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1.
Front Cell Neurosci ; 16: 817198, 2022.
Article in English | MEDLINE | ID: mdl-35401116

ABSTRACT

Induced pluripotent stem cell (iPSC)-based generation of tyrosine hydroxylase-positive (TH+) dopaminergic neurons (DNs) is a powerful method for creating patient-specific in vitro models to elucidate mechanisms underlying Parkinson's disease (PD) at the cellular and molecular level and to perform drug screening. However, currently available differentiation paradigms result in highly heterogeneous cell populations, often yielding a disappointing fraction (<50%) of the PD-relevant TH+ DNs. To facilitate the targeted analysis of this cell population and to characterize their electrophysiological properties, we employed CRISPR/Cas9 technology and generated an mCherry-based human TH reporter iPSC line. Subsequently, reporter iPSCs were subjected to dopaminergic differentiation using either a "floor plate protocol" generating DNs directly from iPSCs or an alternative method involving iPSC-derived neuronal precursors (NPC-derived DNs). To identify the strategy with the highest conversion efficiency to mature neurons, both cultures were examined for a period of 8 weeks after triggering neuronal differentiation by means of immunochemistry and single-cell electrophysiology. We confirmed that mCherry expression correlated with the expression of endogenous TH and that genetic editing did neither affect the differentiation process nor the endogenous TH expression in iPSC- and NPC-derived DNs. Although both cultures yielded identical proportions of TH+ cells (≈30%), whole-cell patch-clamp experiments revealed that iPSC-derived DNs gave rise to larger currents mediated by voltage-gated sodium and potassium channels, showed a higher degree of synaptic activity, and fired trains of mature spontaneous action potentials more frequently compared to NPC-derived DNs already after 2 weeks in differentiation. Moreover, spontaneous action potential firing was more frequently detected in TH+ neurons compared to the TH- cells, providing direct evidence that these two neuronal subpopulations exhibit different intrinsic electrophysiological properties. In summary, the data reveal substantial differences in the electrophysiological properties of iPSC-derived TH+ and TH- neuronal cell populations and that the "floor plate protocol" is particularly efficient in generating electrophysiologically mature TH+ DNs, which are the most vulnerable neuronal subtype in PD.

2.
Sci Adv ; 8(10): eabj9229, 2022 03 11.
Article in English | MEDLINE | ID: mdl-35275727

ABSTRACT

Mutations in the brain-specific ß-tubulin 4A (TUBB4A) gene cause a broad spectrum of diseases, ranging from dystonia (DYT-TUBB4A) to hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). Currently, the mechanisms of how TUBB4A variants lead to this pleiotropic manifestation remain elusive. Here, we investigated whether TUBB4A mutations causing either DYT-TUBB4A (p.R2G and p.Q424H) or H-ABC (p.R2W and p.D249N) exhibit differential effects at the molecular and cellular levels. Using live-cell imaging of disease-relevant oligodendrocytes and total internal reflection fluorescence microscopy of whole-cell lysates, we observed divergent impact on microtubule polymerization and microtubule integration, partially reflecting the observed pleiotropy. Moreover, in silico simulations demonstrated that the mutants rarely adopted a straight heterodimer conformation in contrast to wild type. In conclusion, for most of the examined variants, we deciphered potential molecular disease mechanisms that may lead to the diverse clinical manifestations and phenotype severity across and within each TUBB4A-related disease.


Subject(s)
Dystonia , Tubulin , Basal Ganglia/metabolism , Basal Ganglia/pathology , Cerebellum/metabolism , Dystonia/genetics , Dystonia/pathology , Humans , Mutation , Tubulin/genetics , Tubulin/metabolism
3.
Mol Biol Rep ; 47(5): 3993-4001, 2020 May.
Article in English | MEDLINE | ID: mdl-32239467

ABSTRACT

DYT-TOR1A is the most common inherited dystonia caused by a three nucleotide (GAG) deletion (dE) in the TOR1A gene. Death early after birth and cortical anomalies of the full knockout in rodents underscore its developmental importance. We therefore explored the timed effects of TOR1A-wt and TOR1A-dE during differentiation in a human neural in vitro model. We used lentiviral tet-ON expression of TOR1A-wt and -dE in induced neural stem cells derived from healthy donors. Overexpression was induced during proliferation of neural precursors, during differentiation and after differentiation into mature neurons. Overexpression of both wildtype and mutated protein had no effect on the viability and cell number of neural precursors as well as mature neurons when initiated before or after differentiation. However, if induced during differentiation, overexpression of TOR1A-wt and -dE led to a pronounced reduction of mature neurons in a dose dependent manner. Our data underscores the importance of physiological expression levels of TOR1A as crucial for proper neuronal differentiation. We did not find evidence for a specific impact of the mutated TOR1A on neuronal maturation.


Subject(s)
Molecular Chaperones/biosynthesis , Neural Stem Cells/metabolism , Neurons/metabolism , Cell Differentiation/physiology , Dystonia/genetics , Dystonia/metabolism , Dystonia/pathology , HEK293 Cells , Humans , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mutation , Neural Stem Cells/pathology , Neurons/pathology
5.
Hum Mutat ; 39(12): 1901-1915, 2018 12.
Article in English | MEDLINE | ID: mdl-30079973

ABSTRACT

Mutations in TUBB4A have been identified to cause a wide phenotypic spectrum of diseases ranging from hereditary generalized dystonia with whispering dysphonia (DYT-TUBB4A) and hereditary spastic paraplegia (HSP) to leukodystrophy hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). TUBB4A encodes the brain-specific ß-tubulin isotype, ß-tubulin 4A. To elucidate the pathogenic mechanisms conferred by TUBB4A mutations leading to the different phenotypes, we functionally characterized three pathogenic TUBB4A variants (c.4C>G,p.R2G; c.745G>A,p.D249N; c.811G>A, p.A271T) as representatives of the mutational and disease spectrum) in human neuroblastoma cells and human induced pluripotent stem cell (iPSC)-derived neurons. We showed that mRNA stability was not affected by any of the TUBB4A variants. Although two mutations (p.R2G and p.D249N) are located at the α/ß-tubulin interdimer interface, we confirmed incorporation of all TUBB4A mutants into the microtubule network. However, we showed that the mutations p.D249N and p.A271T interfered with motor protein binding to microtubules and impaired neurite outgrowth and microtubule dynamics. Finally, TUBB4A mutations, as well as heterozygous knockout of TUBB4A, disrupted mitochondrial transport in iPSC-derived neurons. Taken together, our findings suggest that functional impairment of microtubule-associated transport is a shared pathogenic mechanism by which the TUBB4A mutations studied here cause a spectrum of diseases.


Subject(s)
Kinesins/metabolism , Mitochondria/metabolism , Mutation , Neuroblastoma/genetics , Neurons/cytology , Tubulin/genetics , CRISPR-Cas Systems , Cell Culture Techniques , Cell Line, Tumor , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Microtubules/metabolism , Neuroblastoma/metabolism , Neuronal Outgrowth , Neurons/metabolism , Phenotype , RNA Stability , RNA, Messenger/chemistry , Tubulin/chemistry , Tubulin/metabolism
6.
Mov Disord ; 33(5): 730-741, 2018 05.
Article in English | MEDLINE | ID: mdl-29644727

ABSTRACT

This first comprehensive MDSGene review is devoted to the 3 autosomal recessive Parkinson's disease forms: PARK-Parkin, PARK-PINK1, and PARK-DJ1. It followed MDSGene's standardized data extraction protocol and screened a total of 3652 citations and is based on fully curated phenotypic and genotypic data on >1100 patients with recessively inherited PD because of 221 different disease-causing mutations in Parkin, PINK1, or DJ1. All these data are also available in an easily searchable online database (www.mdsgene.org), which also provides descriptive summary statistics on phenotypic and genetic data. Despite the high degree of missingness of phenotypic features and unsystematic reporting of genotype data in the original literature, the present review recapitulates many of the previously described findings including early onset (median age at onset of ∼30 years for carriers of at least 2 mutations in any of the 3 genes) of an overall clinically typical form of PD with excellent treatment response, dystonia and dyskinesia being relatively common and cognitive decline relatively uncommon. However, when comparing actual data with common expert knowledge in previously published reviews, we detected several discrepancies. We conclude that systematic reporting of phenotypes is a pressing need in light of increasingly available molecular genetic testing and the emergence of first gene-specific therapies entering clinical trials. © 2018 International Parkinson and Movement Disorder Society.


Subject(s)
Genetic Association Studies , Parkinson Disease/genetics , Protein Deglycase DJ-1/genetics , Protein Kinases/genetics , Ubiquitin-Protein Ligases/genetics , Animals , Humans , Parkinson Disease/physiopathology
7.
Neuroscience ; 365: 137-145, 2017 Dec 04.
Article in English | MEDLINE | ID: mdl-28987508

ABSTRACT

The polypeptide ghrelin is an endogenous ligand at the growth hormone secretagogue receptor 1a. To ghrelin multiple functions have been ascribed including promotion of gastrointestinal motility. Postprandial ghrelin levels have been reported to be reduced in patients suffering from Parkinson disease (PD). Experimental studies revealed neuroprotective effects of ghrelin in different PD models. The purpose of the present study was (i) to further elucidate the mechanism underlying the neuroprotective action of ghrelin and (ii) to determine whether these effects occur with both the acylated and the unacylated form. The study was conducted in primary mesencephalic cultures treated with mitochondrial complex I and complex II inhibitors. We show that protective effects of ghrelin against complex I inhibition with MPP+ were independent of the acylation status of ghrelin, although acylated ghrelin appeared to be more potent. Protection by both forms was also observed when neurons were exposed to the complex II inhibitor 3-NP. Both forms led to higher oxygen consumption rates upon electron transport chain uncoupling, indicating that the two peptides may exert uncoupling effects themselves. We demonstrate that the rescue provided by ghrelin required calcium influx through L-type voltage-gated calcium channels. Whereas the protective effects of acylated ghrelin required receptor binding, effects of the unacylated form remained unaffected by treatment with a ghrelin receptor antagonist. Importantly, inhibition of ghrelin O-acyltransferase failed to reduce the activity of unacylated ghrelin. Overall, our data suggest that both acylated and unacylated ghrelin afford protection to dopamine neurons but through mechanisms that only partially overlap.


Subject(s)
Ghrelin/pharmacology , Mesencephalon/cytology , Neurons/drug effects , Neuroprotective Agents/pharmacology , 1-Methyl-4-phenylpyridinium/pharmacology , Acylation/physiology , Animals , Calcium Channel Blockers/pharmacology , Cells, Cultured , Dose-Response Relationship, Drug , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Nicardipine/pharmacology , Nitro Compounds/pharmacology , Peptides/pharmacology , Phosphopyruvate Hydratase/metabolism , Propionates/pharmacology , Rats , Rats, Wistar , Tyrosine 3-Monooxygenase/metabolism
8.
Parkinsonism Relat Disord ; 41: 118-120, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28655586

ABSTRACT

Mutations in TUBB4A have been identified to cause a wide phenotypic spectrum ranging from hereditary generalized dystonia with whispering dysphonia (DYT4) to the leukodystrophy hypomyelination syndrome with atrophy of the basal ganglia and cerebellum (H-ABC). To test for the contribution of TUBB4A mutations in different ethnicities (Spanish, Italian, Korean, Japanese), we screened 492 isolated dystonia cases for mutations in this gene and for the first time determined TUBB4A copy number variations in 336 dystonia patients. A potentially pathogenic rare 3bp-in-frame deletion was found in a patient with cervical dystonia but no copy number variations were detected in this study, suggesting that TUBB4A mutations exceedingly rarely contribute to the etiology of isolated dystonia.


Subject(s)
Dystonia/diagnosis , Dystonia/genetics , Mutation/genetics , Tubulin/genetics , Adult , Age of Onset , Aged , DNA Mutational Analysis , Dystonia/pathology , Female , Genetic Association Studies , Humans , International Cooperation , Male , Middle Aged
9.
J Mol Neurosci ; 62(1): 11-16, 2017 May.
Article in English | MEDLINE | ID: mdl-28299530

ABSTRACT

Mutations in the THAP1 gene encoding the transcription factor THAP1 have been shown to cause DYT6 dystonia. THAP1 contains a highly conserved THAP zinc finger at its N-terminal region which allows specific binding to its target sequences as well as a coiled-coil domain (amino acids 139-190) towards its C-terminus postulated as a protein-protein-binding motif. While several DYT6-causing mutations within the THAP domain were shown to decrease THAP1 activity in transcriptional regulation and DNA-binding, the role of mutations within the coiled-coil domain is rather unknown. Therefore, assigning a function to this domain may enable functional testing of mutations in this region. Notably, THAP1 and other THAP proteins form homodimers; however, the responsible domain has not been elucidated in detail. We show that the region of amino acids 139-185 is involved in formation of THAP1 homodimers by using yeast-two-hybrid, GST pull-down, and cross-linking assays. Surprisingly, all nine reported DYT6-causing missense mutations within this region had no effect on dimerization of THAP1 in GST pull-down and formaldehyde cross-linking assays. In conclusion, we demonstrated that a region of 47 amino acids is involved in THAP1 homodimerization but mutations in this region seem not to impair this mechanism.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , DNA-Binding Proteins/metabolism , Dystonia/genetics , Mutation , Nuclear Proteins/metabolism , Protein Multimerization , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , HEK293 Cells , Humans , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Protein Domains
11.
J Neurol Sci ; 356(1-2): 129-36, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26104567

ABSTRACT

Gaucher disease is an autosomal recessive disease, caused by a lack or functional deficiency of the lysosomal enzyme, glucocerebrosidase (GCase). Recently, mutations in the glucocerebrosidase gene (GBA) have been associated with Parkinson's disease (PD) and GBA mutations are now considered the most important genetic vulnerability factor for PD. In this study, we have investigated (i) in vivo whether inhibition of the enzyme glucosylceramide synthase by miglustat may protect C57Bl/6 mice against subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication and (ii) in vitro whether a decrease of GCase activity may render dopaminergic neurons susceptible to MPP(+) (1-methyl-4-phenylpyridinium) or alpha-synuclein (α-Syn) toxicity and amenable to miglustat treatment. We could demonstrate that reduction of glucocerebroside by inhibition of glucosylceramide synthase partially protects mice against MPTP-induced toxicity. Conversely, we could show that inhibition of GCase activity with conduritol-B-epoxide (CBE) enhances both α-Syn and MPP(+) induced toxicity in vitro. However, only CBE-induced enhancement of MPP(+) toxicity could be reversed by miglustat. Moreover, we were unable to reveal any alterations of complex I activity or cell respiration upon treatment with either CBE or miglustat. Our findings suggest that the reduction of GCase activity rather than an accumulation of glucocerebroside increases aSyn toxicity.


Subject(s)
Gaucher Disease/etiology , Mitochondrial Diseases/etiology , Parkinsonian Disorders/complications , 1-Deoxynojirimycin/analogs & derivatives , 1-Deoxynojirimycin/therapeutic use , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Animals , Brain/drug effects , Brain/metabolism , Brain/pathology , Cell Count , Cells, Cultured , Disease Models, Animal , Embryo, Mammalian , Gaucher Disease/drug therapy , Gaucher Disease/prevention & control , Glycoside Hydrolase Inhibitors/therapeutic use , Histocompatibility Antigens/metabolism , Inositol/analogs & derivatives , Inositol/metabolism , L-Lactate Dehydrogenase/metabolism , Male , Mesencephalon/metabolism , Mice , Mice, Inbred C57BL , Neurons/drug effects , Neurons/metabolism , Parkinsonian Disorders/prevention & control , Tyrosine 3-Monooxygenase/metabolism
12.
Brain ; 138(Pt 2): 398-413, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25524711

ABSTRACT

Neuroprotective approaches for central nervous system regeneration have not been successful in clinical practice so far and compounds that enhance remyelination are still not available for patients with multiple sclerosis. The objective of this study was to determine potential regenerative effects of the substance cytidine-5'-diphospho (CDP)-choline in two different murine animal models of multiple sclerosis. The effects of exogenously applied CDP-choline were tested in murine myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. In addition, the cuprizone-induced mouse model of de- and remyelination was used to specifically test the hypothesis that CDP-choline directly increases remyelination. We found that CDP-choline ameliorated the disease course of experimental autoimmune encephalomyelitis and exerted beneficial effects on myelin, oligodendrocytes and axons. After cuprizone-induced demyelination, CDP-choline effectively enhanced myelin regeneration and reversed motor coordination deficits. The increased remyelination arose from an increase in the numbers of proliferating oligodendrocyte precursor cells and oligodendrocytes. Further in vitro studies suggest that this process is regulated by protein kinase C. We thus identified a new mechanism to enhance central nervous system remyelination via the choline pathway. Due to its regenerative action combined with an excellent safety profile, CDP-choline could become a promising substance for patients with multiple sclerosis as an add-on therapy.


Subject(s)
Choline/metabolism , Demyelinating Diseases/chemically induced , Demyelinating Diseases/metabolism , Myelin Sheath/drug effects , Animals , Cell Proliferation/drug effects , Chelating Agents , Cuprizone , Cytidine Diphosphate Choline/pharmacology , Mice , Mice, Inbred C57BL , Neural Stem Cells/drug effects , Neuroglia/drug effects , Nootropic Agents/pharmacology , Oligodendroglia/drug effects , Rats , Rats, Sprague-Dawley , T-Lymphocytes/drug effects
13.
Hum Mutat ; 35(9): 1114-22, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24931141

ABSTRACT

A three-nucleotide (GAG) deletion (ΔE) in TorsinA (TOR1A) has been identified as the most common cause of dominantly inherited early-onset torsion dystonia (DYT1). TOR1A encodes a chaperone-like AAA+-protein localized in the endoplasmic reticulum. Currently, only three additional, likely mutations have been reported in single dystonia patients. Here, we report two new, putative TOR1A mutations (p.A14_P15del and p.E121K) that we examined functionally in comparison with wild-type (WT) protein and two known mutations (ΔE and p.R288Q). While inclusion formation is a characteristic feature for ΔE TOR1A, elevated levels of aggregates for other mutations were not observed when compared with WT TOR1A. WT and mutant TOR1A showed preferred degradation through the autophagy-lysosome pathway, which is most pronounced for p.A14_P15del, p.R288Q, and ΔE TOR1A. Notably, blocking of the autophagy pathway with bafilomycin resulted in a significant increase in inclusion formation in p.E121K TOR1A. In addition, all variants had an influence on protein stability. Although the p.A14_P15del mutation affects the proposed oligomerization domain of TOR1A, this mutation did not disturb the ability to dimerize. Our findings demonstrate functional changes for all four mutations on different levels. Thus, both diagnostic and research genetic screening of dystonia patients should not be limited to testing for the ∆E mutation.


Subject(s)
Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Phenotype , Adult , Age of Onset , Autophagy , Cell Line , Dystonia Musculorum Deformans/diagnosis , Dystonia Musculorum Deformans/genetics , Dystonia Musculorum Deformans/metabolism , Female , Gene Frequency , Humans , Intracellular Space/metabolism , Lysosomes/metabolism , Male , Middle Aged , Molecular Chaperones/chemistry , Mutation , Polymorphism, Single Nucleotide , Protein Multimerization , Protein Stability , Protein Transport , Proteolysis , Signal Transduction , Young Adult
14.
J Neurochem ; 127(6): 782-92, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23802648

ABSTRACT

The uricosuric agent probenecid is co-administered with the dopaminergic neurotoxin MPTP to produce a chronic mouse model of Parkinson's disease. It has been proposed that probenecid serves to elevate concentrations of MPTP in the brain by reducing renal elimination of the toxin. However, this mechanism has never been formally demonstrated to date and is questioned by our previous data showing that intracerebral concentrations of MPP(+), the active metabolite of MPTP, are not modified by co-injection of probenecid. In this study, we investigated the potentiating effects of probenecid in vivo and in vitro arguing against the possibility of altered metabolism or impaired renal elimination of MPTP. We find that probenecid (i) is toxic in itself to several neuronal populations apart from dopaminergic neurons, and (ii) that it also potentiates the effects of other mitochondrial complex I inhibitors such as rotenone. On a mechanistic level, we show that probenecid is able to lower intracellular ATP concentrations and that its toxic action on neuronal cells can be reversed by extracellular ATP. Probenecid can potentiate the effect of mitochondrial toxins due to its impact on ATP metabolism and could therefore be useful to model atypical parkinsonian syndromes.


Subject(s)
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine , 1-Methyl-4-phenylpyridinium/metabolism , Dopamine Agents/toxicity , Neurotoxins/toxicity , Parkinson Disease/pathology , Probenecid/toxicity , Uricosuric Agents/toxicity , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/metabolism , Animals , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Corpus Striatum/pathology , Disease Models, Animal , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Drug Synergism , Electron Transport Complex I/antagonists & inhibitors , Energy Metabolism , Mice , Parkinson Disease/etiology , Parkinson Disease/metabolism , Rotenone/toxicity
15.
PLoS One ; 8(4): e61700, 2013.
Article in English | MEDLINE | ID: mdl-23637888

ABSTRACT

Bee venom has recently been suggested to possess beneficial effects in the treatment of Parkinson disease (PD). For instance, it has been observed that bilateral acupoint stimulation of lower hind limbs with bee venom was protective in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. In particular, a specific component of bee venom, apamin, has previously been shown to have protective effects on dopaminergic neurons in vitro. However, no information regarding a potential protective action of apamin in animal models of PD is available to date. The specific goals of the present study were to (i) establish that the protective effect of bee venom for dopaminergic neurons is not restricted to acupoint stimulation, but can also be observed using a more conventional mode of administration and to (ii) demonstrate that apamin can mimic the protective effects of a bee venom treatment on dopaminergic neurons. Using the chronic mouse model of MPTP/probenecid, we show that bee venom provides sustained protection in an animal model that mimics the chronic degenerative process of PD. Apamin, however, reproduced these protective effects only partially, suggesting that other components of bee venom enhance the protective action of the peptide.


Subject(s)
Apamin/pharmacology , Bee Venoms/pharmacology , Dopaminergic Neurons/drug effects , Neuroprotective Agents/pharmacology , Parkinson Disease/prevention & control , Acupuncture Points , Animals , Behavior, Animal/drug effects , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Disease Models, Animal , Exploratory Behavior/drug effects , MPTP Poisoning/prevention & control , Male , Mice , Mice, Inbred C57BL , Tumor Necrosis Factor-alpha/metabolism
16.
JAMA Neurol ; 70(6): 783-7, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23700088

ABSTRACT

IMPORTANCE: We sought to unravel the genetic cause in a consanguineous Pakistani family with a complex neurological phenotype. OBSERVATIONS: Neurological and ophthalmological examination, including videotaping and fundoscopy, and genetic investigations, including homozygosity mapping and exome sequencing, were performed at the University of the Punjab and the University of Lübeck. Participants included 2 severely affected cousins from consanguineous parents, 10 of their reportedly unaffected relatives, and 342 Pakistani controls. Motor symptoms in the 2 patients started at the age of 3 to 4 years and included chorea, cerebellar ataxia, dystonia, and pyramidal tract signs. Genome-wide genotyping delineated 2 regions of homozygosity on chromosomes 13q12.11 to 13q12.13 and 19q12 to 19q13.41. Exome sequencing revealed 2 rare, homozygous variants (c.32 T>A [p.L11Q] in OPA3 and c.941 C>G [p.A314G] in TSHZ3) that segregated with the disease. Only the OPA3 variant was absent in the control subjects and predicted to be damaging. Subsequent ophthalmological assessment revealed bilateral optic atrophy in both patients. CONCLUSIONS AND RELEVANCE: Mutations in OPA3 have been reported in Costeff optic atrophy syndrome. We identify a novel missense mutation in OPA3 as the cause of a complex neurological disorder, expanding the OPA3 -linked phenotype by early-onset pyramidal tract signs and marked lower limb dystonia. Investigation of optic atrophy was initiated only after genetic analysis, a phenomenon referred to as reverse phenotyping.


Subject(s)
Chorea/diagnosis , Chorea/genetics , Exome/genetics , Metabolism, Inborn Errors/diagnosis , Metabolism, Inborn Errors/genetics , Mutation, Missense/genetics , Optic Atrophy/diagnosis , Optic Atrophy/genetics , Phenotype , Proteins/genetics , Sequence Analysis, DNA/methods , Spastic Paraplegia, Hereditary/diagnosis , Spastic Paraplegia, Hereditary/genetics , Adolescent , Child , Dystonia/diagnosis , Dystonia/genetics , Female , Genome-Wide Association Study/methods , Humans , Male , Nervous System Diseases/diagnosis , Nervous System Diseases/genetics , Pedigree
17.
Neurotox Res ; 24(2): 244-50, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23392957

ABSTRACT

Cuprizone [bis(cyclohexylidenehydrazide)]-induced toxic demyelination is an experimental animal model commonly used to study de- and remyelination in the central nervous system. In this model, mice are fed with the copper chelator cuprizone which leads to oligodendrocyte death with subsequent demyelination. The underlying mechanisms of cuprizone-induced oligodendrocyte death are still unknown, and appropriate in vitro investigations to study these mechanisms are not available. Thus, we studied cuprizone effects on rat primary glial cell cultures and on the neuroblastoma cell line SH-SY5Y. Treatment of cells with different concentrations of cuprizone failed to show effects on the proliferation and survival of SH-SY5Y cells, microglia, astrocytes, and oligodendrocyte precursor cells (OPC). In contrast, differentiated mature oligodendrocytes (OL) were found to be significantly affected by cuprizone treatment. This was accompanied by a reduced mitochondrial potential in cuprizone-treated OL. These results demonstrate that the main toxic target for cuprizone is mature OL, whilst other glial cells including OPC are not or only marginally affected. This explains the selective demyelination induced by cuprizone in vivo.


Subject(s)
Cell Differentiation/drug effects , Chelating Agents/toxicity , Cuprizone/toxicity , Oligodendroglia/drug effects , Oligodendroglia/pathology , Animals , Animals, Newborn , Cell Differentiation/physiology , Cell Line, Tumor , Cells, Cultured , Dose-Response Relationship, Drug , Humans , Rats , Rats, Sprague-Dawley
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