Slobodanka Vukosavic

Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) damages dopaminergic neurons as seen in Parkinson disease. Here we show that after administration of MPTP to mice, there was a robust gliosis in the substantia nigra pars compacta associated with significant upregulation of inducible nitric oxide synthase (iNOS). These changes preceded or paralleled MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking the iNOS gene were significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that iNOS is important in the MPTP neurotoxic process and indicates that inhibitors of iNOS may provide protective benefit in the treatment of Parkinson disease.

α-Synuclein Up-Regulation in Substantia Nigra Dopaminergic Neurons Following Administration of the Parkinsonian Toxin MPTP

Abstract: Mutations in α‐synuclein cause a form of familial Parkinson’s disease (PD), and wild‐type α‐synuclein is a major component of the intraneuronal inclusions called Lewy bodies, a pathological hallmark of PD. These observations suggest a pathogenic role for α‐synuclein in PD. Thus far, however, little is known about the importance of α‐synuclein in the nigral dopaminergic pathway in either normal or pathological situations. Herein, we studied this question by assessing the expression of synuclein‐1, the rodent homologue of human α‐synuclein, in both normal and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐intoxicated mice. In normal mice, detectable levels of synuclein mRNA and protein were seen in all brain regions studied and especially in ventral midbrain. In the latter, there was a dense synuclein‐positive nerve fiber network, which predominated over the substantia nigra, and only few scattered synuclein‐positive neurons. After a regimen of MPTP that kills dopaminergic neurons by apoptosis, synuclein mRNA and protein levels were increased significantly in midbrain extracts; the time course of these changes paralleled that of MPTP‐induced dopaminergic neurodegeneration. In these MPTP‐injected mice, there was also a dramatic increase in the number of synuclein‐immunoreactive neurons exclusively in the substantia nigra pars compacta; all synuclein‐positive neurons were tyrosine hydroxylase‐positive, but none coexpressed apoptotic features. These data indicate that synuclein is highly expressed in the nigrostriatal pathway of normal mice and that it is up‐regulated following MPTP‐induced injury. In light of the synuclein alterations, it can be suggested that, by targeting this protein, one may modulate MPTP neurotoxicity and, consequently, open new therapeutic avenues for PD.

Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages dopaminergic neurons in the substantia nigra pars compacta (SNpc) as seen in Parkinsons disease. Here, we show that the pro-apoptotic protein Bax is highly expressed in the SNpc and that its ablation attenuates SNpc developmental neuronal apoptosis. In adult mice, there is an up-regulation of Bax in the SNpc after MPTP administration and a decrease in Bcl-2. These changes parallel MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking Bax are significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that Bax plays a critical role in the MPTP neurotoxic process and suggests that targeting Bax may provide protective benefit in the treatment of Parkinsons disease.

Inducible Nitric Oxide Synthase Up-Regulation in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

Abstract: Mutations in copper/zinc superoxide dismutase (SOD1) are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS‐like syndrome. Here we show that, during the course of the disease, the spinal cord of transgenic mice expressing mutant SOD1 (mSOD1) is the site not only of a progressive loss of motor neurons, but also of a dramatic gliosis characterized by reactive astrocytes and activated microglial cells. These changes are absent from the spinal cord of age‐matched transgenic mice expressing normal SOD1 and of wild‐type mice. We also demonstrate that, during the course of the disease, the expression of inducible nitric oxide synthase (iNOS) increases. In both early symptomatic and end‐stage transgenic mSOD1 mice, numerous cells with the appearance of glial cells are strongly iNOS‐immunoreactive. In addition, iNOS mRNA level and catalytic activity are increased significantly in the spinal cord of these transgenic mSOD1 mice. None of these alterations are seen in the cerebellum of these animals, a region un‐affected by mSOD1. Similarly, no up‐regulation of iNOS is detected in the spinal cord of age‐matched transgenic mice expressing normal SOD1 or of wild‐type mice. The time course of the spinal cord gliosis and iNOS up‐regulation parallels that of motor neuronal loss in transgenic mSOD1 mice. Neuronal nitric oxide synthase expression is only seen in neurons in the spinal cord of transgenic mSOD1 mice, regardless of the stage of the disease, and of age‐matched transgenic mice expressing normal SOD1 and wild‐type mice. Collectively, these data suggest that the observed alterations do not initiate the death of motor neurons, but may contribute to the propagation of the neurodegenerative process. Furthermore, the up‐regulation of iNOS, which in turn may stimulate the production of nitric oxide, provides further support to the presumed deleterious role of nitric oxide in the pathogenesis of ALS. This observation also suggests that iNOS may represent a valuable target for the development of new therapeutic avenues for ALS.

Oxidative post-translational modifications of α-synuclein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease

Structural and functional alterations of α‐synuclein is a presumed culprit in the demise of dopaminergic neurons in Parkinsons disease (PD). α‐Synuclein mutations are found in familial but not in sporadic PD, raising the hypothesis that effects similar to those of familial PD‐linked α‐synuclein mutations may be achieved by oxidative post‐translational modifications. Here, we show that wild‐type α‐synuclein is a selective target for nitration following peroxynitrite exposure of stably transfected HEK293 cells. Nitration of α‐synuclein also occurs in the mouse striatum and ventral midbrain following administration of the parkinsonian neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). Conversely, β‐synuclein and synaptophysin were not nitrated in MPTP‐intoxicated mice. Our data demonstrate that α‐synuclein is a target for tyrosine nitration, which, by disrupting its biophysical properties, may be relevant to the putative role of α‐synuclein in the neurodegeneration associated with MPTP toxicity and with PD.

Bax and Bcl‐2 Interaction in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

Abstract : It has been proposed that mutations in copper/zinc‐superoxide dismutase (SOD1), the only proven cause of amyotrophic lateral sclerosis (ALS), induce the disease by a toxic property that promotes apoptosis. Consistent with this, we have demonstrated that overexpression of Bcl‐2, a protein that inhibits apoptosis, attenuates neurodegeneration produced by the familial ALS‐linked SOD1 mutant G93A (mSOD1). Herein, we assessed the status of key members of the Bcl‐2 family in the spinal cord of transgenic mSOD1 mice at different stages of the disease. In asymptomatic transgenic mSOD1 mice, expression of Bcl‐2, Bcl‐XL, Bad, and Bax does not differ from that in nontransgenic mice. In contrast, in symptomatic mice, expression of Bcl‐2 and Bcl‐XL, which inhibit apoptosis, is reduced, whereas expression of Bad and Bax, which stimulate apoptosis, is increased. These alterations are specific to affected brain regions and are caused by the mutant and not by the normal SOD1 enzyme. Relevant to the neuroprotective effects of Bcl‐2 in transgenic mSOD1 mice, overexpression of Bcl‐2 increases the formation of Bcl‐2 : Bax heterodimers, which abolish the Bax proapoptotic property. This study demonstrates significant alterations in the expression of key members of the Bcl‐2 family associated with mSOD1 deleterious effects. That these changes contribute to the neurodegenerative process in this model of ALS is supported by our observations in double transgenic mSOD1/Bcl‐2 mice in which the pernicious increase of Bax is tempered by an increase in formation of Bcl‐2 : Bax heterodimers. Based on these findings, it may be concluded that Bcl‐2 family members appear as invaluable targets for the development of new neuroprotective therapies in ALS.

GAP-43 mRNA expression in early development of human nervous system

The temporal and spatial distribution of GAP-43 mRNA in early human development, from 6 to 23 gestational weeks (g.w.), was examined by in situ hybridization histochemistry. GAP-43 mRNA was expressed as early as 6 g.w. in all regions of developing nervous system, the spinal cord, brainstem, cerebellum, diencephalic and telencephalic regions. Although the pronounced level of expression persisted during the entire examined period, the intensity of expression varied along the spatial axis over time. Analysis at the cellular level revealed that early on in development (6 g.w.) GAP-43 mRNA was expressed in the entire neuroblast population. With the onset of differentiation, at 13-23 g.w., GAP-43 mRNA expression had switched to the neurons that are in the process outgrowth. The highest level of GAP-43 mRNA expression was localized in the regions consisting of differentiating neurons, such as the cortical plate and intermediate zone of the telencephalic wall, and several delineated subcortical and thalamic nuclei. The spatial and temporal pattern of GAP-43 mRNA expression obtained suggests a possible dual role of GAP-43 in the development of the human nervous system: in the embryonic brain it could be involved in fundamental processes underlying cell proliferation; in the fetal brain its expression is specifically correlated with differentiation and the outgrowth of axons.

Frequency analysis and clinical characterization of different types of spinocerebellar ataxia in Serbian patients

The relative frequencies of different spinocerebellar ataxias (SCAs) vary widely among different ethnic groups, presumably due to a founder effect. We investigated the relative prevalence of SCA1–3, 6–8, 12, 17; dentate–rubro–pallidoluysian atrophy; and Friedreichs ataxia (FRDA) in Serbian patients with adult‐onset (>20 years of age) hereditary and sporadic SCAs, and compared clinical features of patients with genetically confirmed SCAs. A total of 108 patients from 54 families (38 apparently dominant [ADCA] and 16 apparently recessive) with adult‐onset hereditary ataxia and 75 apparently sporadic patients were assessed. Of 38 families with ADCA, 13 (34%) were positive for an expansion in an SCA1 and 5 families (13%) for an expansion in an SCA2 allele. In 20 families (53%), no expansions have been identified in any of the analyzed genes. Gaze palsy, spasticity, and hyperreflexia were significantly more common in SCA1, whereas slow saccades, hypotonia, hyporeflexia, and dystonia prevailed in SCA2 patients. Among the 16 families with an apparently recessive mode of ataxia inheritance, 4 (25%) were identified as having the FRDA mutation. Ataxia‐causing mutations were identified in 8 (10.6%) of patients with apparently sporadic adult‐onset ataxia.

CTG repeat polymorphism in DMPK gene in healthy Yugoslav population.

Objectives– Myotonic dystrophy type 1 (DM1) is caused by large expansions of cytosine‐thymine‐guanine (CTG)‐repeats in myotonic dystrophy protein kinase (DMPK)‐gene. This gene is highly polymorphic in healthy individuals. It has been proposed that expanded alleles originated from the group of large sized normal alleles. If this is correct, one should expect a positive correlation between the frequency of large sized normal alleles and a prevalence of this disorder in a population. In this paper we determined the distribution of alleles of DMPK gene in healthy Yugoslav population. Material and methods– A sample of 235 healthy individuals of Yugoslav origin have been genotyped for the alleles of DMPK locus. Results– We found 22 different alleles, ranging in size from 5 to 29 repeats. Among 470 chromosomes studied, 41 chromosomes had more than 18 repeats (8.72%). Conclusions– Relatively high frequency of large sized normal alleles found in our population, suggest that prevalence of DM1 in Yugoslavia should not be different from the prevalence in other European populations.

Yugoslav HD phenocopies analyzed on the presence of mutations in PrP, ferritin, and Jp-3 genes.

Huntington disease (HD) is a well-defined autosomal dominant neurodegenerative disease caused by CAG repeat expansions in HD gene. There are a significant number of HD cases where this mutation was not found and such cases are named HD-like phenotype (HDL). This article reports 48 patients with HDL phenotype. Patients were analyzed on the presence of mutations in prion (PrP), ferritin and junctophilin-3 (JP-3) genes. None of the patients showed the presence of the mutation in analyzed genes. This could suggest that there is some other gene/genes where the mutation can cause the disease with clinical features of HD.