Nitration-mediated activation of the small GTPase RhoA stimulates cellular glycolysis through enhanced mitochondrial fission.

Mitochondrial fission and a Warburg phenotype of increased cellular glycolysis are involved in the pathogenesis of pulmonary hypertension (PH). The purpose of this study was to determine whether increases in mitochondrial fission are involved in a glycolytic switch in pulmonary arterial endothelial cells (PAECs). Mitochondrial fission is increased in PAEC isolated from a sheep model of PH induced by pulmonary overcirculation (Shunt PAEC). In Shunt PAEC we identified increases in the S616 phosphorylation responsible for dynamin-related protein 1 (Drp1) activation, the mitochondrial redistribution of Drp1, and increased cellular glycolysis. Reducing mitochondrial fission attenuated cellular glycolysis in Shunt PAEC. In addition, we observed nitration-mediated activation of the small GTPase RhoA in Shunt PAEC, and utilizing a nitration-shielding peptide, NipR1 attenuated RhoA nitration and reversed the Warburg phenotype. Thus, our data identify a novel link between RhoA, mitochondrial fission, and cellular glycolysis and suggest that targeting RhoA nitration could have therapeutic benefits for treating PH.

Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice.

The sex and APOE4 genotype are significant risk factors for Alzheimer's disease (AD); however, the mechanism(s) responsible for this interaction are still a matter of debate. Here, we assess the responses of mixed-sex and sex-specific APOE3 and APOE4 primary microglia (PMG) to lipopolysaccharide and interferon-gamma. In our investigation, inflammatory cytokine profiles were assessed by qPCR and multiplex ELISA assays. Mixed-sex APOE4 PMG exhibited higher basal mRNA expression and secreted levels of TNFa and IL1b. In sex-specific cultures, basal expression and secreted levels of IL1b, TNFa, IL6, and NOS2 were 2−3 fold higher in APOE4 female PMG compared to APOE4 males, with both higher than APOE3 cells. Following an inflammatory stimulus, the expression of pro-inflammatory cytokines and the secreted cytokine level were upregulated in the order E4 female > E4 male > E3 female > E3 male in sex-specific cultures. These data indicate that the APOE4 genotype and female sex together contribute to a greater inflammatory response in PMG isolated from targeted replacement humanized APOE mice. These data are consistent with clinical data and indicate that sex-specific PMG may provide a platform for exploring mechanisms of genotype and sex differences in AD related to neuroinflammation and neurodegeneration.

Exosomes in Parkinson disease.

Parkinson disease (PD) is a prevalent neurodegenerative disease, in which the formation of misfolded and aggregated α-synuclein is a key neuropathological hallmark. Recent studies reveal that extracellular vesicles such as exosomes present a potential mechanism for propagation of pathological α-synuclein throughout the brain. The ability of exosomes to transport proteins and genetic material between cells, including mRNA and microRNAs which have been implicated in PD pathology, provides critical insights as to how exosomes may contribute to pathological progression in PD. Advances have also been made in the investigation of exosomes as potential tools for the modulation of Parkinson's pathology; their detection extracellularly may facilitate their use as biomarkers, while their small size could be utilised as vectors for the delivery of therapeutics. The aim of this review was to highlight our current knowledge of the role of exosomes in PD and potential clinical application.

Microglial exosomes facilitate α-synuclein transmission in Parkinson's disease.

Accumulation of neuronal α-synuclein is a prominent feature in Parkinson's disease. More recently, such abnormal protein aggregation has been reported to spread from cell to cell and exosomes are considered as important mediators. The focus of such research, however, has been primarily in neurons. Given the increasing recognition of the importance of non-cell autonomous-mediated neurotoxicity, it is critical to investigate the contribution of glia to α-synuclein aggregation and spread. Microglia are the primary phagocytes in the brain and have been well-documented as inducers of neuroinflammation. How and to what extent microglia and their exosomes impact α-synuclein pathology has not been well delineated. We report here that when treated with human α-synuclein preformed fibrils, exosomes containing α-synuclein released by microglia are fully capable of inducing protein aggregation in the recipient neurons. Additionally, when combined with microglial proinflammatory cytokines, these exosomes further increased protein aggregation in neurons. Inhibition of exosome synthesis in microglia reduced α-synuclein transmission. The in vivo significance of these exosomes was demonstrated by stereotaxic injection of exosomes isolated from α-synuclein preformed fibrils treated microglia into the mouse striatum. Phosphorylated α-synuclein was observed in multiple brain regions consistent with their neuronal connectivity. These animals also exhibited neurodegeneration in the nigrostriatal pathway in a time-dependent manner. Depleting microglia in vivo dramatically suppressed the transmission of α-synuclein after stereotaxic injection of preformed fibrils. Mechanistically, we report here that α-synuclein preformed fibrils impaired autophagy flux by upregulating PELI1, which in turn, resulted in degradation of LAMP2 in activated microglia. More importantly, by purifying microglia/macrophage derived exosomes in the CSF of Parkinson's disease patients, we confirmed the presence of α-synuclein oligomer in CD11b+ exosomes, which were able to induce α-synuclein aggregation in neurons, further supporting the translational aspect of this study. Taken together, our study supports the view that microglial exosomes contribute to the progression of α-synuclein pathology and therefore, they may serve as a promising therapeutic target for Parkinson's disease.

PINK1 signalling rescues amyloid pathology and mitochondrial dysfunction in Alzheimer's disease.

Mitochondrial dysfunction and synaptic damage are early pathological features of the Alzheimer's disease-affected brain. Memory impairment in Alzheimer's disease is a manifestation of brain pathologies such as accumulation of amyloid-ß peptide and mitochondrial damage. The underlying pathogenic mechanisms and effective disease-modifying therapies for Alzheimer's disease remain elusive. Here, we demonstrate for the first time that decreased PTEN-induced putative kinase 1 (PINK1) expression is associated with Alzheimer's disease pathology. Restoring neuronal PINK1 function strikingly reduces amyloid-ß levels, amyloid-associated pathology, oxidative stress, as well as mitochondrial and synaptic dysfunction. In contrast, PINK1-deficient mAPP mice augmented cerebral amyloid-ß accumulation, mitochondrial abnormalities, impairments in learning and memory, as well as synaptic plasticity at an earlier age than mAPP mice. Notably, gene therapy-mediated PINK1 overexpression promotes the clearance of damaged mitochondria by augmenting autophagy signalling via activation of autophagy receptors (OPTN and NDP52), thereby alleviating amyloid-ß-induced loss of synapses and cognitive decline in Alzheimer's disease mice. Loss of PINK1 activity or blockade of PINK1-mediated signalling (OPTN or NDP52) fails to reverse amyloid-ß-induced detrimental effects. Our findings highlight a novel mechanism by which PINK1-dependent signalling promotes the rescue of amyloid pathology and amyloid-ß-mediated mitochondrial and synaptic dysfunctions in a manner requiring activation of autophagy receptor OPTN or NDP52. Thus, activation of PINK1 may represent a new therapeutic avenue for combating Alzheimer's disease.

Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity.

Adenosine is a potent anticonvulsant acting on excitatory synapses through A1 receptors. Cellular release of ATP, and its subsequent extracellular enzymatic degradation to adenosine, could provide a powerful mechanism for astrocytes to control the activity of neural networks during high-intensity activity. Despite adenosine's importance, the cellular source of adenosine remains unclear. We report here that multiple enzymes degrade extracellular ATP in brain tissue, whereas only Nt5e degrades AMP to adenosine. However, endogenous A1 receptor activation during cortical seizures in vivo or heterosynaptic depression in situ is independent of Nt5e activity, and activation of astrocytic ATP release via Ca(2+) photolysis does not trigger synaptic depression. In contrast, selective activation of postsynaptic CA1 neurons leads to release of adenosine and synaptic depression. This study shows that adenosine-mediated synaptic depression is not a consequence of astrocytic ATP release, but is instead an autonomic feedback mechanism that suppresses excitatory transmission during prolonged activity.

Paraquat neurotoxicity is mediated by the dopamine transporter and organic cation transporter-3.

The herbicide paraquat (PQ) has increasingly been reported in epidemiological studies to enhance the risk of developing Parkinson's disease (PD). Furthermore, case-control studies report that individuals with genetic variants in the dopamine transporter (DAT, SLC6A) have a higher PD risk when exposed to PQ. However, it remains a topic of debate whether PQ can enter dopamine (DA) neurons through DAT. We report here a mechanism by which PQ is transported by DAT: In its native divalent cation state, PQ(2+) is not a substrate for DAT; however, when converted to the monovalent cation PQ(+) by either a reducing agent or NADPH oxidase on microglia, it becomes a substrate for DAT and is accumulated in DA neurons, where it induces oxidative stress and cytotoxicity. Impaired DAT function in cultured cells and mutant mice significantly attenuated neurotoxicity induced by PQ(+). In addition to DAT, PQ(+) is also a substrate for the organic cation transporter 3 (Oct3, Slc22a3), which is abundantly expressed in non-DA cells in the nigrostriatal regions. In mice with Oct3 deficiency, enhanced striatal damage was detected after PQ treatment. This increased sensitivity likely results from reduced buffering capacity by non-DA cells, leading to more PQ(+) being available for uptake by DA neurons. This study provides a mechanism by which DAT and Oct3 modulate nigrostriatal damage induced by PQ(2+)/PQ(+) redox cycling.

Toxin-Induced Animal Models of Parkinson's Disease.

The debilitating motor symptoms of Parkinson's disease (PD) result primarily from the degenerative nigrostriatal dopaminergic pathway. To elucidate pathogenic mechanisms and evaluate therapeutic strategies for PD, numerous animal models have been developed. Understanding the strengths and limitations of these models can significantly impact the choice of model, experimental design, and data interpretation. Herein, we systematically review the literature over the past decade. Some models no longer serve the purpose of PD models. The primary objectives of this review are: First, to assist new investigators in navigating through available animal models and making appropriate selections based on the objective of the study. Emphasis will be placed on common toxin-induced murine models. And second, to provide an overview of basic technical requirements for assessing the nigrostriatal pathway's pathology, structure, and function.

A partial Drp1 knockout improves autophagy flux independent of mitochondrial function.

BACKGROUND: Dynamin-related protein 1 (Drp1) plays a critical role in mitochondrial dynamics. Partial inhibition of this protein is protective in experimental models of neurological disorders such as Parkinson's disease and Alzheimer's disease. The protective mechanism has been attributed primarily to improved mitochondrial function. However, the observations that Drp1 inhibition reduces protein aggregation in such neurological disorders suggest the involvement of autophagy. To investigate this potential novel protective mechanism of Drp1 inhibition, a model with impaired autophagy without mitochondrial involvement is needed. METHODS: We characterized the effects of manganese (Mn), which causes parkinsonian-like symptoms in humans, on autophagy and mitochondria by performing dose-response studies in two cell culture models (stable autophagy HeLa reporter cells and N27 rat immortalized dopamine neuronal cells). Mitochondrial function was assessed using the Seahorse Flux Analyzer. Autophagy flux was monitored by quantifying the number of autophagosomes and autolysosomes, as well as the levels of other autophagy proteins. To strengthen the in vitro data, multiple mouse models (autophagy reporter mice and mutant Drp1+/- mice and their wild-type littermates) were orally treated with a low chronic Mn regimen that was previously reported to increase α-synuclein aggregation and transmission via exosomes. RNAseq, laser captured microdissection, immunofluorescence, immunoblotting, stereological cell counting, and behavioural studies were used. RESULTS IN VITRO: data demonstrate that at low non-toxic concentrations, Mn impaired autophagy flux but not mitochondrial function and morphology. In the mouse midbrain, RNAseq data further confirmed autophagy pathways were dysregulated but not mitochondrial related genes. Additionally, Mn selectively impaired autophagy in the nigral dopamine neurons but not the nearby nigral GABA neurons. In cells with a partial Drp1-knockdown and Drp1+/- mice, Mn induced autophagic impairment was significantly prevented. Consistent with these observations, Mn increased the levels of proteinase-K resistant α-synuclein and Drp1-knockdown protected against this pathology. CONCLUSIONS: This study demonstrates that improved autophagy flux is a separate mechanism conferred by Drp1 inhibition independent of its role in mitochondrial fission. Given that impaired autophagy and mitochondrial dysfunction are two prominent features of neurodegenerative diseases, the combined protective mechanisms targeting these two pathways conferred by Drp1 inhibition make this protein an attractive therapeutic target.

Mitochondrial hyperfusion induces metabolic remodeling in lung endothelial cells by modifying the activities of electron transport chain complexes I and III.

OBJECTIVE: Pulmonary hypertension (PH) is a progressive disease with vascular remodeling as a critical structural alteration. We have previously shown that metabolic reprogramming is an early initiating mechanism in animal models of PH. This metabolic dysregulation has been linked to remodeling the mitochondrial network to favor fission. However, whether the mitochondrial fission/fusion balance underlies the metabolic reprogramming found early in PH development is unknown. METHODS: Utilizing a rat early model of PH, in conjunction with cultured pulmonary endothelial cells (PECs), we utilized metabolic flux assays, Seahorse Bioassays, measurements of electron transport chain (ETC) complex activity, fluorescent microscopy, and molecular approaches to investigate the link between the disruption of mitochondrial dynamics and the early metabolic changes that occur in PH. RESULTS: We observed increased fusion mediators, including Mfn1, Mfn2, and Opa1, and unchanged fission mediators, including Drp1 and Fis1, in a two-week monocrotaline-induced PH animal model (early-stage PH). We were able to establish a connection between increases in fusion mediator Mfn1 and metabolic reprogramming. Using an adenoviral expression system to enhance Mfn1 levels in pulmonary endothelial cells and utilizing 13C-glucose labeled substrate, we found increased production of 13C lactate and decreased TCA cycle metabolites, revealing a Warburg phenotype. The use of a 13C5-glutamine substrate showed evidence that hyperfusion also induces oxidative carboxylation. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels secondary to the disruption of cellular bioenergetics and higher levels of mitochondrial reactive oxygen species (mt-ROS). The elevation in mt-ROS correlated with attenuated ETC complexes I and III activities. Utilizing a mitochondrial-targeted antioxidant to suppress mt-ROS, limited HIF-1α protein levels, which reduced cellular glycolysis and reestablished mitochondrial membrane potential. CONCLUSIONS: Our data connects mitochondrial fusion-mediated mt-ROS to the Warburg phenotype in early-stage PH development.

PINK1-dependent recruitment of Parkin to mitochondria in mitophagy.

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARK2/Parkin mutations cause autosomal recessive forms of Parkinson's disease. Upon a loss of mitochondrial membrane potential (DeltaPsi(m)) in human cells, cytosolic Parkin has been reported to be recruited to mitochondria, which is followed by a stimulation of mitochondrial autophagy. Here, we show that the relocation of Parkin to mitochondria induced by a collapse of DeltaPsi(m) relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal DeltaPsi(m). We also show that once at the mitochondria, Parkin is in close proximity to PINK1, but we find no evidence that Parkin catalyzes PINK1 ubiquitination or that PINK1 phosphorylates Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into mitochondrial aggregates and/or large perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin, together with PINK1, modulates mitochondrial trafficking, especially to the perinuclear region, a subcellular area associated with autophagy. Thus by impairing this process, mutations in either Parkin or PINK1 may alter mitochondrial turnover which, in turn, may cause the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinson's disease.

Manganese-Induced Parkinsonism: Evidence from Epidemiological and Experimental Studies.

Manganese (Mn) exposure has evolved from acute, high-level exposure causing manganism to low, chronic lifetime exposure. In this latter scenario, the target areas extend beyond the globus pallidus (as seen with manganism) to the entire basal ganglia, including the substantia nigra pars compacta. This change of exposure paradigm has prompted numerous epidemiological investigations of the occurrence of Parkinson's disease (PD), or parkinsonism, due to the long-term impact of Mn. In parallel, experimental research has focused on the underlying pathogenic mechanisms of Mn and its interactions with genetic susceptibility. In this review, we provide evidence from both types of studies, with the aim to link the epidemiological data with the potential mechanistic interpretation.

Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Astrocytes from Humanized Targeted Replacement Mice.

Apolipoprotein E4 (APOE4) genotype and sex are significant risk factors for Alzheimer's disease (AD), with females demonstrating increased risk modulated by APOE genotype. APOE is predominantly expressed in astrocytes, however, there is a lack of comprehensive assessments of sex differences in astrocytes stratified by APOE genotype. Here, we examined the response of mixed-sex and sex-specific neonatal APOE3 and APOE4 primary mouse astrocytes (PMA) to a cytokine mix of IL1b, TNFa, and IFNg. Pro-inflammatory and anti-inflammatory cytokine profiles were assessed by qRT-PCR and Meso Scale Discovery multiplex assay. Mixed-sex APOE4 PMA were found to have higher basal messenger RNA expression of several pro-inflammatory cytokines including Il6, Tnfa, Il1b, Mcp1, Mip1a, and Nos2 compared to APOE3 PMA, which was accompanied by increased levels of these secreted cytokines. In sex-specific cultures, basal expression of Il1b, Il6, and Nos2 was 1.5 to 2.5 fold higher in APOE4 female PMA compared to APOE4 males, with both being higher than APOE3 PMA. Similar results were found for secreted levels of these cytokines. Together, these findings indicate that APOE4 genotype and female sex, contribute to a greater inflammatory response in primary astrocytes and these data may provide a framework for investigating the mechanisms contributing to genotype and sex differences in AD-related neuroinflammation.

Metabolic reprogramming, oxidative stress, and pulmonary hypertension.

Mitochondria are highly dynamic organelles essential for cell metabolism, growth, and function. It is becoming increasingly clear that endothelial cell dysfunction significantly contributes to the pathogenesis and vascular remodeling of various lung diseases, including pulmonary arterial hypertension (PAH), and that mitochondria are at the center of this dysfunction. The more we uncover the role mitochondria play in pulmonary vascular disease, the more apparent it becomes that multiple pathways are involved. To achieve effective treatments, we must understand how these pathways are dysregulated to be able to intervene therapeutically. We know that nitric oxide signaling, glucose metabolism, fatty acid oxidation, and the TCA cycle are abnormal in PAH, along with alterations in the mitochondrial membrane potential, proliferation, and apoptosis. However, these pathways are incompletely characterized in PAH, especially in endothelial cells, highlighting the urgent need for further research. This review summarizes what is currently known about how mitochondrial metabolism facilitates a metabolic shift in endothelial cells that induces vascular remodeling during PAH.

A partial Drp1 knockout improves autophagy flux independent of mitochondrial function.

Dynamin-related protein 1 (Drp1) is typically known for its role in mitochondrial fission. A partial inhibition of this protein has been reported to be protective in experimental models of neurodegenerative diseases. The protective mechanism has been attributed primarily to improved mitochondrial function. Herein, we provide evidence showing that a partial Drp1-knockout improves autophagy flux independent of mitochondria. First, we characterized in cell and animal models that at low non-toxic concentrations, manganese (Mn), which causes parkinsonian-like symptoms in humans, impaired autophagy flux but not mitochondrial function and morphology. Furthermore, nigral dopaminergic neurons were more sensitive than their neighbouring GABAergic counterparts. Second, in cells with a partial Drp1-knockdown and Drp1 +/- mice, autophagy impairment induced by Mn was significantly attenuated. This study demonstrates that autophagy is a more vulnerable target than mitochondria to Mn toxicity. Furthermore, improving autophagy flux is a separate mechanism conferred by Drp1 inhibition independent of mitochondrial fission.

The organic cation transporter-3 is a pivotal modulator of neurodegeneration in the nigrostriatal dopaminergic pathway.

Toxic organic cations can damage nigrostriatal dopaminergic pathways as seen in most parkinsonian syndromes and in some cases of illicit drug exposure. Here, we show that the organic cation transporter 3 (Oct3) is expressed in nondopaminergic cells adjacent to both the soma and terminals of midbrain dopaminergic neurons. We hypothesized that Oct3 contributes to the dopaminergic damage by bidirectionally regulating the local bioavailability of toxic species. Consistent with this view, Oct3 deletion and pharmacological inhibition hampers the release of the toxic organic cation 1-methyl-4-phenylpyridinium from astrocytes and protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration in mice. Furthermore, Oct3 deletion impairs the removal of the excess extracellular dopamine induced by methamphetamine and enhances striatal dopaminergic terminal damage caused by this psychostimulant. These results may have far-reaching implications for our understanding of the mechanism of cell death in a wide range of neurodegenerative diseases and may open new avenues for neuroprotective intervention.

Relationship of Blood and Urinary Manganese Levels with Cognitive Function in Elderly Individuals in the United States by Race/Ethnicity, NHANES 2011-2014.

Manganese (Mn) is an essential metal with a biphasic relationship with health outcomes. High-level exposure to Mn is associated with manganism, but few data explore the effects of chronic, lower-level Mn on cognitive function in adults. We sought to determine the relationship between blood/urinary manganese levels and cognitive function in elderly individuals using 2011-2014 data from the National Health and Nutrition Examination Survey (NHANES). Weighted multivariate regression models were used to determine correlations, adjusting for several covariates. Blood Mn was inversely associated with the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) immediate learning of new verbal information (p-value = 0.04), but lost significance after adjusting for medical history (p-value = 0.09). In addition, blood Mn was inversely associated with Animal Fluency scores after adjusting for all covariates. Urinary Mn was inversely associated with CERAD immediate learning after adjusting for all covariates (p-value = 0.01) and inversely associated with the Digit Symbol Substitution Test scores (p-value = 0.0002), but lost significance after adjusting for medical history (p-value = 0.13). Upon stratifying by race/ethnicity, other Races and Non-Hispanic (NH)-Blacks had significantly higher blood Mn levels when compared to NH-Whites. Collectively, these findings suggest that increased blood and urinary Mn levels are associated with poorer cognitive function in an elderly US population.

NAMPT-associated gene signature in the prediction of severe sepsis.

OBJECTIVE: Sepsis is a life-threatening condition of severe organ dysfunction induced by uncontrolled infection and dysregulated host response. However, standardized clinical biomarkers for sepsis are needed to improve patient care, especially in intensive care units (ICUs). Nicotinamide phosphoribosyltransferase (NAMPT) regulates the activity of nicotinamide adenine dinucleotide (NAD)-dependent enzymes and modulates multiple metabolic pathways. Elevated NAMPT gene expression is a risk factor in the pathogenesis and development of sepsis, which is strongly linked to patient morbidity and ICU mortality. At present, there is no identified NAMPT gene signature for prognosis of sepsis patients. METHODS: By analyzing gene expression profiles in peripheral blood mononuclear cells, this study was designed to establish a NAMPT-associated biomarker that effectively predicts survival in sepsis patients. RESULTS: We obtained 19 common genes by intersecting NAMPT-associated genes and sepsis survival-related genes, and this 19-gene signature is significantly enriched in metabolic pathways and NF-κB pathways related to sepsis development. Notably, this 19-gene NAMPT signature was able to discriminate high-risk sepsis from low-risk sepsis in both discovery and validation cohorts. Furthermore, we confirmed that this 19-gene NAMPT signature performed significantly better for sepsis prognosis than random gene sets with 19 genes. CONCLUSIONS: We identified a novel NAMPT gene signature with effective prognostic power for sepsis. Further studies focusing on these biomarkers may also provide an early intervention system for sepsis treatment.

Loss of SOX18/CLAUDIN5 disrupts the pulmonary endothelial barrier in ventilator-induced lung injury.

Mechanical strain contributes to ventilator-induced lung injury (VILI) through multi-factorial and complex mechanisms that remain unresolved. Prevailing evidence suggests that the loss of pulmonary endothelial tight junctions (TJs) plays a critical role. TJs are dynamically regulated by physiologic and hemodynamic forces to stabilize the endothelial barrier. The transcription factor sex-determining region Y-box (SOX)-18 is important in regulating blood vessel development and vascular permeability through its ability to regulate the transcription of Claudin-5, an endothelial TJ protein. Previously, we demonstrated that SOX18 expression is increased by shear stress in the pulmonary endothelium. Therefore, in this study, we investigated how mechanical strain mediated through cyclic stretch affects the SOX18/Claudin-5 regulatory axis. Our data demonstrate that SOX18 and Claudin-5 are downregulated in human lung microvascular endothelial cells (HLMVEC) exposed to cyclic stretch and the mouse lung exposed to high tidal mechanical ventilation. Overexpression of SOX18 reduced the loss of Claudin-5 expression in HLMVEC with cyclic stretch and preserved endothelial barrier function. Additionally, overexpression of Claudin-5 in HLMVEC ameliorated barrier dysfunction in HLMVEC exposed to cyclic stretch, although SOX18 expression was not enhanced. Finally, we found that the targeted overexpression of SOX18 in the pulmonary vasculature preserved Claudin-5 expression in the lungs of mice exposed to HTV. This, in turn reduced lung vascular leak, attenuated inflammatory lung injury, and preserved lung function. Together, these data suggest that enhancing SOX18 expression may prove a useful therapy to treat patients with ventilator-induced lung injury.

Perturbations in mitochondrial dynamics induced by human mutant PINK1 can be rescued by the mitochondrial division inhibitor mdivi-1.

Mutations in the mitochondrial encoded protein PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive Parkinson disease (PD). In mammalian cells, mutant PINK1 has been reported to promote fission or inhibit fusion in mitochondria; however, the mechanism by which this process occurs remains elusive. Using an ecdysone-inducible expression system in mammalian dopaminergic neuronal cells, we report here that human mutant PINK1 (L347P and W437X) mediates an overall fission effect by increasing the ratio of mitochondrial fission over fusion proteins, leading to excessive dysfunctional fragmented mitochondria. Knocking down endogenous Pink1 produces similar effects. In contrast, overexpressing human wild type PINK1 produces a pro-fusion effect by increasing the ratio of mitochondrial fusion/fission proteins without resulting in functionally compromised mitochondria. Parkin knockdown blocks the imbalance in fission/fusion proteins. Furthermore, overexpressing parkin and ubiquitin increases degradation of the mitochondrial fission hFis1 protein, suggesting PINK1 and parkin maintain proper mitochondrial function and integrity via the fission/fusion machinery. Through genetic manipulations and treatment with the small molecule mitochondrial division inhibitor (mdivi-1), which inhibits DLP1/Drp1, both structural and functional mitochondrial defects induced by mutant PINK1 were attenuated, highlighting a potential novel therapeutic avenue for Parkinson disease.