ABSTRACT
Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease despite higher mitochondrial content. We sought to illuminate noncanonical, cell-specific roles for CoQ, independently of the electron transport chain (ETC). Here, we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway rather than ETC dysfunction. Single-nucleus RNA-Seq from kidneys of Pdss2kd/kd mice with nephrotic syndrome and global CoQ deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound, ameliorated kidney disease in Pdss2kd/kd mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.
Subject(s)
Ataxia/metabolism , Indenes/pharmacology , Kidney Diseases/metabolism , Lipid Peroxidation/drug effects , MAP Kinase Signaling System/drug effects , Mitochondrial Diseases/metabolism , Muscle Weakness/metabolism , Podocytes/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Pyrazoles/pharmacology , Ubiquinone/deficiency , Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/metabolism , Animals , Ataxia/drug therapy , Ataxia/genetics , Ataxia/pathology , Drug Delivery Systems , HEK293 Cells , Humans , Kidney Diseases/drug therapy , Kidney Diseases/genetics , Kidney Diseases/pathology , Lipid Peroxidation/genetics , MAP Kinase Signaling System/genetics , Mice , Mitochondrial Diseases/drug therapy , Mitochondrial Diseases/genetics , Mitochondrial Diseases/pathology , Muscle Weakness/drug therapy , Muscle Weakness/genetics , Muscle Weakness/pathology , Podocytes/pathology , Proto-Oncogene Proteins B-raf/genetics , RNA-Seq , Ubiquinone/genetics , Ubiquinone/metabolismABSTRACT
Drug repurposing has the advantage of identifying potential treatments on a shortened timescale. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high-content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce mucin-1 (MUC1) protein abundance. Elevated MUC1 levels predict the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and correlate with poor clinical outcomes. Our screen identifies fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, fostamatinib reduces MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by the active metabolite R406 promotes MUC1 removal from the cell surface. Our work suggests fostamatinib as a repurposing drug candidate for ALI.
ABSTRACT
Upon infection or brain damage, microglia are activated to play roles in immune responses, including phagocytosis and soluble factor release. However, little is known whether the event of phagocytosis could be a trigger for releasing soluble factors from microglia. In this study, we tested if microglia secrete a neurovascular mediator matrix metalloproteinase-9 (MMP-9) after phagocytosis in vitro. Primary microglial cultures were prepared from neonatal rat brains. Cultured microglia phagocytosed Escherichia coli bioparticles within 2 hr after incubation and started to secrete MMP-9 at around 12 hr after the phagocytosis. A TLR4 inhibitor TAK242 suppressed the E. coli-bioparticle-induced MMP-9 secretion. However, TAK242 did not change the engulfment of E. coli bioparticles in microglial cultures. Because lipopolysaccharides (LPS), the major component of the outer membrane of E. coli, also induced MMP-9 secretion in a dose-response manner and because the response was inhibited by TAK242 treatment, we assumed that the LPS-TLR4 pathway, which was activated by adhering to the substance, but not through the engulfing process of phagocytosis, would play a role in releasing MMP-9 from microglia after E. coli bioparticle treatment. To support the finding that the engulfing step would not be a critical trigger for MMP-9 secretion after the event of phagocytosis in microglia, we confirmed that cell debris and amyloid beta were both captured into microglia via phagocytosis, but neither of them induced MMP-9 secretion from microglia. Taken together, these data demonstrate that microglial response in MMP-9 secretion after phagocytosis differs depending on the types of particles/substances that microglia encountered.
Subject(s)
Amyloid beta-Peptides/metabolism , Matrix Metalloproteinase 9/metabolism , Microglia/metabolism , Phagocytosis/physiology , Animals , Cells, Cultured , Escherichia coli/metabolism , Lipopolysaccharides/pharmacology , RatsABSTRACT
OBJECTIVE: To determine if CSF and plasma levels of soluble vascular endothelial (sVE)-cadherin are associated with functional outcome after subarachnoid hemorrhage (SAH) and to investigate sVE-cadherin effects on microglia. METHODS: Serial CSF and plasma were collected from prospectively enrolled patients with nontraumatic SAH from a ruptured aneurysm in the anterior circulation and who required an external ventricular drain for clinical indications. Patients with normal-pressure hydrocephalus without SAH served as controls. For prospective assessment of long-term outcomes at 3 and 6 months after SAH, modified Rankin Scale scores (mRS) were obtained and dichotomized into good (mRS ≤ 2) vs poor (mRS > 2) outcome groups. For SAH severity, Hunt and Hess grade was assessed. Association of CSF sVE-cadherin levels with long-term outcomes, HH grade, and CSF tumor necrosis factor (TNF)-α levels were evaluated. sVE-cadherin effects on microglia were also studied. RESULTS: sVE-cadherin levels in CSF, but not in plasma, were higher in patients with SAH and were associated with higher clinical severity and higher CSF TNF-α levels. Patients with SAH with higher CSF sVE-cadherin levels over time were more likely to develop worse functional outcome at 3 months after SAH. Incubation of cultured microglia with sVE-cadherin resulted in increased inducible nitric oxide synthase, interleukin-1ß, reactive oxygen species, cell soma size, and metabolic activity, consistent with microglia activation. Microinjection of sVE-cadherin fragments into mouse brain results in an increased number of microglia surrounding the injection site, compared to injection of denatured vascular endothelial-cadherin fragments. CONCLUSIONS: These results support the existence of a novel pathway by which sVE-cadherin, released from injured endothelium after SAH, can shift microglia into a more proinflammatory phenotype and contribute to neuroinflammation and poor outcome in SAH.