Journal of Clinical Investigation最新文献

Polymorphisms in Nos3 increases risk for glaucoma, the leading cause of irreversible blindness worldwide. A key modifiable risk factor for glaucoma is intraocular pressure (IOP), which is regulated by nitric oxide (NO), a product of nitric oxide synthase-3 (Nos3) in Schlemm's canal of the conventional outflow pathway. We studied the effects of a conditional, endothelial-specific postnatal deletion of Nos3 (Endo-SclCre-ERT;Nos3flox/flox) on tissues of the outflow pathway. We observed that Cre-ERT expression spontaneously and gradually increased with time in vascular endothelia including Schlemm's canal, beginning at P10, with complete Nos3 deletion occurring around P90. Unlike the reduced outflow resistance in global Nos3 knockout mice, outflow resistance and IOP in Endo-SclCre-ERT;Nos3flox/flox mice were normal. Coinciding with Nos3 deletion, we observed recruitment of macrophages to, and induction of both ELAM-1 and NOS2 expression by endothelia in the distal portion of the outflow pathway, which increased vessel diameter. These adjustments reduced outflow resistance to maintain IOP in these Endo-SclCre-ERT;Nos3flox/flox mice. Selective inhibition of iNOS by 1400W resulted in narrowing of distal vessels and IOP elevation. Together, results emphasize the pliability of the outflow system, the importance of NO signaling in IOP control and implicates an important role for macrophages in IOP homeostasis.

Chimeric Antigen Receptor (CAR) T cell therapy shows promise for various diseases. Our studies in humanized mice and non-human primates (NHPs) demonstrate that hematopoietic stem cell (HSCs) modified with anti-HIV CAR achieve lifelong engraftment, providing functional anti-viral CAR-T cells that reduce viral rebound after ART withdrawal. However, T cell exhaustion due to chronic immune activation remains a key obstacle for sustained CAR-T efficacy, necessitating additional measures to achieve functional cure. We recently showed that low dose rapamycin treatment reduced inflammation and improved anti-HIV T cell function in HIV-infected humanized mice. Here, we report that rapamycin improved CAR-T cell function both in vitro and in vivo. In vitro treatment with rapamycin enhanced CAR-T cell mitochondria respiration and cytotoxicity. In vivo treatment with low-dose rapamycin in HIV-infected, CAR-HSC mice decreased chronic inflammation, prevented exhaustion of CAR-T cells and improved CAR-T control of viral replication. RNAseq analysis of CAR-T cells from humanized mice showed that rapamycin downregulated multiple checkpoint inhibitors and the upregulated key survival genes. Mice treated with CAR-HSCs and rapamycin had delayed viral rebound post-ART and reduced HIV reservoir compared to CAR-HSCs alone. These findings suggest that HSCs-based anti-HIV CAR-T combined with rapamycin treatment is a promising approach for treating persistent inflammation and improving immune control of HIV replication.

Myotonic Dystrophy Type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the DMPK gene. The expanded CUG repeat RNA (CUGexp RNA) transcribed from the mutant allele sequesters the muscleblind-like (MBNL) family of RNA-binding proteins, causing their loss of function and disrupting regulated pre-mRNA processing. We used a DM1 heart mouse model that inducibly expresses CUGexp RNA to test the contribution of MBNL loss to DM1 cardiac abnormalities and explore MBNL restoration as a potential therapy. AAV9-mediated overexpression of MBNL1 and/or MBNL2 significantly rescued DM1 cardiac phenotypes including conduction delays, contractile dysfunction, hypertrophy, and mis-regulated alternative splicing and gene expression. While robust, rescue was partial compared to reduced CUGexp RNA and plateaued with increased exogenous MBNL expression. These findings demonstrate that MBNL loss is a major contributor to DM1 cardiac manifestations, and suggest that additional mechanisms play a role, highlighting the complex nature of DM1 pathogenesis.

Merkel Cell Carcinoma (MCC) is an aggressive neuroendocrine cutaneous malignancy arising from either ultraviolet-induced mutagenesis or Merkel cell polyomavirus (MCPyV) integration. Despite extensive research, our understanding of the molecular mechanisms driving the transition from normal cells to MCC remains limited. To address this knowledge gap, we assessed the impact of inducible MCPyV T antigens on normal human fibroblasts by performing RNA sequencing. Our data uncovered changes in expression and regulation of Wnt signaling pathway members. Building on this observation, we bioinformatically evaluated various Wnt pathway perturbagens for their ability to reverse the MCC gene expression signature and identified pyrvinium pamoate, an FDA-approved anthelminthic drug known for its anti-tumor activity in other cancers. Leveraging transcriptomic, network, and molecular analyses, we found that pyrvinium targets multiple MCC vulnerabilities. Pyrvinium not only reverses the neuroendocrine features of MCC by modulating canonical and non-canonical Wnt signaling but also inhibits cancer cell growth by activating p53-mediated apoptosis, disrupting mitochondrial function, and inducing endoplasmic reticulum stress. Finally, we demonstrated that pyrvinium reduces tumor growth in an MCC mouse xenograft model. These findings offer a new understanding of the role of Wnt signaling in MCC and highlight the utility of pyrvinium as a potential treatment for MCC.

Protein aggregates are emerging therapeutic targets in rare monogenic causes of cardiomyopathy and amyloid heart disease, but their role in more prevalent heart failure syndromes remains mechanistically unexamined. We observed mis-localization of desmin and sarcomeric proteins to aggregates in human myocardium with ischemic cardiomyopathy and in mouse hearts with post-myocardial infarction ventricular remodeling, mimicking findings of autosomal-dominant cardiomyopathy induced by R120G mutation in the cognate chaperone protein, CRYAB. In both syndromes, we demonstrate increased partitioning of CRYAB phosphorylated on serine-59 to NP40-insoluble aggregate-rich biochemical fraction. While CRYAB undergoes phase separation to form condensates, the phospho-mimetic mutation of serine-59 to aspartate (S59D) in CRYAB mimics R120G-CRYAB mutants with reduced condensate fluidity, formation of protein aggregates and increased cell death. Conversely, changing serine to alanine (phosphorylation-deficient mutation) at position 59 (S59A) restored condensate fluidity, and reduced both R120G-CRYAB aggregates and cell death. In mice, S59D CRYAB knock-in was sufficient to induce desmin mis-localization and myocardial protein aggregates, while S59A CRYAB knock-in rescued left ventricular systolic dysfunction post-myocardial infarction and preserved desmin localization with reduced myocardial protein aggregates. 25-Hydroxycholesterol attenuated CRYAB serine-59 phosphorylation and rescued post-myocardial infarction adverse remodeling. Thus, targeting CRYAB phosphorylation-induced condensatopathy is an attractive strategy to counter ischemic cardiomyopathy.

Glioblastoma (GBM) is a highly aggressive form of brain tumor characterized by dysregulated metabolism. Increased fatty acid oxidation (FAO) protects tumor cells from lipid peroxidation-induced cell death, although the precise mechanisms involved remain unclear. Herein, we report that loss of tumor necrosis factor receptor-associated factor 3 (TRAF3) in GBM critically regulates lipid peroxidation and tumorigenesis by controlling the oxidation of polyunsaturated fatty acids (PUFAs). TRAF3 is frequently repressed in GBM due to promoter hypermethylation. TRAF3 interacts with enoyl-CoA hydratase 1 (ECH1), an enzyme catalyzing the isomerization of unsaturated fatty acids (UFAs), and mediates K63-linked ubiquitination of ECH1 at Lys214. ECH1 ubiquitination impedes TOMM20-dependent mitochondrial translocation of ECH1, which otherwise promotes the oxidation of UFAs, preferentially the PUFAs, and limits lipid peroxidation. Overexpression of TRAF3 enhances the sensitivity of GBM to ferroptosis and anti-PD-L1 immunotherapy in mice. Thus, the TRAF3-ECH1 axis plays a key role in the metabolism of PUFAs, and is crucial for lipid peroxidation damage and immune elimination in GBM.

The osteogenic environment promotes vascular calcium phosphate deposition and aggregation of unfolded and misfolded proteins, resulting in endoplasmic reticulum (ER) stress in chronic renal disease (CKD). Controlling ER stress through genetic intervention is a promising approach for treating vascular calcification. In this study, we demonstrated a positive correlation between ER stress-induced tribble 3 (TRIB3) expression and progression of vascular calcification in human and rodent CKD. Increased TRIB3 expression promoted vascular smooth muscle cell (VSMC) calcification by interacting with the C2 domain of the E3 ubiquitin-protein ligase Smurf1, facilitating its K48-related self-ubiquitination at Lys381 and Lys383 and subsequent dissociation from the plasma membrane and nuclei. This degeneration of Smurf1 accelerated the stabilization of the osteogenic transcription factors RUNX Family Transcription Factor 2 (Runx2) and SMAD Family Member 1 (Smad1). C/EBP homologous protein and activating transcription factor 4 are upstream transcription factors of TRIB3 in an osteogenic environment. Genetic knockout of TRIB3 or rescue of Smurf1 ameliorated VSMC and vascular calcification by stabilizing Smurf1 and enhancing the degradation of Runx2 and Smad1. Our findings shed light on the vital role of TRIB3 as a scaffold in ER stress and vascular calcification and offer a potential therapeutic option for chronic renal disease.

Mammalian injury responses are predominantly characterized by fibrosis and scarring rather than functional regeneration. This limited regenerative capacity in mammals could reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we screened transcripts in human subjects following laser rejuvenation treatment and compared them to mice with enhanced Wound Induced Hair Neogenesis (WIHN), a rare example of mammalian organogenesis. We found that Rnasel-/- mice exhibit an increased regenerative capacity, with elevated WIHN through enhanced IL-36α. Consistent with RNase L's known role to stimulate caspase-1, we found that pharmacologic inhibition of caspases promoted regeneration in an IL-36 dependent manner in multiple epithelial tissues. We identified a negative feedback loop, where RNase L activated caspase-1 restrains the pro-regenerative dsRNA-TLR3 signaling cascade through the cleavage of toll-like adaptor protein TRIF. Through integrated single-cell RNA sequencing and spatial transcriptomic profiling, we confirmed Oas & Il36 genes to be highly expressed at the site of wounding and are elevated in Rnasel-/- mice wounds. This work suggests that RNase L functions as a regeneration repressor gene, in a functional tradeoff that tempers immune hyper-activation during viral infection at the cost of inhibiting regeneration.

Sleep disturbance is bidirectionally associated with increased risks of Alzheimer's disease and other tauopathies. While the sleep-wake cycle regulates interstitial and cerebrospinal fluid (CSF) tau levels, the underlying mechanisms remain unknown. Understanding these mechanisms is crucial given evidence indicates that tau pathology spreads through neuron-to-neuron transfer, involving the secretion and internalization of pathological tau forms. Here, we combine in vitro, in vivo and clinical methods to reveal a pathway by which changes in body temperature (BT) over the sleep-wake cycle modulate extracellular tau levels. In mice, higher BT during wakefulness and sleep-deprivation increased CSF and plasma tau levels, while also upregulating unconventional protein secretion pathway-I (UPS-I) components, including (i) intracellular tau dephosphorylation, (ii) caspase-3-mediated cleavage of tau (TauC3) and (iii) its membrane translocation through binding to PIP2 and syndecan-3. In humans, the increase in CSF and plasma tau levels observed post-wakefulness correlated with BT increase during wakefulness. By demonstrating that sleep-wake variation in BT regulates extracellular tau levels, our findings highlight the importance of thermoregulation in linking sleep disturbances to tau-mediated neurodegeneration, and the preventative potential of thermal interventions.