帕金森病与ATP13A2基因

ATP13A2基因是帕金森病(PD)的致病基因,早发型帕金森病(EOPD)和Kufor-Rakeb综合征(KRS)患者中均发现ATP13A2基因突变。基因突变类型与疾病的严重程度和发病年龄相关,PD患者中黑质多巴胺能神经元也存在ATP13A2基因mRNA表达升高。因此,对ATP13A2基因的研究将有助于该病的基因诊断、病理生理学机制的阐明和治疗。     

帕金森病与ATP13A2基因的研究进展

 帕金森病（Parkinson’s disease, PD）是一种患病率仅次于老年痴呆,年龄相关的神经退行性疾病,环境因素与遗传因素共同参与PD的发病。目前已定位16个基因位点并克隆了11个致病基因,其中位于PARK9上的ATP13A2基因与Kufor-Rakeb综合征（Kufor-Rakeb syndrome, KRS）和早发型帕金森病（Early-onset Parkinson’s disease, EOPD）相关。本文将对PD患者中ATP13A2基因突变和功能研究进行综述。     

ATP13A2 mutations impair mitochondrial function in fibroblasts from patients with Kufor-Rakeb syndrome

Mutations in ATP13A2 cause autosomal-recessive parkinsonism (Kufor-Rakeb syndrome; KRS). Because several other parkinsonism-associated proteins have been connected to mitochondrial function and mitophagy, we studied the impact of endogenous mutations in ATPase type 13A2 (ATP13A2) on mitochondria in fibroblasts from KRS patients compared with controls. In patients, we detected decreased adenosine triphosphate (ATP) synthesis rates, increased mitochondrial DNA levels, a higher frequency of mitochondrial DNA lesions, increased oxygen consumption rates, and increased fragmentation of the mitochondrial network. Importantly, overexpression of wild-type ATP13A2 rescued the respiration phenotype. These findings collectively suggest that ATP13A2 contributes to the maintenance of a healthy mitochondrial pool, supporting the hypothesis that impaired mitochondrial clearance represents an important pathogenic mechanism underlying KRS.     

Pathogenic effects of novel mutations in the P-type ATPase ATP13A2 (PARK9) causing Kufor-Rakeb syndrome, a form of early-onset parkinsonism

Kufor-Rakeb syndrome (KRS) is a rare form of autosomal recessive juvenile or early-onset, levodopa responsive parkinsonism and has been associated with mutations in ATP13A2(also known as PARK9), a lysosomal type 5 P-type ATPase. Recently, we identified novel compound heterozygous mutations, c.3176T>G (p.L1059R) and c.3253delC (p.L1085WfsX1088) in ATP13A2 of two siblings affected with KRS. When overexpressed, wild-type ATP13A2 localized to Lysotracker-positive and LAMP2-positive lysosomes while both truncating and missense mutated ATP13A2 were retained in the endoplasmic reticulum (ER). Both mutant proteins were degraded by the proteasomal but not the lysosomal pathways. In addition, ATP13A2 mRNA with c.3253delC was degraded by nonsense-mediated mRNA decay (NMD), which was protected by cycloheximide treatment. To validate our findings in a biologically relevant setting, we used patient-derived human olfactory neurosphere cultures and fibroblasts and demonstrated persistent ER stress by detecting upregulation of unfolded protein response-related genes in the patient-derived cells. We also confirmed NMD degraded ATP13A2 c.3253delC mRNA in the cells. These findings indicate that these novel ATP13A2 mutations are indeed pathogenic and support the notion that mislocalization of the mutant ATP13A2, resultant ER stress, alterations in the proteasomal pathways and premature degradation of mutant ATP13A2 mRNA contribute to the aetiology of KRS.     

Mutant Atp13a2 proteins involved in parkinsonism are degraded by ER-associated degradation and sensitize cells to ER-stress induced cell death

Mutations in ATP13A2 (PARK9) have been linked to juvenile parkinsonism with dementia or Kufor-Rakeb syndrome (KRS). The ATP13A2 gene encodes at least three protein isoforms that arise by alternate splicing. A previous study indicated the Atp13a2(Isoform-1) protein is localized to lysosomes, whereas three separate mutations involved in disease cause retention of the protein in the ER. One speculation is that the mutant Atp13a2(Isoform-1) proteins are misfolded and eliminated by the ER-associated degradation pathway (ERAD), which involves the dislocation of proteins from the ER to the cytoplasm for proteasome degradation. We examined whether Atp13a2 proteins are degraded by ERAD and whether the Atp13a2(Isoform-3) protein has similar localization to the Atp13a2(Isoform-1) protein. Through analysis of protein turnover and by disrupting different steps in the ERAD pathway we demonstrate that mutant Atp13a2(Isoform-1) proteins are indeed eliminated by ERAD. Thus, siRNA-mediated knockdown of erasin, a platform for assembly of an ERAD complex, or expression of a dominant negative form of p97/VCP, a protein essential for dislocation of ERAD substrates, or inhibition of the proteasome all slowed degradation of the mutant Atp13a2(Isoform-1) proteins, but not the wild-type Atp13a2(Isoform-1) protein. Immunoprecipitation assays confirmed that the Atp13a2 proteins are ubiquitinated in accord with degradation by ERAD. In contrast to Atp13a2(Isoform-1), we show Atp13a2(Isoform-3) is localized to the ER and rapidly degraded. Lastly, we show Atp13a2 mutants have increased cytotoxicity and predispose cells to ER-stress-induced cell death. These results provide new insight into the properties of wild-type and mutant Atp13a2 proteins involved in KRS.     

The genetic spectrum of human neuronal ceroid-lipofuscinoses

The neuronal ceroid lipofuscinoses (NCL), also known as Batten disease, are a group of inherited severe neurodegenerative disorders primarily affecting children. They are characterised by the accumulation of autofluorescent storage material in many cells. Children suffer from visual failure, seizures, progressive physical and mental decline and premature death, associated with the loss of cortical neurones. Six genes have been identified that cause human NCL (CLN1, CLN2, CLN3, CLN5, CLN6, CLN8), and approximately 150 mutations have been described. The majority of mutations result in a characteristic disease course for each gene. However, mutations associated with later disease onset or a more protracted disease course have also been described. At least seven common mutations exist, either with a world-wide distribution or associated with families from specific countries. All mutations are described in the NCL Mutation Database (http://www.uc.ac.uk/ncl).     

ATP13A2 variability in Parkinson disease

Recessively inherited mutations in ATP13A2 result in Kufor‐Rakeb syndrome (KRS), whereas genetic variability and elevated ATP13A2 expression have been implicated in Parkinson disease (PD). Given this background, ATP13A2 was comprehensively assessed to support or refute its contribution to PD. Sequencing of ATP13A2 exons and intron‐exon boundaries was performed in 89 probands with familial parkinsonism from Tunisia. The segregation of mutations with parkinsonism was subsequently assessed within pedigrees. The frequency of genetic variants and evidence for association was also examined in 240 patients with nonfamilial PD and 372 healthy controls. ATP13A2 mRNA expression was also quantified in brain tissues from 38 patients with nonfamilial PD and 38 healthy subjects from the United States. Sequencing analysis revealed 37 new variants; seven missense, six silent, and 24 that were noncoding. However, no single ATP13A2 mutation segregated with familial parkinsonism in either a dominant or recessive manner. Four markers showed marginal association with nonfamilial PD, prior to correction for multiple testing. ATP13A2 mRNA expression was marginally decreased in PD brains compared with tissue from control subjects. In conclusion, neither ATP13A2 genetic variability nor quantitative gene expression in brain appears to contribute to familial parkinsonism or nonfamilial PD. Hum Mutat 0, 1–5, 2008. © 2008 Wiley‐Liss, Inc.     

Anomalies of mitochondrial ATP synthase regulation in four different types of neuronal ceroid lipofuscinosis

Several neuronal ceroid lipofuscinoses (NCL) show storage of subunit c of mitochondrial ATP synthase. The neurodegenerative process, however, remains obscure. We previously reported a decreased basal ATP synthase activity in fibroblasts from late-infantile NCL (CLN2) and juvenile NCL (CLN3) patients. We have now extended the study of the ATP synthase system to an ovine NCL (a model for the late-infantile NCL variant, CLN6) and the infantile NCL (CLN1). In fibroblasts from healthy sheep, active regulation of ATP synthase in response to cellular energy demand was present similar to human cells: ATP synthase was down-regulated under conditions of anoxia or functional uncoupling and was up-regulated in response to calcium. In fibroblasts from NCL sheep, basal ATP synthase activity was slightly elevated and down-regulation in response to anoxia or uncoupling of mitochondria also occurred. Calcium produced an unexpected down-regulation to 55% of basal activity. Activities of respiratory chain enzymes did not differ between healthy and NCL sheep. In fibroblasts from CLN1 patients, basal ATP synthase activity was reduced and regulation of the enzyme was absent. Activities of respiratory chain complexes II and IV were reduced. The defect of ATP synthase regulation found in fibroblasts from NCL sheep and infantile NCL patients is different from the ATP synthase deficiencies demonstrated in late-infantile and juvenile NCL, but problems of mitochondrial energy production, if also expressed in brain, would be a common feature of several NCL forms. Deficient ATP supply could result in degeneration of neurons, especially in those with high energy requirements.     

Analysis of ATP13A2 in large neurodegeneration with brain iron accumulation (NBIA) and dystonia-parkinsonism cohorts

Several causative genes have been identified for both dystonia-parkinsonism and neurodegeneration with brain iron accumulation (NBIA), yet many patients do not have mutations in any of the known genes. Mutations in the ATP13A2 lead to Kufor Rakeb disease, a form of autosomal recessive juvenile parkinsonism that also features oromandibular dystonia. More recently, evidence of iron deposition in the caudate and putamen have been reported in patients with ATP13A2 mutations. We set out to determine the frequency of ATP13A2 mutations in cohorts of idiopathic NBIA and dystonia-parkinsonism. We screened for large deletions using whole genome arrays, and sequenced the entire coding region in 92 cases of NBIA and 76 cases of dystonia-parkinsonism. A number of coding and non-coding sequence variants were identified in a heterozygous state, but none were predicted to be pathogenic based on in silico analyses. Our results indicate that ATP13A2 mutations are a rare cause of both NBIA and dystonia-parkinsonism.     

Identification of novel ATP13A2 interactors and their role in α-synuclein misfolding and toxicity

Lysosomes are responsible for degradation and recycling of bulky cell material, including accumulated misfolded proteins and dysfunctional organelles. Increasing evidence implicates lysosomal dysfunction in several neurodegenerative disorders, including Parkinson's disease and related synucleinopathies, which are characterized by the accumulation of α-synuclein (α-syn) in Lewy bodies. Studies of lysosomal proteins linked to neurodegenerative disorders present an opportunity to uncover specific molecular mechanisms and pathways that contribute to neurodegeneration. Loss-of-function mutations in a lysosomal protein, ATP13A2 (PARK9), cause Kufor-Rakeb syndrome that is characterized by early-onset parkinsonism, pyramidal degeneration and dementia. While loss of ATP13A2 function plays a role in α-syn misfolding and toxicity, the normal function of ATP13A2 in the brain remains largely unknown. Here, we performed a screen to identify ATP13A2 interacting partners, as a first step toward elucidating its function. Utilizing a split-ubiquitin membrane yeast two-hybrid system that was developed to identify interacting partners of full-length integral membrane proteins, we identified 43 novel interactors that primarily implicate ATP13A2 in cellular processes such as endoplasmic reticulum (ER) translocation, ER-to-Golgi trafficking and vesicular transport and fusion. We showed that a subset of these interactors modified α-syn aggregation and α-syn-mediated degeneration of dopaminergic neurons in Caenorhabditis elegans, further suggesting that ATP13A2 and α-syn are functionally linked in neurodegeneration. These results implicate ATP13A2 in vesicular trafficking and provide a platform for further studies of ATP13A2 in neurodegeneration.     

Lack of association between ATP13A2 Ala746Thr variant and Parkinson's disease in Han population of mainland China

ATP13A2 (PARK9) mutations are related to Kufor-Rakeb syndrome (KRS). We performed genetic analysis of the Ala746Thr variant in an independent cohort of the patients with PD and healthy controls from mainland China. The Ala746Thr variant was present in 1/532 (0.19%) of PD compared with 1/480 (0.21%) of healthy controls (odds ratio = 0.90, 95% CI 0.06, 14.39, P = 1.00). The two subjects carried the heterozygous genotype. Subset analysis in the group ≤50 years of age revealed a prevalence of 0.7% in PD compared with 0% in healthy controls and in the group >50 years of age showed 0% in PD versus 0.3% in healthy controls. We did not observe a significant association between Ala746Thr and Parkinson's disease in Han Chinese population, even after stratification by age at onset. The results suggested that Ala746Thr variant was not a major susceptible factor for PD in Han Chinese people.     

Clinico-pathological manifestations of variant late infantile neuronal ceroid lipofuscinosis (vLINCL) caused by a novel mutation in MFSD8 gene

Neuronal ceroid lipofuscinosis (NCL) refers to a growing heterogeneous group of neurodegenerative disorders characterized by lysosomal accumulation of abnormal autofluorescent material. NCLs are traditionally classified clinically according to their age of onset. Variable late infantile NCL (vLINCL) is the most genetically heterogeneous subtype as it has been shown to be caused by mutations in at least six genes. We report on 5 patients of a consanguineous family who presented in early childhood with intractable seizures, severe cognitive and motor decline, behavioral impairment and progressive retinal degeneration. Disease course was severe; all patients were in a vegetative state by the second decade of life, and eventually die prematurely (except in one case). Ultrastructural studies of brain and rectal mucosa disclosed accumulation of storage material in various patterns including fingerprint, curvilinear, and granular osmiophilic deposits consistent with the diagnosis of NCL. Brain pathologic features from a living patient are first reported here and shed light on disease progression and pathogenesis. Using a combination of whole genome autozygosity mapping and candidate gene direct sequencing, we identified a mutation in MFSD8, c.472G>A (p.Gly158Ser), which was found to segregate with the disease phenotype in the family.This study underscores the importance of a combined clinic-molecular workup in NCLs and other neurodegenerative conditions.     

Clinical and molecular analysis of Japanese patients with neuronal ceroid lipofuscinosis

Neuronal ceroid lipofuscinosis (NCL) is one of the most common inherited neurological diseases in childhood. It occurs every 12,500 births in northern-European populations. Mental retardation, visual impairment, and seizures are common symptoms. The prevalence of NCL is variable depending upon the races or countries. Although a wealth information is available in Caucasian populations, there is little information about NCL in Asian people. Because a nationwide survey in Japanese patients with NCL has never been performed, we pursued an epidemiological survey. We identified 36 NCL patients in Japan. Patients with infantile, late infantile, juvenile, and adult type accounted for 2, 15, 15, and 4 cases, respectively. Seizures were a major initial symptom in the late infantile type. In the juvenile type, visual failure was present in 73% at onset. Recently, the juvenile NCL (Batten disease) gene has been isolated. Studies of the mutations in this gene demonstrated that a 1.02-kb deletion was the most prevalent mutation among Caucasian patients, accounting for 81% of total alleles. To determine the prevalence of this 1.02-kb deletion in Japanese patients, we performed a rapid allele-specific polymerase chain reaction test. No 1.02-kb major deletion was detected in 5 Japanese juvenile NCL cases. These data suggest that the distribution of NCL and clinical findings are similar to those of Caucasian subjects; however, prevalence of mutations in Japanese patients with NCL would be distinct from that observed in Caucasians.     

ATP13A2 variability in Taiwanese Parkinson's disease

Mutations in ATP13A2 have been reported to associate with Parkinson's disease (PD). This study investigates the contribution of genetic variants in ATP13A2 to Taiwanese PD. ATP13A2 cDNA fragments from 65 early onset PD (onset <50 years) were sequenced. The identified variants were validated in a cohort of PD (n = 493) and ethnically matched controls (n = 585). A novel heterozygous G1014S, located at the conserved seventh transmembrane domain of ATP13A2 protein, was identified in an early onset PD patient, which was absent in 585 normal controls. Additionally, a reported heterozygous A746T was found in two PD patients and four controls. The clinical features and 99mTc‐TRODAT‐1 single photon emission computed tomography (SPECT) image of the patients carrying G1014S and A746T were similar to that of idiopathic PD. One normal control with A746T showed an asymmetric reduction of 99mT TRODAT‐1 uptake in the right striatum. Under oxidative stress or apoptotic stimulus, lymphoblastoid cells carrying either A764T or G1014S showed increased caspase 3 activity compared with the controls. The rates of decay for G1014S and A746T proteins were more or less reduced in cycloheximide chase experiment. In silico modeling of G1014S exhibited a more stable feature than wild‐type, and G1014S is mislocalized mainly in the intralysosomal space, which is coherent with the prediction of prohibiting N‐myristoylation and membrane association. We therefore hypothesize that rare variants of ATP13A2 may contribute to PD susceptibility in Taiwan. The role played by ATP13A2 variants in PD remains to be clarified. © 2011 Wiley‐Liss, Inc.     

Neuronal ceroid lipofuscinosis. Closing chapter of a long story

Neuronal ceroid lipofuscinoses represent a group of diseases which has until quite recently resisted definite elucidation of the underlying defect(s) on the molecular level. The common feature of all the NCLs is a serious and progressive neurological disorder, accompanied, with only few exceptions, by retinal degeneration. Visceral symptoms, despite the presence of the storage process, are absent, or minimal. There are many clinical variants of the disease process, among which a set of standard, historical phenotypes exists found to be linked to specific genotypes. The disorder is inherited and transmitted as an autosomal recessive trait. At the cellular level, it is featured by lyzosomal storage of autofluorescent hydrophobic material, the substantial part of which consists of hydrophobic proteins and esterified dolichol. The dominant protein is the subunit c of mitochondria ATP synthase. In one NCL type (NCL1) the dominant proteins are saposins A and D. Ultrastructural appearance is membranous with several relatively specific patterns with some tendency to condensation or, namely in NCL3 to vacuolar distension. Amorphous appearance is associated with NCL1. The impact of the disease process on the cell biology differs substantially depending on the cell type. The brain neurons are most seriously affected and degenerate, whereas other cell types mostly survive without detectable deterioration. Pathogenesis at the molecular level is now being elucidated using the modern molecular biology techniques, which have already enabled unravelling of a set of genes controlling majority of the standard historical phenotypes. The original infantile form of NCL (NCL1) is now defined as palmitoyl protein thioesterase deficiency (gen at the 1p32 locus), the late infantile form (NCL2) as pepstatin resistant proteinase deficiency (gen at the 11p15.5 locus) and the original juvenile form (NCL3) as a defect of the specific gene (locus 16p11.2-12.3), the product of which, the NCL3 protein, still lacks functional characterization. Two gene loci have been identified in the so-called early juvenile, or variant late infantile NCL. One of them is in the 13q21 locus (NCL5 or Finnish variant late infantile form), the second is in the 15q21-23 one (NCL6). Kufs form remains the least defined form of NCL. Recently two novel NCL variants were described with specific loci. Thanks to introduction of molecular genetic based diagnosis it was possible to recognize, besides the standard phenotype, existence of further phenotypic variants. The phenotype based scheme of NCL has thus been definitely substituted by classification based on genotype and biochemistry.     

Retention of lysosomal protein CLN5 in the endoplasmic reticulum causes neuronal ceroid lipofuscinosis in Asian Sibship

The neuronal ceroid lipofuscinoses (NCLs) form a group of autosomal recessively inherited neurodegenerative disorders that mainly affect children. Ten NCL forms can be distinguished by age at onset, clinicopathologic features, and genetics. In eight of these forms, the underlying genes have been identified. At present, approximately 10% of all patients do not fall into one of the eight known genetic forms of NCL. We have identified two Asian families with two novel homozygous mutations in the CLN5 gene. In the first Pakistani family, two children developed symptoms of an early juvenile NCL. After exclusion of mutations in genes known to be associated with this age of onset in families from many different countries (CLN1, CLN2, CLN3, CLN6, CLN8 and CLN10) SNP array‐based homozygosity mapping led to the identification of a novel homozygous mutation c.1072_1073delTT (p.Leu358AlafsX4) in CLN5. In the second Afghan family, two children developed symptoms of a late infantile NCL. The mutation c.1137G>T (p.Trp379Cys) in CLN5 was identified. The affected children in these families represent the first reported CLN5 patients originating in Asian sibships. Expression analysis showed that mutant p.Leu358AlafsX4 CLN5 is truncated and lacks a used N‐glycosylation site at Asn401. The missense mutation p.Trp379Cys affected neither the size nor glycosylation of the CLN5 protein. Double immunofluorescence microscopy showed that while the wild‐type CLN5 protein is localized in lysosomes, both mutant CLN5 proteins are retained in the endoplasmic reticulum rather than reaching the lysosome. © 2009 Wiley‐Liss, Inc.     

The clinical and genetic epidemiology of neuronal ceroid lipofuscinosis in Newfoundland

The neuronal ceroid lipofuscinoses (NCLs) are the commonest neurodegenerative disorders of children. The aims of this study were to determine the incidence of NCL in Newfoundland, identify the causative genes, and analyze the relationship between phenotype and genotype. Patients with NCL diagnosed between 1960 and 2005 were ascertained through the provincial genetics and pediatric neurology clinics. Fifty-two patients from 34 families were identified. DNA was obtained from 28/34 (82%) families; 18 families had mutations in the CLN2 gene, comprising five different mutations of which two were novel. One family had a CLN3 mutation, another had a novel mutation in CLN5 , and five families shared the same mutation in CLN6 . One family was misdiagnosed, and in two, molecular testing was inconclusive. Disease from CLN2 mutations had an earlier presentation (p = 0.003) and seizure onset (p < 0.001) compared with CLN6 mutation. There was a slower clinical course for those with CLN5 mutation compared with CLN2 mutation. NCL in Newfoundland has a high incidence, 1 in 7353 live births, and shows extensive genetic heterogeneity. The incidence of late infantile NCL, 9.0 per 100,000 (or 1 in 11,161) live births, is the highest reported in the world.     

The intracellular location and function of proteins of neuronal ceroid lipofuscinoses

Neuronal ceroid lipofuscinoses are a group of diseases characterized by accumulation of hydrophobic proteins in lysosomes of neurons and other types of cells. NCLs are caused by at least 8 mutant genes (CLN1-CLN8), though CLN4 and CLN7 have not yet been identified. Except for Cln1p, the protein encoded by CLN1, the defective proteins are associated with lysosomal accumulation of mitochondrial ATP synthase subunit c. Cln1p and Cln2p are soluble lysosomal enzymes, targeted to lysosomes in a mannose 6-phosphate dependent manner. Mutations in the lysosomal protease cathepsin D cause another NCL. Cln3p, Cln5p, Cln6p and Cln8p are thought to be transmembrane proteins. Cln3p and Cln5p are localized in the endosome-lysosomal compartment. Deficiency of endosomal membrane protein CLC-3, a member of the chloride channel family, causes NCL-like phenotype and lysosomal storage of subunit c. Herein, we review the features of NCL and NCL-related proteins and discuss the involvement of the proteins in lysosomal degradation of subunit c.     

The neuropsychology of Kuf's Disease: a case of atypical early onset dementia.

Adult Neuronal Ceroid Lipofuscinosis (Adult NCL), also known as Kuf's Disease, is a rare progressive encephalopathic disease that results in dementia. This report describes the presentation, diagnosis, neuroanatomy, and management of Adult NCL. A neuropsychological case study of Adult NCL is presented, emphasizing early onset of psychiatric symptoms, motor involvement, and severe diffuse cognitive impairment. The patient's neuropsychological course is documented over a 2-year period, with findings interpreted within the context of current models of cortical and subcortical dementia. The biopsychosocial impact of misdiagnosis and pharmaceutical management are discussed.