SnapShot: Biology of Genetic Ataxias.

Genetic ataxias are a clinically important group of disabling, mostly neurodegenerative, diseases of the cerebellum. This SnapShot shows that the vast majority of established monogenic causes of dominant and recessive ataxias can be captured by a limited number of affected cellular components and biological processes in the cerebellum. To view this SnapShot, open or download the PDF.

Biallelic loss-of-function variations in PRDX3 cause cerebellar ataxia.

Peroxiredoxin 3 (PRDX3) belongs to a superfamily of peroxidases that function as protective antioxidant enzymes. Among the six isoforms (PRDX1-PRDX6), PRDX3 is the only protein exclusively localized to the mitochondria, which are the main source of reactive oxygen species. Excessive levels of reactive oxygen species are harmful to cells, inducing mitochondrial dysfunction, DNA damage, lipid and protein oxidation and ultimately apoptosis. Neuronal cell damage induced by oxidative stress has been associated with numerous neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Leveraging the large aggregation of genomic ataxia datasets from the PREPARE (Preparing for Therapies in Autosomal Recessive Ataxias) network, we identified recessive mutations in PRDX3 as the genetic cause of cerebellar ataxia in five unrelated families, providing further evidence for oxidative stress in the pathogenesis of neurodegeneration. The clinical presentation of individuals with PRDX3 mutations consists of mild-to-moderate progressive cerebellar ataxia with concomitant hyper- and hypokinetic movement disorders, severe early-onset cerebellar atrophy, and in part olivary and brainstem degeneration. Patient fibroblasts showed a lack of PRDX3 protein, resulting in decreased glutathione peroxidase activity and decreased mitochondrial maximal respiratory capacity. Moreover, PRDX3 knockdown in cerebellar medulloblastoma cells resulted in significantly decreased cell viability, increased H2O2 levels and increased susceptibility to apoptosis triggered by reactive oxygen species. Pan-neuronal and pan-glial in vivo models of Drosophila revealed aberrant locomotor phenotypes and reduced survival times upon exposure to oxidative stress. Our findings reveal a central role for mitochondria and the implication of oxidative stress in PRDX3 disease pathogenesis and cerebellar vulnerability and suggest targets for future therapeutic approaches.

O-GlcNAcase contributes to cognitive function in Drosophila.

O-GlcNAcylation is an abundant post-translational modification in neurons. In mice, an increase in O-GlcNAcylation leads to defects in hippocampal synaptic plasticity and learning. O-GlcNAcylation is established by two opposing enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the role of OGA in elementary learning, we generated catalytically inactive and precise knockout Oga alleles (OgaD133N and OgaKO , respectively) in Drosophila melanogaster Adult OgaD133N and OgaKO flies lacking O-GlcNAcase activity showed locomotor phenotypes. Importantly, both Oga lines exhibited deficits in habituation, an evolutionarily conserved form of learning, highlighting that the requirement for O-GlcNAcase activity for cognitive function is preserved across species. Loss of O-GlcNAcase affected a number of synaptic boutons at the axon terminals of larval neuromuscular junction. Taken together, we report behavioral and neurodevelopmental phenotypes associated with Oga alleles and show that Oga contributes to cognition and synaptic morphology in Drosophila.

Integrative network and brain expression analysis reveals mechanistic modules in ataxia.

BACKGROUND: Genetic forms of ataxia are a heterogenous group of degenerative diseases of the cerebellum. Many causative genes have been identified. We aimed to systematically investigate these genes to better understand ataxia pathophysiology. METHODS: A manually curated catalogue of 71 genes involved in disorders with progressive ataxias as a major clinical feature was subjected to an integrated gene ontology, protein network and brain gene expression profiling analysis. RESULTS: We found that genes mutated in ataxias operate in networks with significantly enriched protein connectivity, demonstrating coherence on a global level, independent of inheritance mode. Moreover, elevated expression specifically in the cerebellum predisposes to ataxia. Genes expressed in this pattern are significantly over-represented among genes mutated in ataxia and are enriched for ion homeostasis/synaptic functions. The majority of genes mutated in ataxia, however, does not show elevated cerebellar expression that could account for region-specific degeneration. For these, we identified defective cellular stress responses as a major common biological theme, suggesting that the defence pathways against stress are more critical to maintain cerebellar integrity than integrity of other brain regions. Approximately half of the genes mutated in ataxia, mostly part of the stress module, show higher expression at embryonic stages, which argues for a developmental predisposition. CONCLUSION: Genetic defects in ataxia predominantly affect neuronal homeostasis, to which the cerebellum appears to be excessively susceptible. Based on the identified modules, it is conceivable to propose common therapeutic interventions that target deregulated calcium and reactive oxygen species levels, or mechanisms that can decrease the harmful downstream effects of these deleterious insults.

GDAP2 mutations implicate susceptibility to cellular stress in a new form of cerebellar ataxia.

Autosomal recessive cerebellar ataxias are a group of rare disorders that share progressive degeneration of the cerebellum and associated tracts as the main hallmark. Here, we report two unrelated patients with a new subtype of autosomal recessive cerebellar ataxia caused by biallelic, gene-disruptive mutations in GDAP2, a gene previously not implicated in disease. Both patients had onset of ataxia in the fourth decade. Other features included progressive spasticity and dementia. Neuropathological examination showed degenerative changes in the cerebellum, olive inferior, thalamus, substantia nigra, and pyramidal tracts, as well as tau pathology in the hippocampus and amygdala. To provide further evidence for a causative role of GDAP2 mutations in autosomal recessive cerebellar ataxia pathophysiology, its orthologous gene was investigated in the fruit fly Drosophila melanogaster. Ubiquitous knockdown of Drosophila Gdap2 resulted in shortened lifespan and motor behaviour anomalies such as righting defects, reduced and uncoordinated walking behaviour, and compromised flight. Gdap2 expression levels responded to stress treatments in control flies, and Gdap2 knockdown flies showed increased sensitivity to deleterious effects of stressors such as reactive oxygen species and nutrient deprivation. Thus, Gdap2 knockdown in Drosophila and GDAP2 loss-of-function mutations in humans lead to locomotor phenotypes, which may be mediated by altered responses to cellular stress.

Gene identification in the congenital disorders of glycosylation type I by whole-exome sequencing.

Congenital disorders of glycosylation type I (CDG-I) form a growing group of recessive neurometabolic diseases. Identification of disease genes is compromised by the enormous heterogeneity in clinical symptoms and the large number of potential genes involved. Until now, gene identification included the sequential application of biochemical methods in blood samples and fibroblasts. In genetically unsolved cases, homozygosity mapping has been applied in consanguineous families. Altogether, this time-consuming diagnostic strategy led to the identification of defects in 17 different CDG-I genes. Here, we applied whole-exome sequencing (WES) in combination with the knowledge of the protein N-glycosylation pathway for gene identification in our remaining group of six unsolved CDG-I patients from unrelated non-consanguineous families. Exome variants were prioritized based on a list of 76 potential CDG-I candidate genes, leading to the rapid identification of one known and two novel CDG-I gene defects. These included the first X-linked CDG-I due to a de novo mutation in ALG13, and compound heterozygous mutations in DPAGT1, together the first two steps in dolichol-PP-glycan assembly, and mutations in PGM1 in two cases, involved in nucleotide sugar biosynthesis. The pathogenicity of the mutations was confirmed by showing the deficient activity of the corresponding enzymes in patient fibroblasts. Combined with these results, the gene defect has been identified in 98% of our CDG-I patients. Our results implicate the potential of WES to unravel disease genes in the CDG-I in newly diagnosed singleton families.

Unraveling the brain's response to COVID-19: How SARS-CoV-2 afflicts dopaminergic neurons.

COVID-19 patients often display dysfunctions of the nervous system, indicating an effect of SARS-CoV-2 on neural cells. Yang et al. now show that human stem-cell-derived dopaminergic neurons are susceptible to SARS-CoV-2, triggering inflammation and senescence. The study further identifies three FDA-approved drugs capable of reversing these cellular phenotypes.

Pre-clinical evaluation of clinically relevant iPS cell derived neuroepithelial stem cells as an off-the-shelf cell therapy for spinal cord injury.

Preclinical transplantations using human neuroepithelial stem (NES) cells in spinal cord injury models have exhibited promising results and demonstrated cell integration and functional improvement in transplanted animals. Previous studies have relied on the generation of research grade cell lines in continuous culture. Using fresh cells presents logistic hurdles for clinical transition regarding time and resources for maintaining high quality standards. In this study, we generated a good manufacturing practice (GMP) compliant human iPS cell line in GMP clean rooms alongside a research grade iPS cell line which was produced using standardized protocols with GMP compliant chemicals. These two iPS cell lines were differentiated into human NES cells, from which six batches of cell therapy doses were produced. The doses were cryopreserved, thawed on demand and grafted in a rat spinal cord injury model. Our findings demonstrate that NES cells can be directly grafted post-thaw with high cell viability, maintaining their cell identity and differentiation capacity. This opens the possibility of manufacturing off-the-shelf cell therapy products. Moreover, our manufacturing process yields stable cell doses with minimal batch-to-batch variability, characterized by consistent expression of identity markers as well as similar viability of cells across the two iPS cell lines. These cryopreserved cell doses exhibit sustained viability, functionality, and quality for at least 2 years. Our results provide proof of concept that cryopreserved NES cells present a viable alternative to transplanting freshly cultured cells in future cell therapies and exemplify a platform from which cell formulation can be optimized and facilitate the transition to clinical trials.

Ataxia-associated DNA repair genes protect the Drosophila mushroom body and locomotor function against glutamate signaling-associated damage.

The precise control of motor movements is of fundamental importance to all behaviors in the animal kingdom. Efficient motor behavior depends on dedicated neuronal circuits - such as those in the cerebellum - that are controlled by extensive genetic programs. Autosomal recessive cerebellar ataxias (ARCAs) provide a valuable entry point into how interactions between genetic programs maintain cerebellar motor circuits. We previously identified a striking enrichment of DNA repair genes in ARCAs. How dysfunction of ARCA-associated DNA repair genes leads to preferential cerebellar dysfunction and impaired motor function is however unknown. The expression of ARCA DNA repair genes is not specific to the cerebellum. Only a limited number of animal models for DNA repair ARCAs exist, and, even for these, the interconnection between DNA repair defects, cerebellar circuit dysfunction, and motor behavior is barely established. We used Drosophila melanogaster to characterize the function of ARCA-associated DNA repair genes in the mushroom body (MB), a structure in the Drosophila central brain that shares structural features with the cerebellum. Here, we demonstrate that the MB is required for efficient startle-induced and spontaneous motor behaviors. Inhibition of synaptic transmission and loss-of-function of ARCA-associated DNA repair genes in the MB affected motor behavior in several assays. These motor deficits correlated with increased levels of MB DNA damage, MB Kenyon cell apoptosis and/or alterations in MB morphology. We further show that expression of genes involved in glutamate signaling pathways are highly, specifically, and persistently elevated in the postnatal human cerebellum. Manipulation of glutamate signaling in the MB induced motor defects, Kenyon cell DNA damage and apoptosis. Importantly, pharmacological reduction of glutamate signaling in the ARCA DNA repair models rescued the identified motor deficits, suggesting a role for aberrant glutamate signaling in ARCA-DNA repair disorders. In conclusion, our data highlight the importance of ARCA-associated DNA repair genes and glutamate signaling pathways to the cerebellum, the Drosophila MB and motor behavior. We propose that glutamate signaling may confer preferential cerebellar vulnerability in ARCA-associated DNA repair disorders. Targeting glutamate signaling could provide an exciting therapeutic entry point in this large group of so far untreatable disorders.

Intracellular Lipid Accumulation and Mitochondrial Dysfunction Accompanies Endoplasmic Reticulum Stress Caused by Loss of the Co-chaperone DNAJC3.

Recessive mutations in DNAJC3, an endoplasmic reticulum (ER)-resident BiP co-chaperone, have been identified in patients with multisystemic neurodegeneration and diabetes mellitus. To further unravel these pathomechanisms, we employed a non-biased proteomic approach and identified dysregulation of several key cellular pathways, suggesting a pathophysiological interplay of perturbed lipid metabolism, mitochondrial bioenergetics, ER-Golgi function, and amyloid-beta processing. Further functional investigations in fibroblasts of patients with DNAJC3 mutations detected cellular accumulation of lipids and an increased sensitivity to cholesterol stress, which led to activation of the unfolded protein response (UPR), alterations of the ER-Golgi machinery, and a defect of amyloid precursor protein. In line with the results of previous studies, we describe here alterations in mitochondrial morphology and function, as a major contributor to the DNAJC3 pathophysiology. Hence, we propose that the loss of DNAJC3 affects lipid/cholesterol homeostasis, leading to UPR activation, ß-amyloid accumulation, and impairment of mitochondrial oxidative phosphorylation.

The CHD8/CHD7/Kismet family links blood-brain barrier glia and serotonin to ASD-associated sleep defects.

Sleep disturbances in autism and neurodevelopmental disorders are common and adversely affect patient's quality of life, yet the underlying mechanisms are understudied. We found that individuals with mutations in CHD8, among the highest-confidence autism risk genes, or CHD7 suffer from disturbed sleep maintenance. These defects are recapitulated in Drosophila mutants affecting kismet, the sole CHD8/CHD7 ortholog. We show that Kismet is required in glia for early developmental and adult sleep architecture. This role localizes to subperineurial glia constituting the blood-brain barrier. We demonstrate that Kismet-related sleep disturbances are caused by high serotonin during development, paralleling a well-established but genetically unsolved autism endophenotype. Despite their developmental origin, Kismet's sleep architecture defects can be reversed in adulthood by a behavioral regime resembling human sleep restriction therapy. Our findings provide fundamental insights into glial regulation of sleep and propose a causal mechanistic link between the CHD8/CHD7/Kismet family, developmental hyperserotonemia, and autism-associated sleep disturbances.

Conserved regulation of neurodevelopmental processes and behavior by FoxP in Drosophila.

FOXP proteins form a subfamily of evolutionarily conserved transcription factors involved in the development and functioning of several tissues, including the central nervous system. In humans, mutations in FOXP1 and FOXP2 have been implicated in cognitive deficits including intellectual disability and speech disorders. Drosophila exhibits a single ortholog, called FoxP, but due to a lack of characterized mutants, our understanding of the gene remains poor. Here we show that the dimerization property required for mammalian FOXP function is conserved in Drosophila. In flies, FoxP is enriched in the adult brain, showing strong expression in ~1000 neurons of cholinergic, glutamatergic and GABAergic nature. We generate Drosophila loss-of-function mutants and UAS-FoxP transgenic lines for ectopic expression, and use them to characterize FoxP function in the nervous system. At the cellular level, we demonstrate that Drosophila FoxP is required in larvae for synaptic morphogenesis at axonal terminals of the neuromuscular junction and for dendrite development of dorsal multidendritic sensory neurons. In the developing brain, we find that FoxP plays important roles in α-lobe mushroom body formation. Finally, at a behavioral level, we show that Drosophila FoxP is important for locomotion, habituation learning and social space behavior of adult flies. Our work shows that Drosophila FoxP is important for regulating several neurodevelopmental processes and behaviors that are related to human disease or vertebrate disease model phenotypes. This suggests a high degree of functional conservation with vertebrate FOXP orthologues and established flies as a model system for understanding FOXP related pathologies.

Feeding difficulties, a key feature of the Drosophila NDUFS4 mitochondrial disease model.

Mitochondrial diseases are associated with a wide variety of clinical symptoms and variable degrees of severity. Patients with such diseases generally have a poor prognosis and often an early fatal disease outcome. With an incidence of 1 in 5000 live births and no curative treatments available, relevant animal models to evaluate new therapeutic regimes for mitochondrial diseases are urgently needed. By knocking down ND-18, the unique Drosophila ortholog of NDUFS4, an accessory subunit of the NADH:ubiquinone oxidoreductase (Complex I), we developed and characterized several dNDUFS4 models that recapitulate key features of mitochondrial disease. Like in humans, the dNDUFS4 KD flies display severe feeding difficulties, an aspect of mitochondrial disorders that has so far been largely ignored in animal models. The impact of this finding, and an approach to overcome it, will be discussed in the context of interpreting disease model characterization and intervention studies.This article has an associated First Person interview with the first author of the paper.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay.

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens. This manuscript describes the "Drosophila Island Assay" and "Island Assay Analysis" algorithms for high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The "Drosophila Island Assay" algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The "Island Assay Analysis" script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology.

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

TRPM7 is required for breast tumor cell metastasis.

TRPM7 encodes a Ca2+-permeable nonselective cation channel with kinase activity. TRPM7 has been implicated in control of cell adhesion and migration, but whether TRPM7 activity contributes to cancer progression has not been established. Here we report that high levels of TRPM7 expression independently predict poor outcome in breast cancer patients and that it is functionally required for metastasis formation in a mouse xenograft model of human breast cancer. Mechanistic investigation revealed that TRPM7 regulated myosin II-based cellular tension, thereby modifying focal adhesion number, cell-cell adhesion and polarized cell movement. Our findings therefore suggest that TRPM7 is part of a mechanosensory complex adopted by cancer cells to drive metastasis formation.