In vivo overexpression of synaptogyrin-3 promotes striatal synaptic dopamine uptake in LRRK2R1441G mutant mouse model of Parkinson's disease.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) mutation is a common genetic risk factor of Parkinson's disease (PD). Presynaptic dysfunction is an early pathogenic event associated with dopamine (DA) dysregulation in striatum of the brain. DA uptake activity of DA uptake transporter (DAT) affects synaptic plasticity and motor and non-motor behavior. Synaptogyrin-3 (SYNGR3) is part of the synaptogyrin family, especially abundant in brain. Previous in vitro studies demonstrated interaction between SYNGR3 and DAT. Reduced SYNGR3 expression was observed in human PD brains with unclear reasons. METHODS: Here, we further explored whether inducing SYNGR3 expression can influence (i) cellular DA uptake using differentiated human SH-SY5Y neuronal cells, (ii) striatal synaptosomal DA uptake in a mutant LRRK2R1441G  knockin mouse model of PD, and (iii) innate rodent behavior using the marble burying test. RESULTS: Young LRRK2 mutant mice exhibited significantly lower SYNGR3 levels in striatum compared to age-matched wild-type (WT) controls, resembling level in aged WT mice. SYNGR3 is spatially co-localized with DAT at striatal presynaptic terminals, visualized by immuno-gold transmission electron microscopy and immunohistochemistry. Their protein-protein interaction was confirmed by co-immunoprecipitation. Transient overexpression of SYNGR3 in differentiated SH-SY5Y cells increased cellular DA uptake activity without affecting total DAT levels. Inducing SYNGR3 overexpression by adeno-associated virus-7 (AAV7) injection in vivo into striatum increased ex vivo synaptosomal DA uptake in LRRK2 mutant mice and improved their innate marble burying behavior. CONCLUSION: Brain SYNGR3 expression may be an important determinant to striatal DA homeostasis and synaptic function. Our preliminary behavioral test showed improved innate behavior after SYNGR3 overexpression in LRRK2 mutant mice, advocating further studies to determine the influence of SYNGR3 in the pathophysiology of DA neurons in PD.

Long-term inhibition of mutant LRRK2 hyper-kinase activity reduced mouse brain α-synuclein oligomers without adverse effects.

Parkinson's disease (PD) is characterized by dopaminergic neurodegeneration in nigrostriatal and cortical brain regions associated with pathogenic α-synuclein (αSyn) aggregate/oligomer accumulation. LRRK2 hyperactivity is a disease-modifying therapeutic target in PD. However, LRRK2 inhibition may be associated with peripheral effects, albeit with unclear clinical consequences. Here, we significantly reduced αSyn oligomer accumulation in mouse striatum through long-term LRRK2 inhibition using GNE-7915 (specific brain-penetrant LRRK2 inhibitor) without causing adverse peripheral effects. GNE-7915 concentrations in wild-type (WT) mouse sera and brain samples reached a peak at 1 h, which gradually decreased over 24 h following a single subcutaneous (100 mg/kg) injection. The same dose in young WT and LRRK2R1441G mutant mice significantly inhibited LRRK2 kinase activity (Thr73-Rab10 and Ser106-Rab12 phosphorylation) in the lung, which dissipated by 72 h post-injection. 14-month-old mutant mice injected with GNE-7915 twice weekly for 18 weeks (equivalent to ~13 human years) exhibited reduced striatal αSyn oligomer and cortical pSer129-αSyn levels, correlating with inhibition of LRRK2 hyperactivity in brain and lung to WT levels. No GNE-7915-treated mice showed increased mortality or morbidity. Unlike reports of abnormalities in lung and kidney at acute high doses of LRRK2 inhibitors, our GNE-7915-treated mice did not exhibit swollen lamellar bodies in type II pneumocytes or abnormal vacuolation in the kidney. Functional and histopathological assessments of lung, kidney and liver, including whole-body plethysmography, urinary albumin-creatinine ratio (ACR), serum alanine aminotransferase (ALT) and serum interleukin-6 (inflammatory marker) did not reveal abnormalities after long-term GNE-7915 treatment. Long-term inhibition of mutant LRRK2 hyper-kinase activity to physiological levels presents an efficacious and safe disease-modifying therapy to ameliorate synucleinopathy in PD.

Transcriptional Regulation of the Synaptic Vesicle Protein Synaptogyrin-3 (SYNGR3) Gene: The Effects of NURR1 on Its Expression.

Synaptogyrin-3 (SYNGR3) is a synaptic vesicular membrane protein. Amongst four homologues (SYNGR1 to 4), SYNGR1 and 3 are especially abundant in the brain. SYNGR3 interacts with the dopamine transporter (DAT) to facilitate dopamine (DA) uptake and synaptic DA turnover in dopaminergic transmission. Perturbed SYNGR3 expression is observed in Parkinson's disease (PD). The regulatory elements which affect SYNGR3 expression are unknown. Nuclear-receptor-related-1 protein (NURR1) can regulate dopaminergic neuronal differentiation and maintenance via binding to NGFI-B response elements (NBRE). We explored whether NURR1 can regulate SYNGR3 expression using an in silico analysis of the 5'-flanking region of the human SYNGR3 gene, reporter gene activity and an electrophoretic mobility shift assay (EMSA) of potential cis-acting sites. In silico analysis of two genomic DNA segments (1870 bp 5'-flanking region and 1870 + 159 bp of first exon) revealed one X Core Promoter Element 1 (XCPE1), two SP1, and three potential non-canonical NBRE response elements (ncNBRE) but no CAAT or TATA box. The longer segment exhibited gene promoter activity in luciferase reporter assays. Site-directed mutagenesis of XCPE1 decreased promoter activity in human neuroblastoma SH-SY5Y (↓43.2%) and human embryonic kidney HEK293 cells (↓39.7%). EMSA demonstrated NURR1 binding to these three ncNBRE. Site-directed mutagenesis of these ncNBRE reduced promoter activity by 11-17% in SH-SY5Y (neuronal) but not in HEK293 (non-neuronal) cells. C-DIM12 (Nurr1 activator) increased SYNGR3 protein expression in SH-SY5Y cells and its promoter activity using a real-time luciferase assay. As perturbed vesicular function is a feature of major neurodegenerative diseases, inducing SYNGR3 expression by NURR1 activators may be a potential therapeutic target to attenuate synaptic dysfunction in PD.

LRRK2, GBA and their interaction in the regulation of autophagy: implications on therapeutics in Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) and glucocerebrosidase (GBA) represent two most common genetic causes of Parkinson's disease (PD). Both genes are important in the autophagic-lysosomal pathway (ALP), defects of which are associated with α-synuclein (α-syn) accumulation. LRRK2 regulates macroautophagy via activation of the mitogen activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) kinase (MEK) and the calcium-dependent adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathways. Phosphorylation of Rab GTPases by LRRK2 regulates lysosomal homeostasis and endosomal trafficking. Mutant LRRK2 impairs chaperone-mediated autophagy, resulting in α-syn binding and oligomerization on lysosomal membranes. Mutations in GBA reduce glucocerebrosidase (GCase) activity, leading to glucosylceramide accumulation, α-syn aggregation and broad autophagic abnormalities. LRRK2 and GBA influence each other: GCase activity is reduced in LRRK2 mutant cells, and LRRK2 kinase inhibition can alter GCase activity in GBA mutant cells. Clinically, LRRK2 G2019S mutation seems to modify the effects of GBA mutation, resulting in milder symptoms than those resulting from GBA mutation alone. However, dual mutation carriers have an increased risk of PD and earlier age of onset compared with single mutation carriers, suggesting an additive deleterious effect on the initiation of PD pathogenic processes. Crosstalk between LRRK2 and GBA in PD exists, but its exact mechanism is unclear. Drugs that inhibit LRRK2 kinase or activate GCase are showing efficacy in pre-clinical models. Since LRRK2 kinase and GCase activities are also altered in idiopathic PD (iPD), it remains to be seen if these drugs will be useful in disease modification of iPD.

Aberrant mitochondrial morphology and function associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in aged mutant Parkinsonian LRRK2R1441G mice.

Mitochondrial dysfunction causes energy deficiency and nigrostriatal neurodegeneration which is integral to the pathogenesis of Parkinson disease (PD). Clearance of defective mitochondria involves fission and ubiquitin-dependent degradation via mitophagy to maintain energy homeostasis. We hypothesize that LRRK2 (leucine-rich repeat kinase 2) mutation disrupts mitochondrial turnover causing accumulation of defective mitochondria in aging brain. We found more ubiquitinated mitochondria with aberrant morphology associated with impaired function in aged (but not young) LRRK2R1441G knockin mutant mouse striatum compared to wild-type (WT) controls. LRRK2R1441G mutant mouse embryonic fibroblasts (MEFs) exhibited reduced MAP1LC3/LC3 activation indicating impaired macroautophagy/autophagy. Mutant MEFs under FCCP-induced (mitochondrial uncoupler) stress showed increased LC3-aggregates demonstrating impaired mitophagy. Using a novel flow cytometry assay to quantify mitophagic rates in MEFs expressing photoactivatable mito-PAmCherry, we found significantly slower mitochondria clearance in mutant cells. Specific LRRK2 kinase inhibition using GNE-7915 did not alleviate impaired mitochondrial clearance suggesting a lack of direct relationship to increased kinase activity alone. DNM1L/Drp1 knockdown in MEFs slowed mitochondrial clearance indicating that DNM1L is a prerequisite for mitophagy. DNM1L knockdown in slowing mitochondrial clearance was less pronounced in mutant MEFs, indicating preexisting impaired DNM1L activation. DNM1L knockdown disrupted mitochondrial network which was more evident in mutant MEFs. DNM1L-Ser616 and MAPK/ERK phosphorylation which mediate mitochondrial fission and downstream mitophagic processes was apparent in WT using FCCP-induced stress but not mutant MEFs, despite similar total MAPK/ERK and DNM1L levels. In conclusion, aberrant mitochondria morphology and dysfunction associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in mutant LRRK2 MEFs and mouse brain.Abbreviations: ATP: adenosine triphosphate; BAX: BCL2-associated X protein; CDK1: cyclin-dependent kinase 1; CDK5: cyclin-dependent kinase 5; CQ: chloroquine; CSF: cerebrospinal fluid; DNM1L/DRP1: dynamin 1-like; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; LAMP2A: lysosomal-associated membrane protein 2A; LRRK2: leucine-rich repeat kinase 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MEF: mouse embryonic fibroblast; MFN1: mitofusin 1; MMP: mitochondrial membrane potential; PAmCherry: photoactivatable-mCherry; PD: Parkinson disease; PINK1: PTEN induced putative kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RAB10: RAB10, member RAS oncogene family; RAF: v-raf-leukemia oncogene; SNCA: synuclein, alpha; TEM: transmission electron microscopy; VDAC: voltage-dependent anion channel; WT: wild type; SQSTM1/p62: sequestosome 1.

Age-dependent accumulation of oligomeric SNCA/α-synuclein from impaired degradation in mutant LRRK2 knockin mouse model of Parkinson disease: role for therapeutic activation of chaperone-mediated autophagy (CMA).

Parkinson disease (PD) is an age-related neurodegenerative disorder associated with misfolded SNCA/α-synuclein accumulation in brain. Impaired catabolism of SNCA potentiates formation of its toxic oligomers. LRRK2 (leucine-rich repeat kinase-2) mutations predispose to familial and sporadic PD. Mutant LRRK2 perturbs chaperone-mediated-autophagy (CMA) to degrade SNCA. We showed greater age-dependent accumulation of oligomeric SNCA in striatum and cortex of aged LRRK2R1441G knockin (KI) mice, compared to age-matched wildtype (WT) by 53% and 31%, respectively. Lysosomal clustering and accumulation of CMA-specific LAMP2A and HSPA8/HSC70 proteins were observed in aged mutant striatum along with increased GAPDH (CMA substrate) by immunohistochemistry of dorsal striatum and flow cytometry of ventral midbrain cells. Using our new reporter protein clearance assay, mutant mouse embryonic fibroblasts (MEFs) expressing either SNCA or CMA recognition 'KFERQ'-like motif conjugated with photoactivated-PAmCherry showed slower cellular clearance compared to WT by 28% and 34%, respectively. However, such difference was not observed after the 'KFERQ'-motif was mutated. LRRK2 mutant MEFs exhibited lower lysosomal degradation than WT indicating lysosomal dysfunction. LAMP2A-knockdown reduced total lysosomal activity and clearance of 'KFERQ'-substrate in WT but not in mutant MEFs, indicating impaired CMA in the latter. A CMA-specific activator, AR7, induced neuronal LAMP2A transcription and lysosomal activity in MEFs. AR7 also attenuated the progressive accumulation of both intracellular and extracellular SNCA oligomers in prolonged cultures of mutant cortical neurons (DIV21), indicating that oligomer accumulation can be suppressed by CMA activation. Activation of autophagic pathways to reduce aged-related accumulation of pathogenic SNCA oligomers is a viable disease-modifying therapeutic strategy for PD.Abbreviations: 3-MA: 3-methyladenine; AR7: 7-chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine; CMA: chaperone-mediated autophagy; CQ: chloroquine; CSF: cerebrospinal fluid; DDM: n-dodecyl ß-D-maltoside; DIV: days in vitro; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GWAS: genome-wide association studies; HSPA8/HSC70: heat shock protein 8; KFERQ: CMA recognition pentapeptide; KI: knockin; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LDH: lactate dehydrogenase; LRRK2: leucine-rich repeat kinase 2; MEF: mouse embryonic fibroblast; NDUFS4: NADH:ubiquinone oxidoreductase core subunit S4; NE: novel epitope; PD: Parkinson disease; RARA/RARα: retinoic acid receptor, alpha; SNCA: synuclein, alpha; TUBB3/TUJ1: tubulin, beta 3 class III; WT: wild-type.

Combined LRRK2 mutation, aging and chronic low dose oral rotenone as a model of Parkinson's disease.

Aging, genetics and environmental toxicity are important etiological factors in Parkinson's disease (PD). However, its pathogenesis remains unclear. A major obstacle is the lack of an appropriate experimental model which incorporates genetic susceptibility, aging and prolonged environmental toxicity. Here, we explored the interplay amongst these factors using mutant LRRK2R1441G (leucine-rich-repeat-kinase-2) knockin mice. We found that mutant primary cortical and mesencephalic dopaminergic neurons were more susceptible to rotenone-induced ATP deficiency and cell death. Compared with wild-type controls, striatal synaptosomes isolated from young mutant mice exhibited significantly lower dopamine uptake after rotenone toxicity, due to reduced striatal synaptosomal mitochondria and synaptic vesicular proton pump protein (V-ATPase H) levels. Mutant mice developed greater locomotor deficits in open-field tests than wild-type mice following low oral rotenone doses given twice weekly over 50 weeks (half their lifespan). The increased locomotor deficit was associated with specific reduction in striatal mitochondrial Complex-I (NDUFS4) in rotenone-treated mutant but not in similarly treated wild-type mice. Our unique experimental model which incorporates genetic effect, natural aging and prolonged oral environmental toxicity administered to mutant knockin LRRK2 mice over half their life span, with observable and measurable phenotype, is invaluable in further studies of the pathogenic process and therapeutics of PD.

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

BACKGROUND: 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. METHODS: As PMCA4 removes cytosolic calcium, we measured transient changes and the time-dependent decay of cytosolic calcium level as visualized by using fura-2 fluorescent dye with confocal microscopy in human SH-SY5Y neuroblastoma cells overexpressing either wild-type or R268Q mutant PMCA4. RESULTS: Overexpressing both wild-type and R268Q PMCA4 significantly reduced maximum calcium surge after KCl-induced depolarization as compared with vector control cells. However, cells overexpressing mutant PMCA4 protein demonstrated significantly higher level of calcium surge when compared with wild-type. Furthermore, the steady-state cytosolic calcium concentration in these mutant cells remained markedly higher than the wild-type after SERCA inhibition by thapsigargin. CONCLUSION: Our result showed that p.R268Q mutation in PMCA4 resulted in functional changes in calcium homeostasis in human neuronal cells. This suggests that calcium dysregulation may be associated with the pathogenesis of FSP.

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

OBJECTIVE: Mutations in leucine-rich repeat kinase 2 (LRRK2) pose a significant genetic risk in familial and sporadic Parkinson's 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. METHODS: To explore the pathogenic effects of LRRK2(R1441G) mutation on nigrostriatal synaptic nerve terminals and locomotor activity, we generated C57BL/6N mice with homozygous LRRK2(R1441G) knockin (KI) mutation, and examined for early changes in nigrostriatal region, striatal synaptosomal [(3)H]-DA uptake and locomotor activity after reserpine-induced DA depletion. RESULTS: Under normal conditions, mutant mice showed no differences, (1) in amount and morphology of nigrostriatal DA neurons and neurites, (2) tyrosine hydroxylase (TH), DA uptake transporter (DAT), vesicular monoamine transporter-2 (VMAT2) expression in striatum, (3) COX IV, LC3B, Beclin-1 expression in midbrain, (4) LRRK2 expression in total cell lysate from whole brain, (5) α-synuclein, ubiquitin, and tau protein immunostaining in midbrain, (6) locomotor activity, compared to wild-type controls. However, after a single intraperitoneal reserpine dose, striatal synaptosomes from young 3-month-old mutant mice demonstrated significantly lower DA uptake with impaired locomotor activity and significantly slower recovery from the effects of reserpine. INTERPRETATION: Although no abnormal phenotype was observed in mutant LRRK2(R1441G) mice, the KI mutation increases vulnerability to reserpine-induced striatal DA depletion and perturbed DA homeostasis resulting in presynaptic dysfunction and locomotor deficits with impaired recovery from reserpine. This subtle nigrostriatal synaptic vulnerability may reflect one of the earliest pathogenic processes in LRRK2-associated PD.

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.

Assessment of cellular estrogenic activity based on estrogen receptor-mediated reduction of soluble-form catechol-O-methyltransferase (COMT) expression in an ELISA-based system.

Xenoestrogens are either natural or synthetic compounds that mimic the effects of endogenous estrogen. These compounds, such as bisphenol-A (BPA), and phthalates, are commonly found in plastic wares. Exposure to these compounds poses major risk to human health because of the potential to cause endocrine disruption. There is huge demand for a wide range of chemicals to be assessed for such potential for the sake of public health. Classical in vivo assays for endocrine disruption are comprehensive but time-consuming and require sacrifice of experimental animals. Simple preliminary in vitro screening assays can reduce the time and expense involved. We previously demonstrated that catechol-O-methyltransferase (COMT) is transcriptionally regulated by estrogen via estrogen receptor (ER). Therefore, detecting corresponding changes of COMT expression in estrogen-responsive cells may be a useful method to estimate estrogenic effects of various compounds. We developed a novel cell-based ELISA to evaluate cellular response to estrogenicity by reduction of soluble-COMT expression in ER-positive MCF-7 cells exposed to estrogenic compounds. In contrast to various existing methods that only detect bioactivity, this method elucidates direct physiological effect in a living cell in response to a compound. We validated our assay using three well-characterized estrogenic plasticizers - BPA, benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP). Cells were exposed to either these plasticizers or 17ß-estradiol (E2) in estrogen-depleted medium with or without an ER-antagonist, ICI 182,780, and COMT expression assayed. Exposure to each of these plasticizers (10(-9)-10(-7)M) dose-dependently reduced COMT expression (p<0.05), which was blocked by ICI 182,780. Reduction of COMT expression was readily detectable in cells exposed to picomolar level of E2, comparable to other in vitro assays of similar sensitivity. To satisfy the demand for in vitro assays targeting different cellular components, a cell-based COMT assay provides useful initial screening to supplement the current assessments of xenoestrogens for potential estrogenic activity.

UCP4 is a target effector of the NF-κB c-Rel prosurvival pathway against oxidative stress.

Mitochondrial uncoupling protein-4 (UCP4) enhances neuronal survival in 1-methyl-4-phenylpyridinium (MPP(+)) toxicity by suppressing oxidative stress and preserving intracellular ATP and mitochondrial membrane potential (MMP). NF-κB regulates neuronal viability via its complexes, p65 mediating cell death and c-Rel promoting cell survival. We reported previously that NF-κB mediates UCP4 neuroprotection against MPP(+) toxicity. Here, we investigated its link with the NF-κB c-Rel prosurvival pathway in alleviating mitochondrial dysfunction and oxidative stress. We overexpressed a c-Rel-encoding plasmid in SH-SY5Y cells and showed that c-Rel overexpression induced NF-κB activity without affecting p65 level. Overexpression of c-Rel increased UCP4 promoter activity and protein expression. Electrophoretic mobility shift assay showed that H(2)O(2) increased NF-κB binding to the UCP4 promoter and that NF-κB complexes were composed of p50/p50 and p50/c-Rel dimers. Under H(2)O(2)-induced oxidative stress, UCP4 knockdown significantly increased superoxide levels, decreased reduced glutathione (GSH) levels, and increased oxidized glutathione levels, compared to controls. UCP4 expression induced by c-Rel overexpression significantly decreased superoxide levels and preserved GSH levels and MMP under similar stress. These protective effects of c-Rel overexpression in H(2)O(2)-induced oxidative stress were significantly reduced after UCP4 knockdown, indicating that UCP4 is a target effector gene of the NF-κB c-Rel prosurvival pathway to mitigate the effects of oxidative stress.

Uncoupling protein-4 (UCP4) increases ATP supply by interacting with mitochondrial Complex II in neuroblastoma cells.

Mitochondrial uncoupling protein-4 (UCP4) protects against Complex I deficiency as induced by 1-methyl-4-phenylpyridinium (MPP(+)), but how UCP4 affects mitochondrial function is unclear. Here we investigated how UCP4 affects mitochondrial bioenergetics in SH-SY5Y cells. Cells stably overexpressing UCP4 exhibited higher oxygen consumption (10.1%, p<0.01), with 20% greater proton leak than vector controls (p<0.01). Increased ATP supply was observed in UCP4-overexpressing cells compared to controls (p<0.05). Although state 4 and state 3 respiration rates of UCP4-overexpressing and control cells were similar, Complex II activity in UCP4-overexpressing cells was 30% higher (p<0.05), associated with protein binding between UCP4 and Complex II, but not that of either Complex I or IV. Mitochondrial ADP consumption by succinate-induced respiration was 26% higher in UCP4-overexpressing cells, with 20% higher ADP:O ratio (p<0.05). ADP/ATP exchange rate was not altered by UCP4 overexpression, as shown by unchanged mitochondrial ADP uptake activity. UCP4 overexpression retained normal mitochondrial morphology in situ, with similar mitochondrial membrane potential compared to controls. Our findings elucidate how UCP4 overexpression increases ATP synthesis by specifically interacting with Complex II. This highlights a unique role of UCP4 as a potential regulatory target to modulate mitochondrial Complex II and ATP output in preserving existing neurons against energy crisis.

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).

Transcriptional regulation of UCP4 by NF-kappaB and its role in mediating protection against MPP+ toxicity.

Mitochondrial uncoupling protein-4 (UCP4) enhances neuronal cell survival in MPP(+)-induced toxicity by suppressing oxidative stress and preserving intracellular ATP and mitochondrial membrane potential. UCP4 expression is increased by MPP(+), but its regulation is unknown. Using serial human UCP4 promoter-luciferase reporter gene constructs, we identified and characterized several cis-acting elements that can regulate UCP4 expression. Core promoter activity exists within 100 bp upstream of the transcription initiation site (TIS=+1). Both CAAT box (-33/-27) and Sp1 (-62/-49) elements are crucial and act synergistically in its transcription. We identified a NF-kappaB putative binding site at -507/-495. Mutation of this site significantly decreased UCP4 promoter activity. Activation of NF-kappaB by TNFalpha or cycloheximide increased, whereas its inhibition by 4-hydroxy-2-nonenal or transfection of pIkappaBalphaM suppressed, UCP4 promoter activity. NF-kappaB inhibition significantly suppressed the MPP(+)-induced increase in UCP4 expression. MPP(+) increased specific binding of NF-kappaB protein complexes to this site in electrophoretic mobility shift assay. Both UCP4 knockdown and NF-kappaB inhibition exacerbated MPP(+)-induced cell death. We present the first direct evidence that UCP4 is regulated by NF-kappaB, mediated via a functional NF-kappaB site in its promoter region, and that UCP4 has a significant role in NF-kappaB prosurvival signaling, mediating its protection against MPP(+) toxicity.

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.

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.

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.