Glucocerebrosidase inhibition causes mitochondrial dysfunction and free radical damage

Mutations of the gene for glucocerebrosidase 1 (GBA) cause Gaucher disease (GD), an autosomal recessive lysosomal storage disorder. Individuals with homozygous or heterozygous (carrier) mutations of GBA have a significantly increased risk for the development of Parkinson’s disease (PD), with clinical and pathological features that mirror the sporadic disease. The mechanisms whereby GBA mutations induce dopaminergic cell death and Lewy body formation are unknown. There is evidence of mitochondrial dysfunction and oxidative stress in PD and so we have investigated the impact of glucocerebrosidase (GCase) inhibition on these parameters to determine if there may be a relationship of GBA loss-of-function mutations to the known pathogenetic pathways in PD. We have used exposure to a specific inhibitor (conduritol-β-epoxide, CβE) of GCase activity in a human dopaminergic cell line to identify the biochemical abnormalities that follow GCase inhibition. We show that GCase inhibition leads to decreased ADP phosphorylation, reduced mitochondrial membrane potential and increased free radical formation and damage, together with accumulation of alpha-synuclein. Taken together, inhibition of GCase by CβE induces abnormalities in mitochondrial function and oxidative stress in our cell culture model. We suggest that GBA mutations and reduced GCase activity may increase the risk for PD by inducing these same abnormalities in PD brain.     

The role of mitochondria in sepsis-induced cardiomyopathy

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Myocardial dysfunction, often termed sepsis-induced cardiomyopathy, is a frequent complication and is associated with worse outcomes. Numerous mechanisms contribute to sepsis-induced cardiomyopathy and a growing body of evidence suggests that bioenergetic and metabolic derangements play a central role in its development; however, there are significant discrepancies in the literature, perhaps reflecting variability in the experimental models employed or in the host response to sepsis. The condition is characterised by lack of significant cell death, normal tissue oxygen levels and, in survivors, reversibility of organ dysfunction. The functional changes observed in cardiac tissue may represent an adaptive response to prolonged stress that limits cell death, improving the potential for recovery. In this review, we describe our current understanding of the pathophysiology underlying myocardial dysfunction in sepsis, with a focus on disrupted mitochondrial processes.     

Preconditioning protects by inhibiting the mitochondrial permeability transition

Mitochondrial permeability transition (mPT) is a crucial event in the progression to cell death in the setting of ischemia-reperfusion. We have used a model system in which mPT can be reliably and reproducibly induced to test the hypothesis that the profound protection associated with the phenomenon of myocardial preconditioning is mediated by suppression of the mPT. Adult rat myocytes were loaded with the fluorescent probe tetramethylrhodamine methyl ester, which generates oxidative stress on laser illumination, thus inducing the mPT (indicated by collapse of the mitochondrial membrane potential) and ATP depletion, seen as rigor contracture. The known inhibitors of the mPT, cyclosporin A (0.2 microM) and N-methyl-4-valine-cyclosporin A (0.4 microM), increased the time taken to induce the mPT by 1.8- and 2.9-fold, respectively, compared with control (P < 0.001) and rigor contracture by 1.5-fold compared with control (P < 0.001). Hypoxic preconditioning (HP) and pharmacological preconditioning, using diazoxide (30 microM) or nicorandil (100 microM), also increased the time taken to induce the mPT by 2.0-, 2.1-, and 1.5-fold, respectively (P < 0.001), and rigor contracture by 1.9-, 1.7-, and 1.5-fold, respectively, compared with control (P < 0.001). Effects of HP, diazoxide, and nicorandil were abolished in the presence of mitochondrial ATP-sensitive K(+) (K(ATP)) channel blockers glibenclamide (10 microM) and 5-hydroxydecanoate (100 microM) but were maintained in the presence of the sarcolemmal K(ATP) channel blocker HMR-1098 (10 microM). In conclusion, preconditioning protects the myocardium by reducing the probability of the mPT, which normally occurs during ischemia-reperfusion in response to oxidative stress.     

Knockdown of aquaporin-8 induces mitochondrial dysfunction in 3T3-L1 cells

BackgroundAquaporin-8 (AQP8), a member of the aquaporin water channel family, is expressed in various tissue and cells, including liver, testis, and pancreas. AQP8 appears to have functions on the plasma membrane and/or on the mitochondrial inner membrane. Mitochondrial AQP8 with permeability for water, H₂O₂ and NH₃ has been expected to have important role in various cells, but its information is limited to a few tissues and cells including liver and kidney. In the present study, we found that AQP8 was expressed in the mitochondria in mouse adipose tissues and 3T3-L1 preadipocytes, and investigated its role by suppressing its gene expression.MethodsAQP8-knocked down (shAQP8) cells were established using a vector expressing short hairpin RNA. Cellular localization of AQP8 was examined by western blotting and immunocytochemistry. Mitochondrial function was assessed by measuring mitochondrial membrane potential, oxygen consumption and ATP level measurements.ResultsIn 3T3-L1 cells, AQP8 was expressed in the mitochondria. In shAQP8 cells, mRNA and protein levels of AQP8 were decreased by about 75%. The shAQP8 showed reduced activities of complex IV and ATP synthase; it is probable that the impaired mitochondrial water handling in shAQP8 caused suppression of the electron transport and ADP phosphorylation through inhibition of the two steps which yield water. The reduced activities of the last two steps of oxidative phosphorylation in shAQP8 cause low routine and maximum capacity of respiration and mitochondrial hyperpolarization.ConclusionMitochondrial AQP8 contributes to mitochondrial respiratory function probably through maintenance of water homeostasis.General significanceThe AQP8-knocked down cells we established provides a model system for the studies on the relationships between water homeostasis and mitochondrial function.     

Membrane cholesterol content plays a key role in the neurotoxicity of β-amyloid: implications for Alzheimer's disease

Beta amyloid (βA) plays a central role in the pathogenesis of the most common and devastating neurodegenerative disorder, Alzheimer's disease (AD). The mechanisms of βA neurotoxicity remain controversial, but include dysregulation of calcium homeostasis and oxidative stress. A large body of data suggest that cholesterol plays a significant role in AD. In mixed cultures containing hippocampal neurons and astrocytes, we have shown that neurotoxic βA peptides (1-42 and 25-35) cause sporadic cytosolic calcium ([Ca(2+) ](c) ) signals in astrocytes but not in neurons, initiating a cascade that ends in neuronal death. We now show, using the cholesterol-sensitive fluorescent probe, Filipin, that membrane cholesterol is significantly higher in astrocytes than in neurons and mediates the selective response of astrocytes to βA. Thus, lowering [cholesterol] using mevastatin, methyl-β-cyclodextrin or filipin prevented the βA-induced [Ca(2+) ](c) signals, while increased membrane [cholesterol] increased βA-induced [Ca(2+) ](c) signals in both neurons and astrocytes. Addition of βA to lipid bilayers caused the appearance of a conductance that was significantly higher in membranes containing cholesterol. Increasing membrane [cholesterol] significantly increased βA-induced neuronal and astrocytic death. We conclude that a high membrane [cholesterol] promotes βA incorporation into membranes and increased [Ca(2+) ](c) leading to cell death.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

Mitochondria modulate the spatio-temporal properties of intra- and intercellular Ca signals in cochlear supporting cells

In the cochlea, cell damage triggers intercellular Ca²⁺ waves that propagate through the glial-like supporting cells that surround receptor hair cells. These Ca²⁺ waves are thought to convey information about sensory hair cell-damage to the surrounding supporting cells within the cochlear epithelium. Mitochondria are key regulators of cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt), and yet little is known about their role during the propagation of such intercellular Ca²⁺ signalling. Using neonatal rat cochlear explants and fluorescence imaging techniques, we explore how mitochondria modulate supporting cell [Ca²⁺]_cyt signals that are triggered by ATP or by hair cell damage. ATP application (0.1–50 μM) caused a dose dependent increase in [Ca²⁺]_cyt which was accompanied by an increase in mitochondrial calcium. Blocking mitochondrial Ca²⁺ uptake by dissipating the mitochondrial membrane potential using CCCP and oligomycin or using Ru360, an inhibitor of the mitochondrial Ca²⁺ uniporter, enhanced the peak amplitude and duration of ATP-induced [Ca²⁺]_cyt transients. In the presence of Ru360, the mean propagation velocity, amplitude and extent of spread of damage-induced intercellular Ca²⁺ waves was significantly increased. Thus, mitochondria function as spatial Ca²⁺ buffers during agonist-evoked [Ca²⁺]_cyt signalling in cochlear supporting cells and play a significant role in regulating the spatio-temporal properties of intercellular Ca²⁺ waves.     

Selective Inhibition of the Mitochondrial Permeability Transition Pore Protects against Neurodegeneration in Experimental Multiple Sclerosis

The mitochondrial permeability transition pore is a recognized drug target for neurodegenerative conditions such as multiple sclerosis and for ischemia-reperfusion injury in the brain and heart. The peptidylprolyl isomerase, cyclophilin D (CypD, PPIF), is a positive regulator of the pore, and genetic down-regulation or knock-out improves outcomes in disease models. Current inhibitors of peptidylprolyl isomerases show no selectivity between the tightly conserved cyclophilin paralogs and exhibit significant off-target effects, immunosuppression, and toxicity. We therefore designed and synthesized a new mitochondrially targeted CypD inhibitor, JW47, using a quinolinium cation tethered to cyclosporine. X-ray analysis was used to validate the design concept, and biological evaluation revealed selective cellular inhibition of CypD and the permeability transition pore with reduced cellular toxicity compared with cyclosporine. In an experimental autoimmune encephalomyelitis disease model of neurodegeneration in multiple sclerosis, JW47 demonstrated significant protection of axons and improved motor assessments with minimal immunosuppression. These findings suggest that selective CypD inhibition may represent a viable therapeutic strategy for MS and identify quinolinium as a mitochondrial targeting group for in vivo use.     

Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids

Mutations in mitochondrial DNA (mtDNA) can cause mitochondrial disease, a group of metabolic disorders that affect both children and adults. Interestingly, individual mtDNA mutations can cause very different clinical symptoms, however the factors that determine these phenotypes remain obscure. Defects in mitochondrial oxidative phosphorylation can disrupt cell signaling pathways, which may shape these disease phenotypes. In particular, mitochondria participate closely in cellular calcium signaling, with profound impact on cell function. Here, we examined the effects of a homoplasmic m.13565C>T mutation in MT-ND5 on cellular calcium handling using transmitochondrial cybrids (ND5 mutant cybrids). We found that the oxidation of NADH and mitochondrial membrane potential (Δψ_m) were significantly reduced in ND5 mutant cybrids. These metabolic defects were associated with a significant decrease in calcium uptake by ND5 mutant mitochondria in response to a calcium transient. Inhibition of glycolysis with 2-deoxy-D-glucose did not affect cytosolic calcium levels in control cybrids, but caused an increase in cytosolic calcium in ND5 mutant cybrids. This suggests that glycolytically-generated ATP is required not only to maintain Δψ_m in ND5 mutant mitochondria but is also critical for regulating cellular calcium homeostasis. We conclude that the m.13565C>T mutation in MT-ND5 causes defects in both mitochondrial oxidative metabolism and mitochondrial calcium sequestration. This disruption of mitochondrial calcium handling, which leads to defects in cellular calcium homeostasis, may be an important contributor to mitochondrial disease pathogenesis.