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

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 17β-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

Transcriptional Regulation of the Synaptic Vesicle Protein Synaptogyrin-3 (<i>SYNGR3</i>) 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

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&rsquo;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&prime;-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&prime;-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 (&darr;43.2%) and human embryonic kidney HEK293 cells (&darr;39.7%). EMSA demonstrated NURR1 binding to these three ncNBRE. Site-directed mutagenesis of these ncNBRE reduced promoter activity by 11&ndash;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

The 6 predicted pathogenic in the prioritized short list.

<p><b>Notes</b>: 1: Reference allele and alternative allele; 2: The maximal frequency of the alternative allele in one of reference datasets; 3: The 4850 curated gene sets from Curated gene sets from MSigDB 3.1 (<a href="http://www.broadinstitute.org/gsea/msigdb/genesets.jsp?collection=C2" target="_blank">http://www.broadinstitute.org/gsea/msigdb/genesets.jsp?collection=C2</a>) were used; 4: It is a posterior probability given their deleteriousness and functional scores and the prior probability 0.05.</p

Number of sequence variants after the step-by-step filtration and prioritization in KGGSeq.

<p><b>Notes</b>: 1: Dominant mode only considered with variants in heterozygous genotypes and with shared alleles between the two patients; 2: The rare variants referred to variants with MAF≤1% in the datasets; 3: This category includes missense, stopgain, stoploss and splicing single nucleotide variants and insertions/deletions causing frameshift, nonframeshift, stoploss, stopgain and splicing differences; 4: Knowledge-related variants/genes refer to those variants' genes having PPI(s) or sharing pathway(s) with at least one known causal gene of FSP and those variants fell into gene(s) which were co-mentioned in the titles or abstracts of papers in the PubMed database.</p

Identification of a Chinese family with autosomal dominant spastic paraplegia.

<p>(a) Pedigree. Filled and unfilled symbols indicate affected and unaffected individuals, respectively. Squares and circles represent males and females, respectively. Slashed symbols indicate deceased subjects. (b) DNA sequencing showing PMCA4 (or ATP2B4) R268Q mutation.</p

Computational modeling of R268Q ATP2B4 mutant protein.

<p>(a) Local tertiary molecular structure of, (b–i) wild-type and (b–ii) mutant PMCA4 (or ATP2B4) protein. The red dashed line denotes hydrogen bond.</p

Three common plasticizers (bisphenol-A (BPA), benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP)) dose-dependently decreased COMT expression via estrogen receptor (ER) in MCF-7 cells.

<p>Cells were treated with graded doses of each of the three compounds, in the absence or presence of ICI 182,780 (ER antagonist). The resultant changes of soluble-COMT (S-COMT; 23kDa) protein expression were determined by Western blotting. Equal loading of samples were normalized by actin (42kDa). Results are mean ± SEM of four separate experiments (n=4). Statistical significance at the level of *p<0.05 or **p<0.01, as compared to untreated controls. ^## p<0.01, as compared between the two designated treatment groups.</p

Level of proton leak and integrity of isolated mitochondria from UCP4-overexpressing and vector control cells.

<p>(a) Oxygraphs showing changes in dissolved oxygen in isolated mitochondria suspension from these cells under substrates-induced respiration. Maximum rate of oxygen consumption was recorded for 2 min after addition of substrates containing 5 mM glutamate & malate, 5 mM succinate, and 0.5 mM ADP. ATP synthase inhibitor, oligomycin (2.5 µg/ml) was then added to block ATP synthesis by Complex V. The rate of oxygen consumption with oligomycin was recorded for an additional 2 min, indicating Complex V-insensitive proton leak. Numbers in brackets represent the rate of oxygen consumption in µmol O₂/min/mg mitochondrial protein. (b) The level of proton leak was defined as the ratio of the rate of oxygen consumption in the presence of oligomycin to the rate of substrates-stimulated oxygen consumption (<i>state 3</i> respiration). Results are expressed as mean ratios ± SEM based on at least three independent trials. ** represents statistical significance at p<0.01, compared to the vector control. (c) Representative oxygraphs reflecting integrity of the outer mitochondrial membrane of isolated mitochondria isolated from both vector control and UCP4-overexpressing cells. Exogenous cytochrome c (Cyt <i>c</i>) did not further increase mitochondrial oxygen consumption. Intact and functional mitochondria were demonstrated by <i>state 3</i> respiration as stimulated by addition of substrates (glutamate/malate/succinate) and ADP.</p