Michelle Hiu-Wai Kung

Mitochondrial UCP4 attenuates MPP+-and dopamine-induced oxidative stress, mitochondrial depolarization, and ATP deficiency in neurons and is interlinked with UCP2 expression

Mitochondrial uncoupling proteins (UCPs) uncouple oxidative phosphorylation from ATP synthesis. We explored the neuroprotective role of UCP4 with its stable overexpression in SH-SY5Y cells, after exposure to either MPP(+) or dopamine to induce ATP deficiency and oxidative stress. Cells overexpressing UCP4 proliferated faster in normal cultures and after exposure to MPP(+) and dopamine. Differentiated UCP4-overexpressing cells survived better when exposed to MPP(+) with decreased LDH release. Contrary to the mild uncoupling hypothesis, UCP4 overexpression resulted in increased absolute ATP levels (with ADP/ATP ratios similar to those of controls under normal conditions and ADP supplementation) associated with increased respiration rate. Under MPP(+) toxicity, UCP4 overexpression preserved ATP levels and mitochondrial membrane potential (MMP) and reduced oxidative stress; the preserved ATP level was not due to increased glycolysis. Under MPP(+) toxicity, the induction of UCP2 expression in vector controls was absent in UCP4-overexpressing cells, suggesting that UCP4 may compensate for UCP2 expression. UCP4 function does not seem to adhere to the mild uncoupling hypothesis in its neuroprotective mechanisms under oxidative stress and ATP deficiency. UCP4 overexpression increases cell survival by inducing oxidative phosphorylation, preserving ATP synthesis and MMP, and reducing oxidative stress.

Mitochondrial UCP5 is neuroprotective by preserving mitochondrial membrane potential, ATP levels, and reducing oxidative stress in MPP+ and dopamine toxicity.

We explored the protective mechanisms of human neuronal mitochondrial uncoupling protein-5 (UCP5) in MPP(+)- and dopamine-induced toxicity after its stable overexpression in SH-SY5Y cells. We raised specific polyclonal antibodies. Overexpressed UCP5 localized in mitochondria but not in cytosol. UCP5 overexpression increased proton leak, decreased mitochondrial membrane potential (MMP), reduced ATP production, and increased overall oxygen consumption (demonstrating uncoupling activity). UCP5 overexpression did not affect other neuronal UCP expression (UCP2 and UCP4). Overexpressing UCP5 is protective against MPP(+)- and dopamine-induced toxicity. MPP(+) and dopamine exposure for 6h reduced MMP and increased superoxide levels. ATP levels in UCP5-overexpressing cells were preserved under MPP(+) and dopamine toxicity, comparable to levels in untreated vector controls. At 24h, UCP5 overexpression preserved MMP, ATP levels, and cell survival; attenuated superoxide generation; and maintained oxidative phosphorylation as indicated by lower lactate levels. MPP(+) and dopamine exposure induced UCP5 mRNA transcription but did not decrease transcript degradation, as inhibition of transcription by actinomycin-D abolished induction by either toxin. Compared with our previous studies on UCP4, we observed functional differences between UCP4 and UCP5 in enhancing mitochondrial efficiency. These neuronal UCP homologues may work synergistically to maintain oxidative balance (through uncoupling activities) and ATP production (by modifying MMP).

Knockdown of uncoupling protein-5 in neuronal SH-SY5Y cells: Effects on MPP+-induced mitochondrial membrane depolarization, ATP deficiency, and oxidative cytotoxicity.

Uncoupling proteins (UCPs) uncouple oxidative phosphorylation from ATP synthesis by dissipating proton gradient across mitochondrial inner membrane. The physiological role of neuronal specific UCP5 is unknown. We explored the effects of reduced UCP5 expression on mitochondrial membrane potential (MMP), oxidative stress, ATP levels, and cell viability, under normal and MPP+‐induced cytotoxic conditions, in human catecholaminergic SH‐SY5Y cells. UCP5 expression was reduced by 56% by siRNA, compared to scrambled‐siRNA controls. UCP5 knockdown induced apoptosis but did not affect basal levels of ATP, oxidative stress and MMP in the cells under normal conditions. However, UCP5 knockdown increased MPP+‐induced cytotoxicity by 15% and oxidative stress levels by 40%, and partially restored MPP+‐induced mitochondrial depolarization by 57%. UCP2 and UCP4 expression were unaffected by UCP5 knockdown. Exacerbation of cytotoxicity, oxidative stress and modification of MMP with reduced UCP5 expression in the face of MPP+ toxicity suggest that UCP5 might be physiologically important in the pathology of oxidative stress‐induced neurodegeneration.

Neuromyelitis optica-IgG in idiopathic inflammatory demyelinating disorders amongst Hong Kong Chinese

Background:  Idiopathic inflammatory demyelinating disorders (IIDD) affect the central nervous system. In classical multiple sclerosis (CMS), brain, optic nerves [optic neuritis (ON)] and spinal cord [acute transverse myelitis (ATM)] are affected. In neuromyelitis optica (NMO), optic nerves and spinal cord are predominantly affected. NMO‐IgG, an autoantibody targeting aquaporin‐4, is a marker for NMO. We studied the frequency and clinical relevance of NMO‐IgG seropositivity in IIDD patients.

Methyl-4-phenylpyridinium ion modulates expression of mitochondrial uncoupling proteins 2, 4, and 5 in catecholaminergic (SK-N-SH) cells

Methyl‐4‐phenylpyridinium ion (MPP+), a specific dopaminergic neurotoxin, inhibits mitochondrial complex I activity, generates reactive oxygen species (ROS), reduces ATP production, and induces cell death. We explored changes in expression of uncoupling proteins (UCPs 2, 4, and 5) following MPP+‐induced toxicity in SK‐N‐SH cells over 72 hr at the transcriptional (quantification of mRNA by real‐time RT‐PCR) and translational (Western analysis) levels. UCP5 mRNA and protein were markedly up‐regulated (1 mM MPP+ at 72 hr caused a twofold increase, P < 0.01), as was UCP4 mRNA, albeit to a much lesser extent. Surprisingly, UCP2 mRNA levels decreased at 24 hr (P < 0.05) but thereafter significantly increased to greater than control levels at 72 hr (P < 0.05), although UCP2 protein levels were decreased throughout (1 mM MPP+ at 72 hr caused a reduction of 50%, P < 0.01). The increase in ROS production may be attenuated by UCP4 and UCP5 up‐regulation. The consequence of decreased UCP2 levels is unclear, although this may represent an adaptive response to declines in ATP levels, the subsequent increase in mRNA being a response to further increases in oxidative stress.

PMCA4 (ATP2B4) Mutation in Familial Spastic Paraplegia

Familial spastic paraplegia (FSP) is a heterogeneous group of disorders characterized primarily by progressive lower limb spasticity and weakness. More than 50 disease loci have been described with different modes of inheritance. In this study, we identified a novel missense mutation (c.803G>A, p.R268Q) in the plasma membrane calcium ATPase (PMCA4, or ATP2B4) gene in a Chinese family with autosomal dominant FSP using whole-exome sequencing and confirmed with Sanger sequencing. This mutation co-segregated with the phenotype in the six family members studied and is predicted to be pathogenic when multiple deleteriousness predictions were combined. This novel R268Q mutation was not present in over 7,000 subjects in public databases, and over 1,000 Han Chinese in our database. Prediction of potential functional consequence of R268Q mutation on PMCA4 by computational modeling revealed that this mutation is located in protein aggregation-prone segment susceptible to protein misfolding. Analysis for thermodynamic protein stability indicated that this mutation destabilizes the PMCA4 protein structure with higher folding free energy. As PMCA4 functions to maintain neuronal calcium homeostasis, our result showed that calcium dysregulation may be associated with the pathogenesis of FSP.

Aquaporin-4 water channel expression by thymoma of patients with and without myasthenia gravis.

BACKGROUND Neuromyelitis optica (NMO) is a serious idiopathic inflammatory demyelinating disorder characterized by acute transverse myelitis and optic neuritis. A significant proportion of NMO patients are seropositive for NMO-IgG, an autoantibody targeting aquaporin-4 (AQP4) water channel. Paraneoplastic NMO associated various tumors were recently reported. AIM We studied the expression of AQP4 by thymoma from patients with and without myasthenia gravis (MG). METHODS Thymoma obtained from thymomectomy in patients with and without MG were studied by immunohistochemistry and western blot. RESULTS Ten thymoma patients (9 with MG) and two control patients without thymoma or MG were studied. Immunohistochemistry revealed AQP4 immunoreactivity in cell membrane of thymoma cells from all ten thymoma specimens whereas thymic tissues from patients without thymoma or MG were negative for AQP4 immunoreactivity. Western blot revealed that lysates of nine of the ten thymoma specimens reacted with anti-human AQP4 antibody with a band of ~30 kDa compatible with the molecular weight of AQP4. Interestingly, immunofluorescence revealed that IgG isolated from 2 NMO patients seropositive for NMO-IgG bound to cell membrane of thymoma cells from all ten thymoma specimens while IgG from healthy control subject did not. CONCLUSION Thymoma cells of patients with and without MG express AQP4. AQP4 autoantibodies from serum of NMO patients bound to AQP4 expressed on thymoma cell membrane.

PMCA4 (ATP2B4) mutation in familial spastic paraplegia causes delay in intracellular calcium extrusion

Familial spastic paraplegia (FSP) is a heterogeneous group of disorders characterized primarily by progressive lower limb spasticity and weakness. More than 50 disease loci have been described with different modes of inheritance. Recently, we described a novel missense mutation (c.803G>A, p.R268Q) in the plasma membrane calcium ATPase (PMCA4, or ATP2B4) gene in a Chinese family with autosomal dominant FSP. Further to this finding, here we describe the functional effect of this mutation.

Estrogenic phenol and catechol metabolites of PCBs modulate catechol-o-methyltransferase expression via the estrogen receptor: Potential contribution to cancer risk

Commercial PCB mixtures have been shown to induce liver tumors in female rats and this effect has been attributed to the effects of PCBs on estrogen metabolism. Catechol metabolites of PCBs are potent inhibitors of COMT activity and are likely to contribute significantly to reduced clearance of genotoxic catechol metabolites of estrogen. The effect of PCB metabolites on COMT expression in cultured cells was investigated to explore potential mechanisms by which PCB exposure alters catechol estrogen clearance. We hypothesize that estrogenic PCB metabolites may contribute to reduction of COMT expression via interaction with the estrogen receptor. To test this hypothesis, human MCF-7 cells were exposed to PCB analogues and the expression of COMT determined. Western blot analysis demonstrated that COMT protein levels were statistically significantly reduced by both the phenolic and the catechol compounds, an effect which was abolished by the anti-estrogen, ICI182780. The above suggests that COMT levels may be reduced by estrogenic PCB metabolites, via interactions between PCB metabolites and the ER. It supports the hypothesis that both phenolic and catechol metabolites of PCBs may contribute to PCB-mediated carcinogenesis through reduction of COMT levels and activities and subsequent reduction in clearance of endogenous and xenobiotic catechols.

LRRK2 R1441G mice are more liable to dopamine depletion and locomotor inactivity

Mutations in leucine‐rich repeat kinase 2 (LRRK2) pose a significant genetic risk in familial and sporadic Parkinsons disease (PD). R1441 mutation (R1441G/C) in its GTPase domain is found in familial PD. How LRRK2 interacts with synaptic proteins, and its role in dopamine (DA) homeostasis and synaptic vesicle recycling remain unclear.