit proteins for the sodium channel. We combined 1,680 vertebrate species sequences of family members, protein modeling, molecular dynamic simulation of lipid embedded protein, screening of human variants (gnomAD, ClinVar, Geno2MP), and analysis of a large number of human RNAseq and single-cell RNA-seq to understand the function of each amino acid in the human proteins and how the amino acids are conserved across family members to contribute to sodium channel functions within diverse cells of human. There are 610 amino acids conserved across the alpha subunit family, representing the most conserved amino acids for each member. These amino acids drive the structural fold, membrane interactions, and channel opening. A cluster of amino acids is conserved between a subset of family members that defines subclusters of alpha subunit proteins. Expression analysis of each member reveals clusters of members co-expressed in various cells, where heart and brain datasets show the specificity of cell types that contribute to genotype-to-phenotype associations of each member. Further, to support variant characterizations, the ortholog alignment data provides a unique ability to discover loss vs. gain of function variants found between members, allowing for known pathogenic variants of one member to aid in the characterization of another member's uncertain variants. For example, a clinical variant of SCN8A linked to seizures was supported as loss-of-function through variants within SCN1A (epilepsy), SCN2A (seizures), SCN4A (Familial hyperkalemic periodic paralysis), and SCN5A (Brugada syndrome). Overall, we show an exciting and promising new tool for ortholog genomic mapping that can support clinical variant insights into physiological outcomes (genotype-to-phenotype relationship), which should be applied to other cellular ion channels and transporters. This research was funded by the Gerber Foundation (to JWP), National Institutes of Health (K01ES025435 to JWP), and Michigan State University. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Physiology research must be guided moving forward by our significant investments represented by deposited data. We hypothesize that integrating these resources, namely our investments into genomics, makes it possible to discover many aspects of well-studied physiological pathways. Within this work, we build a genomics integration for the Insulin gene/protein, which serves as an example of data integrations that can be applied to any physiological system. The INS gene region has 2,076 unique variants from population genetics of hundreds of thousands of individuals (gnomAD, ClinVar, Geno2MP, Avada, dbSNP). Several variants are found near the transcriptional start site, enhancers, and following the INS transcripts that might influence the readthrough fusion-transcript INS- IGF2. This INS- IGF2 transcript splice site is confirmed within hundreds of pancreas RNAseq samples, lacks drift based on human genome sequencing, and has a possible elevated expression due to viral regulation within the pancreas/liver. Moreover, a rare, poorly characterized African-specific variant of INS-IGF2 results in a loss of the stop codon. INS transcript UTR variants rs689 and rs3842753, highly associated with Type 1 Diabetes, are found in hundreds of pancreas RNAseq datasets with an elevation of the 3’UTR alternatively spliced INS transcript. Finally, by combining literature, evolutionary profiling, and structural biology, we map rare missense variants involved in type-1 diabetes, type-2 diabetes, and monogenic/neonatal diabetes that influence Pre-Pro-Insulin translation, Pro-Insulin processing, dimer/hexamer secretory storage, receptor activation, and C-peptide detection for quasi-Insulin blood measurements. Finally, through these complex Insulin pathways and multiple animal model species, we highlight the many genes involved and how environmental factors such as cellular stress may interact with genetics to increase the prevalence of diabetes. Overall, our data integration strategy yields a platform for merging our investments into animal physiological model systems, human clinical genomics, and growing investments into genome sequencing to highlight the many exciting future research needs of physiological pathways. This research was funded by the Gerber Foundation (to JWP), National Institutes of Health (K01ES025435 to JWP), and Michigan State University. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Throughout the last few years, understanding the physiological mechanisms to viral infections has highlighted individuals' unique outcomes. While SARS-CoV-2 and other pathogens can cause hospitalization and mortality, some individuals remain asymptomatic, and others will suffer from years of altered diverse physiological pathways (such as long COVID). Our team, through transcriptomics, has built a hypothesis that many of the individual-level responses are due to the interaction of a virus with a process known as nonsense-mediated decays (NMD) and how suppression of NMD interacts with an individual's genomic variants to drive diverse physiological response. This hypothesis has been built on nearly five hundred clinical transcriptomes of infants with RSV infection, newborns with genomic disorders, kids with multiple organ dysfunction syndrome, and adults with hospitalized COVID-19 paired with >16,000 publicly deposited blood transcriptomes of broad pathologies. Several virus interactions with human genomics have been observed within our work. First, a patient with EBV had an acute activation of viral-induced genetics through suppression of NMD, resulting in elevated RNASEH2B dominant-negative human protein resulting in Hemophagocytic lymphohistiocytosis (HLH), a mechanism of a rare transient disorders. Second, we have observed multiple individuals with viral infections that resulted in either the activation or suppression of interferon cascades correlated to common variant alleles altered by NMD changes, such as ISG15 and cytokines. Third, we show that several of these cytokine modulation genetics can elevate the risk of NMD-regulated Neutrophile Extracellular Trap (NET) production associated with long-term endocrine and hemodynamic modulation. Finally, we show that when a virus suppresses NMD (SARS-CoV-2, HIV, EBV), multiple individuals with latent infections can have an elevation of the dormant virus transcripts, including EBV and Torque teno virus. Thus, individuals can have multiple viruses activated that change physiological outcomes simultaneously. Overall, it is essential to physiology and medicine to refine each individual's precision and unique outcomes based on the complex interaction of a virus with NMD and genomic variants to modify cellular, systems, and inflammatory responses. This research was funded by the Gerber Foundation, National Institutes of Health (K01ES025435 and R01AI171984), Michigan Department of Health and Human Services, and Michigan State University. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Idiopathic Pulmonary Fibrosis (IPF) is a pathological condition of unknown etiology which results from injury to the lung and an ensuing fibrotic response that leads to the thickening of the alveolar walls and obliteration of the alveolar space. The pathogenesis is not clear and there are currently no effective therapies for IPF. Small airway disease and mucus accumulation are prominent features in IPF lungs, similar to Cystic Fibrosis (CF) lung disease. The ATP12A gene encodes the alpha-subunit of the non-gastric H+, K+-ATPase, which functions to acidify the airway surface liquid (ASL) and impairs mucociliary transport function in cystic fibrosis patients. Hypothesis: We hypothesize that the ATP12A protein may play a role in the pathogenesis of IPF. Methods: ATP12A expression level was evaluated by immunohistochemical staining and RNAscope in situ hybridization in normal and IPF human distal lungs. Primary human small airway culture model was used to elucidate the potential roles of ATP12A in the activation of latent TGF-beta activation. Viral vector mediated ATP12A overexpression was used in the bleomycin-induced lung fibrosis mouse model. A potassium-competitive proton pump blocker, vonoprazan was used to block ATP12A functions both in vitro and in vivo. Results: Our studies demonstrate that ATP12A protein is overexpressed in distal small airways from IPF patient lungs compared to normal human lungs. Potassium competitive proton pump blocker vonoprazan decreased airway surface liquid (ASL) pH and TGF-β1 activation in IPF small airway epithelial cells. In addition, overexpression of the ATP12A protein in mouse lungs worsened the Bleomycin (BLEO)-induced experimental pulmonary fibrosis. This was prevented by a potassium-competitive proton pump blocker, vonoprazan. Conclusion: Those data support that the ATP12A protein plays an important role in the pathogenesis of lung fibrosis. Inhibition of the ATP12A protein has the potential as a novel therapeutic strategy in IPF. This work was funded by the National Institutes of Health (HL153165-01A1 to X.L.), the Cystic Fibrosis Foundation (LI19XX0), Cystic Fibrosis Research Institute, and Spectrum Health-MSU Alliance Corporation funds. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Introduced mutations in BAP1 that are predicted to disrupt the helix structure of the BAP1-ULD domain dramatically disassociate its binding to the ASXL2-AB box. The GST-UCH association assay was performed with highly purified recombinant ULD-WT, L-P, or F-P mutant proteins and/or ASXL2-AB box proteins.

Deleterious mutations of the ubiquitin carboxy-terminal hydrolase BAP1 found in cancers are predicted to encode inactive truncated proteins, suggesting that loss of enzyme function is a primary tumorigenic mechanism. However, many tumors exhibit missense mutations or in-frame deletions or insertions, often outside the functionally critical UCH domain in this tumor suppressor protein. Thus, precisely how these mutations inactivate BAP1 is unknown. Here, we show how these mutations affect BAP1 interactions with the Polycomb group-like protein, ASXL2, using combinations of computational modeling technology, molecular biology, and in vitro reconstitution biochemistry. We found that the BAP1–ASXL2 interaction is direct and high affinity, occurring through the ASXH domain of ASXL2, an obligate partner for BAP1 enzymatic activity. The ASXH domain was the minimal domain for binding the BAP1 ULD domain, and mutations on the surfaces of predicted helices of ASXH abolished BAP1 association and stimulation of BAP1 enzymatic activity. The BAP1-UCH, BAP1-ULD, and ASXH domains formed a cooperative stable ternary complex required for deubiquitination. We defined four classes of alterations in BAP1 outside the UCH domain, each failing to productively recruit ASXH to the wild-type BAP1 catalytic site via the ULD, resulting in loss of BAP1 ubiquitin hydrolase activity. Our results indicate that many BAP1 mutations act allosterically to inhibit ASXH binding, thereby leading to loss of enzyme activity. Small-molecule approaches to reactivate latent wild-type UCH activity of these mutants might be therapeutically viable.Significance: Combined computational and biochemical approaches demonstrate that the BAP1–ASXL2 interaction is direct and high affinity and that many BAP1 mutations act allosterically to inhibit BAP1–ASXL2 binding. Cancer Res; 78(5); 1200–13. ©2017 AACR.

The deletion R666-H669 in the ULD domain of BAP1 corresponding to that observed in a uveal melanoma completely abolished ASXL2 binding in vivo. HEK 293 cells were co-transiently transfected with plasmids expressing WT or mutant BAP1 and ASXL2 (261-649aa) proteins. At 48 h post-transfection, the cells were collected and whole cell lysates were used for co-IP with ANTI-Flag M2 Affinity Gel. Western blot analyses were performed with BAP1 (rAb) and Flag (rAb).

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IPF is a condition in which an injury to the lung leads to the accumulation of scar tissue. This fibrotic tissue reduces lung compliance and impairs gas exchange. Studies have shown that infection with COVID-19 significantly worsens the clinical outcomes of IPF patients. The exact etiology of IPF is unknown, but recent evidence suggests that the distal small airways, (those having a diameter less than 2 mm in adults), play a role in the early pathogenesis of IPF. TGF-β1 is a main driver of fibrosis in a variety of tissues; the binding of TGF-β1 to its receptor triggers a signaling cascade that results in inflammatory signaling, accumulation of collagen and other components of the extracellular matrix, and immune system activation. This study aimed to investigate possible mechanisms that contribute to worsening lung fibrosis in IPF patients after being diagnosed with COVID-19, with a particular focus on the role of TGF-β1. Small airway cell cultures derived from IPF and post-COVID-19 IPF patient transplant tissues were submitted for RNA-sequencing and differential gene expression analysis. The genetic signatures for each disease state were determined by comparing the differentially expressed genes present in the cells cultured under control conditions to cells cultured with TGF-β1. The genes shared between the culture conditions laid the framework for determining the genetic signatures of each disease. Our data found that genes associated with pulmonary fibrosis appeared to be more highly expressed in the post-COVID fibrosis samples, under both control and TGF-β1-treated conditions. A similar trend was noted for genes involved in the TGF-β1 signaling pathway; the post-COVID fibrosis cell cultures seemed to be more responsive to treatment with TGF-β1. Gene expression analysis, RT-PCR, and immunohistochemistry confirmed increased levels of BMP signaling in the IPF small airway cell cultures. These findings suggest that TGF-β1 signaling in IPF small airway cells could be inhibited by BMP signaling, leading to the differences in genetic signatures between IPF and post-COVID fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a pathological condition of unknown etiology which results from injury to the lung and an ensuing fibrotic response that leads to thickening of the alveolar walls and obliteration of the alveolar space. The pathogenesis is not clear and there are currently no effective therapies for IPF. Small airway disease and mucus accumulation are prominent features in IPF lungs, similar to Cystic Fibrosis (CF) lung disease. The ATP12A gene encodes the alpha-subunit of the non-gastric H+, K+-ATPase, which functions to acidify the airway surface fluid and impairs mucociliary transport function in cystic fibrosis patients. We hypothesize that the ATP12A protein may play a role in the pathogenesis of IPF. Our studies demonstrate that ATP12A protein is overexpressed in distal small airways from IPF patient lungs compared to normal human lungs. In addition, overexpression of the ATP12A protein in mouse lungs worsened the bleomycin (BLEO)-induced experimental pulmonary fibrosis. This was prevented by a potassium-competitive proton pump blocker, vonoprazan (VON). This data supports the concept that the ATP12A protein plays an important role in the pathogenesis of lung fibrosis. Inhibition of the ATP12A protein has the potential as a novel therapeutic strategy in IPF.