Ceramide accumulation induces mitophagy and impairs ß-oxidation in PINK1 deficiency.

Energy production via the mitochondrial electron transport chain (ETC) and mitophagy are two important processes affected in Parkinson's disease (PD). Interestingly, PINK1, mutations of which cause early-onset PD, plays a key role in both processes, suggesting that these two mechanisms are connected. However, the converging link of both pathways currently remains enigmatic. Recent findings demonstrated that lipid aggregation, along with defective mitochondria, is present in postmortem brains of PD patients. In addition, an increasing body of evidence shows that sphingolipids, including ceramide, are altered in PD, supporting the importance of lipids in the pathophysiology of PD. Here, we identified ceramide to play a crucial role in PINK1-related PD that was previously linked almost exclusively to mitochondrial dysfunction. We found ceramide to accumulate in mitochondria and to negatively affect mitochondrial function, most notably the ETC. Lowering ceramide levels improved mitochondrial phenotypes in pink1-mutant flies and PINK1-deficient patient-derived fibroblasts, showing that the effects of ceramide are evolutionarily conserved. In addition, ceramide accumulation provoked ceramide-induced mitophagy upon PINK1 deficiency. As a result of the ceramide accumulation, ß-oxidation in PINK1 mutants was decreased, which was rescued by lowering ceramide levels. Furthermore, stimulation of ß-oxidation was sufficient to rescue PINK1-deficient phenotypes. In conclusion, we discovered a cellular mechanism resulting from PD-causing loss of PINK1 and found a protective role of ß-oxidation in ETC dysfunction, thus linking lipids and mitochondria in the pathophysiology of PINK1-related PD. Furthermore, our data nominate ß-oxidation and ceramide as therapeutic targets for PD.

THAP1, the gene mutated in DYT6 dystonia, autoregulates its own expression.

THAP1 encodes a transcription factor but its regulation is largely elusive. TOR1A was shown to be repressed by THAP1 in vitro. Notably, mutations in both of these genes lead to dystonia (DYT6 or DYT1). Surprisingly, expressional changes of TOR1A in THAP1 mutation carriers have not been detected indicating additional levels of regulation. Here, we investigated whether THAP1 is able to autoregulate its own expression. Using in-silico prediction, luciferase reporter gene assays, and (quantitative) chromatin immunoprecipitation (ChIP), we defined the THAP1 minimal promoter to a 480bp-fragment and demonstrated specific binding of THAP1 to this region which resulted in repression of the THAP1 promoter. This autoregulation was disturbed by different DYT6-causing mutations. Two mutants (Ser6Phe, Arg13His) were shown to be less stable than wildtype THAP1 adding to the effect of reduced binding to the THAP1 promoter. Overexpressed THAP1 is preferably degraded through the proteasome. Notably, endogenous THAP1 expression was significantly reduced in cells overexpressing wildtype THAP1 as demonstrated by quantitative PCR. In contrast, higher THAP1 levels were detected in induced pluripotent stem cell (iPS)-derived neurons from THAP1 mutation carriers. Thus, we identified a feedback-loop in the regulation of THAP1 expression and demonstrated that mutant THAP1 leads to higher THAP1 expression levels. This compensatory autoregulation may contribute to the mean age at onset in the late teen years or even reduced penetrance in some THAP1 mutation carriers.

Whispering dysphonia (DYT4 dystonia) is caused by a mutation in the TUBB4 gene.

OBJECTIVE: A study was undertaken to identify the gene underlying DYT4 dystonia, a dominantly inherited form of spasmodic dysphonia combined with other focal or generalized dystonia and a characteristic facies and body habitus, in an Australian family. METHODS: Genome-wide linkage analysis was carried out in 14 family members followed by genome sequencing in 2 individuals. The index patient underwent a detailed neurological follow-up examination, including electrophysiological studies and magnetic resonance imaging scanning. Biopsies of the skin and olfactory mucosa were obtained, and expression levels of TUBB4 mRNA were determined by quantitative real-time polymerase chain reaction in 3 different cell types. All exons of TUBB4 were screened for mutations in 394 unrelated dystonia patients. RESULTS: The disease-causing gene was mapped to a 23cM region on chromosome 19p13.3-p13.2 with a maximum multipoint LOD score of 5.338 at markers D9S427 and D9S1034. Genome sequencing revealed a missense variant in the TUBB4 (tubulin beta-4; Arg2Gly) gene as the likely cause of disease. Sequencing of TUBB4 in 394 unrelated dystonia patients revealed another missense variant (Ala271Thr) in a familial case of segmental dystonia with spasmodic dysphonia. mRNA expression studies demonstrated significantly reduced levels of mutant TUBB4 mRNA in different cell types from a heterozygous Arg2Gly mutation carrier compared to controls. INTERPRETATION: A mutation in TUBB4 causes DYT4 dystonia in this Australian family with so-called whispering dysphonia, and other mutations in TUBB4 may contribute to spasmodic dysphonia. Given that TUBB4 is a neuronally expressed tubulin, our results imply abnormal microtubule function as a novel mechanism in the pathophysiology of dystonia.

Exome sequencing in a family with restless legs syndrome.

BACKGROUND: Restless legs syndrome (RLS) has a high familial aggregation. To date, several loci and genetic risk factors have been identified, but no causative gene mutation has been found. METHODS: We evaluated a German family with autosomal dominantly inherited RLS in 7 definitely and 2 possibly affected members by genome-wide linkage analysis and exome sequencing. RESULTS: We identified three novel missense and one splice site variant in the PCDHA3, WWC2, ATRN, and FAT2 genes that segregated with RLS in the family. All four exons of the PCDHA3 gene, the most plausible candidate, were sequenced in 64 unrelated RLS cases and 250 controls. This revealed three additional rare missense variants (frequency <1%) of unknown pathogenicity in 2 patients and 1 control. CONCLUSIONS: We present the first next-generation sequencing study on RLS and suggest PCDHA3 as a candidate gene for RLS.

The dystonia gene DYT1 is repressed by the transcription factor THAP1 (DYT6).

Mutations in THAP1 have been associated with dystonia 6. THAP1 encodes a transcription factor with mostly unknown targets. We tested the hypothesis that THAP1 regulates the expression of DYT1 (TOR1A), another dystonia-causing gene. After characterization of the TOR1A promoter, we demonstrate that THAP1 binds to the core promoter of TOR1A. Further, we report that wild type THAP1 represses the expression of TOR1A, whereas dystonia 6-associated mutant THAP1 results in decreased repression of TOR1A. Our data demonstrate that THAP1 regulates the transcription of TOR1A, suggesting transcriptional dysregulation as a cause of dystonia.

Clinical neuroimaging and electrophysiological assessment of three DYT6 dystonia families.

The purpose of the study was to delineate clinical and electrophysiological characteristics as well as laryngoscopical and transcranial ultrasound (TCS) findings in THAP1 mutation carriers (MutC). According to recent genetic studies, DYT6 (THAP1) gene mutations are an important cause of primary early-onset dystonia. In contrast to DYT1 mutations, THAP1 mutations are associated with primary early-onset segmental or generalised dystonia frequently involving the craniocervical region and the larynx. Blood samples from twelve individuals of three German families with DYT6 positive index cases were obtained to test for THAP1 mutations. Eight THAP1 MutC were identified. Of these, six (three symptomatic and three asymptomatic) THAP1 MutC could be clinically evaluated. Laryngoscopy was performed to evaluate laryngeal dysfunction in patients. Brainstem echogenicity was investigated in all MutC using TCS. Two of the patients had undergone bilateral pallidal DBS. In all three symptomatic MutC, early-onset laryngeal dystonia was a prominent feature. Laryngeal assessment demonstrated adductor-type dystonia in all of them. On clinical examination, the three asymptomatic MutC also showed subtle signs of focal or segmental dystonia. TCS revealed increased substantia nigra (SN) hyperechogenicity in all MutC. Intraoperative microelectrode recordings under general anesthesia in two of the patients showed no difference between THAP1 and previously operated DYT1 MutC. The presence of spasmodic dysphonia in patients with young-onset segmental or generalised dystonia is a hallmark of DYT6 dystonia. SN hyperechogenicity on TCS may represent an endophenotype in these patients. Pallidal DBS in two patients was unsatisfactory.

Childhood-onset restless legs syndrome: clinical and genetic features of 22 families.

Restless legs syndrome (RLS) is a sensory-motor disorder that is underdiagnosed in children and often misclassified as attention deficit hyperactivity disorder. Five different gene loci (RLS1-5) and three susceptibility loci have been identified in adult-onset RLS. We included 23 children with RLS (age at onset < or =14 years) from 22 families. In 14 families, we performed linkage and genotype analyses. Of the 23 RLS patients, only seven (30.4%) were admitted for a suspected diagnosis of RLS. Five patients had a retrospectively established onset at an age as early as 1 year. The most frequent complaint in patients were sleep problems (21 of 23; 91%) resulting in fatigue in 14 children (60.9%). Twelve of the 19 tested cases (63.2%) exhibited an index of periodic limb movements in sleep greater than 5. Dopaminergic therapy was successful in 12 of 14 treated patients (85.7%). Family history for RLS was positive in 20 of 23 children (87.0%) and compatible with an autosomal dominant inheritance pattern. Linkage analysis excluded all five loci in two families. A trend for an association at two of the three reported susceptibility regions was observed. RLS symptoms can occur in early childhood. The positive family history suggests a genetic cause in most families with at least one additional RLS gene locus.

Cardiolipin promotes electron transport between ubiquinone and complex I to rescue PINK1 deficiency.

PINK1 is mutated in Parkinson's disease (PD), and mutations cause mitochondrial defects that include inefficient electron transport between complex I and ubiquinone. Neurodegeneration is also connected to changes in lipid homeostasis, but how these are related to PINK1-induced mitochondrial dysfunction is unknown. Based on an unbiased genetic screen, we found that partial genetic and pharmacological inhibition of fatty acid synthase (FASN) suppresses toxicity induced by PINK1 deficiency in flies, mouse cells, patient-derived fibroblasts, and induced pluripotent stem cell-derived dopaminergic neurons. Lower FASN activity in PINK1 mutants decreases palmitate levels and increases the levels of cardiolipin (CL), a mitochondrial inner membrane-specific lipid. Direct supplementation of CL to isolated mitochondria not only rescues the PINK1-induced complex I defects but also rescues the inefficient electron transfer between complex I and ubiquinone in specific mutants. Our data indicate that genetic or pharmacologic inhibition of FASN to increase CL levels bypasses the enzymatic defects at complex I in a PD model.

Clinical spectrum of homozygous and heterozygous PINK1 mutations in a large German family with Parkinson disease: role of a single hit?

BACKGROUND: Although homozygous mutations in the PTEN-induced putative kinase 1 (PINK1) gene have been unequivocally associated with early-onset Parkinson disease (PD), the role of single heterozygous PINK1 mutations is less clear. OBJECTIVE: To investigate the role of homozygous and heterozygous PINK1 mutations in a large German pedigree (family W). DESIGN: Mutation analysis of PINK1 and results of standardized neurological and motor examination by 3 independent movement disorder specialists, including blinded video rating. SETTINGS: University of Lübeck. PARTICIPANTS: Twenty family members. MAIN OUTCOME MEASURES: The PINK1 genotype and PD status of all family members. RESULTS: The index patient of family W carried a homozygous nonsense mutation (c.1366C>T; p.Q456X) and presented with a phenotype closely resembling idiopathic PD but with an onset at 39 years of age. The family included a total of 4 affected homozygous members (age, 60-71 years; age at onset, 39-61 years), 6 members with slight or mild signs of PD (affected) and a heterozygous mutation (age, 31-49 years), and 5 unaffected heterozygous mutation carriers (age, 34-44 years). Although none of the heterozygous affected family members was aware of their signs (asymptomatic), the clinical findings were unequivocal and predominantly or exclusively present on their dominant right-hand side, eg, unilaterally reduced or absent arm swing and unilateral rigidity. The heterozygous members were all considerably younger than the affected homozygous mutation carriers. CONCLUSIONS: Heterozygous PINK1 mutations may predispose to PD, as was previously suggested by the presence of dopamine hypometabolism in asymptomatic mutation carriers. Long-term follow-up of our large family W provides an excellent opportunity to further evaluate the role of single heterozygous PINK1 mutations later in life, which will have major implications on genetic counseling.

The role of mutations in COL6A3 in isolated dystonia.

Specific mutations in COL6A3 have recently been reported as the cause of isolated recessive dystonia, which is a rare movement disorder. In all patients, at least one mutation was located in Exons 41 and 42. In an attempt to replicate these findings, we assessed by direct sequencing the frequency of rare variants in Exons 41 and 42 of COL6A3 in 955 patients with isolated or combined dystonia or with another movement disorder with dystonic features. We identified nine heterozygous carriers of rare variants including five different missense mutations and an extremely rare synonymous variant. In these nine patients, we sequenced the remaining 41 coding exons of COL6A3 to test for a second mutation in the compound heterozygous state. In only one of them, a second rare variant was identified (Thr732Met + Pro3082Arg). Of note, this patient had been diagnosed with Parkinson´s disease (with dystonic posturing) due to homozygous PINK1 mutations. The COL6A3 mutations clearly did not segregate with the disease in the four affected siblings of this family. Further, there was no indication for a disease-modifying effect of the COL6A3 mutations since disease severity or age at onset did not correlate with the number of COL6A3 mutated alleles in this family. In conjunction with the relatively high frequency of homozygous carriers of reported mutations in publically available databases, our data call a causal role for variants in COL6A3 in isolated dystonia into question.

PINK1, Parkin, and DJ-1 mutations in Italian patients with early-onset parkinsonism.

Recessively inherited early-onset parkinsonism (EOP) has been associated with mutations in the Parkin, DJ-1, and PINK1 genes. We studied the prevalence of mutations in all three genes in 65 Italian patients (mean age of onset: 43.2+/-5.4 years, 62 sporadic, three familial), selected by age at onset equal or younger than 51 years. Clinical features were compatible with idiopathic Parkinson's disease in all cases. To detect small sequence alterations in Parkin, DJ-1, and PINK1, we performed a conventional mutational analysis (SSCP/dHPLC/sequencing) of all coding exons of these genes. To test for the presence of exon rearrangements in PINK1, we established a new quantitative duplex PCR assay. Gene dosage alterations in Parkin and DJ-1 were excluded using previously reported protocols. Five patients (8%; one woman/four men; mean age at onset: 38.2+/-9.7 (range 25-49) years) carried mutations in one of the genes studied: three cases had novel PINK1 mutations, one of which occurred twice (homozygous c.1602_1603insCAA; heterozygous c.1602_1603insCAA; heterozygous c.836G>A), and two patients had known Parkin mutations (heterozygous c.734A>T and c.924C>T; heterozygous c.924C>T). Family history was negative for all mutation carriers, but one with a history of tremor. Additionally, we detected one novel polymorphism (c.344A>T) and four novel PINK1 changes of unknown pathogenic significance (-21G/A; IVS1+97A/G; IVS3+38_40delTTT; c.852C>T), but no exon rearrangements. No mutations were found in the DJ-1 gene. The number of mutation carriers in both the Parkin and the PINK1 gene in our cohort is low but comparable, suggesting that PINK1 has to be considered in EOP.

Recessive dystonia-ataxia syndrome in a Turkish family caused by a COX20 (FAM36A) mutation.

DYTCA is a syndrome that is characterized by predominant dystonia and mild cerebellar ataxia. We examined two affected siblings with healthy, consanguineous, Turkish parents. Both patients presented with a combination of childhood-onset cerebellar ataxia, dystonia, and sensory axonal neuropathy. In the brother, dystonic features were most pronounced in the legs, while his sister developed torticollis. Routine diagnostic investigations excluded known genetic causes. Biochemical analyses revealed a mitochondrial respiratory chain complex IV and a coenzyme Q10 deficiency in a muscle biopsy. By exome sequencing, we identified a homozygous missense mutation (c.154A >C; p.Thr52Pro) in both patients in exon 2 of the COX20 (FAM36A) gene, which encodes a complex IV assembly factor. This variant was confirmed by Sanger sequencing, was heterozygous in both parents, and was absent from 427 healthy controls. The exact same mutation was recently reported in a patient with ataxia and muscle hypotonia. Among 128 early-onset dystonia and/or ataxia patients, we did not detect any other patient with a COX20 mutation. cDNA sequencing and semi-quantitative analysis were performed in fibroblasts from one of our homozygous mutation carriers and six controls. In addition to the exchange of an amino acid, the mutation led to a shift in splicing. In conclusion, we extend the phenotypic spectrum of a recently identified mutation in COX20 to a recessively inherited, early-onset dystonia-ataxia syndrome that is characterized by reduced complex IV activity. Further, we confirm a pathogenic role of this mutation in cerebellar ataxia, but this mutation seems to be a rather rare cause.

Mutations in GNAL: a novel cause of craniocervical dystonia.

IMPORTANCE: Mutations in the GNAL gene have recently been shown to cause primary torsion dystonia. The GNAL-encoded protein (Gαolf) is important for dopamine D1 receptor function and odorant signal transduction. We sequenced all 12 exons of GNAL in 461 patients from Germany, Serbia, and Japan, including 318 patients with dystonia (190 with cervical dystonia), 51 with hyposmia and Parkinson disease, and 92 with tardive dyskinesia or acute dystonic reactions. OBSERVATIONS: We identified the following two novel heterozygous putative mutations in GNAL: p.Gly213Ser in a German patient and p.Ala353Thr in a Japanese patient. These variants were predicted to be pathogenic in silico, were absent in ethnically matched control individuals, and impaired Gαolf coupling to D1 receptors in a bioluminescence energy transfer (BRET) assay. Two additional variants appeared to be benign because they behaved like wild-type samples in the BRET assay (p.Ala311Thr) or were detected in ethnically matched controls (p.Thr92Ala). Both patients with likely pathogenic mutations had craniocervical dystonia with onset in the fifth decade of life. No pathogenic mutations were detected in the patients with hyposmia and Parkinson disease, tardive dyskinesias, or acute dystonic reactions. CONCLUSIONS AND RELEVANCE: Mutations in GNAL can cause craniocervical dystonia in different ethnicities. The BRET assay may be a useful tool to support the pathogenicity of identified variants in the GNAL gene.

Identification and functional analysis of novel THAP1 mutations.

Mutations in THAP1 have been associated with dystonia 6 (DYT6). THAP1 encodes a transcription factor that represses the expression of DYT1. To further evaluate the mutational spectrum of THAP1 and its associated phenotype, we sequenced THAP1 in 567 patients with focal (n = 461), segmental (n = 68), or generalized dystonia (n = 38). We identified 10 novel variants, including six missense substitutions within the DNA-binding Thanatos-associated protein domain (Arg13His, Lys16Glu, His23Pro, Lys24Glu, Pro26Leu, Ile80Val), a 1bp-deletion downstream of the nuclear localization signal (Asp191Thrfs*9), and three alterations in the untranslated regions. The effect of the missense variants was assessed using prediction tools and luciferase reporter gene assays. This indicated the Ile80Val substitution as a benign variant. The subcellular localization of Asp191Thrfs*9 suggests a disturbed nuclear import for this mutation. Thus, we consider six of the 10 novel variants as pathogenic mutations accounting for a mutation frequency of 1.1%. Mutation carriers presented mainly with early onset dystonia (<12 years in five of six patients). Symptoms started in an arm or neck and spread to become generalized in three patients or segmental in two patients. Speech was affected in four mutation carriers. In conclusion, THAP1 mutations are rare in unselected dystonia patients and functional analysis is necessary to distinguish between benign variants and pathogenic mutations.

Frequency of heterozygous Parkin mutations in healthy subjects: need for careful prospective follow-up examination of mutation carriers.

The role of single heterozygous mutations in the putatively recessive Parkin gene in Parkinson disease (PD) is a vividly debated issue, partly caused by the largely unknown frequency of these mutations in healthy individuals. We investigated mutations in all 12 Parkin exons in 356 controls from two European populations including individuals from South Tyrol and Germany. None of the controls carried a homozygous or compound heterozygous mutation. Seventeen carriers of rare heterozygous alterations were detected, of which 13 (13/356; 3.7%) are considered to alter protein structure including four different gene dosage alterations, four missense mutations, and two frameshift mutations. Two of the mutations occurred recurrently in the South Tyrolean population. There was no obvious difference in the mutation frequency between the two populations. One of the presumably healthy mutation carrier was available for re-examination at the age of 67 years. He presented with mild signs of parkinsonism but not fulfilling diagnostic criteria for definite PD. To elucidate the role of heterozygosity is important for genetic testing and counseling of mutation carriers. A detailed clinical prospective and follow-up examination of mutation carriers is required for a better understanding of the role of heterozygous Parkin mutations.

Heterozygous PINK1 mutations: a susceptibility factor for Parkinson disease?

PINK1 mutations cause recessively inherited early-onset Parkinson's disease (EOPD). We comprehensively tested 75 Serbian and 17 South Tyrolean EOPD patients for mutations in this gene and found three heterozygous mutation carriers. Two of these patients shared mutations with their affected relatives, further suggesting that heterozygous PINK1 mutations may act as a susceptibility factor for EOPD.

Co-occurrence of restless legs syndrome and Parkin mutations in two families.

Recent studies have suggested an association between restless legs syndrome (RLS) and Parkinson's disease (PD). We present a large multigenerational family and a smaller family with RLS. A Parkin mutation was found in 10 of 20 patients from both families with idiopathic RLS but was not considered causative. The clinical phenotype did not differ between RLS patients with and without a Parkin mutation. Inheritance of RLS was consistent with autosomal dominant transmission, and linkage analysis excluded all three known loci for RLS.