CAG Repeat Size Influences the Progression Rate of Spinocerebellar Ataxia Type 3.

OBJECTIVE: In spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD), the expanded cytosine adenine guanine (CAG) repeat in ATXN3 is the causal mutation, and its length is the main factor in determining the age at onset (AO) of clinical symptoms. However, the contribution of the expanded CAG repeat length to the rate of disease progression after onset has remained a matter of debate, even though an understanding of this factor is crucial for experimental data on disease modifiers and their translation to clinical trials and their design. METHODS: Eighty-two Dutch patients with SCA3/MJD were evaluated annually for 15 years using the International Cooperative Ataxia Rating Scale (ICARS). Using linear growth curve models, ICARS progression rates were calculated and tested for their relation to the length of the CAG repeat expansion and to the residual age at onset (RAO): The difference between the observed AO and the AO predicted on the basis of the CAG repeat length. RESULTS: On average, ICARS scores increased 2.57 points/year of disease. The length of the CAG repeat was positively correlated with a more rapid ICARS progression, explaining 30% of the differences between patients. Combining both the length of the CAG repeat and RAO as comodifiers explained up to 47% of the interpatient variation in ICARS progression. INTERPRETATION: Our data imply that the length of the expanded CAG repeat in ATXN3 is a major determinant of clinical decline, which suggests that CAG-dependent molecular mechanisms similar to those responsible for disease onset also contribute to the rate of disease progression in SCA3/MJD. ANN NEUROL 2021;89:66-73.

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.

Cortical pencil lining on SWI MRI in NBIA and healthy aging.

BACKGROUND: Neurodegeneration with brain iron accumulation (NBIA) is characterized by pathological iron accumulation in the subcortical nuclei and the cortex. As age-related iron accumulation studies in these structures are lacking in healthy aging, we aimed to characterize the dynamics of age-dependent iron accumulation in subcortical nuclei in healthy aging and selected NBIA cases. This is fundamental to understand the natural age-related iron deposition in the healthy brain prior to using this marker as a potential prognostic or diagnostic tool in neurodegenerative disorders. METHODS: Susceptibility-weighted imaging (SWI) scans from 81 healthy volunteers (0-79 years) and four genetically confirmed patients suffering from NBIA (2-14 years) were obtained. We scored the presence or absence of pencil lining of the motor cortex and putamen and analyzed the normalized SWI signal intensity ratio (NSIR) in five subcortical nuclei. RESULTS: In healthy subjects, an age-dependent increase of pencil lining occurred starting from the second decade of life and was present in all cases at the age of 50. In their first decade, NBIA patients showed no cortical pencil lining, but we did observe putaminal pencil lining at this stage. In healthy subjects, age and NSIR of all nuclei correlated positively and was particularly dynamic in early childhood until young adulthood in the globus pallidus, dentate nucleus and red nucleus, but not in the caudate nucleus and putamen. NBIA patients showed an increased NSIR in the globus pallidus only and not in the other subcortical nuclei compared to age-matched healthy subjects. CONCLUSIONS: Cortical pencil lining is part of healthy aging. This should be considered when assessing this as a potential marker in NBIA diagnosis and prognosis. Putaminal pencil lining has the potential to become a specific marker for some subtypes of NBIA in the first decade of life, as it was only observed in NBIA and not in age-matched healthy subjects. NSIR in the subcortical nuclei during healthy aging was shown to be dynamic, accentuating the importance of having an age-dependent baseline.

Neuronal Expression of Opioid Gene is Controlled by Dual Epigenetic and Transcriptional Mechanism in Human Brain.

Molecular mechanisms that define patterns of neuropeptide expression are essential for the formation and rewiring of neural circuits. The prodynorphin gene (PDYN) gives rise to dynorphin opioid peptides mediating depression and substance dependence. We here demonstrated that PDYN is expressed in neurons in human dorsolateral prefrontal cortex (dlPFC), and identified neuronal differentially methylated region in PDYN locus framed by CCCTC-binding factor binding sites. A short, nucleosome size human-specific promoter CpG island (CGI), a core of this region may serve as a regulatory module, which is hypomethylated in neurons, enriched in 5-hydroxymethylcytosine, and targeted by USF2, a methylation-sensitive E-box transcription factor (TF). USF2 activates PDYN transcription in model systems, and binds to nonmethylated CGI in dlPFC. USF2 and PDYN expression is correlated, and USF2 and PDYN proteins are co-localized in dlPFC. Segregation of activatory TF and repressive CGI methylation may ensure contrasting PDYN expression in neurons and glia in human brain.

Altered secondary structure of Dynorphin A associates with loss of opioid signalling and NMDA-mediated excitotoxicity in SCA23.

Spinocerebellar ataxia type 23 (SCA23) is caused by missense mutations in prodynorphin, encoding the precursor protein for the opioid neuropeptides α-neoendorphin, Dynorphin (Dyn) A and Dyn B, leading to neurotoxic elevated mutant Dyn A levels. Dyn A acts on opioid receptors to reduce pain in the spinal cord, but its cerebellar function remains largely unknown. Increased concentration of or prolonged exposure to Dyn A is neurotoxic and these deleterious effects are very likely caused by an N-methyl-d-aspartate-mediated non-opioid mechanism as Dyn A peptides were shown to bind NMDA receptors and potentiate their glutamate-evoked currents. In the present study, we investigated the cellular mechanisms underlying SCA23-mutant Dyn A neurotoxicity. We show that SCA23 mutations in the Dyn A-coding region disrupted peptide secondary structure leading to a loss of the N-terminal α-helix associated with decreased κ-opioid receptor affinity. Additionally, the altered secondary structure led to increased peptide stability of R6W and R9C Dyn A, as these peptides showed marked degradation resistance, which coincided with decreased peptide solubility. Notably, L5S Dyn A displayed increased degradation and no aggregation. R6W and wt Dyn A peptides were most toxic to primary cerebellar neurons. For R6W Dyn A, this is likely because of a switch from opioid to NMDA- receptor signalling, while for wt Dyn A, this switch was not observed. We propose that the pathology of SCA23 results from converging mechanisms of loss of opioid-mediated neuroprotection and NMDA-mediated excitotoxicity.

Exome sequencing and network analysis identifies shared mechanisms underlying spinocerebellar ataxia.

The autosomal dominant cerebellar ataxias, referred to as spinocerebellar ataxias in genetic nomenclature, are a rare group of progressive neurodegenerative disorders characterized by loss of balance and coordination. Despite the identification of numerous disease genes, a substantial number of cases still remain without a genetic diagnosis. Here, we report five novel spinocerebellar ataxia genes, FAT2, PLD3, KIF26B, EP300, and FAT1, identified through a combination of exome sequencing in genetically undiagnosed families and targeted resequencing of exome candidates in a cohort of singletons. We validated almost all genes genetically, assessed damaging effects of the gene variants in cell models and further consolidated a role for several of these genes in the aetiology of spinocerebellar ataxia through network analysis. Our work links spinocerebellar ataxia to alterations in synaptic transmission and transcription regulation, and identifies these as the main shared mechanisms underlying the genetically diverse spinocerebellar ataxia types.

Spinocerebellar ataxia: miRNAs expose biological pathways underlying pervasive Purkinje cell degeneration.

Recent work has demonstrated the importance of miRNAs in the pathogenesis of various brain disorders including the neurodegenerative disorder spinocerebellar ataxia (SCA). This review focuses on the role of miRNAs in the shared pathogenesis of the different SCA types. We examine the novel findings of a recent cell-type-specific RNA-sequencing study in mouse brain and discuss how the identification of Purkinje-cell-enriched miRNAs highlights biological pathways that expose the mechanisms behind pervasive Purkinje cell degeneration in SCA. These key pathways are likely to contain targets for therapeutic development and represent potential candidate genes for genetically unsolved SCAs.

CACNA1B mutation is linked to unique myoclonus-dystonia syndrome.

Using exome sequencing and linkage analysis in a three-generation family with a unique dominant myoclonus-dystonia-like syndrome with cardiac arrhythmias, we identified a mutation in the CACNA1B gene, coding for neuronal voltage-gated calcium channels CaV2.2. This mutation (c.4166G>A;p.Arg1389His) is a disruptive missense mutation in the outer region of the ion pore. The functional consequences of the identified mutation were studied using whole-cell and single-channel patch recordings. High-resolution analyses at the single-channel level showed that, when open, R1389H CaV2.2 channels carried less current compared with WT channels. Other biophysical channel properties were unaltered in R1389H channels including ion selectivity, voltage-dependent activation or voltage-dependent inactivation. CaV2.2 channels regulate transmitter release at inhibitory and excitatory synapses. Functional changes could be consistent with a gain-of-function causing the observed hyperexcitability characteristic of this unique myoclonus-dystonia-like syndrome associated with cardiac arrhythmias.

Opioid precursor protein isoform is targeted to the cell nuclei in the human brain.

BACKGROUND: Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain. METHODS: Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence-activated nuclei sorting (FANS) from postmortem human striatal tissue. Immunofluorescence staining and confocal microscopy was performed for human caudate nucleus. RESULTS: Two novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging. CONCLUSIONS AND GENERAL SIGNIFICANCE: High levels of alternatively spliced ∆SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum.

Elevated mutant dynorphin A causes Purkinje cell loss and motor dysfunction in spinocerebellar ataxia type 23.

Spinocerebellar ataxia type 23 is caused by mutations in PDYN, which encodes the opioid neuropeptide precursor protein, prodynorphin. Prodynorphin is processed into the opioid peptides, α-neoendorphin, and dynorphins A and B, that normally exhibit opioid-receptor mediated actions in pain signalling and addiction. Dynorphin A is likely a mutational hotspot for spinocerebellar ataxia type 23 mutations, and in vitro data suggested that dynorphin A mutations lead to persistently elevated mutant peptide levels that are cytotoxic and may thus play a crucial role in the pathogenesis of spinocerebellar ataxia type 23. To further test this and study spinocerebellar ataxia type 23 in more detail, we generated a mouse carrying the spinocerebellar ataxia type 23 mutation R212W in PDYN. Analysis of peptide levels using a radioimmunoassay shows that these PDYN(R212W) mice display markedly elevated levels of mutant dynorphin A, which are associated with climber fibre retraction and Purkinje cell loss, visualized with immunohistochemical stainings. The PDYN(R212W) mice reproduced many of the clinical features of spinocerebellar ataxia type 23, with gait deficits starting at 3 months of age revealed by footprint pattern analysis, and progressive loss of motor coordination and balance at the age of 12 months demonstrated by declining performances on the accelerating Rotarod. The pathologically elevated mutant dynorphin A levels in the cerebellum coincided with transcriptionally dysregulated ionotropic and metabotropic glutamate receptors and glutamate transporters, and altered neuronal excitability. In conclusion, the PDYN(R212W) mouse is the first animal model of spinocerebellar ataxia type 23 and our work indicates that the elevated mutant dynorphin A peptide levels are likely responsible for the initiation and progression of the disease, affecting glutamatergic signalling, neuronal excitability, and motor performance. Our novel mouse model defines a critical role for opioid neuropeptides in spinocerebellar ataxia, and suggests that restoring the elevated mutant neuropeptide levels can be explored as a therapeutic intervention.

Spinocerebellar ataxia type 19/22 mutations alter heterocomplex Kv4.3 channel function and gating in a dominant manner.

The dominantly inherited cerebellar ataxias are a heterogeneous group of neurodegenerative disorders caused by Purkinje cell loss in the cerebellum. Recently, we identified loss-of-function mutations in the KCND3 gene as the cause of spinocerebellar ataxia type 19/22 (SCA19/22), revealing a previously unknown role for the voltage-gated potassium channel, Kv4.3, in Purkinje cell survival. However, how mutant Kv4.3 affects wild-type Kv4.3 channel functioning remains unknown. We provide evidence that SCA19/22-mutant Kv4.3 exerts a dominant negative effect on the trafficking and surface expression of wild-type Kv4.3 in the absence of its regulatory subunit, KChIP2. Notably, this dominant negative effect can be rescued by the presence of KChIP2. We also found that all SCA19/22-mutant subunits either suppress wild-type Kv4.3 current amplitude or alter channel gating in a dominant manner. Our findings suggest that altered Kv4.3 channel localization and/or functioning resulting from SCA19/22 mutations may lead to Purkinje cell loss, neurodegeneration and ataxia.

First de novo KCND3 mutation causes severe Kv4.3 channel dysfunction leading to early onset cerebellar ataxia, intellectual disability, oral apraxia and epilepsy.

BACKGROUND: Identification of the first de novo mutation in potassium voltage-gated channel, shal-related subfamily, member 3 (KCND3) in a patient with complex early onset cerebellar ataxia in order to expand the genetic and phenotypic spectrum. METHODS: Whole exome sequencing in a cerebellar ataxia patient and subsequent immunocytochemistry, immunoblotting and patch clamp assays of the channel were performed. RESULTS: A de novo KCND3 mutation (c.877_885dupCGCGTCTTC; p.Arg293_Phe295dup) was found duplicating the RVF motif and thereby adding an extra positive charge to voltage-gated potassium 4.3 (Kv4.3) in the voltage-sensor domain causing a severe shift of the voltage-dependence gating to more depolarized voltages. The patient displayed a severe phenotype with early onset cerebellar ataxia complicated by intellectual disability, epilepsy, attention deficit hyperactivity disorder, strabismus, oral apraxia and joint hyperlaxity. CONCLUSIONS: We identified a de novo KCND3 mutation causing the most marked change in Kv4.3's channel properties reported so far, which correlated with a severe and unique spinocerebellar ataxia (SCA) type 19/22 disease phenotype.

SCA14 mutation V138E leads to partly unfolded PKCγ associated with an exposed C-terminus, altered kinetics, phosphorylation and enhanced insolubilization.

The protein kinase C γ (PKCγ) undergoes multistep activation and participates in various cellular processes in Purkinje cells. Perturbations in its phosphorylation state, conformation or localization can disrupt kinase signalling, such as in spinocerebellar ataxia type 14 (SCA14) that is caused by missense mutations in PRKCG encoding for PKCγ. We previously showed that SCA14 mutations enhance PKCγ membrane translocation upon stimulation owing to an altered protein conformation. As the faster translocation did not result in an increased function, we examined how SCA14 mutations induce this altered conformation of PKCγ and what the consequences of this conformational change are on PKCγ life cycle. Here, we show that SCA14-related PKCγ-V138E exhibits an exposed C-terminus as shown by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy in living cells, indicative of its partial unfolding. This conformational change was associated with faster phorbol 12-myristate 13-acetate-induced translocation and accumulation of fully phosphorylated PKCγ in the insoluble fraction, which could be rescued by coexpressing PDK1 kinase that normally triggers PKCγ autophosphorylation. We propose that the SCA14 mutation V138E causes unfolding of the C1B domain and exposure of the C-terminus of the PKCγ-V138E molecule, resulting in a decrease of functional kinase in the soluble fraction. Here, we show that the mutation V138E of the protein kinase C γ (PKCγ) C1B domain (PKCγ-V138E), which is implicated in spinocerebellar ataxia type 14, exhibits a partially unfolded C-terminus. This leads to unusually fast phorbol 12-myristate 13-acetate-induced membrane translocation and accumulation of phosphorylated PKCγ-V138E in the insoluble fraction, causing loss of the functional kinase. In contrast to general chaperones, coexpression of PKCγ's 'natural chaperone', PDK1 kinase, could rescue the PKCγ-V138E phenotype.

Comprehensive analysis of genetic risk loci uncovers novel candidate genes and pathways in the comorbidity between depression and Alzheimer's disease.

There is growing evidence of a shared pathogenesis between Alzheimer's disease and depression. Therefore, we aimed to further investigate their shared disease mechanisms. We made use of publicly available brain-specific eQTL data and gene co-expression networks of previously reported genetic loci associated with these highly comorbid disorders. No direct genetic overlap was observed between Alzheimer's disease and depression in our dataset, but we did detect six shared brain-specific eQTL genes: SRA1, MICA, PCDHA7, PCDHA8, PCDHA10 and PCDHA13. Several pathways were identified as shared between Alzheimer's disease and depression by conducting clustering pathway analysis on hippocampal co-expressed genes; synaptic signaling and organization, myelination, development, and the immune system. This study highlights trans-synaptic signaling and synaptoimmunology in the hippocampus as main shared pathomechanisms of Alzheimer's disease and depression.

Cross-species analysis uncovers the mitochondrial stress response in the hippocampus as a shared mechanism in mouse early life stress and human depression.

Depression, or major depressive disorder, poses a significant burden for both individuals and society, affecting approximately 10.8% of the general population. This psychiatric disorder leads to approximately 800,000 deaths per year. A combination of genetic and environmental factors such as early life stress (ELS) increase the risk for development of depression in humans, and a clear role for the hippocampus in the pathophysiology of depression has been shown. Nevertheless, the underlying mechanisms of depression remain poorly understood, resulting in a lack of effective treatments. To better understand the core mechanisms underlying the development of depression, we used a cross-species design to investigate shared hippocampal pathophysiological mechanisms in mouse ELS and human depression. Mice were subjected to ELS by a maternal separation paradigm, followed by RNA sequencing analysis of the adult hippocampal tissue. This identified persistent transcriptional changes linked to mitochondrial stress response pathways, with oxidative phosphorylation and protein folding emerging as the main mechanisms affected by maternal separation. Remarkably, there was a significant overlap between the pathways involved in mitochondrial stress response we observed and publicly available RNAseq data from hippocampal tissue of depressive patients. This cross-species conservation of changes in gene expression of mitochondria-related genes suggests that mitochondrial stress may play a pivotal role in the development of depression. Our findings highlight the potential significance of the hippocampal mitochondrial stress response as a core mechanism underlying the development of depression. Further experimental investigations are required to expand our understanding of these mechanisms.

Prodynorphin mutations cause the neurodegenerative disorder spinocerebellar ataxia type 23.

Spinocerebellar ataxias (SCAs) are dominantly inherited neurodegenerative disorders characterized by progressive cerebellar ataxia and dysarthria. We have identified missense mutations in prodynorphin (PDYN) that cause SCA23 in four Dutch families displaying progressive gait and limb ataxia. PDYN is the precursor protein for the opioid neuropeptides, α-neoendorphin, and dynorphins A and B (Dyn A and B). Dynorphins regulate pain processing and modulate the rewarding effects of addictive substances. Three mutations were located in Dyn A, a peptide with both opioid activities and nonopioid neurodegenerative actions. Two of these mutations resulted in excessive generation of Dyn A in a cellular model system. In addition, two of the mutant Dyn A peptides induced toxicity above that of wild-type Dyn A in cultured striatal neurons. The fourth mutation was located in the nonopioid PDYN domain and was associated with altered expression of components of the opioid and glutamate system, as evident from analysis of SCA23 autopsy tissue. Thus, alterations in Dyn A activities and/or impairment of secretory pathways by mutant PDYN may lead to glutamate neurotoxicity, which underlies Purkinje cell degeneration and ataxia. PDYN mutations are identified in a small subset of ataxia families, indicating that SCA23 is an infrequent SCA type (∼0.5%) in the Netherlands and suggesting further genetic SCA heterogeneity.

Mutations in potassium channel kcnd3 cause spinocerebellar ataxia type 19.

OBJECTIVE: To identify the causative gene for the neurodegenerative disorder spinocerebellar ataxia type 19 (SCA19) located on chromosomal region 1p21-q21. METHODS: Exome sequencing was used to identify the causal mutation in a large SCA19 family. We then screened 230 ataxia families for mutations located in the same gene (KCND3, also known as Kv4.3) using high-resolution melting. SCA19 brain autopsy material was evaluated, and in vitro experiments using ectopic expression of wild-type and mutant Kv4.3 were used to study protein localization, stability, and channel activity by patch-clamping. RESULTS: We detected a T352P mutation in the third extracellular loop of the voltage-gated potassium channel KCND3 that cosegregated with the disease phenotype in our original family. We identified 2 more novel missense mutations in the channel pore (M373I) and the S6 transmembrane domain (S390N) in 2 other ataxia families. T352P cerebellar autopsy material showed severe Purkinje cell degeneration, with abnormal intracellular accumulation and reduced protein levels of Kv4.3 in their soma. Ectopic expression of all mutant proteins in HeLa cells revealed retention in the endoplasmic reticulum and enhanced protein instability, in contrast to wild-type Kv4.3 that was localized on the plasma membrane. The regulatory ß subunit Kv channel interacting protein 2 was able to rescue the membrane localization and the stability of 2 of the 3 mutant Kv4.3 complexes. However, this either did not restore the channel function of the membrane-located mutant Kv4.3 complexes or restored it only partially. INTERPRETATION: KCND3 mutations cause SCA19 by impaired protein maturation and/or reduced channel function.