Mitochondrial Uncoupling Protein-2 (UCP2) Mediates Leptin Protection Against MPP+ Toxicity in Neuronal Cells

Mitochondrial dysfunction is involved in the pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD). Uncoupling proteins (UCPs) delink ATP production from biofuel oxidation in mitochondria to reduce oxidative stress. UCP2 is expressed in brain, and has neuroprotective effects under various toxic insults. We observed induction of UCP2 expression by leptin in neuronal cultures, and hypothesize that leptin may preserve neuronal survival via UCP2. We showed that leptin preserved cell survival in neuronal SH-SY5Y cells against MPP+ toxicity (widely used in experimental Parkinsonian models) by maintaining ATP levels and mitochondrial membrane potential (MMP); these effects were accompanied by increased UCP2 expression. Leptin had no effect in modulating reactive oxygen species levels. Stable knockdown of UCP2 expression reduced ATP levels, and abolished leptin protection against MPP+-induced mitochondrial depolarization, ATP deficiency, and cell death, indicating that UCP2 is critical in mediating these neuroprotective effects of leptin against MPP+ toxicity. Interestingly, UCP2 knockdown increased UCP4 expression, but not of UCP5. Our findings show that leptin preserves cell survival by maintaining MMP and ATP levels mediated through UCP2 in MPP+-induced toxicity

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

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

Bioenergetic characterization of SH-SY5Y cells stably overexpressing UCP4.

<p>(a) Human UCP4 protein expression level was significantly higher in UCP4-overexpressing cells than vector controls. (b) Total intracellular ATP level in UCP4-overexpressing cells was higher than vector control cells, as determined by luciferase bioluminescent assay in total cell lysates. (c) Rate of respiration in vector control and UCP4-overexpressing cells. Under normal culture condition, oxygen consumption rate of UCP4-overexpressing cells was higher than vector controls. Results are expressed as mean ± SEM based on at least three independent trials. ** represents statistical significance at p<0.01, * represents p<0.05, compared to the vector control cells.</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

Complex II-specific oxygen consumption and ATP production of isolated mitochondria from UCP4-overexpressing and vector control cells.

<p>(a) Oxygraph showing specificity of Complex II-mediated respiration in isolated mitochondria induced by succinate. Addition of rotenone (10 µM) completely blocked Complex I activity, because addition of Complex I substrates (glutamate & malate) and ADP did not induce oxygen consumption. Subsequently, addition of Complex II substrate, succinate, induced oxygen consumption by <i>state 3</i> respiration. (b) Amount of ATP produced in isolated mitochondria from vector and UCP4-overexpressing cells utilizing either Complex I or Complex II substrates. UCP4 overexpression facilitates ATP production using Complex II substrate (succinate). There was no difference in amount of ATP produced when using Complex I substrate (glutamate/malate). Basal value of ATP produced in vector control: glutamate/malate (Complex I) = <i>11.42 nmol ATP/mg mitochondria protein/nmol O₂</i>; succinate (Complex II): <i>6.68 nmol ATP/mg mitochondria protein/nmol O₂</i>. Results are expressed as mean ± SEM based on at least four independent trials. ** represents statistical significance at p<0.01, compared to the vector control cells. NS: not significant.</p

Mitochondrial oxygen consumption and ADP∶oxygen (ADP∶O) ratio of isolated mitochondria from vector control and UCP4-overexpressing cells.

<p>ADP-stimulated (<i>state 3</i>) and resting (<i>state 4</i>) respiration rates are shown for mitochondria maintained in normoxia at 25°C utilizing either (a–b) Complex I or (c–d) Complex II substrates (i.e. glutamate & malate or succinate, respectively). UCP4-overexpressing mitochondria demonstrated similar ADP∶O ratio utilizing (b) Complex I substrates, but was significantly higher when utilizing (d) Complex II substrates, as compared to vector control mitochondria. Results are expressed as mean ± SEM based on at least six independent measurements. * represents statistical significance at p<0.05, compared to the vector control. NS: not significant.</p