Franca Vulinovic

Cuprizone [Bis(Cyclohexylidenehydrazide)] is Selectively Toxic for Mature Oligodendrocytes

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.

Bee Venom and Its Component Apamin as Neuroprotective Agents in a Parkinson Disease Mouse Model

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.

A Novel OPA3 Mutation Revealed by Exome Sequencing: An Example of Reverse Phenotyping

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.

Glucocerebrosidase deficiency and mitochondrial impairment in experimental Parkinson disease

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 Parkinsons 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.

Unraveling cellular phenotypes of novel TorsinA/TOR1A mutations.

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.

Probenecid potentiates MPTP/MPP+ toxicity by interference with cellular energy metabolism.

The uricosuric agent probenecid is co‐administered with the dopaminergic neurotoxin MPTP to produce a chronic mouse model of Parkinsons 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.

Genotype-Phenotype Relations for the Parkinson's Disease Genes Parkin, PINK1, DJ1: MDSGene Systematic Review: MDSGene REVIEW: Parkin, PINK1, DJ1

This first comprehensive MDSGene review is devoted to the 3 autosomal recessive Parkinsons disease forms: PARK‐Parkin, PARK‐PINK1, and PARK‐DJ1. It followed MDSGenes 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.

Acylated and unacylated ghrelin confer neuroprotection to mesencephalic neurons

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.

Mutations in TUBB4A and spastic paraplegia

We note with great interest the article published by Kancheva and colleagues reporting a TUBB4A mutation in a family with complicated hereditary spastic paraplegia (HSP). Whole exome sequencing was performed in a consanguineous family with five affected children and a presumed homozygous mutation in the context of recessive inheritance. Instead, the authors identified a novel heterozygous c.568C>T (p.H190Y) TUBB4A mutation originating de novo in the asymptomatic, mosaic mother. The clinical phenotype in this family included early-onset spastic paraplegia, ataxia, and brain hypomyelination. Complicated HSP, the authors concluded, could be added to the spectrum of TUBB4A-associated neurological disorders. Mutations in this gene have previously been implicated in hereditary whispering dysphonia (DYT4 dystonia), hypomyelination with atrophy of the basal ganglia and cerebellum, or isolated hypomyelination. To further explore the relationship between TUBB4A mutations and HSP, Sanger sequencing of the TUBB4A gene was performed as previously described in 76 probands with HSP recruited from the Royal North Shore Hospital, Sydney, Australia. All patients had undergone brain magnetic resonance imaging (MRI), and none had prominent white matter changes consistent with a leukodystrophy, in keeping with the recommendation that alternative conditions such as a leukodystrophy should be excluded before the diagnosis of HSP is made. The study was approved by the local institutional ethics committees, and written informed consent was obtained from each subject. The characteristics of the patient sample are included in Table 1. Patients with HSP had undergone genetic testing as reported, and patients with known genetic forms of HSP also were included to investigate for a confounding effect from TUBB4A variants. Screening of the HSP samples did not reveal any pathogenic variants in the TUBB4A gene. Our screening study suggests that TUBB4A mutations are an uncommon cause of HSP in the absence of hypomyelination on MRI. Thus, whether specific screening of TUBB4A should be undertaken on a clinical basis for HSP without white matter abnormalities on MRI is uncertain. Although some white matter lesions can be a feature of HSP, the MRI findings in this family were described as “confluent.” One can argue that the neuroimaging findings in this situation at least partially fit with the pattern of a hypomyelinating leukodystrophy, rather than HSP per se. This disorder may be part of the spectrum of hypomyelination with atrophy of the basal ganglia and cerebellum syndrome, which is known to have a broad range of clinical manifestations, including spasticity. This underscores the difficulty in determining whether a disorder should be categorized as “hereditary spastic paraplegia,” given the marked phenotypic overlap between HSP and other disorders. Hypomyelination is appearing to be the core radiological feature of TUBB4A-related disorders and is the main clinical indication for directed genetic testing for a TUBB4A mutation. Despite these caveats, microtubule dysfunction may be a shared pathogenic theme between the most common form of autosomal dominant HSP (SPG4, caused by mutations in the microtubule-severing protein spastin) and TUBB4A-related disorders. Further studies investigating the molecular mechanisms underlying TUBB4A-related disorders are now required.

Screening study of TUBB4A in isolated dystonia

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.