Alcohol dehydrogenase 1 is a tubular mitophagy-dependent apoptosis inhibitor against septic acute kidney injury.

Alcohol dehydrogenase 1 (ADH1) is an alcohol-oxidizing enzyme with poorlydefined biology. Here we report that ADH1 is highly expressed in kidneys of mice with lethal endotoxemia and is transcriptionally upregulated in tubular cells by lipopolysaccharide (LPS) stimuli through TLR4/NF-κB cascade. The Adh1 knockout (Adh1KO) mice with lethal endotoxemia displayed increased susceptibility to acute kidney injury (AKI) but not systemic inflammatory response. Adh1KO mice develop more severe tubular cell apoptosis in comparison to Adh1 wild-type (Adh1WT) mice during course of lethal endotoxemia. ADH1 deficiency facilitates the LPS-induced tubular cell apoptosis in a caspase-dependent manner. Mechanistically, ADH1 deficiency dampens tubular mitophagy that relies on PINK1-Parkin pathway characterized by the reduced membrane potential, reactive oxygen species (ROS) and release of fragmented mtDNA to cytosol. Kidney-specific overexpression of PINK1 and Parkin by adeno-associated viral vector 9 (AAV9) delivery ameliorates AKI exacerbation in Adh1KO mice with lethal endotoxemia. Our study supports the notion that ADH1 is critical for blockade of tubular apoptosis mediated by mitophagy, allowing the rapid identification and targeting of alcohol-metabolic route applicable to septic AKI.

Ascorbate uptake enables tubular mitophagy to prevent septic AKI by PINK1-PARK2 axis.

Ascorbate (Vitamin C) has been proposed as a promising therapeutic agent against sepsis in clinical trials, but there is little experimental evidence on its anti-septic efficacy. We report that Toll-like receptor 4 (TLR4) activation by LPS stimuli augments ascorbate uptake in murine and human tubular cells through upregulation of two ascorbate transporters SVCT-1 and -2 mediated by Fn14/SCFFbxw7α cascade. Ascorbate restriction, or knockout of SVCT-1 and -2, the circumstance reminiscent to blockade of ascorbate uptake, endows tubular cells more vulnerable to the LPS-inducible apoptosis, whereas exogenous administration of ascorbate overrides the ruin execution, for which the PINK1-PARK2, rather than BNIP3-NIX axis is required. Ascorbate increases, while SVCT-1 and -2 knockout or ascorbate restriction dampens tubular mitophagy upon LPS stimuli. Treatment of endotoxemic mice with high-dose ascorbate confers mitophagy and substantial protection against mortality and septic acute kidney injury (AKI). Our work provides a rationale for clinical management of septic AKI with high doses of ascorbate.

Selenoprotein T Promotes Proliferation and G1-to-S Transition in SK-N-SH Cells: Implications in Parkinson's Disease.

BACKGROUND: Selenium is prioritized to the brain mainly for selenoprotein expression. Selenoprotein T (SELENOT) protects dopaminergic, postmitotic neurons in a mouse model of Parkinson's disease (PD). OBJECTIVE: We hypothesized a proliferative role of SELENOT in neural cells. METHODS: To assess SELENOT status in PD, sedated male C57BL/6 mice at 10-12 wk of age were injected with 6-hydroxydopamine in neurons, and human peripheral blood mononuclear cells were isolated from 9 healthy subjects (56% men, 68-y-old) and 11 subjects with PD (64% men, 63-y-old). Dopaminergic neural progenitor-like SK-N-SH cells with transient SELENOT overexpression or knockdown were maintained in the presence or absence of the antioxidant N-acetyl-l-cysteine and the calcium channel blocker nimodipine. Cell cycle, proliferation, and signaling parameters were determined by immunoblotting, qPCR, and flow cytometry. RESULTS: SELENOT mRNA abundance was increased (P < 0.05) in SK-N-SH cells treated with 1-methyl-4-phenylpyridinium iodide (3.5-fold) and peripheral blood mononuclear cells from PD patients (1.6-fold). Likewise, SELENOT was expressed in tyrosine hydroxylase-positive dopaminergic neurons of 6-hydroxydopamine-injected mice. Knockdown of SELENOT in SK-N-SH cells suppressed (54%; P < 0.05) 5-ethynyl-2'-deoxyuridine incorporation but induced (17-47%; P < 0.05) annexin V-positive cells, CASPASE-3 cleavage, and G1/S cell cycle arrest. SELENOT knockdown and overexpression increased (88-120%; P < 0.05) and reduced (37-42%; P < 0.05) both forkhead box O3 and p27, but reduced (51%; P < 0.05) and increased (1.2-fold; P < 0.05) cyclin-dependent kinase 4 protein abundance, respectively. These protein changes were diminished by nimodipine or N-acetyl-l-cysteine treatment (24 h) at steady-state levels. While the N-acetyl-l-cysteine treatment did not influence the reduction in the amount of calcium (13%; P < 0.05) by SELENOT knockdown, the nimodipine treatment reversed the decreased amount of reactive oxygen species (33%; P < 0.05) by SELENOT overexpression. CONCLUSIONS: These cellular and mouse data link SELENOT to neural proliferation, expanding our understanding of selenium protection in PD.

Mitochondrial DNA variants modulate genetic susceptibility to Parkinson's disease in Han Chinese.

It is well recognized that mitochondrial dysfunction is involved in the pathogenesis of Parkinson's disease (PD). The mtDNA displacement loop (D-loop) region is known to accumulate structural alterations and mutations. To understand how mtDNA variants contribute to the susceptibility to sporadic PD in Chinese, a total of 500 PD patients and 505 controls were recruited from East China, and their D-loop regions were sequenced. A total of 389 variants were detected out of the 1005 subjects. There were 91 variants with frequencies >1%, which included 88 single nucleotide polymorphisms (SNPs), 2 deletions and 1 insertion. Amongst, 6 SNPs were significantly associated with sporadic PD. Specifically, the SNPs 151T/C, 189G/A, 16086C/T and 16271C/T contributed to increased susceptibility, while 318C/T and 16134T/C were associated with reduced risk for PD. Further analyses of mtDNA haplogroups and their risk for PD occurrence showed that subjects carrying haplogroup A5 were susceptible while haplogroup B5 carriers were more resistant to the disease. In summary, our study for the first time systematically analyzed mtDNA variants by sequencing the D-loop region in a Chinese population to understand their associations with PD. These results demonstrate that mtDNA variants modulate risk for sporadic PD.

Auto- and paracrine rewiring of NIX-mediated mitophagy by insulin-like growth factor-binding protein 7 in septic AKI escalates inflammation-coupling tubular damage.

AIMS: Inflammation-coupling tubular damage (ICTD) contributes to pathogenesis of septic acute kidney injury (AKI), in which insulin-like growth factor-binding protein 7 (IGFBP-7) serves as a biomarker for risk stratification. The current study aims to discern how IGFBP-7 signalling influences ICTD, the mechanisms that underlie this process and whether blockade of the IGFBP-7-dependent ICTD might have therapeutic value for septic AKI. MATERIALS AND METHODS: In vivo characterization was carried out in B6/JGpt-Igfbp7em1Cd1165/Gpt mice subjected to cecal ligation and puncture (CLP). Transmission electron microscopy, immunofluorescence, flow cytometry, immunoblotting, ELISA, RT-qPCR and dual-luciferase reporter assays were used to determine mitochondrial functions, cell apoptosis, cytokine secretion and gene transcription. KEY FINDINGS: ICTD augments the transcriptional activity and protein secretion of tubular IGFBP-7, which enables an auto- and paracrine signalling via deactivation of IGF-1 receptor (IGF-1R). Genetic knockout (KO) of IGFBP-7 provides renal protection, improves survival and resolves inflammation in murine models of cecal ligation and puncture (CLP), while administering recombinant IGFBP-7 aggravates ICTD and inflammatory invasion. IGFBP-7 perpetuates ICTD in a NIX/BNIP3-indispensable fashion through dampening mitophagy that restricts redox robustness and preserves mitochondrial clearance programs. Adeno-associated viral vector 9 (AAV9)-NIX short hairpin RNA (shRNA) delivery ameliorates the anti-septic AKI phenotypes of IGFBP-7 KO. Activation of BNIP3-mediated mitophagy by mitochonic acid-5 (MA-5) effectively attenuates the IGFBP-7-dependent ICTD and septic AKI in CLP mice. SIGNIFICANCE: Our findings identify IGFBP-7 is an auto- and paracrine manipulator of NIX-mediated mitophagy for ICTD escalation and propose that targeting the IGFBP-7-dependent ICTD represents a novel therapeutic strategy against septic AKI.

Interruption of neutrophil extracellular traps formation dictates host defense and tubular HOXA5 stability to augment efficacy of anti-Fn14 therapy against septic AKI.

The immunosuppressive, inflammatory microenvironment orchestrated by neutrophil extracellular traps (NETs) plays a principal role in pathogenesis of sepsis. Fibroblast growth factor-inducible molecule 14 (Fn14) has been established as a potential target for septic acute kidney injury (AKI), making further therapeutic benefits from combined NETs and Fn14 blockade possible. Methods: The concurrence of NETs and Fn14 in mice and patients with septic AKI were assessed by immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and in silico studies. Survival, histopathological and biochemical analyses of wild-type and PAD4-deficient CMV-Cre; PAD4 fl/fl mice with septic AKI were applied to evaluate the efficacy of either pharmacological or genetic NETs interruption in combination with Fn14 blockade. Molecular mechanisms underlying such effects were determined by CRISPR technology, fluorescence-activated cell sorter analysis (FACS), cycloheximide (CHX) pulse-chase, luciferase reporter and chromatin immunoprecipitation (ChIP) assay. Results: NETs formation is concurred with Fn14 upregulation in murine AKI models of abdominal, endotoxemic, multidrug-resistant sepsis as well as in serum samples of patients with septic AKI. Pharmacological or genetic interruption of NETs formation synergizes with ITEM-2, a monoclonal antibody (mAb) of Fn14, to prolong mice survival and provide renal protection against abdominal sepsis, the effects that could be abrogated by elimination of macrophages. Interrupting NETs formation predominantly perpetuates infiltration and survival of efferocytic growth arrest-specific protein 6+ (GAS6+) macrophages in combination with ITEM-2 therapy and enhances transcription of tubular cell-intrinsic Fn14 in a DNA methyltransferase 3a (DNMT3a)-independent manner through dismantling the proteasomes-mediated turnover of homeobox protein Hox-A5 (HOXA5) upon abdominal sepsis challenge or LPS stimuli. Pharmacological NETs interruption potentiates the anti-septic AKI efficacy of ITEM-2 in murine models of endotoxemic and multidrug-resistant sepsis. Conclusion: Our preclinical data propose that interrupting NETs formation in combination with Fn14 mAb might be a feasible therapeutic strategy for septic AKI.

MicroRNA-19a Targets Fibroblast Growth Factor-Inducible Molecule 14 and Prevents Tubular Damage in Septic AKI.

Fibroblast growth factor-inducible molecule 14 (Fn14) plays a principal role in triggering tubular damage during septic acute kidney injury (AKI). Here, we explore the mechanism underlying Fn14 deregulation in septic AKI. We identify Fn14 as a bona fide target of miR-19a, which directly binds to 3' UTR of Fn14 for repression independent of cylindromatosis (CYLD), the deubiquitinase (DUB) downstream of miR-19a, and thereby antagonizes the LPS-induced tubular cell apoptosis. Genetic ablation of Fn14, but not of CYLD, abolishes the ability of miR-19a to antagonize the tubular apoptosis by lipopolysaccharide (LPS). In mice, systemic delivery of miR-19a confers protection against septic AKI. Our findings implicate that miR-19a may serve as a promising therapeutic candidate in the prevention of septic AKI.

Redox DAPK1 destabilizes Pellino1 to govern inflammation-coupling tubular damage during septic AKI.

Tubular damage initiated by inflammatory response and ischemic/hypoxic stress is a hallmark of septic acute kidney injury (AKI), albeit the molecular mechanism coupling the two events remains unclear. We investigated the intrinsic nature of tubular damage with respect to inflammatory/hypoxic stress during septic AKI. Methods: The apoptotic response of tubular cells to LPS stimuli was analyzed before and after hypoxia exposure. Cellular ubiquitination, co-immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and CRISPR technology were adopted to determine the molecular mechanism underlying this process. In vivo characterization was performed in wild-type and DAPK1-/- mice models of cecal ligation and puncture (CLP). Results: We found that the MyD88-dependent inflammatory response couples to tubular damage during LPS stimuli under hypoxia in a Fn14/SCFFbxw7α-dispensable manner via recruitment of caspase-8 with TRIF-RIP1 signalosome mediated by DAPK1, which directly binds to and phosphorylates Pellino1 at Ser39, leading to Pellino1 poly-ubiquitination and turnover. Either pharmacological deactivation or genetic ablation of DAPK1 makes tubular cells refractory to the LPS-induced damage in the context of hypoxia, while kinase activity of DAPK1 is essential for ruin execution. Targeting DAPK1 effectively protects mice against septic AKI and potentiates the efficacy of a MyD88 homodimerization inhibitor, ST2825. Conclusion: Our findings provide a rationale for the mechanism whereby inflammation intersects with hypoxic tubular damage during septic AKI through a previously unappreciated role of DAPK1-inducible Ser39 phosphorylation in Pellino1 turnover and underscore that combined targeting DAPK1 and MyD88 might be a feasible strategy for septic AKI management.

Comparison of the efficacy of four cholinesterase inhibitors in combination with memantine for the treatment of Alzheimer's disease.

BACKGROUND: Combined use of memantine and acetylcholinesterase inhibitors (AChEIs) has shown improved outcomes in patients with Alzheimer's disease (AD). However, it is not clear which AChEI is the optimal for the combined treatment with memantine. METHODS: A total of 110 AD patients were randomized to receive memantine and one of the following add-on drugs: placebo, donepezil, rivastigmine, galantamine, and huperzine A for 24 weeks (n=22). At baseline, 12 weeks, and 24 weeks, the patients were evaluated using mini-mental state examination (MMSE) and Alzheimer Disease Cooperative Study-Activities of Daily Living (ADCS-ADL) scales. Adverse events were recorded to analyze the safety profile. RESULTS: The MMSE scores were significantly increased and the ADL scores were significantly decreased at 12 weeks and 24 weeks in all five groups compared with baseline (all P<0.01). At 24 weeks, patients treated with memantine+huperzine A showed better MMSE and ADL scores than those treated with memantine+placebo. CONCLUSIONS: Huperzine A may be an optimal choice for the combined therapy with memantine in treating AD.

Neuroinflammation and neuronal autophagic death were suppressed via Rosiglitazone treatment: New evidence on neuroprotection in a rat model of global cerebral ischemia.

Ischemic stroke is one of the leading causes of mortality and disability with documented high incidence and relapse rate. Accumulating evidence indicates that autophagy participated in neuronal cell death and functional loss induced following ischemia/reperfusion (I/R) injury. The peroxisome proliferating activating receptor-γ (PPAR-γ) agonist, Rosiglitazone (RSG), is known for its anti-inflammatory actions. Previous studies have demonstrated that RSG can exert neuroprotection in animal models of both chronic brain injuries and acute brain insults. However, whether RSG treatment is involved in the autophagic neuronal death following I/R injury remains totally unclear. The present study aimed to hypothesize that treatment of RSG could induce neuroprotective properties in a rat model of global cerebral ischemia (GCI), and thereby to investigate the underline mechanisms. We found that a single injection of RSG immediately following GCI significantly reduced cerebral infarct volume and brain edema, as well as increased neuron survival rate and function recovery. These effects correlate with a decrease of inflammatory cytokines and autophagy-associated proteins expression in the hippocampus region. Our results provide in vivo evidence that RSG significantly protected rats against I/R injury induced brain injury, and the mechanism might associate with inhibiting the processes of neuroinflammation and thereby attenuated of neuronal autophagic death. All data suggest that RSG can be further developed as a clinical neuroprotective candidate in ischemic stroke.