Defective synaptic transmission is a prominent pathology that underlies cognitive deficits in Alzheimer's disease (AD), highlighting the need to elucidate molecular mechanisms of synaptic failure. Vacuolar H+-ATPase (V-ATPase), a proton-pumping enzyme, is essential for synaptic vesicle acidification and neurotransmitter loading. However, whether SV-associated V-ATPase is vulnerable to AD remains unclear. Here, using SV-rich fractions from postmortem brain tissues, we identified SV-associated V-ATPase deficits, including decreased enzymatic activity, impaired complex assembly, and altered expression of its key subunits in AD. SV-associated V-ATPase dysfunction was further associated with pathological and clinical characteristics of AD. Genetic downregulation of the V-ATPase V1D subunit, a component reduced in AD brains, disrupted V-ATPase proton transport and impaired SV acidification. Further experiments using 5 & times;FAD mice, which exhibited AD-like SV-associated V-ATPase deficits, demonstrated the deleterious impact of V-ATPase dysfunction on SV acidification and synaptic transmission including presynaptic neurotransmitter release. In addition, ex vivo studies identified amyloid beta-induced oxidative stress as a driver of V1D loss and V-ATPase disassembly, linking AD pathology to SV-associated V-ATPase dysfunction. These findings indicate that SV-associated V-ATPase dysfunction contributes to synaptic failure and cognitive deficits in AD. Therapeutic avenues to mitigate V-ATPase dysfunction have the potential to attenuate synaptic failure for the management of AD.
Persistent monocyte activation contributes to HIV-associated neurocognitive disorders (HAND), yet biomarkers that predict neurocognitive impairment before and after antiretroviral therapy (ART) remain incompletely defined. We evaluated monocyte subsets and activation markers in participants from the SEARCH007 cohort before ART initiation and at 6 and 12 months following treatment. Increased frequencies of CD14+CD16+ monocytes and elevated CD163 expression were associated with worsening neurocognitive performance and HAND severity. Plasma soluble CD163 levels increased with neurocognitive impairment and correlated with plasma HIV RNA levels, while CCR2 expression was associated with a composite neuropsychological performance Z-score (NPZglobal). Notably, CD169 expression was elevated across all monocyte subsets and demonstrated a stepwise increase with worsening neurocognitive impairment. Although ART reduced overall monocyte activation, elevated CD169 expression persisted in some individuals despite virologic suppression. Bayesian kernel machine regression and random forest analyses identified CD169 expression as one of the strongest predictors of cognitive impairment, surpassing plasma viral load, CD4+ T cell count, and several established monocyte activation markers. These findings identify monocyte CD169 expression as a biomarker of neurocognitive dysfunction before and during the first year of ART and support further investigation of its role in HAND pathogenesis.
Essential thrombocythemia (ET) and myelofibrosis (MF) are myeloproliferative neoplasms (MPN) that can result from heterozygous or homozygous mutations in the calreticulin (CRT) gene ( CALR ). Mutant CRT binds to the thrombopoietin receptor to induce signaling that drives ET and MF. CRT is also a chaperone for the assembly of the highly polymorphic human leukocyte antigen (HLA) class I molecules. The effects of heterozygous versus homozygous MPN CALR mutations on the expression of HLA class I variants are unknown. To assess mutant CRT-induced changes to HLA class I expression, we generated CRISPR-based mutant CALR knock-in cell lines and CALR -knockout cell lines reconstituted with wild-type or MPN mutant CALR and also undertook measurements in MPN patient platelets and monocytes. Collectively, our findings indicate that heterozygous CALR mutations preserve HLA class I surface expression, whereas full loss of wild-type CRT results in marked, allele-dependent reductions in HLA class I expression, which are rescued by wild-type CRT but not MPN mutants. Reduced expression of CRT-dependent HLA class I in cells lacking a functional CRT induces natural killer (NK) cell activation. These studies identify specific HLA class I alleles and homozygous CALR mutations as prerequisites for low HLA class I expression in MPN.
TGF-β-activating integrins promote solid-organ fibrosis, suggesting their potential as disease markers. Chronic lung allograft dysfunction (CLAD), a progressive fibrotic complication that limits lung transplant survival, is driven by intragraft TGF-β activation. However, TGF-β-activating integrin expression patterns remain undefined in lung transplants. Single-cell RNA sequencing and proteomic analysis of tissue specimens from a mouse CLAD model and human explanted lungs with CLAD revealed high levels of the TGF-β-activating integrin αvβ6, primarily localized to fibrosis-associated Keratin 8 + alveolar transitional epithelial cells, which were greatly reduced in tolerant lungs. Molecular imaging with a newly optimized positron emission tomography radiotracer specific for αvβ6, [ 64 Cu]Cu-DOTA-A20-K16R, showed significantly higher uptake in CLAD than in tolerant transplants. [ 64 Cu]Cu-DOTA-A20-K16R activity was also reduced by experimental treatments that lowered αvβ6 expression and inhibited CLAD severity. Finally, [ 64 Cu]Cu-DOTA-A20-K16R autoradiographic analysis of human explanted lungs with CLAD showed elevated activity that correlated with increased αvβ6 expression. Collectively, these findings illustrate the potential utility of αvβ6 molecular imaging for detecting CLAD pathogenesis.
Inflammatory bowel diseases (IBD) including Crohn’s disease (CD) are characterized by impaired intestinal epithelial barrier (IEB) function. Alterations in the desmosomal cadherin desmoglein 2 (Dsg2) have been linked to loss of IEB and intestinal inflammation in IBD. We investigated the pathogenic impact of a cytoplasmatic-truncating mutation in the desmosomal cadherin desmoglein 2 gene (DSG2) in an IBD patient. Genetic analysis of a CD patient revealed a likely pathogenic DSG2 mutation leading to a truncated protein lacking part of the intracellular domain. Generation of an enterocyte-specific mouse model to recapitulate the human mutation resulted in a lethal intestinal barrier defect which was evident in homozygous but not in heterozygous mice. In vitro, barrier defects correlated with changes in Dsg2 mobility and upregulation of pore-forming tight junction protein claudin-2. Organoids derived from heterozygous Dsg2 truncated mice exhibited increased epithelial susceptibility to proinflammatory cytokines. Bulk RNAseq followed by gene ontology analysis in homozygous mice revealed a skewed IL17/IL23 response overlapping with transcriptional changes in patients with CD. Furthermore, 16S rRNA sequencing revealed alterations in microbiome composition in animals that carried the Dsg2 truncation. In summary, a patient-derived mutation in the DSG2 gene causing cytoplasmic truncation in homozygous state induced a lethal intestinal barrier breakdown in mice paralleled by altered gut microbiota and intestinal immune response.
