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M Ruberg

Publications and source records attributed to M Ruberg.

At least 37 records · Page 2Linked to original sources
PubMed2000

Evolution of changes in neuronal activity in the subthalamic nucleus of rats with unilateral lesion of the substantia nigra assessed by metabolic and electrophysiological measurements.

Cellular expression of cytochrome oxidase subunit I (COI) mRNA has recently been used as a metabolic marker for neuronal activity to study the functional changes in the subthalamic nucleus (STN) in parkinsonism. The previous experimental studies have been performed when the pathological state was stabilized at a maximal level. In order to determine the evolution of changes in neuronal activity in the STN after nigrostriatal denervation, we analysed by in situ hybridization the cellular expression of COI mRNA in the subthalamic neurons at different times, from 6 h to 14 days, after unilateral intranigral microinjection of 6-hydroxydopamine (6-OHDA) in rats. In parallel, the time-dependent changes of the unit neuronal activity of subthalamic neurons have been recorded. Levels of COI mRNA increased by 41% in subthalamic neurons from 24 h after 6-OHDA intoxication, to 14 days (+26%). Similarly, electrical activity started to increase slightly 24 h after lesion (+20%) and remained significantly higher at 14 days after the lesion (+189%). Changes in neuronal mean discharge rate were associated with changes in the pattern of spiking activity, from a regular firing pattern to an irregular one with a high bursting activity. These results show that: (i) the hyperactivity of the STN represents a very early phenomenon in the physiopathology of parkinsonian syndromes; and (ii) that changes in COI mRNA expression slightly precede changes in electrical neuronal activity.

Animals↗
PubMed2000

Distribution of ataxin-7 in normal human brain and retina.

Spinocerebellar ataxia 7 (SCA7) is a neurodegenerative disease caused by the expansion of a CAG repeat encoding a polyglutamine tract in the protein ataxin-7. We developed antibodies directed against two different parts of the ataxin-7 protein and studied its distribution in brain and peripheral tissue from healthy subjects. Normal ataxin-7 was widely expressed in brain, retina and peripheral tissues, including striated muscle, testis and thyroid gland. In the brain, expression of ataxin-7 was not limited to areas in which neurones degenerate, and the level of expression was not related to the severity of neuronal loss. Immunoreactivity was low in some vulnerable populations of neurones, such as Purkinje cells. In neurones, ataxin-7 was found in the cell bodies and in processes. Nuclear labelling was also observed in some neurones, but was not related to the distribution of intranuclear inclusions observed in an SCA7 patient. In this patient, the proportion of neurones with nuclear labelling was higher, on average, in regions with neuronal loss. Double immunolabelling coupled with confocal microscopy showed that ataxin-7 colocalized with BiP, a marker of the endoplasmic reticulum, but not with markers of mitochondria or the trans-Golgi network.

Adult↗
PubMed1999

Survival factors promote BDNF protein expression in mesencephalic dopaminergic neurons.

The aim of the present study was to characterize signals and/or molecules which regulate BDNF protein expression in mesencephalic dopaminergic neurons. Treatment of mesencephalic cells with dibutyryl-cAMP (dbcAMP), 30 mM K+ (HK+), or the antimitotic ara-C not only promoted the survival of tyrosine hydroxylase expressing (TH+) neurons but also increased the proportion of these cells that were immunopositive for BDNF. The effect of dbcAMP was mimicked by forskolin, a known adenylate cyclase activator. It was not antagonized by PKA inhibitors. Increases in BDNF expression resulting from K+-induced depolarization or ara-C treatment were abolished, respectively, by the L-type calcium channel blocker nifedipine and the deoxynucleotide dCTP. BDNF added exogenously to the cultures improved the survival of TH+ neurons. However, induction of the expression of BDNF in these neurons by dbcAMP, HK+ or ara-C was apparently not responsible for survival promotion by these factors.

Animals↗
PubMed1999

Mechanisms of apoptosis in PC12 cells irreversibly differentiated with nerve growth factor and cyclic AMP.

PC12 cells treated with cAMP become irreversibly differentiated and die by apoptosis when deprived of trophic support, instead of dedifferentiating and reentering the cell cycle. To approach the molecular mechanism underlying the cAMP-induced switch from differentiation/proliferation to apoptosis, we compared three sequential markers of a candidate apoptogenic signal transduction pathway (ceramide, free radicals and NF-kappaB), after trophic factor withdrawal in PC12 cells before and after irreversible differentiation. Serum withdrawal increased ceramide and free radical production regardless of the state of differentiation of the cells. It was followed by cell death, however, only in the absence of NGF and/or cAMP, and was no longer required for apoptosis in NGF/cAMP-differentiated cells. NGF and cAMP withdrawal sufficed. NF-kappaB was activated by NGF withdrawal in reversibly differentiated PC12 cells during dedifferentiation and reentry into the cell cycle, whereas in NGF/cAMP-differentiated cells, it was activated, at a late stage of the apoptotic process, concomitantly with cell death. These results show that a serum factor inhibits ceramide-dependent apoptosis upstream of ceramide and free radical production, whereas NGF- and cAMP-dependent mechanisms inhibit apoptosis either downstream or parallel to these events. After terminal differentiation by cAMP, apoptosis appears to be initiated from the second site, consistent with the serum independence of these cells and the absence of ceramide and free radical production when the NGF/cAMP-dependent inhibitions are released. The differential regulation of NF-kappaB appears to be an important step in the switch from mitosis to apoptosis that occurs during irreversible differentiation of PC12 cells by cAMP.

Animals↗
PubMed1999

Adenosine prevents the death of mesencephalic dopaminergic neurons by a mechanism that involves astrocytes.

The purinergic nucleoside adenosine effectively prevented the death of dopaminergic neurons that occurs spontaneously and progressively in cultures of rat mesencephalon. Adenosine also significantly increased dopamine uptake, attesting to the state of differentiation and functional integrity of the neurons that were rescued. The effects of adenosine were (a) specific to the dopaminergic neurons in these cultures, (b) long-lived, (c) still observed when the treatment was delayed after plating, (d) potentiated by inhibition of adenosine deaminase, and (e) abolished when this enzyme was added in excess to the culture medium. The action of adenosine was mimicked by 5'-(N-ethylcarboxamido)adenosine and dibutyryl-cyclic AMP, but not by CGS-21680, suggesting the possible involvement of A2B subtype purinergic receptors. ATP was also neuroprotective, but its action resulted principally from conversion to adenosine by ectonucleotidases. Several anticancer drugs, including cytosine arabinoside, have been shown previously to prevent apoptosis in cultured dopaminergic neurons by a mechanism that requires the inhibition of proliferating astrocytes. In the presence of adenosine, astrocytes were more differentiated, and their proliferation rate was significantly reduced, suggesting that the neurotrophic effect of the adenine nucleoside resulted also from the repression of the astroglial cells. We did not find evidence of a trophic intermediary in adenosine-treated cultures, however, leading to the hypothesis that limitation of astrocyte replication in itself and/or ensuing changes in the glial phenotype were crucial. Our results suggest that molecules that modulate adenine nucleotide/nucleoside release may be useful for the treatment of chronic neurodegenerative conditions affecting dopaminergic neurons, such as Parkinson's disease.

Adenosine↗
PubMed1999

Systemic administration of NMDA and AMPA receptor antagonists reverses the neurochemical changes induced by nigrostriatal denervation in basal ganglia.

In Parkinson's disease, nigrostriatal denervation leads to an overactivity of the subthalamic nucleus and its target areas, which is responsible of the clinical manifestations of the disease. Because the subthalamic nucleus uses glutamate as neurotransmitter and is innervated by glutamatergic fibers, pharmacological blockade of glutamate transmission might be expected to restore the cascade of neurochemical changes induced by a dopaminergic denervation within the basal ganglia. To test this hypothesis, two types of glutamate antagonists, the NMDA receptor antagonist MK-801 and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist LY293558, were administered systemically, either alone or in combination with L-DOPA, in rats with a unilateral 6-hydroxydopamine lesion of the nigrostriatal dopamine pathway. The effect of treatment was assessed neurochemically by analyzing at the cellular level the functional activity of basal ganglia output structures and the subthalamic nucleus using the expression levels of the mRNAs coding for glutamic acid decarboxylase and cytochrome oxidase, respectively, as molecular markers of neuronal activity. The present study shows that treatment with glutamate antagonists, and particularly with AMPA antagonists, alone or in combination with L-DOPA, reverses the overactivity of the subthalamic nucleus and its target areas induced by nigrostriatal denervation. These results furnish the neurochemical basis for the potential use of glutamate antagonists as therapeutic agents in Parkinson's disease.

Animals↗
PubMed1999

Homologous DNA exchanges in humans can be explained by the yeast double-strand break repair model: a study of 17p11.2 rearrangements associated with CMT1A and HNPP.

Rearrangements in 17p11.2, responsible for the 1.5 Mb duplications and deletions associated, respectively, with autosomal dominant Charcot-Marie-Tooth type 1A disease (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are a suitable model for studying human recombination. Rearrangements in 17p11.2 are caused by unequal crossing-over between two homologous 24 kb sequences, the CMT1A-REPs, that flank the disease locus and occur in most cases within a 1.7 kb hotspot. We sequenced this hotspot in 28 de novo patients (25 CMT1A and three HNPP), in order to localize precisely, at the DNA sequence level, the crossing-overs. We show that some chimeric CMT1A-REPs in de novo patients (10/28) present conversion of DNA segments associated with the crossing-over. These rearrangements can be explained by the double-strand break (DSB) repair model described in yeast. Fine mapping of the de novo rearrangements provided evidence that the successive steps of this model, heteroduplex DNA formation, mismatch correction and gene conversion, occurred in patients. Furthermore, the model explains 17p11.2 recombinations between chromosome homologues as well as between sister chromatids. In addition, defective mismatch repair of the heteroduplex DNA, observed in two patients, resulted in two heterozygous chimeric CMT1A-REPs which can be explained, as in yeast, by post-meiotic segregation. This work supports the hypothesis that the DSB repair model of DNA exchange may apply universally from yeasts to humans.

Charcot-Marie-Tooth Disease↗
PubMed1998

Fine mapping of de novo CMT1A and HNPP rearrangements within CMT1A-REPs evidences two distinct sex-dependent mechanisms and candidate sequences involved in recombination.

The molecular mechanism resulting in the duplication or deletion of a 1.5 Mb region of 17p11.2-p12, associated, respectively, with Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), has been proposed to be an unequal crossing-over during meiosis between the two chromosome 17 homologues generated by misalignment of the proximal and distal CMT1A-REP repeats, two homologous sequences flanking the 1.5 Mb CMT1A/HNPP monomer unit. In a recent study of a large series of de novo cases of CMT1A and HNPP, two distinct sex-dependent mechanisms were identified. Rearrangements of paternal origin, essentially duplications, were indeed generated by unequal meiotic crossing-over between the two chromosome 17 homologues, but duplications and deletions of maternal origin resulted from an intrachromosomal process, either unequal sister chromatid exchange or, in the case of deletion, excision of an intrachromatidal loop. In order to determine how these recombinations occur, 24 de novo crossover breakpoints were localized within the 1.7 kb rearrangement hot spot by comparing the sequences of the parental CMT1A-REPs with the chimeric copy in affected offspring. Nineteen out of 21 paternal crossovers were found in a 741 bp hot spot. All the breakpoints of maternal origin (n = 3), however, were located outside this interval, but in closely flanking sequences, supporting the hypothesis that two distinct sex-dependent mechanisms are involved. Several putative recombination promoting sequences in the hot spot, which are rare or absent in the surrounding 7.8 kb, were identified.

Charcot-Marie-Tooth Disease↗
PubMed1998

De novo expansion of intermediate alleles in spinocerebellar ataxia 7.

Spinocerebellar ataxia 7 (SCA7) is the eighth neurodegenerative disorder caused by a translated CAG repeat expansion. Normal SCA7 alleles carry from four to 35 CAG repeats, whereas pathological alleles carry from 37 to approximately 200. Intermediate alleles (IAs), with 28-35 repeats in the SCA7 gene are exceedingly rare in the general population and are not associated with the SCA7 phenotype, although they have been found among relatives of four SCA7 families. In two of these families, IAs bearing 35 and 28 CAG repeats gave rise, during paternal transmission, to SCA7 expansions of 57 and 47 repeats, respectively, that were confirmed by haplotype reconstructions in one case and by inference in the other. Furthermore, the four haplotypes segregating with IAs were identical to the expanded alleles in each kindred, but differed among the families, indicating multiple origins of the SCA7 mutation in these families with different geographical origins. Our results provide the first evidence of de novo SCA7 expansions from IAs that are not associated with the phenotype but can expand to the pathological range during some paternal transmissions. IAs that segregate in unaffected branches of the pedigrees might, therefore, constitute a reservoir for future de novo mutations that occur in a recurrent but random manner. This would explain the persistence of the disease in spite of the great anticipation (approximately 20 years/generation) characteristic of SCA7. So far, de novo expansions among the disorders caused by polyglutamine repeats have only been demonstrated in Huntington's disease.

Alleles↗
PubMed1997

Nuclear translocation of NF-kappaB is increased in dopaminergic neurons of patients with parkinson disease.

Evidence from postmortem studies suggest an involvement of oxidative stress in the degeneration of dopaminergic neurons in Parkinson disease (PD) that have recently been shown to die by apoptosis, but the relationship between oxidative stress and apoptosis has not yet been elucidated. Activation of the transcription factor NF-kappaB is associated with oxidative stress-induced apoptosis in several nonneuronal in vitro models. To investigate whether it may play a role in PD, we looked for the translocation of NF-kappaB from the cytoplasm to the nucleus, evidence of its activation, in melanized neurons in the mesencephalon of postmortem human brain from five patients with idiopathic PD and seven matched control subjects. In PD patients, the proportion of dopaminergic neurons with immunoreactive NF-kappaB in their nuclei was more than 70-fold that in control subjects. A possible relationship between the nuclear localization of NF-kappaB in mesencephalic neurons of PD patients and oxidative stress in such neurons has been shown in vitro with primary cultures of rat mesencephalon, where translocation of NF-kappaB is preceded by a transient production of free radicals during apoptosis induced by activation of the sphingomyelin-dependent signaling pathway with C2-ceramide. The data suggest that this oxidant-mediated apoptogenic transduction pathway may play a role in the mechanism of neuronal death in PD.

Aged↗
PubMed1997

Consequences of nigrostriatal denervation on the functioning of the basal ganglia in human and nonhuman primates: an in situ hybridization study of cytochrome oxidase subunit I mRNA.

To examine the consequences of nigrostriatal denervation and chronic levodopa (L-DOPA) treatment on functional activity of the basal ganglia, we analyzed, using in situ hybridization, the cellular expression of the mRNA encoding for cytochrome oxidase subunit I (COI mRNA), a molecular marker for functional neuronal activity, in the basal ganglia. This analysis was performed in monkeys rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Intoxication, some of which had been receiving L-DOPA, and in patients with Parkinson's disease (PD). In MPTP-intoxicated monkeys compared with control animals, COI mRNA expression was increased in the subthalamic nucleus (STN) and in the output nuclei of the basal ganglia, i.e., the internal segment of the globus pallidus and the substantia nigra pars reticulata. This increase was partially reversed by L-DOPA treatment. COI mRNA expression remained unchanged in the external segment of the globus pallidus (GPe). In PD patients, all of whom had been treated chronically by L-DOPA, COI mRNA expression in the analyzed basal ganglia structures was similar to that in control subjects. These results are in agreement with the accepted model of basal ganglia organization, to the extent that the output nuclei of the basal ganglia are considered to be overactive after nigrostriatal denervation, partly because of increased activity of excitatory afferents from the STN. Yet, our results would also seem to contradict this model, because the overactivity of the STN does not seem to be attributable to a hypoactivation of the GPe.

Aged↗
PubMed1997

Decreased TrkA gene expression in cholinergic neurons of the striatum and basal forebrain of patients with Alzheimer's disease.

In addition to cortical pathology, Alzheimer's disease is characterized by a loss of cholinergic neurons in the basal forebrain and the ventral striatum. Since cholinergic neurons which degenerate in Alzheimer's disease are sensitive to nerve growth factor, a link between nerve growth factor sensitivity and the vulnerability of cholinergic neurons has been suspected. The purpose of this study was to determine, in cholinergic neurons, the level of expression of TrkA, the high affinity receptor for nerve growth factor, in control subjects and Alzheimer patients. The study was performed by in situ hybridization using a 35S-labeled RNA probe complementary to human TrkA mRNA on immunohistochemically identified cholinergic neurons of the nucleus basalis of Meynert, the ventral striatum, and the putamen in postmortem brains of patients with clinically and neuropathologically confirmed Alzheimer's disease and control subjects. In patients with Alzheimer's disease, a decrease in TrkA mRNA expression was observed in the nucleus basalis of Meynert (-75%, P < 0.001) and the ventral striatum (-41%, P < 0.01), where the cholinergic neurons degenerate, and also in the anterior (-43%, P < 0.01) and posterior (-51%, P < 0.01) parts of the putamen, where they are spared but display precocious signs of cell alterations. These results, taken in conjunction with the reduced choline acetyltransferase activity and our previously published data showing a loss of high affinity nerve growth factor binding in both the dorsal and the ventral striatum of patients with Alzheimer's disease, indicate that receptor loss and the consequent decrease in trophic support may be associated with the degeneration of cholinergic neurons during Alzheimer's disease.

Aged↗
PubMed1997

Re-evaluation of the functional anatomy of the basal ganglia in normal and Parkinsonian states.

In the late 1980s, a functional and anatomical model of basal ganglia organization was proposed in order to explain the clinical syndrome of Parkinson's disease. According to this model, the pathological overactivity observed in the subthalamic nucleus and the output station of the basal ganglia plays a crucial role in the pathophysiology of the motor signs of Parkinson's disease. The hyperactivity of subthalamic neurons in Parkinsonism is viewed as a direct consequence of a pathological hypoactivity of the external segment of the pallidum. This article reviews recent data from different experimental approaches that challenge the established model of basal ganglia organization by reinterpreting the functional interaction between the external segment of the pallidum and the subthalamic nucleus in both the normal and pathological state. Indeed, recent neurobiochemical studies have rather unexpectedly shown that the GABAergic and metabolic activities of the external pallidum are not decreased in human and non-human primates with Parkinsonism. This absence of any decrease in activity might be explained by the functionally antagonistic influences of the striatal and subthalamic afferences within the external pallidum, as suggested by several anatomical studies. In addition, there are clues from electrophysiological studies to suggest that the hyperactivity found in the subthalamic neurons in Parkinsonism may not depend solely on the level of activity in the external pallidum. In such a framework, the hyperactivity of the subthalamic neurons would have to be explained, at least in part, by other sources of excitation or disinhibition. However, any explanation for the origin of the subthalamic overactivity in Parkinsonism remains speculative.

Animals↗
PubMed1997

Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion.

The gene for spinocerebellar ataxia 7 (SCA7) has been mapped to chromosome 3p12-13. By positional cloning, we have identified a new gene of unknown function containing a CAG repeat that is expanded in SCA7 patients. On mutated alleles, CAG repeat size is highly variable, ranging from 38 to 130 repeats, whereas on normal alleles it ranges from 7 to 17 repeats. Gonadal instability in SCA7 is greater than that observed in any of the seven known neuro-degenerative diseases caused by translated CAG repeat expansions, and is markedly associated with paternal transmissions. SCA7 is the first such disorder in which the degenerative process also affects the retina.

Adult↗
PubMed1997

Is differential regulation of mitochondrial transcripts in Parkinson's disease related to apoptosis?

A cDNA library of substantia nigra pars compacta from a patient with Parkinson's disease (PD) was differentially screened with probes of normal and parkinsonian substantia nigra enriched in neuronal transcripts. Fifty-eight clones were isolated; 39 were subunits of mitochondrial respiratory complexes I and IV. Parallel screening of a cDNA library derived from normal substantia nigra confirmed differential representation of the transcripts in the substantia nigra pars compacta. In situ hybridization in postmortem brain from parkinsonian and control subjects, with representative complex I and complex IV probes, showed increased labeling, at the cellular level, of the complex I subunit ND1 in neurons of the lateral substantia nigra, where cell death is greatest in PD, but decreased labeling in the medial substantia nigra where fewer cells die. Expression of a complex IV subunit, COXI, increased, however, in both parts of the structure. Increased expression of ND1 and COXI was also observed in nerve growth factor-differentiated PC12 cells undergoing apoptosis induced by tumor necrosis factor-alpha, suggesting that the differential regulation of certain mitochondrial mRNAs may be associated with this form of cell death. This in vitro model of apoptosis is potentially relevant to the death of dopaminergic neurons in PD, because these cells express the tumor necrosis factor-alpha receptor, and neighboring microglial cells in patients synthesize the cytokine.

Aged↗
PubMed1997

Rescue of mesencephalic dopamine neurons by anticancer drug cytosine arabinoside.

Nanomolar concentrations of cytosine arabinoside (ara-C), a structural analogue of 2'-deoxycytidine (2'dC) used in the chemotherapy of cancer, proved to be highly effective in preventing the death of postmitotic dopaminergic neurons that occurs spontaneously by apoptosis in mesencephalic cultures. The rescued cells were totally functional and highly differentiated. The trophic/neuroprotective effects of ara-C were (1) specific for dopaminergic neurons; (2) long-lived, remaining detectable several days after withdrawal of the nucleoside analogue from the culture medium; (3) still observed when the treatment was delayed after plating; (4) abolished by an excess of 2'dC or dCTP, or by exposure to the neurotoxin 1-methyl-4-phenylpyridinium; and (5) mimicked by ara-CTP, 5-fluoro-2'-deoxyuridine, and aphidicolin. Autoradiographic studies revealed that ara-C was incorporated exclusively into astrocyte nuclei, suggesting that the dopaminotrophic activity was indirect and resulted from the antiproliferative action of the modified nucleoside on glial cells at concentrations that were not neurotoxic. No evidence was found for putative deleterious or trophic molecules secreted by proliferating or ara-C-treated astrocytes, respectively, suggesting that neuroglial contact may play a role. Our results suggest a possible mechanism underlying neurodegeneration in Parkinson's disease, where selective loss of dopaminergic neurons in the mesencephalon is accompanied by astrogliosis.

1-Methyl-4-phenylpyridinium↗
PubMed1997

Mitochondrial free radical signal in ceramide-dependent apoptosis: a putative mechanism for neuronal death in Parkinson's disease.

Activation of the apoptogenic sphingomyelin-dependent signaling pathway in neuronally differentiated PC12 cells with cell-permeant C2-ceramide resulted in a transient and short-lived emission of reactive oxygen species that was maximal 6 h after the beginning of treatment, followed immediately by nuclear translocation of the transcription factor nuclear factor kappaB. The production of reactive oxygen species was necessary for cell death to occur. The origin of the reactive oxygen species was identified as complex I of the mitochondrial electron transport chain. The mitochondria were not dysfunctional, however. They maintained normal membrane potentials and ATP synthesis until the cells began to die and the cell nuclei to condense and to fragment, approximately 12 h after the beginning of treatment. We conclude that a mitochondrial free radical signal plays a role in the sphingomyelin-dependent transduction pathway. Convergent data from postmortem brain suggest that this signaling pathway may be activated in the dopaminergic neurons that die in patients with Parkinson's disease and would provide a mechanism for oxidative stress implicating the mitochondria, both of which have long been hypothesized to play a role in the pathogenesis of this disease.

Animals↗