Mutations in PINK1 and parkin cause autosomal recessive parkinsonism, a neurodegenerative disorder characterized by the loss of dopaminergic neurons. To highlight potential therapeutic pathways we have identified factors that genetically interact with parkin/PINK1. Here we report that overexpression of the translation inhibitor 4E-BP can suppress all pathologic phenotypes including degeneration of dopaminergic neurons in Drosophila. 4E-BP is activated in vivo by the TOR inhibitor rapamycin, which we find can potently suppress pathology in PINK1/parkin mutants. Rapamycin also ameliorates mitochondrial defects in cells from parkin-mutant patients. Recently, 4E-BP was shown to be inhibited by the most common cause of parkinsonism, dominant mutations in LRRK2. Here we further show that loss of the Drosophila LRRK2 homolog activates 4E-BP and is also able to suppress PINK1/parkin pathology. Thus, in conjunction with recent findings our results suggest that pharmacologic stimulation of 4E-BP activity may represent a viable therapeutic approach for multiple forms of parkinsonism.
Objective There are marked mitochondrial abnormalities in parkin-knock out drosophila and other model systems. The aim of our study was to determine mitochondrial function and morphology in parkin-mutant patients. We also investigated whether pharmacological rescue of impaired mitochondrial function may be possible in parkin-mutant human tissue. Methods We used three sets of techniques, namely biochemical measurements of mitochondrial function, quantitative morphology and live cell imaging of functional connectivity to assess the mitochondrial respiratory chain, the outer shape and connectivity of the mitochondria and their functional inner connectivity in fibroblasts from patients with homozygous or compound heterozygous parkin mutations. Results Parkin-mutant cells had lower mitochondrial complex I activity and complex I linked ATP-production which correlated with a higher degree of mitochondrial branching, suggesting that the functional and morphological effects of parkin are related. Knockdown of parkin in control fibroblasts confirmed that parkin deficiency is sufficient to explain these mitochondrial effects. In contrast, 50% knockdown of parkin, mimicking haploinsufficiency in human patient tissue, did not result in impaired mitochondrial function or morphology. Fluorescence recovery after photobleaching (FRAP) assays demonstrated a lower level of functional connectivity of the mitochondrial matrix which further worsened after rotenone exposure. Treatment with experimental neuroprotective compounds resulted in a rescue of the mitochondrial membrane potential. Interpretation Our study demonstrates marked abnormalities of mitochondrial function and morphology in parkin-mutant patients and provides proof of principle data for the potential usefulness of this new model system as a tool to screen for disease-modifying compounds in genetically homogenous parkinsonian disorders.
Our results provide evidence for impaired mitochondrial function and morphology in LRRK2(G2019S) mutant patient tissue. Further studies are required to determine whether the impaired mitochondrial function is due to increased LRRK2 kinase activity or other mechanisms such as LRRK2 haploinsufficiency.
BackgroundAmyotrophic lateral sclerosis (ALS), a common late-onset neurodegenerative disease, is associated with fronto-temporal dementia (FTD) in 3–10% of patients. A mutation in CHMP2B was recently identified in a Danish pedigree with autosomal dominant FTD. Subsequently, two unrelated patients with familial ALS, one of whom also showed features of FTD, were shown to carry missense mutations in CHMP2B. The initial aim of this study was to determine whether mutations in CHMP2B contribute more broadly to ALS pathogenesis.Methodology/Principal FindingsSequencing of CHMP2B in 433 ALS cases from the North of England identified 4 cases carrying 3 missense mutations, including one novel mutation, p.Thr104Asn, none of which were present in 500 neurologically normal controls. Analysis of clinical and neuropathological data of these 4 cases showed a phenotype consistent with the lower motor neuron predominant (progressive muscular atrophy (PMA)) variant of ALS. Only one had a recognised family history of ALS and none had clinically apparent dementia. Microarray analysis of motor neurons from CHMP2B cases, compared to controls, showed a distinct gene expression signature with significant differential expression predicting disassembly of cell structure; increased calcium concentration in the ER lumen; decrease in the availability of ATP; down-regulation of the classical and p38 MAPK signalling pathways, reduction in autophagy initiation and a global repression of translation. Transfection of mutant CHMP2B into HEK-293 and COS-7 cells resulted in the formation of large cytoplasmic vacuoles, aberrant lysosomal localisation demonstrated by CD63 staining and impairment of autophagy indicated by increased levels of LC3-II protein. These changes were absent in control cells transfected with wild-type CHMP2B.Conclusions/SignificanceWe conclude that in a population drawn from North of England pathogenic CHMP2B mutations are found in approximately 1% of cases of ALS and 10% of those with lower motor neuron predominant ALS.We provide a body of evidence indicating the likely pathogenicity of the reported gene alterations. However, absolute confirmation of pathogenicity requires further evidence, including documentation of familial transmission in ALS pedigrees which might be most fruitfully explored in cases with a LMN predominant phenotype.
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