Systematic analysis of PINK1 variants of unknown significance shows intact mitophagy function for most variants.

Pathogenic variants in PINK1 cause early-onset Parkinson's disease. Although many PINK1 variants have been reported, the clinical significance is uncertain for the majority of them. To gain insights into the consequences of PINK1 missense variants in a systematic manner, we selected 50 PINK1 missense variants from patient- and population-wide databases and systematically classified them using Sherloc, a comprehensive framework for variant interpretation based on ACMG-AMP guidelines. We then performed functional experiments, including mitophagy and Parkin recruitment assays, to assess the downstream consequences of PINK1 variants. Analysis of PINK1 missense variants based on Sherloc showed that the patient databases over-annotate variants as likely pathogenic. Furthermore, our study shows that pathogenic PINK1 variants are most often linked to a loss-of-function for mitophagy and Parkin recruitment, while this is not observed for variants of unknown significance. In addition to the Sherloc framework, the added layer of evidence of our functional tests suggests a reclassification of 9/50 missense variants. In conclusion, we suggest the assessment of multiple layers of evidence, including functional data on top of available clinical and population-based data, to support the clinical classification of a variant and show that the presence of a missense variant in PINK1 in a Parkinson's disease case does not automatically imply pathogenicity.

Parkinson's disease-associated VPS35 mutant reduces mitochondrial membrane potential and impairs PINK1/Parkin-mediated mitophagy.

BACKGROUND: Mitochondrial dysfunction plays a prominent role in the pathogenesis of Parkinson's disease (PD), and several genes linked to familial PD, including PINK1 (encoding PTEN-induced putative kinase 1 [PINK1]) and PARK2 (encoding the E3 ubiquitin ligase Parkin), are directly involved in processes such as mitophagy that maintain mitochondrial health. The dominant p.D620N variant of vacuolar protein sorting 35 ortholog (VPS35) gene is also associated with familial PD but has not been functionally connected to PINK1 and PARK2. METHODS: To better mimic and study the patient situation, we used CRISPR-Cas9 to generate heterozygous human SH-SY5Y cells carrying the PD-associated D620N variant of VPS35. These cells were treated with a protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce the PINK1/Parkin-mediated mitophagy, which was assessed using biochemical and microscopy approaches. RESULTS: Mitochondria in the VPS35-D620N cells exhibited reduced mitochondrial membrane potential and appeared to already be damaged at steady state. As a result, the mitochondria of these cells were desensitized to the CCCP-induced collapse in mitochondrial potential, as they displayed altered fragmentation and were unable to accumulate PINK1 at their surface upon this insult. Consequently, Parkin recruitment to the cell surface was inhibited and initiation of the PINK1/Parkin-dependent mitophagy was impaired. CONCLUSION: Our findings extend the pool of evidence that the p.D620N mutation of VPS35 causes mitochondrial dysfunction and suggest a converging pathogenic mechanism among VPS35, PINK1 and Parkin in PD.

Comparative Studies in the A30P and A53T α-Synuclein C. elegans Strains to Investigate the Molecular Origins of Parkinson's Disease.

The aggregation of α-synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression. We report two C. elegans models of PD (PDA30P and PDA53T), which express these mutational variants in the muscle cells, and probed their behavior relative to animals expressing the wild-type protein (PDWT). PDA30P worms showed a reduced speed of movement and an increased paralysis rate, control worms, but no change in the frequency of body bends. By contrast, in PDA53T worms both speed and frequency of body bends were significantly decreased, and paralysis rate was increased. α-Synuclein was also observed to be less well localized into aggregates in PDA30P worms compared to PDA53T and PDWT worms, and amyloid-like features were evident later in the life of the animals, despite comparable levels of expression of α-synuclein. Furthermore, squalamine, a natural product currently in clinical trials for treating symptomatic aspects of PD, was found to reduce significantly the aggregation of α-synuclein and its associated toxicity in PDA53T and PDWT worms, but had less marked effects in PDA30P. In addition, using an antibody that targets the N-terminal region of α-synuclein, we observed a suppression of toxicity in PDA30P, PDA53T and PDWT worms. These results illustrate the use of these two C. elegans models in fundamental and applied PD research.

Cerebellar developmental deficits underlie neurodegenerative disorder spinocerebellar ataxia type 23.

Spinocerebellar ataxia type 23 (SCA23) is a late-onset neurodegenerative disorder characterized by slowly progressive gait and limb ataxia, for which there is no therapy available. It is caused by pathogenic variants in PDYN, which encodes prodynorphin (PDYN). PDYN is processed into the opioid peptides α-neoendorphin and dynorphins (Dyn) A and B; inhibitory neurotransmitters that function in pain signaling, stress-induced responses and addiction. Variants causing SCA23 mostly affect Dyn A, leading to loss of secondary structure and increased peptide stability. PDYNR212W mice express human PDYN containing the SCA23 variant p.R212W. These mice show progressive motor deficits from 3 months of age, climbing fiber (CF) deficits from 3 months of age, and Purkinje cell (PC) loss from 12 months of age. A mouse model for SCA1 showed similar CF deficits, and a recent study found additional developmental abnormalities, namely increased GABAergic interneuron connectivity and non-cell autonomous disruption of PC function. As SCA23 mice show a similar pathology to SCA1 mice in adulthood, we hypothesized that SCA23 may also follow SCA1 pathology during development. Examining PDYNR212W cerebella during development, we uncovered developmental deficits from 2 weeks of age, namely a reduced number of GABAergic synapses on PC soma, possibly leading to the observed delay in early phase CF elimination between 2 and 3 weeks of age. Furthermore, CFs did not reach terminal height, leaving proximal PC dendrites open to be occupied by parallel fibers (PFs). The observed increase in vGlut1 protein-a marker for PF-PC synapses-indicates that PFs indeed take over CF territory and have increased connectivity with PCs. Additionally, we detected altered expression of several critical Ca2+ channel subunits, potentially contributing to altered Ca2+ transients in PDYNR212W cerebella. These findings indicate that developmental abnormalities contribute to the SCA23 pathology and uncover a developmental role for PDYN in the cerebellum.

A Gain-of-Function Variant in Dopamine D2 Receptor and Progressive Chorea and Dystonia Phenotype.

BACKGROUND: We describe a 4-generation Dutch pedigree with a unique dominantly inherited clinical phenotype of a combined progressive chorea and cervical dystonia carrying a novel heterozygous dopamine D2 receptor (DRD2) variant. OBJECTIVES: The objective of this study was to identify the genetic cause of the disease and to further investigate the functional consequences of the genetic variant. METHODS: After detailed clinical and neurological examination, whole-exome sequencing was performed. Because a novel variant in the DRD2 gene was found as the likely causative gene defect in our pedigree, we sequenced the DRD2 gene in a cohort of 121 Huntington-like cases with unknown genetic cause (Germany). Moreover, functional characterization of the DRD2 variant included arrestin recruitment, G protein activation, and G protein-mediated inhibition of adenylyl cyclase determined in a cell model, and G protein-regulated inward-rectifying potassium channels measured in midbrain slices of mice. RESULT: We identified a novel heterozygous variant c.634A > T, p.Ile212Phe in exon 5 of DRD2 that cosegregated with the clinical phenotype. Screening of the German cohort did not reveal additional putative disease-causing variants. We demonstrated that the D2S/L -I212 F receptor exhibited increased agonist potency and constitutive activation of G proteins in human embryonic kidney 239 cells as well as significantly reduced arrestin3 recruitment. We further showed that the D2S -I212 F receptor exhibited aberrant receptor function in mouse midbrain slices. CONCLUSIONS: Our results support an association between the novel p.Ile212Phe variant in DRD2, its modified D2 receptor activity, and the hyperkinetic movement disorder reported in the 4-generation pedigree. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Fast Fluorescence Lifetime Imaging Reveals the Aggregation Processes of α-Synuclein and Polyglutamine in Aging Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans has emerged as an important model organism in the study of the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation, and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterization of different types of aggregates. Specifically, in longitudinal studies of C. elegans models of Parkinson's and Huntington's diseases, we observed marked differences in the aggregation kinetics and the nature of the protein inclusions formed by α-synuclein and polyglutamine. In particular, we found that α-synuclein inclusions do not display amyloid-like features until late in the life of the worms, whereas polyglutamine forms amyloid characteristics rapidly in early adulthood. Furthermore, we show that the TG-FLIM method is capable of imaging live and non-anaesthetized worms moving in specially designed agarose microchambers. Taken together, our results show that the TG-FLIM method enables high-throughput functional imaging of living C. elegans that can be used to study in vivo mechanisms of protein aggregation and that has the potential to aid the search for therapeutic modifiers of protein aggregation and toxicity.

Crystal structure of truncated human coatomer protein complex subunit ζ1 (Copζ1).

The majority of modern anticancer approaches target DNA/protein targets involved in tumour-cell proliferation. Such approaches have a major drawback, as nonproliferating cancer cells remain unaffected and may cause relapse or remission. Human coatomer protein complex I (COPI) subunit ζ (Copζ), a component of the coat protein involved in cell apoptosis and intracellular trafficking, has recently been proposed as a potential anticancer drug target. Previous studies have shown that two different isoforms of the Copζ subunit exist in mammalian cells. While normal cells express both Copζ1 and Copζ2 isoforms, various types of tumour cells display a loss of Copζ2 expression and rely solely on Copζ1 for growth and survival. Subsequent knockdown of Copζ1 results in specific inhibition of both proliferating and dormant tumour-cell populations, with no adverse growth effects on normal cells. Therefore, a Copζ1-targeting therapy was proposed to bypass the problem of dormant cancer cells that are resistant to conventional antiproliferative drugs, which is the major cause of tumour relapse. In order to aid in structure-based inhibitor design, a crystal structure is required. In this article, the recombinant expression, purification, crystallization and crystal structure of Copζ1, as well as the expression and purification of Copζ2, are reported.