Johann Hagenah

Phosphatase and Tensin Homolog (PTEN)-induced Putative Kinase 1 (PINK1)-dependent Ubiquitination of Endogenous Parkin Attenuates Mitophagy STUDY IN HUMAN PRIMARY FIBROBLASTS AND INDUCED PLURIPOTENT STEM CELL-DERIVED NEURONS

Background: The Parkinson disease-related proteins PINK1 and Parkin initiate mitophagy of damaged mitochondria. Results: Endogenous Parkin is not sufficient to induce mitophagy due to PINK1-dependent ubiquitination of Parkin. Conclusion: Mitophagy is detectable only with supraphysiological levels of Parkin and differs between fibroblasts and iPS-derived neurons. Significance: Stresses the importance of future studies in Parkinson disease-relevant tissue, i.e., dopaminergic neurons. Mutations in the E3 ubiquitin ligase Parkin and the mitochondrial PTEN-induced putative kinase 1 (PINK1) have been identified to cause autosomal recessive forms of familial Parkinson disease, with PINK1 functioning upstream of Parkin in a pathway important for the maintenance of mitochondrial function and morphology. Upon the loss of the mitochondrial membrane potential, Parkin translocates to mitochondria in a PINK1-dependent manner to ubiquitinate mitochondrial proteins. Parkin-mediated polyubiquitination of outer mitochondrial membrane (OMM) proteins recruits the ubiquitin- and LC3-binding adaptor protein p62 to mitochondria and induces mitophagy. Although previous studies examined mitophagy in established cell lines through overexpression approaches, there is an imperative to study the role of endogenous Parkin and PINK1 in human-derived and biologically relevant cell models. Here, we demonstrate in human primary fibroblasts and induced pluripotent stem-derived neurons from controls and PINK1 mutation carriers that endogenous levels of Parkin are not sufficient to initiate mitophagy upon loss of the mitochondrial membrane potential, caused by its (self-)ubiquitination, followed by degradation via the ubiquitin proteasome system. Next, we showed differential PINK1-dependent, Parkin-mediated ubiquitination of OMM proteins, which is Parkin dose-dependent and affects primarily OMM proteins of higher molecular mass. In contrast to the situation fibroblasts, we did not detect mitophagy in induced pluripotent stem-derived neurons even upon overexpression of Parkin. Taken together, our data demonstrate that mitophagy differs between human non-neuronal and neuronal cells and between “endogenous” and “Parkin-overexpressing” cellular models.

α-Synuclein and Parkinson disease susceptibility

Background: Mutations in the α-synuclein (SNCA) gene have been shown to be responsible for a rare familial form of Parkinson disease (PD). Furthermore, polymorphic variants in multiple regions of the gene have been associated with susceptibility to idiopathic PD in different populations. Objective: To evaluate and to confirm the role of SNCA variants in PD pathogenesis. Methods: We included 667 subjects (397 cases with idiopathic PD and 270 healthy, ethnically matched controls) of Northern Central and Southeastern European origin. We analyzed genotypes at 14 markers spanning the SNCA locus and its major haplotype blocks and conducted a haplotype analysis for four promoter markers including the microsatellite marker Rep1. Results: The three single nucleotide polymorphisms (SNPs) of the promoter region (rs2583988, rs2619364, rs2619363) and a SNP in the 3′UTR (rs356165) of the SNCA gene showed the greatest evidence for an association with PD (p ≤ 0.003), with significant pairwise values for linkage disequilibrium (D′ ≥ 0.74, r 2 ≥ 0.29). The promoter haplotype “261-T-G-T” (Rep1-rs2583988-rs2619364-rs2619363) was associated with disease (p = 0.032). The most significant association with PD was generated by excluding Rep1 (p = 0.008). This association remained significant when analyzing the Serbian patients separately and was of borderline significance for the German patients. Conclusions: Our findings confirm that genetic variability within the SNCA locus is associated with susceptibility to idiopathic Parkinson disease (PD). We found evidence for disease association with single nucleotide polymorphisms at both the 5′ and the 3′ end of the gene with pairwise linkage disequilibrium between them. The association was independent of the Rep1 status, and one major SNCA promoter haplotype class seems to be associated with PD susceptibility. GLOSSARY: SNCA = α-synuclein; CI = confidence interval; HT = haplotype-tagging; LD = linkage disequilibrium; OR = odds ratio; PD = Parkinson disease; SNP = single nucleotide polymorphism.

Altered Resting State Brain Networks in Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder affecting dopaminergic neurons in the substantia nigra leading to dysfunctional cortico-striato-thalamic-cortical loops. In addition to the characteristic motor symptoms, PD patients often show cognitive impairments, affective changes and other non-motor symptoms, suggesting system-wide effects on brain function. Here, we used functional magnetic resonance imaging and graph-theory based analysis methods to investigate altered whole-brain intrinsic functional connectivity in PD patients (n = 37) compared to healthy controls (n = 20). Global network properties indicated less efficient processing in PD. Analysis of brain network modules pointed to increased connectivity within the sensorimotor network, but decreased interaction of the visual network with other brain modules. We found lower connectivity mainly between the cuneus and the ventral caudate, medial orbitofrontal cortex and the temporal lobe. To identify regions of altered connectivity, we mapped the degree of intrinsic functional connectivity both on ROI- and on voxel-level across the brain. Compared to healthy controls, PD patients showed lower connectedness in the medial and middle orbitofrontal cortex. The degree of connectivity was also decreased in the occipital lobe (cuneus and calcarine), but increased in the superior parietal cortex, posterior cingulate gyrus, supramarginal gyrus and supplementary motor area. Our results on global network and module properties indicated that PD manifests as a disconnection syndrome. This was most apparent in the visual network module. The higher connectedness within the sensorimotor module in PD patients may be related to compensation mechanism in order to overcome the functional deficit of the striato-cortical motor loops or to loss of mutual inhibition between brain networks. Abnormal connectivity in the visual network may be related to adaptation and compensation processes as a consequence of altered motor function. Our analysis approach proved sensitive for detecting disease-related localized effects as well as changes in network functions on intermediate and global scale.

Different types of repeat expansion in the TATA-binding protein gene are associated with a new form of inherited ataxia.

A novel neurological syndrome has recently been described to be associated with an expanded polyglutamine domain. The expansion results from partial duplication within the TATA-binding protein (TBP). By investigation of 604 sporadic and familial cases with various forms of neurological syndromes and 157 unaffected individuals, we found repeat expansions in the TBP in four patients of two families with autosomal dominant inheritance of ataxia, dystonia, and intellectual decline. Two different genotypes for the repetitive sequence could be demonstrated which led to elongated polyglutamine stretches between 50 and 55 residues, whereas normal alleles with 27 to a maximum of 44 glutamine residues were found in this study. The expansion to 50 or more glutamine residues results in a pathological phenotype and confirms the report of a new polyglutamine disease.

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

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.

High mutation rate in dopa-responsive dystonia: detection with comprehensive GCHI screening.

Mutations in GTP cyclohydrolase I (GCHI) are found in 50 to 60% of cases with dopa-responsive dystonia (DRD). Heterozygous GCHI exon deletions, undetectable by sequencing, have recently been described in three DRD families. We tested 23 individuals with DRD for the different mutation types by conventional and quantitative PCR analyses and found mutations, including two large exon deletions, in 87%. The authors attribute this high mutation rate to rigorous inclusion criteria and comprehensive mutational analysis.

Etiology of musician’s dystonia: Familial or environmental?

Objective: To test the hypothesis that there is familial aggregation of dystonia and other movement disorders in relatives of patients with musician’s dystonia (MD) and to identify possible environmental triggers. Methods: The families of 28 index patients with MD (14 with a reported positive family history of focal task-specific dystonia [FTSD] and 14 with no known family history [FH−]) underwent a standardized telephone screening interview using a modified version of the Beth Israel Dystonia Screen. Videotaped neurologic examinations were performed on all participants who screened positive and consensus diagnoses established. All patients were investigated for DYT1 dystonia and suitable families were tested for linkage to DYT7. All family members were administered questionnaires covering potential triggers of FTSD. Results: A diagnosis of dystonia was established in all 28 index patients and in 19/97 examined relatives (MD: n = 8, other FTSD: n = 9, other dystonias: n = 2), 5 of whom were members of FH− families. In 27 of the 47 affected individuals, additional forms of dystonia were seen; other movement disorders were observed in 23 patients. In total, 18 families were multiplex families with two to four affected members. Autosomal dominant inheritance was compatible in at least 12 families. The GAG deletion in DYT1 was absent in all patients. Linkage to DYT7 could be excluded in 1 of the 11 informative families. With respect to potential environmental triggers, there was no significant difference between patients with MD/FTSD compared to unaffected family members. Conclusion: Our results suggest a genetic contribution to musician’s dystonia with phenotypic variability including focal task-specific dystonia. BIDS = Beth Israel Dystonia Screen; FH+ = reported positive family history of focal task-specific dystonia; FH− = no known family history of focal task-specific dystonia; FTSD = focal task-specific dystonia; MD = musician’s dystonia; WC = writer’s cramp.

Mutations in DYT1 Extension of the phenotypic and mutational spectrum

Background: Most cases of early-onset primary torsion dystonia (PTD) are caused by the same three–base pair (bp) (GAG) deletion in the DYT1 gene. Exon rearrangements are a common mutation type in other genes and have not yet been tested for in DYT1. Several lines of evidence suggest a relationship of the DYT1 gene with Parkinson disease (PD). Objective: To investigate the frequency and type of DYT1 mutations and explore the associated phenotypes in a mixed movement disorders patient cohort and in controls. Methods: The authors screened 197 patients with dystonia (generalized: n = 5; focal/segmental: n = 126; myoclonus-dystonia: n = 34; neuroleptic-induced: n = 32), 435 with PD, and 42 with various other movement disorders, along with 812 healthy controls, for small deletions in exon 5 of DYT1 and tested for exon rearrangements by quantitative, duplex PCR in 51 GAG deletion-negative dystonia cases. Results: The GAG deletion was detected in five patients: three with early-onset PTD, one with generalized jerky or clonic dystonia, and one with generalized dystonia and additional features (developmental delay, pyramidal syndrome). A novel out-of-frame four-bp deletion (934_937delAGAG) in exon 5 of the DYT1 gene was found in a putatively healthy blood donor. No exon rearrangements were identified in DYT1. Conclusions: In this mixed patient sample, the GAG deletion was rare and in two out of five cases associated with an unusual phenotype. In addition, a novel DYT1 truncating mutation of unknown clinical relevance was found in a putatively unaffected individual. DYT1 exon rearrangements, however, do not seem to be associated with PTD.

Reproducibility and diagnostic accuracy of substantia nigra sonography for the diagnosis of Parkinson's disease

Objective Transcranial sonography (TCS) shows characteristic hyperechogenicity of the substantia nigra (SN) in patients with Parkinsons disease (PD). Although this feature is well established, sufficient observer reliability and diagnostic accuracy are prerequisites for advancements of this method. Methods The authors investigated both aspects in a cross-sectional study with four blinded TCS raters in 22 PD patients and 10 healthy controls. Results As expected, the authors found significant bilateral SN hyperechogenicity in PD patients. Quantitative computerised SN planimetry had a substantial intra- (intraclass correlation coefficient (ICC) 0.97 and 0.93 respectively for both hemispheres) and inter-rater reliability (ICC 0.84 and 0.89), while visual semiquantitative echogenicity grading of the SN revealed a moderate intrarater (weighted kappa 0.80 ipsilateral and 0.74 contralateral) and slight (0.33) to fair (0.51) inter-rater reliability only. Diagnostic accuracy measured as the area under the curve of receiver-operating characteristics plots was highest in TCS of the SN opposite the clinically most affected body side (planimetry 0.821, echogenicity grading 0.792) with a hyperechogenic area of 0.24 cm2 as the optimum cut-off value for the differentiation between PD and controls (sensitivity 79%, specificity 81%). Conclusions The data demonstrate that the observer variability of SN planimetry is low in the hands of experienced investigators. This approach also offers adequate diagnostic accuracy. The authors conclude that reliable SN TCS data on PD can be achieved in clinical routine and multicentre trials when standardised analysis protocols and certain quality criteria of brain parenchyma sonography are met.

Substantia nigra hyperechogenicity correlates with clinical status and number of Parkin mutated alleles

To further evaluate (1) transcranial sonography (TCS) for (pre)clinical diagnosis of Parkinsons disease (PD) and (2) to examine asymptomatic carriers of Parkin mutations we investigated substantia nigra (SN) hyperechogenicity in PD patients and unaffected subjects with and without Parkin mutations. The area (aSN) of the hyperechogenic SN were calculated bilaterally and study subjects were assigned to high versus low value groups. Eleven of the (affected and unaffected) mutation carriers had previously undergone 18-fluoro-dopa-(FDOPA)-PET scans. Fifty-eight individuals were investigated, including 24 with clinically definite and 34 without symptoms or signs of PD. Of the patients, three had one mutated and six had two mutated Parkin alleles. Of the unaffected subjects, 13 carried a single Parkin mutated allele. After dichotomization, 21 subjects had high and 37 subjects low values of mean aSN. Regarding the clinical status, 13 (62%) of the individuals with a high mean aSN had PD,while 26 (70%) of the study subjects with low values did not show signs of PD (p = 0.0393). Similarly, probands with high mean aSN values more frequently carried Parkin mutations (58%) than probands with low values (27%, p = 0.0234). A negative correlation between FDOPA uptake in the posterior putamen and maximum aSN was found in the group of mutation carriers (r = -.809, p = 0.0234). In conclusion, hyperechogenicity of the SN is found in both idiopathic and Parkin-associated PD. Further strengthening the notion of a potential relationship between SN hyperechogenicity and Parkin mutational status, a larger aSN was associated with an increasing number of mutated alleles in our study.