Db Ramsden

Human catechol-O-methyltransferase down-regulation by estradiol

Catechol-O-methyltransferase (COMT) is a crucial enzyme in dopamine and levodopa metabolism. Previously we reported that physiological concentrations of 17beta-estradiol (E2) down-regulated steady-state 1.3-kb COMT mRNA levels in MCF-7 cells. In this study, we investigated whether similar reductions occurred in a glial cell line (U138MG) and whether COMT protein and activity levels paralleled the reduction in COMT mRNA levels in MCF-7 cells. In addition, we explored the mechanism of E2 action. E2 had no effect on COMT mRNA levels in U138MG cells, but significantly reduced COMT protein and activity in MCF-7 cells (activity by 53% at 10(-7) M of E2, by 45% at 10(-8) M, and by 28% at 10(-9) M relative to non-E2-treated cells). A specific estrogen receptor antagonist (ICI 182780) blocked these estrogenic effects. Estrogen receptor in nuclear extracts of MCF-7 cells, which were pretreated with E2 (10(-9) M) for 48 h, bound to the whole proximal and distal promoter regions, as determined by electrophoretic mobility shift analysis (EMSA). We propose that E2 decreased COMT activity through down-regulation of its gene and protein expression mediated via ER interaction with response elements in the promoter region of the gene. Our findings may explain the lower of COMT activity in women compared to that in men, and, in part, the beneficial effects of E2 therapy in post-menopausal Parkinsons disease patients.

Is the abundance of Faecalibacterium prausnitzii relevant to Crohn's disease?

Reports that bacteria within the Firmicutes phylum, especially the species Faecalibacterium prausnitzii, are less abundant in Crohns disease (CD) patients and supernatants from cultures of this bacterium are anti-inflammatory prompted the investigation of the possible correlations between the abundance of F. prausnitzii and the response to treatment in patients with gut diseases and healthy controls. In a randomized, double-blind trial, faeces were collected from healthy volunteers, and from patients with active CD, ulcerative colitis (UC) and irritable bowel syndrome before and after treatment. The levels of F. prausnitzii DNA in faecal suspensions were determined by PCR. Treatment by an elemental diet was effective, resulting in decreases in both the Harvey and Bradshaw index (P<0.001) and the concentrations of serum C-reactive protein (P<0.05). The total levels of F. prausnitzii in faecal samples from CD patients at presentation were lower than those in the other groups both before and after the treatment. There was no correlation between F. prausnitzii abundance and the severity of CD before treatment. Clinical improvement unexpectedly correlated with a significant decrease in the abundance of F. prausnitzii, especially the A2-165 subgroup (P<0.05). Our data suggest that a paucity of F. prausnitzii in the gastrointestinal microbial communities is likely to be a minor aetiological factor in CD: recovery following elemental diet is attributed to lower levels of gut flora.

COX-2-deficient mice are less prone to MPTP-neurotoxicity than wild-type mice.

The primary lesion in Parkinsons disease is the death of dopaminergic neurons in the substantia nigra. The role of cyclooxygenase (COX)-2 in the etiology of Parkinsons disease was explored using COX-2 gene knockout mice. Mortality after injection of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP, a chemical known to cause parkinsonism in humans) in heterozygous COX-2-deficient mice was lower than that in wild-type mice. The number of tyrosine hydroxylase immunoreactive neurons in the substantia nigra pars compacta of MPTP-treated wild-type mice declined to a greater extent than in heterozygous mice. Inhibition of COX-2 protein expression decreased the lesion caused by MPTP and protected the dopaminergic neurons in substantia nigra pars compacta. This result suggested that inhibition of COX-2 has potential therapeutic implications.

Mitochondrial neuronal uncoupling proteins: a target for potential disease-modification in Parkinson's disease

This review gives a brief insight into the role of mitochondrial dysfunction and oxidative stress in the converging pathogenic processes involved in Parkinsons disease (PD). Mitochondria provide cellular energy in the form of ATP via oxidative phosphorylation, but as an integral part of this process, superoxides and other reactive oxygen species are also produced. Excessive free radical production contributes to oxidative stress. Cells have evolved to handle such stress via various endogenous anti-oxidant proteins. One such family of proteins is the mitochondrial uncoupling proteins (UCPs), which are anion carriers located in the mitochondrial inner membrane. There are five known homologues (UCP1 to 5), of which UCP4 and 5 are predominantly expressed in neural cells. In a series of previous publications, we have shown how these neuronal UCPs respond to 1-methyl-4-phenylpyridinium (MPP+; toxic metabolite of MPTP) and dopamine-induced toxicity to alleviate neuronal cell death by preserving ATP levels and mitochondrial membrane potential, and reducing oxidative stress. We also showed how their expression can be influenced by nuclear factor kappa-B (NF-κB) signaling pathway specifically in UCP4. Furthermore, we previously reported an interesting link between PD and metabolic processes through the protective effects of leptin (hormone produced by adipocytes) acting via UCP2 against MPP+-induced toxicity. There is increasing evidence that these endogenous neuronal UCPs can play a vital role to protect neurons against various pathogenic stresses including those associated with PD. Their expression, which can be induced, may well be a potential therapeutic target for various drugs to alleviate the harmful effects of pathogenic processes in PD and hence modify the progression of this disease.

The expression of nicotinamide N-methyltransferase increases ATP synthesis and protects SH-SY5Y neuroblastoma cells against the toxicity of Complex I inhibitors.

NNMT (nicotinamide N-methyltransferase, E.C. 2.1.1.1) catalyses the N-methylation of nicotinamide to 1-methylnicotinamide. NNMT expression is significantly elevated in a number of cancers, and we have previously demonstrated that NNMT expression is significantly increased in the brains of patients who have died of Parkinsons disease. To investigate the cellular effects of NNMT overexpression, we overexpressed NNMT in the SH-SY5Y cell line, a tumour-derived human dopaminergic neuroblastoma cell line with no endogenous expression of NNMT. NNMT expression significantly decreased SH-SY5Y cell death, which correlated with increased intracellular ATP content, ATP/ADP ratio and Complex I activity, and a reduction in the degradation of the NDUFS3 [NADH dehydrogenase (ubiquinone) iron-sulfur protein 3] subunit of Complex I. These effects were replicated by incubation of SH-SY5Y cells with 1-methylnicotinamide, suggesting that 1-methylnicotinamide mediates the cellular effects of NNMT. Both NNMT expression and 1-methylnicotinamide protected SH-SY5Y cells from the toxicity of the Complex I inhibitors MPP+ (1-methyl-4-phenylpyridinium ion) and rotenone by reversing their effects upon ATP synthesis, the ATP/ADP ratio, Complex I activity and the NDUFS3 subunit. The results of the present study raise the possibility that the increase in NNMT expression that we observed in vivo may be a stress response of the cell to the underlying pathogenic process. Furthermore, the results of the present study also raise the possibility of using inhibitors of NNMT for the treatment of cancer.

Abnormal diffusion tensor in nonsymptomatic familial amyotrophic lateral sclerosis with a causative superoxide dismutase 1 mutation

To determine whether diffusion abnormalities can be observed in nonsymptomatic family members with a known causative Cu/Zn superoxide dismutase mutation (asymptomatic familial amyotrophic lateral sclerosis; AFALS+SOD1) in a family with autosomal dominant familial amyotrophic lateral sclerosis (ALS) using diffusion tensor imaging (DTI).

Human neuronal uncoupling proteins 4 and 5 (UCP4 and UCP5): structural properties, regulation, and physiological role in protection against oxidative stress and mitochondrial dysfunction

Uncoupling proteins (UCPs) belong to a large family of mitochondrial solute carriers 25 (SLC25s) localized at the inner mitochondrial membrane. UCPs transport protons directly from the intermembrane space to the matrix. Of five structural homologues (UCP1 to 5), UCP4 and 5 are principally expressed in the central nervous system (CNS). Neurons derived their energy in the form of ATP that is generated through oxidative phosphorylation carried out by five multiprotein complexes (Complexes I–V) embedded in the inner mitochondrial membrane. In oxidative phosphorylation, the flow of electrons generated by the oxidation of substrates through the electron transport chain to molecular oxygen at Complex IV leads to the transport of protons from the matrix to the intermembrane space by Complex I, III, and IV. This movement of protons to the intermembrane space generates a proton gradient (mitochondrial membrane potential; MMP) across the inner membrane. Complex V (ATP synthase) uses this MMP to drive the conversion of ADP to ATP. Some electrons escape to oxygen‐forming harmful reactive oxygen species (ROS). Proton leakage back to the matrix which bypasses Complex V resulting in a major reduction in ROS formation while having a minimal effect on MMP and hence, ATP synthesis; a process termed “mild uncoupling.” UCPs act to promote this proton leakage as means to prevent excessive build up of MMP and ROS formation. In this review, we discuss the structure and function of mitochondrial UCPs 4 and 5 and factors influencing their expression. Hypotheses concerning the evolution of the two proteins are examined. The protective mechanisms of the two proteins against neurotoxins and their possible role in regulating intracellular calcium movement, particularly with regard to the pathogenesis of Parkinsons disease are discussed.

High expression of nicotinamide N-methyltransferase in patients with idiopathic Parkinson's disease

We have previously speculated that elevated levels of nicotinamide N-methyltransferase (NNMT), the primary catabolic enzyme of nicotinamide, may result in reduced Complex I activity in idiopathic Parkinsons disease (IPD) in two ways: (1) reduction in the levels of nicotinamide available for nicotinamide adenine dinucleotide synthesis; and (2) increased methylation of compounds such as tetrahydroisoquinolines and beta-carbolines, which are potent Complex I inhibitors. Expression of NNMT was assessed in 91 cerebella (53 IPD, 38 control) using immunohistochemistry coupled with quantitative digital image analysis. Control cerebella showed a distribution of expression ascribed to low, intermediate and high expressors with ratios of 1:2:1 categories. Expression in the parkinsonian cerebella was significantly higher than in the control group (control group median expression 17%, mean expression 16.6%, range 0-51%, standard deviation 11.4%, standard error 1.9%; IPD group median expression 46%, mean expression 53.7%, range 21-100%, standard deviation 23.4%, standard error 3.2%; P<0.0001; unpaired t-test with Welch correction (parametric) and Mann-Whitney U-test (non-parametric)). These results confirm that NNMT expression is elevated in IPD, which may ultimately lead to neurodegeneration via a reduction in Complex I activity.

Analysis of volatile organic compounds of bacterial origin in chronic gastrointestinal diseases

Background: The aim of this study was to determine whether volatile organic compounds (VOCs) present in the headspace of feces could be used to diagnose or distinguish between chronic diseases of the gastrointestinal tract and apparently healthy volunteers. Methods: A total of 87 people were recruited, divided between 4 categories: healthy volunteers (n = 19), Crohn’s disease (n = 22), ulcerative colitis (n = 20), and irritable bowel syndrome (n = 26). They each supplied fecal samples before, and except for the healthy volunteers, after treatment. Fecal samples were incubated in a sample bag with added purified air at 40°C and headspace samples were taken and concentrated on thermal sorption tubes. Gas chromatography–mass spectrometry then desorbed and analyzed these. The concentrations of a selection of high-abundance compounds were determined and assessed for differences in concentration between the groups. Results: Crohns disease samples showed significant elevations in the concentrations of ester and alcohol derivates of short-chain fatty acids and indole compared with the other groups; indole and phenol were elevated in ulcerative colitis and irritable bowel syndrome but not at a statistically significant level. After treatment, the levels of many of the VOCs were significantly reduced and were more similar to those concentrations in healthy controls. Conclusions: The abundance of a number of VOCs in feces differs markedly between Crohns disease and other gastrointestinal conditions. Following treatment, the VOC profile is altered to more closely resemble that of healthy volunteers.

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.