Parkin mediates the degradation-independent ubiquitination of Hsp70

Mutations in the parkin gene cause autosomal recessive, juvenile-onset parkinsonism. Parkin is an E3 ubiquitin ligase that mediates the ubiquitination of protein substrates. Disease-associated mutations cause a loss-of-function of parkin which may compromise the poly-ubiquitination and proteasomal degradation of specific protein substrates, potentially leading to their deleterious accumulation. Here, we identify the molecular chaperones, Hsp70 and Hsc70, as substrates for parkin. Parkin mediates the ubiquitination of Hsp70 both in vitro and in cultured cells. Parkin interacts with Hsp70 via its second RING finger domain and mutations in/near this domain compromise Hsp70 ubiquitination. Ubiquitination of Hsp70 fails to alter its steady-state levels or turnover, nor does it promote its proteasomal degradation. Consistent with this observation, Hsp70 levels remain unaltered in brains from parkin-deficient autosomal recessive, juvenile-onset parkinsonism subjects, whereas alternatively, Hsp70 levels are elevated in the detergent-insoluble fraction of sporadic Parkinson's disease/dementia with Lewy bodies brains. Parkin mediates the multiple mono-ubiquitination of Hsp70/Hsc70 consistent with a degradation-independent role for this ubiquitin modification. Our observations support a novel functional relationship between parkin and Hsc/Hsp70 and support the notion that parkin is a multi-purpose E3 ubiquitin ligase capable of modifying proteins either via attachment of alternatively linked poly-ubiquitin chains or through multiple mono-ubiquitination to achieve alternate biological outcomes.     

Founder mutations in NDRG1 and HK1 genes are common causes of inherited neuropathies among Roma/Gypsies in Slovakia

Autosomal recessive forms of Charcot–Marie–Tooth disease (CMT) account for less than 10 % of all CMT cases, but are more frequent in the populations with a high rate of consanguinity. Roma (Gypsies) are a transnational minority with an estimated population of 10 to 14 million, in which a high degree of consanguineous marriages is a generally known fact. Similar to the other genetically isolated founder populations, the Roma harbour a number of unique or rare autosomal recessive disorders, caused by “private” founder mutations. There are three subtypes of autosomal recessive CMT with mutations private to the Roma population: CMT4C, CMT4D and CMT4G. We report on the molecular examination of four families of Roma origin in Slovakia with early-onset demyelinating neuropathy and autosomal recessive inheritance. We detected mutation p.R148X (g.631C>T) in the NDRG1 (NM_006096.3) gene in two families and mutation g.9712G>C in the HK1 (NM_033498) gene in the other two families. These mutations cause CMT4D and CMT4G, respectively. The success of molecular genetic analysis in all families confirms that autosomal recessive forms of CMT caused by mutations on the NDRG1 and HK1 genes are common causes of inherited neuropathies among Slovak Roma. Providing genetic analysis of these genes for patients with Roma origin as a common part of diagnostic procedure would contribute to a better rate of diagnosed cases of demyelinating neuropathy in Slovakia and in other countries with a Roma minority.     

Pathogenic mutations inactivate parkin by distinct mechanisms

Loss of parkin function is the major cause of autosomal recessive Parkinson's disease (ARPD). A wide variety of parkin mutations have been identified in patients; however, the pathophysiological mechanisms leading to the inactivation of mutant parkin are poorly understood. In this study we characterized pathogenic C- and N-terminal parkin mutants and found distinct pathways of parkin inactivation. Deletion of the C terminus abrogated the association of parkin with cellular membranes and induced rapid misfolding and aggregation. Four N-terminal missense mutations, located within the ubiquitin-like domain (UBL), decrease the stability of parkin; as a consequence, these mutants are rapidly degraded by the proteasome. Furthermore, we present evidence that a smaller parkin species of 42 kDa, which is present in extracts prepared from human brain and cultured cells, originates from an internal start site and lacks the N-terminal UBL domain.     

Novel monoclonal antibodies demonstrate biochemical variation of brain parkin with age

Autosomal recessive juvenile parkinsonism is a movement disorder associated with the degeneration of dopaminergic neurons in substantia nigra pars compacta. The loss of functional parkin caused by parkin gene mutations is the most common single cause of juvenile parkinsonism. Parkin has been shown to aid in protecting cells from endoplasmic reticulum and oxidative stressors presumably due to ubiquitin ligase activity of parkin that targets proteins for proteasomal degradation. However, studies on parkin have been impeded because of limited reagents specific for this protein. Here we report the generation and characterization of a panel of parkin-specific monoclonal antibodies. Biochemical analyses indicate that parkin is present only in the high salt-extractable fraction of mouse brain, whereas it is present in both the high salt-extractable and RIPA-resistant, SDS-extractable fraction in young human brain. Parkin is present at decreased levels in the high salt-extractable fraction and at increased levels in the SDS-extractable fraction from aged human brain. This shift in the extractability of parkin upon aging is seen in humans but not in mice, demonstrating species-specific differences in the biochemical characteristics of murine versus human parkin. Finally, by using these highly specific anti-parkin monoclonal antibodies, it was not possible to detect parkin in alpha-synuclein-containing lesions in alpha-synucleinopathies, thereby challenging prior inferences about the role of parkin in movement disorders other than autosomal recessive juvenile parkinsonism.     

Impaired dopamine release and synaptic plasticity in the striatum of Parkin−/− mice

Parkin is the most common causative gene of juvenile and early-onset familial Parkinson's diseases and is thought to function as an E3 ubiquitin ligase in the ubiquitin-proteasome system. However, it remains unclear how loss of Parkin protein causes dopaminergic dysfunction and nigral neurodegeneration. To investigate the pathogenic mechanism underlying these mutations, we used parkin −/− mice to study its physiological function in the nigrostriatal circuit. Amperometric recordings showed decreases in evoked dopamine release in acute striatal slices of parkin −/− mice and reductions in the total catecholamine release and quantal size in dissociated chromaffin cells derived from parkin −/− mice. Intracellular recordings of striatal medium spiny neurons revealed impairments of long-term depression and long-term potentiation in parkin −/− mice, whereas long-term potentiation was normal in the Schaeffer collateral pathway of the hippocampus. Levels of dopamine receptors and dopamine transporters were normal in the parkin −/− striatum. These results indicate that Parkin is involved in the regulation of evoked dopamine release and striatal synaptic plasticity in the nigrostriatal pathway, and suggest that impairment in evoked dopamine release may represent a common pathophysiological change in recessive parkinsonism.     

PARK7, a Novel Locus for Autosomal Recessive Early-Onset Parkinsonism, on Chromosome 1p36

Although the role of genetic factors in the origin of Parkinson disease has long been disputed, several genes involved in autosomal dominant and recessive forms of the disease have been localized. Mutations associated with early-onset autosomal recessive parkinsonism have been identified in the Parkin gene, and recently a second gene, PARK6, involved in early-onset recessive parkinsonism was localized on chromosome 1p35-36. We identified a family segregating early-onset parkinsonism with multiple consanguinity loops in a genetically isolated population. Homozygosity mapping resulted in significant evidence for linkage on chromosome 1p36. Multipoint linkage analysis using MAPMAKER-HOMOZ generated a maximum LOD-score of 4.3, with nine markers spanning a disease haplotype of 16 cM. On the basis of several recombination events, the region defining the disease haplotype can be clearly separated, by > or =25 cM, from the more centromeric PARK6 locus on chromosome 1p35-36. Therefore, we conclude that we have identified on chromosome 1 a second locus, PARK7, involved in autosomal recessive, early-onset parkinsonism.     

Genome screen to identify susceptibility genes for Parkinson disease in a sample without parkin mutations

Parkinson disease (PD) is a common neurodegenerative disorder characterized by bradykinesia, resting tremor, muscular rigidity, and postural instability, as well as by a clinically significant response to treatment with levodopa. Mutations in the alpha-synuclein gene have been found to result in autosomal dominant PD, and mutations in the parkin gene produce autosomal recessive juvenile-onset PD. We have studied 203 sibling pairs with PD who were evaluated by a rigorous neurological assessment based on (a) inclusion criteria consisting of clinical features highly associated with autopsy-confirmed PD and (b) exclusion criteria highly associated with other, non-PD pathological diagnoses. Families with positive LOD scores for a marker in an intron of the parkin gene were prioritized for parkin-gene testing, and mutations in the parkin gene were identified in 22 families. To reduce genetic heterogeneity, these families were not included in subsequent genome-screen analysis. Thus, a total of 160 multiplex families without evidence of a parkin mutation were used in multipoint nonparametric linkage analysis to identify PD-susceptibility genes. Two models of PD affection status were considered: model I included only those individuals with a more stringent diagnosis of verified PD (96 sibling pairs from 90 families), whereas model II included all examined individuals as affected, regardless of their final diagnostic classification (170 sibling pairs from 160 families). Under model I, the highest LOD scores were observed on chromosome X (LOD score 2.1) and on chromosome 2 (LOD score 1.9). Analyses performed with all available sibling pairs (model II) found even greater evidence of linkage to chromosome X (LOD score 2.7) and to chromosome 2 (LOD score 2.5). Evidence of linkage was also found to chromosomes 4, 5, and 13 (LOD scores >1.5). Our findings are consistent with those of other linkage studies that have reported linkage to chromosomes 5 and X.     

Localization of a novel locus for autosomal recessive early-onset parkinsonism, PARK6, on human chromosome 1p35-p36

The cause of Parkinson disease (PD) is still unknown, but genetic factors have recently been implicated in the etiology of the disease. So far, four loci responsible for autosomal dominant PD have been identified. Autosomal recessive juvenile parkinsonism (ARJP) is a clinically and genetically distinct entity; typical PD features are associated with early onset, sustained response to levodopa, and early occurrence of levodopa-induced dyskinesias, which are often severe. To date, only one ARJP gene, Parkin, has been identified, and multiple mutations have been detected both in families with autosomal recessive parkinsonism and in sporadic cases. The Parkin-associated phenotype is broad, and some cases are indistinguishable from idiopathic PD. In > or = 50% of families with ARJP that have been analyzed, no mutations could be detected in the Parkin gene. We identified a large Sicilian family with four definitely affected members (the Marsala kindred). The phenotype was characterized by early-onset (range 32-48 years) parkinsonism, with slow progression and sustained response to levodopa. Linkage of the disease to the Parkin gene was excluded. A genomewide homozygosity screen was performed in the family. Linkage analysis and haplotype construction allowed identification of a single region of homozygosity shared by all the affected members, spanning 12.5 cM on the short arm of chromosome 1. This region contains a novel locus for autosomal recessive early-onset parkinsonism, PARK6. A maximum LOD score 4.01 at recombination fraction .00 was obtained for marker D1S199.     

The Drosophila parkin homologue is required for normal mitochondrial dynamics during spermiogenesis

Drosophila parkin, the ortholog of the human parkin gene, responsible for a familiar form of autosomal recessive juvenile parkinsonism, has been shown previously to be involved in Drosophila male fertility. Loss-of-function mutations in the parkin gene cause failure of spermatid individualization by affecting the proper progression of the actin-based investment cones that assemble in the nuclear region, but fail to translocate in synchrony down the cyst. In parkin mutants, the investment cones are scattered along the post-elongated spermatid bundles and fail to act properly in the process of sperm individualization. Using phase-contrast and electron microscopy analysis, we demonstrate that the parkin spermatids assemble a seemingly normal onion-stage nebenkern, but when the axoneme elongates only one mitochondrial derivative unfurls from the nebenkern. This unique mitochondrial derivative undergoes abnormal shaping and condensation during spermatid elongation. Our results indicate that parkin gene function is necessary for mitochondrial morphogenesis during earlier and later phases of spermiogenesis. The failure of cyst individualization may be due to the sensitivity of investment cone movement to the perturbation of mitochondrial morphology during spermatid elongation.     

Mutations of the ATM gene detected in Japanese ataxia-telangiectasia patients: possible preponderance of the two founder mutations 4612del165 and 7883del5

The ATM (A-T, mutated) gene on human chromosome 11q22.3 has recently been identified as the gene responsible for the human recessive disease ataxia-telangiectasia (A-T). In order to define the types of disease-causing ATM mutations in Japanese A-T patients as well as to look for possible mutational hotspots, reverse-transcribed RNA derived from ten patients belonging to eight unrelated Japanese A-T families was analyzed for mutations by the restriction endonuclease fingerprinting method. As has been reported by others, mutations that lead to exon skipping or premature protein truncation were also predominant in our mutants. Six different mutations were identified on 12 of the 16 alleles examined. Four were deletions involving a loss of a single exon: exon 7, exon 16, exon 33 or exon 35. The others were minute deletions, 4649delA in exon 33 and 7883del5 in exon 55. The mutations 4612del165 and 7883del5 were found in more than two unrelated families; 44% (7 of 16) of the mutant alleles had one of the two mutations. The 4612del165 mutations in three different families were all ascribed to the same T→A substitution at the splice donor site in intron 33. Microsatellite genotyping around the ATM locus also indicated that a common haplotype was shared by the mutant alleles in both mutations. This suggests that these two founder mutations may be predominant among Japanese ATM mutant alleles. Received: 15 September 1997 / Accepted: 12 January 1998     

Parkin (PARK 2) Mutations Are Rare in Czech Patients with Early-Onset Parkinson's Disease

Objective

The aim of the study is to determine the frequency of parkin allelic variants in Czech early-onset Parkinson''s disease patients and healthy controls.

Methods

A total of 70 early-onset Parkinson''s disease patients (age at onset ≤40 years) and 75 controls were screened for the sequence variants and exon rearrangements in the parkin gene.

Results

Parkin mutations were identified in five patients (7.1%): the p.R334C point mutation was present in one patient, four patients had exon deletions. The detected mutations were observed in the heterozygous state except one homozygous deletion of the exon 4. No mutations were obtained in control subjects. A novel sequence variant p.V380I (c.1138G>A) was identified in one control. Non-pathogenic polymorphisms p.S167N and p.D394N were seen in similar percentage in patients and controls, polymorphism p.V380L was almost twice as frequent in controls as in patients.

Conclusions

Our study contributes to the growing body of evidence on the low frequency of the parkin mutations in the early-onset Parkinson''s disease suggesting the potential role of other genes in the pathogenesis of the disease.     

Parkin mono-ubiquitinates Bcl-2 and regulates autophagy

Parkin is an E3 ubiquitin ligase that mediates the ubiquitination of protein substrates. The mutations in the parkin gene can lead to a loss of function of parkin and cause autosomal recessive juvenile onset parkinsonism. Recently, parkin was reported to be involved in the regulation of mitophagy. Here, we identify the Bcl-2, an anti-apoptotic and autophagy inhibitory protein, as a substrate for parkin. Parkin directly binds to Bcl-2 via its C terminus and mediates the mono-ubiquitination of Bcl-2, which increases the steady-state levels of Bcl-2. Overexpression of parkin, but not its ligase-deficient forms, decreases autophagy marker LC3 conversion, whereas knockdown of parkin increases LC3 II levels. In HeLa cells, a parkin-deficient cell line, knockdown of parkin does not change LC3 conversion. Moreover, overexpression of parkin enhances the interactions between Bcl-2 and Beclin 1. Our results provide evidence that parkin mono-ubiquitinates Bcl-2 and regulates autophagy via Bcl-2.     

Parkin interacts with LIM Kinase 1 and reduces its cofilin-phosphorylation activity via ubiquitination

Mutations in the PARKIN (PARK2) gene have been found in the majority of early-onset familial Parkinson's disease (PD) patients with autosomal recessive juvenile parkinsonism (ARJP). Parkin protein functions as an ubiquitin (E3) ligase that targets specific proteins for degradation in the 26S proteasome. Here, based on a mass spectrometry analysis of the human dopaminergic neuroblastoma-derived cell line SH-SY5Y that over-expresses parkin, we found that parkin may suppress cofilin phosphorylation. LIM Kinase 1 (LIMK1) is the upstream protein that phosphorylates cofilin, an actin depolymerizing protein. Thus, we postulated a possible connection between parkin and LIMK1. Our studies in other cell lines, using co-transfection assays, demonstrated that LIMK1 and parkin bind each other. LIMK1 also interacted with previously known parkin interactors Hsp70 and CHIP. Parkin enhanced LIMK1-ubiquitination in the human neuroblastoma-derived BE(2)-M17 cell line, but not in the human embryonic kidney-derived HEK293 cell line. In fact, parkin-over-expression reduced the level of LIMK1-induced phosphocofilin in the BE(2)-M17 cells but not in the HEK293 cells. Additionally, in simian kidney-derived COS-7 cells, parkin-over-expression reduced LIMK1-induced actin filament accumulation. LIMK1 in cultured cells regulates parkin reversibly: LIMK1 did not phosphorylate parkin but LIMK1 overexpression reduced parkin self-ubiquitination in vitro and in HEK293 cells. Furthermore, in the cells co-transfected with parkin and p38, LIMK1 significantly decreased p38-ubiquitination by parkin. These findings demonstrate a cell-type dependent functional interaction between parkin and LIMK1 and provide new evidence that links parkin and LIMK1 in the pathogenesis of familial PD.     

Mitochondrial defect and PGC-1α dysfunction in parkin-associated familial Parkinson's disease

Mutations in the parkin gene are expected to play an essential role in autosomal recessive Parkinson's disease. Recent studies have established an impact of parkin mutations on mitochondrial function and autophagy. In primary skin fibroblasts from two patients affected by an early onset Parkinson's disease, we identified a hitherto unreported compound heterozygous mutation del exon2-3/del exon3 in the parkin gene, leading to the complete loss of the full-length protein. In both patients, but not in their heterozygous parental control, we observed severe ultrastructural abnormalities, mainly in mitochondria. This was associated with impaired energy metabolism, deregulated reactive oxygen species (ROS) production, resulting in lipid oxidation, and peroxisomal alteration. In view of the involvement of parkin in the mitochondrial quality control system, we have investigated upstream events in the organelles' biogenesis. The expression of the peroxisome proliferator-activated receptor gamma-coactivator 1-alpha (PGC-1α), a strong stimulator of mitochondrial biogenesis, was remarkably upregulated in both patients. However, the function of PGC-1α was blocked, as revealed by the lack of its downstream target gene induction. In conclusion, our data confirm the role of parkin in mitochondrial homeostasis and suggest a potential involvement of the PGC-1α pathway in the pathogenesis of Parkinson's disease. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.     

Different mutations in the LMNA gene cause autosomal dominant and autosomal recessive Emery-Dreifuss muscular dystrophy

Emery-Dreifuss muscular dystrophy (EMD) is a condition characterized by the clinical triad of early-onset contractures, progressive weakness in humeroperoneal muscles, and cardiomyopathy with conduction block. The disease was described for the first time as an X-linked muscular dystrophy, but autosomal dominant and autosomal recessive forms were reported. The genes for X-linked EMD and autosomal dominant EMD (AD-EMD) were identified. We report here that heterozygote mutations in LMNA, the gene for AD-EMD, may cause diverse phenotypes ranging from typical EMD to no phenotypic effect. Our results show that LMNA mutations are also responsible for the recessive form of the disease. Our results give further support to the notion that different genetic forms of EMD have a common pathophysiological background. The distribution of the mutations in AD-EMD patients (in the tail and in the 2A rod domain) suggests that unique interactions between lamin A/C and other nuclear components exist that have an important role in cardiac and skeletal muscle function.     

Common pathogenic effects of missense mutations in the P-type ATPase ATP13A2 (PARK9) associated with early-onset parkinsonism

Mutations in the ATP13A2 gene (PARK9) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome (KRS), a neurodegenerative disease characterized by parkinsonism. KRS mutations produce truncated forms of ATP13A2 with impaired protein stability resulting in a loss-of-function. Recently, homozygous and heterozygous missense mutations in ATP13A2 have been identified in subjects with early-onset parkinsonism. The mechanism(s) by which missense mutations potentially cause parkinsonism are not understood at present. Here, we demonstrate that homozygous F182L, G504R and G877R missense mutations commonly impair the protein stability of ATP13A2 leading to its enhanced degradation by the proteasome. ATP13A2 normally localizes to endosomal and lysosomal membranes in neurons and the F182L and G504R mutations disrupt this vesicular localization and promote the mislocalization of ATP13A2 to the endoplasmic reticulum. Heterozygous T12M, G533R and A746T mutations do not obviously alter protein stability or subcellular localization but instead impair the ATPase activity of microsomal ATP13A2 whereas homozygous missense mutations disrupt the microsomal localization of ATP13A2. The overexpression of ATP13A2 missense mutants in SH-SY5Y neural cells does not compromise cellular viability suggesting that these mutant proteins lack intrinsic toxicity. However, the overexpression of wild-type ATP13A2 may impair neuronal integrity as it causes a trend of reduced neurite outgrowth of primary cortical neurons, whereas the majority of disease-associated missense mutations lack this ability. Finally, ATP13A2 overexpression sensitizes cortical neurons to neurite shortening induced by exposure to cadmium or nickel ions, supporting a functional interaction between ATP13A2 and heavy metals in post-mitotic neurons, whereas missense mutations influence this sensitizing effect. Collectively, our study provides support for common loss-of-function effects of homozygous and heterozygous missense mutations in ATP13A2 associated with early-onset forms of parkinsonism.     

The 2588G-->C mutation in the ABCR gene is a mild frequent founder mutation in the Western European population and allows the classification of ABCR mutations in patients with Stargardt disease

In 40 western European patients with Stargardt disease (STGD), we found 19 novel mutations in the retina-specific ATP-binding cassette transporter (ABCR) gene, illustrating STGD's high allelic heterogeneity. One mutation, 2588G-->C, identified in 15 (37.5%) patients, shows linkage disequilibrium with a rare polymorphism (2828G-->A) in exon 19, suggesting a founder effect. The guanine at position 2588 is part of the 3' splice site of exon 17. Analysis of the lymphoblastoid cell mRNA of two STGD patients with the 2588G-->C mutation shows that the resulting mutant ABCR proteins either lack Gly863 or contain the missense mutation Gly863Ala. We hypothesize that the 2588G-->C alteration is a mild mutation that causes STGD only in combination with a severe ABCR mutation. This is supported in that the accompanying ABCR mutations in at least five of eight STGD patients are null (severe) and that a combination of two mild mutations has not been observed among 68 STGD patients. The 2588G-->C mutation is present in 1 of every 35 western Europeans, a rate higher than that of the most frequent severe autosomal recessive mutation, the cystic fibrosis conductance regulator gene mutation DeltaPhe508. Given an STGD incidence of 1/10,000, homozygosity for the 2588G-->C mutation or compound heterozygosity for this and other mild ABCR mutations probably does not result in an STGD phenotype.     

Developmental changes in the expression of parkin and UbcR7, a parkin-interacting and ubiquitin-conjugating enzyme, in rat brain.

Parkin is a product of the Park2 gene the mutation of which causes autosomal recessive juvenile parkinsonism (AR-JP) characterized by selective dopaminergic neuronal death and absence of Lewy bodies. Recently we found that parkin is directly linked to the ubiquitin (Ub)-proteasome pathway as a Ub-protein ligase (E3) collaborating with a Ub-conjugating enzyme (E2) UbcH7. Here we analysed by in situ hybridization the expression of mRNAs for parkin and UbcR7 (rat orthologue of human UbcH7) in the developing rat brain. Parkin mRNA increased in parallel with neuronal maturation, but was unevenly distributed in various brain regions after four postnatal days. The expression pattern of the UbcR7 mRNA was almost identical to that of the parkin mRNA in all cases examined. Both parkin and UbcR7 mRNAs were distributed in neurones but not glial cells. Our findings indicate that parkin is expressed not only in the substantia nigra, but also uniformly in various brain regions in a development-dependent manner. Co-expression of UbcR7 with parkin suggests that UbcR7 may interact with parkin in vivo for ubiquitination of yet unidentified target protein(s).