Jm Cooper

Mitochondrial complex I deficiency in Parkinson's disease.

The structure and function of mitochondrial respiratory-chain enzyme proteins were studied postmortem in the substantia nigra of nine patients with Parkinsons disease and nine matched controls. Total protein and mitochondrial mass were similar in the two groups. NADH-ubiquinone reductase (Complex I) and NADH cytochrome c reductase activities were significantly reduced, whereas succinate cytochrome c reductase activity was normal. These results indicated a specific defect of Complex I activity in the substantia nigra of patients with Parkinsons disease. This biochemical defect is the same as that produced in animal models of parkinsonism by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and adds further support to the proposition that Parkinsons disease may be due to an environmental toxin with action(s) similar to those of MPTP.

Biochemical abnormalities and excitotoxicity in Huntington's disease brain

The physiological role of huntingtin and the mechanisms by which the expanded CAG repeat in ITI5 and its polyglutamine stretch in mutant huntingtin induce Huntingtons disease (HD) are unknown. Several techniques have now demonstrated abnormal metabolism in HD brain; direct measurement of respiratory chain enzyme activities has shown severe deficiency of complex II/III and a milder defect of complex IV. We confirm that these abnormalities appear to be confined to the striatum within the HD brain. Analysis of complex II/III activity in HD fibroblasts was normal, despite expression of mutant huntingtin. Although glyceraldehyde 3‐phosphate dehydrogenase (a huntingtin binding protein) activity was normal in all areas studied, aconitase activity was decreased to 8% in HD caudate, 27% in putamen, and 52% in cerebral cortex, but normal in HD cerebellum and fibroblasts. We have demonstrated that although complexes II and III are those parts of the respiratory chain most vulnerable to inhibition in the presence of a nitric oxide (NO•) generator, aconitase activity was even more sensitive to inhibition. The pattern of these enzyme deficiencies and their parallel to the anatomical distribution of HD pathology support an important role for NO• and excitotoxicity in HD pathogenesis. Furthermore, based on the biochemical defects we have described, we suggest that NO• generation produces a graded response, with aconitase inhibition followed by complex II/III inhibition and the initiation of a self‐amplifying cycle of free radical generation and aconitase inhibition, which results in severe ATP depletion. We propose that these events are important in determining neuronal cell death and are critical steps in the pathogenesis of HD. Ann Neurol 1999;45:25–32

Mitochondrial dysfunction and free radical damage in the Huntington R6/2 transgenic mouse.

Huntingtons disease is a progressive neurodegenerative disease caused by an abnormally expanded (>36) CAG repeat within the ITI5 gene encoding a widely expressed 349‐kd protein, huntingtin. The medium spiny neurons of the caudate preferentially degenerate in Huntingtons disease, with the presence of neuronal intranuclear inclusions. Excitotoxicity is thought to be important in the pathogenesis of Huntingtons disease; the recently described mitochondrial respiratory chain and aconitase defects in Huntingtons disease brain are consistent with this hypothesis. A transgenic mouse model (R6/2) of Huntingtons disease develops a movement disorder, muscle wasting, and premature death at about 14 to 16 weeks. Selective neuronal death in these mice is not seen until 14 weeks. Biochemical analysis of R6/2 mouse brain at 12 weeks demonstrated a significant reduction in aconitase and mitochondrial complex IV activities in the striatum and a decrease in complex IV activity in the cerebral cortex. Increased immunostaining for inducible nitric oxide synthase and nitrotyrosine was seen in the transgenic mouse model but not control mouse brains. These results extend the parallels between Huntingtons disease and the transgenic mouse model to biochemical events and suggest complex IV deficiency and elevated nitric oxide and superoxide radical generation precede neuronal death in the R6/2 mouse and contribute to pathogenesis. Ann Neurol 2000; 47:80–86

Chaperone-mediated autophagy markers in Parkinson disease brains.

OBJECTIVE To investigate chaperone-mediated autophagy in the pathogenesis of Parkinson disease (PD). DESIGN Postmortem observational study. SETTING University Department of Clinical Neuroscience, Institute of Neurology, University College London. SUBJECTS Postmortem samples from 7 PD, 6 Alzheimer disease (AD), and 8 control brains. MAIN OUTCOME MEASURE Lysosomal-associated membrane protein 2A (LAMP2A) and heat shock cognate 70 (hsc70) protein levels were compared in the substantia nigra pars compacta and amygdala of PD, AD, and control brain samples. To provide insight into the turnover of α-synuclein, degradation pathways for this protein were studied in a dopaminergic cell line. RESULTS The expression levels of the chaperone-mediated autophagy proteins LAMP2A and hsc70 were significantly reduced in the substantia nigra pars compacta and amygdala of PD brains compared with age-matched AD and control brain samples. Lewy bodies in these regions contained autophagy-related proteins. We demonstrated that decreased LAMP2A levels in dopaminergic cell lines reduced chaperone-mediated autophagy activity and increased the half-life of α-synuclein. CONCLUSIONS These findings suggest that there is reduced chaperone-mediated autophagy activity in the PD brain, provide evidence for the role of autophagy in PD pathogenesis and Lewy body formation, and suggest that this pathway may be a suitable therapeutic target in PD.

Influence of microRNA deregulation on chaperone-mediated autophagy and α-synuclein pathology in Parkinson's disease.

The presence of α-synuclein aggregates in the characteristic Lewy body pathology seen in idiopathic Parkinson’s disease (PD), together with α-synuclein gene mutations in familial PD, places α-synuclein at the center of PD pathogenesis. Decreased levels of the chaperone-mediated autophagy (CMA) proteins LAMP-2A and hsc70 in PD brain samples suggests compromised α-synuclein degradation by CMA may underpin the Lewy body pathology. Decreased CMA protein levels were not secondary to the various pathological changes associated with PD, including mitochondrial respiratory chain dysfunction, increased oxidative stress and proteasomal inhibition. However, decreased hsc70 and LAMP-2A protein levels in PD brains were associated with decreases in their respective mRNA levels. MicroRNA (miRNA) deregulation has been reported in PD brains and we have identified eight miRNAs predicted to regulate LAMP-2A or hsc70 expression that were reported to be increased in PD. Using a luciferase reporter assay in SH-SY5Y cells, four and three of these miRNAs significantly decreased luciferase activity expressed upstream of the lamp-2a and hsc70 3′UTR sequences respectively. We confirmed that transfection of these miRNAs also decreased endogenous LAMP-2A and hsc70 protein levels respectively and resulted in significant α-synuclein accumulation. The analysis of PD brains confirmed that six and two of these miRNAs were significantly increased in substantia nigra compacta and amygdala respectively. These data support the hypothesis that decreased CMA caused by miRNA-induced downregulation of CMA proteins plays an important role in the α-synuclein pathology associated with PD, and opens up a new avenue to investigate PD pathogenesis.

Pramipexole protects against MPTP toxicity in non-human primates

The neurotoxin MPTP induces nigral dopaminergic cell death in primates and produces a partial model of Parkinsons disease (PD). Pramipexole is a D2/D3 dopamine receptor agonist used in the symptomatic treatment of PD, and which also protects neuronal cells against dopaminergic toxins in vitro. We now demonstrate that pramipexole partially prevents MPTP toxicity in vivo in a primate species. Common marmosets were repeatedly treated with pramipexole either before, coincidentally with, or after low‐dose MPTP treatment designed to induce a partial lesion of the substantia nigra. Animals pretreated with pramipexole had a significantly greater number of surviving tyrosine hydroxylase (TH) positive neurones in the pars compacta of the substantia nigra. Pramipexole pretreatment also prevented degeneration of striatal dopamine terminals. Treatment with pramipexole concurrently with MPTP or following MPTP did not prevent TH‐positive cell loss. Pramipexole pretreatment appears to induce adaptive changes that protect against dopaminergic cell loss in primates.

Functional consequences of the 3460-bp mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy

Complex I is the largest of the mitochondrial respiratory chain proteins, and contains subunits encoded by both mitochondrial and nuclear genomes. Lebers hereditary optic neuropathy has been clearly linked to mutations of mitochondrial DNA complex I genes, and variable complex I functional defects have been reported. We have confirmed an approximate 60% defect in mitochondrial NADH CoQ1 reductase activity in cultured fibroblasts bearing the 3460-bp G to A mutation within the ND1 gene. However complex I-linked ATP synthesis was found to be normal in these fibroblasts. A 60% rotenone-induced decrease in complex I activity was shown to reduce ATP synthesis in normal fibroblasts, indicating that this level of complex I activity was below the threshold required to affect ATP synthesis. Although 3460 LHON mitochondria were less sensitive to rotenone inhibition, this did not explain the decreased complex I activity as the rotenone insensitive activity was not increased, nor did the inhibitor diphenyleneiodonium inhibit the NADH CoQ1 reductase activity to a greater extent. Decreased NADH cytochrome c reductase activity in cybrids homoplasmic for the 3460 LHON mtDNA mutation confirmed that the decrease in complex I activity was not specific to the assay used and was not caused by inhibitory effects of ubiquinone analogues used in the NADH CoQ1 reductase assay. These findings have important implications for our understanding of complex I dysfunction in the pathogenesis of 3460 Lebers hereditary optic neuropathy.

MOLECULAR DEFECTS OF NADH-UBIQUINONE OXIDOREDUCTASE (COMPLEX-I) IN MITOCHONDRIAL DISEASES

Defects in Complex I of the mitochondrial respiratory chain have been identified in 38 patients. The clinical and laboratory features are reviewed and the results of recently devised strategies aimed at characterizing the primary molecular and genetic abnormalities are presented. Although not exhaustive, these studies have provided a molecular basis for the contention that defects in Complex I may have their origin in nuclear or in mitochondrial genes.

Mitochondrial Myopathy with a Defect of Mitochondrial-Protein Transport

The clinical and biochemical heterogeneity of the mitochondrial myopathies is now well established.1 , 2 Recent work has focused on identifying the molecular basis of these disorders and has demons...

Differences in toxicity of the catechol-O-methyl transferase inhibitors, tolcapone and entacapone to cultured human neuroblastoma cells

Tolcapone and entacapone are catechol-O-methyltransferase (COMT) inhibitors used as adjuncts to levodopa in the treatment of Parkinsons disease (PD). The use of tolcapone has been limited by its hepatotoxicity, the cause of which remains uncertain. Tolcapone compound is an uncoupler of mitochondrial respiration in isolated mitochondria and this action may be relevant to its effect on liver function. We have examined the actions of COMT inhibitors on cultured cells, comparing them with those of the classical uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), in order to provide insight into their mechanism of potential toxicity. Tolcapone and FCCP were shown to be toxic to human neuroblastoma SH-SY5Y cells and caused a profound reduction in ATP synthesis. Entacapone was not toxic to SH-SY5Y. Tolcapone and FCCP were shown to be equally toxic to cells depleted of mtDNA and thus devoid of a functional respiratory chain. This study demonstrates that tolcapone markedly inhibits ATP synthesis in cultured cells mirroring the effects of a classical uncoupler. However its toxicity may also involve a mechanism independent of its effects upon oxidative phosphorylation.