[Show abstract][Hide abstract] ABSTRACT: Huntington disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cell, suggested that alterations in the ubiquitin-proteasome system (UPS) might contribute to HD pathogenesis. In previous work we reported that in a conditional mouse model of Huntington's disease (HD94 mice), the chymiotrypsin- and trypsin-"like" activities of the proteasome are increased selectivity in the affected and aggregate-containing brain regions: striatum, and cortex. Moreover, in these areas a neuronal increase in the interferon-inducible subunits of the immunoproteasome LMP2 and LPM7 was observed. In order to test if the expression of N-terminal mutant huntingtin (htt) by itself is sufficient to induce the change in proteasome catalytic activities as well as in LMP2 subunit expression, we performed activities of the proteasome and western blot experiments in striatal cultured neurons from HD94 mice free of glial contamination. We found no changes in any of the activities in these cells. Furthermore, western blot analysis performed with specific antibody against LMP2 subunits, revealed no difference in levels of this subunit in striatal neurons from HD94 compared to control cultures were treated with interferon-gamma (IFN-gamma) during 72 hours, a clear increase in LMP2 levels was observed in control neuronal cultures. Interestingly, this increase was much more pronounced (95% higher) in HD94 striatal cultures. These results indicate that although expression of mutant htt is not sufficient to induce the changes in proteasome catalytic core observed in HD, it synergizes the changes induced by IFN-gamma. Furthermore, immunocytochemical studies revealed that HD94 striatal neuron expressing high levels of LMP2 subunit showed a pre-apoptotic appearance. These results suggest that the correlation between neuronal induction of the immunoproteasome and neurodegeneration found in HD brains is secondary to inflammatory processes.
Neurotoxicity Research 02/2004; 6(6):463-8. · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Huntington's disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cells, suggest that an impairment of the ubiquitin-proteasome system (UPS) may be key in HD pathogenesis. To test whether proteasome activity is impaired in vivo, we performed enzymatic assays for the three peptidase activities of the proteasome in brain extracts from the HD94 conditional mouse model of HD. We found no inhibition of any of the activities, suggesting that if UPS impairment happens in vivo, it is not at the level of the proteasome catalytic core. Intriguingly, the chymotrypsin- and trypsin-like activities increased selectively in the affected and aggregate-containing regions: cortex and striatum. Western blot analysis revealed no difference in total proteasome content whereas an increase in the interferon-inducible subunits of the immunoproteasome, LMP2 and LMP7, was observed. These subunits confer to the proteasome catalytic properties that are optimal for MHC-I peptide presentation. Immunohistochemistry in control mouse brain revealed LMP2 and LMP7 mainly in neurons. Accordingly, their increase in HD94 mice predominantly took place in neurons, and 5% of the ubiquitin-positive cortical aggregates were also LMP2-positive. Ultrastructural analysis of neurons with high level of immunoproteasome subunits revealed signs of neurodegeneration like nuclear indentation or fragmentation and dark cell appearance. The neuronal induction of LMP2 and LMP7 and the associated signs of neurodegeneration were also found in HD postmortem brains. Our results indicate that LMP2 and LMP7 participate in normal neuronal physiology and suggest a role in HD neurodegeneration.
Journal of Neuroscience 01/2004; 23(37):11653-61. · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Semaphorins (sema) constitute a family of molecules sharing a common extracellular domain (semaphorin domain). This family includes several types of secreted and membrane-associated molecules that are grouped into eight subclasses (subclasses 1-7 and viral semaphorins). Subclass 3 semaphorins are secreted molecules involved in axonal guidance, mainly through repulsive gradients and induction of growth cone collapse. More recently sema 3 molecules have been identified as positive factors in dependence of the type of neurons. Besides their axonal guidance function, some semaphorins have been implicated in apoptosis and survival. We investigated the effect of sema3C on survival and neurite outgrowth of rat cerebellar granule neurons (CGNs) in culture. 3T3 cells were stably transfected with sema3C. Several clonal lines were established and tested for their neuritogenic activity and one, S3C-8, was selected for the bulk of experiments. S3C-8 was co-cultured with CGNs. Sema3C enhanced CGN viability as assessed in co-cultures of CGNs with monolayers of S3C-8 in comparison with co-cultures of CGNs with control mock-transfected 3T3 cells. Moreover sema3C induced neuritogenesis of cultured CGNs, which express neuropilin-1 and -2. S3C-8 cells, overexpressing sema3C, were significantly more neuritogenic for CGN than poly l-lysine (PLL), a positive substrate for CGNs, as assessed by the measurement of the length of neurites and confirmed by Tau expression along the time of culture. CGNs co-cultured with S3C-8, showed up-regulation of the expression of axonal microtubule-associated proteins (MAPs) such as Tau, phosphorylated MAP2C and mode I-phosphorylated MAP1B compared with neurons cultured on control 3T3 cells. We also found increased expression of a specific marker of neuronal cell bodies and dendrites, high molecular weight MAP2 (HMW-MAP2). Interestingly, there was no accompanying up-regulation of a marker enriched within the neuronal somatodendritic domain, mode II-phosphorylated MAP1B. These data support the idea that secreted sema3C favors survival and neuritogenesis of cultured CGNs.
Journal of Neurochemistry 12/2003; 87(4):879-90. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Unlike normal huntingtin (htt) which is located predominantly in the cytoplasm, mutant htt is also found in the nucleus of affected neurons. Nuclear localization of toxic polyglutamine-containing proteins has been postulated to be necessary for the pathogenesis of triplet repeat disorders. However, little is known about the mechanism by which mutant htt enters the nucleus. We have recently reported exclusive nuclear localization of exon 1 mutant htt in striatal primary neuronal cultures from the HD94 conditional mouse model of HD. This seemed to contradict the predominant cytoplasmic localization of N-terminal htt reported from transfection experiments and prompted us to hypothesize that subcellular localization of the toxic htt fragment might be favoured in nondividing cells. To test this, we analyzed subcellular localization of mutant htt in HD94 mixed neuron-glia cultures. Subconfluent glial cells showed cytoplasmic localization. However, nuclear localization was prompted by confluence, by serum withdrawal, and by treatment with cell cycle progression inhibitors such as Ara C or lactacystin. BrdU labelling experiments further confirmed that nuclear localization does not occur in dividing cells. Our findings offer an explanation for the neuronal specific toxicity of mutant htt despite its ubiquitous expression. Unraveling the mechanism of this cell cycle arrest-dependent entrance into the nucleus may offer new opportunities for therapeutic intervention.
European Journal of Neuroscience 08/2002; 16(2):355-9. · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ephrins are developmentally regulated molecules that may contribute to axonal pathfinding through their binding to Eph receptor tyrosine kinases. In many cases, ephrins act as negative molecules that stimulate growth cone collapse, although some forms may promote axonal growth. Here, we have addressed the role played by ephrin-B1 during rat postnatal cerebellar development. Ephrin-B1 is expressed by both granule and Purkinje neurons whereas EphB is present in granule neurons in early postnatal cerebellum at a time coincident with axonal and dendrite outgrowth. Stably transfected 3T3 cells overexpressing ephrin-B1 enhance survival and neurite growth from cultured cerebellar granule neurons, an effect that is inhibited by the presence of an excess of a soluble EphB protein. Ephrin-B1-induced neuritogenesis is correlated with an increased expression of certain neuronal-specific microtubule-associated proteins (MAPs). Cerebellar granule neurons plated on stably transfected 3T3 cells overexpressing ephrin-B1 show an up-regulation of the expression of axonal MAPs such as Tau and phosphorylated MAP2C compared with neurons cultured on control 3T3 cells. The level of expression of these axonal MAPs is similar to that found in neurons plated on poly-L-lysine. Interestingly, there is a noteworthy up-regulation of somatodendritic MAPs such as high-molecular-weight MAP2 and mode II-phosphorylated MAP1B in neurons cultured on stably transfected 3T3 cells overexpressing ephrin-B1 compared with neurons plated on either control 3T3 cells or poly-L-lysine. In view of these data, we suggest that ephrin-B1 favors dendritogenesis of granule neurons during cerebellum development.
Molecular and Cellular Neuroscience 08/2002; 20(3):429-46. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Data obtained from the basal ganglia of postmortem Huntington's disease (HD) brains have revealed that the level of cannabinoid CB1 receptors in striatal efferent neurons decreases in parallel to the dysfunction and subsequent degeneration of these neurons. These findings, and others from rat models of HD generated by lesions with mitochondrial toxins, suggest that the loss of CB1 receptors may be involved in the pathogenesis of the disease. To explore further the changes in the endocannabinoid system, as well as the potential of endocannabinoid-related compounds, we examined the status of CB1 receptors in the HD94 transgenic mouse model of HD. These mice express huntingtin exon 1 with a polyglutamine tract of 94 repeats in a tissue-specific and conditional manner using the tet regulatable system. They develop many features of HD, such as striatal atrophy, intraneuronal aggregates and progressive dystonia. In these animals, we analyzed mRNA levels for the CB1 receptor, in addition to the number of specific binding sites and the activation of GTP-binding proteins by CB1 receptor agonists. mRNA transcripts of the CB1 receptor were significantly decreased in the caudate-putamen of HD transgenic mice compared to age-matched littermate controls. The decrease concurred with a marked reduction in receptor density in both the caudate-putamen and its projection areas such as the globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata. Furthermore, the efficacy of CB1 receptor activation was reduced in the globus pallidus, as determined by agonist-induced [35S]GTPgammaS binding, and tended towards a decrease in the substantia nigra. None of these changes was seen in the cerebral cortex and hippocampus, despite high levels of expression of the mutant protein in these regions. The decrease in CB1 receptor levels was accompanied by a decrease in the proenkephalin-mRNA levels but not in substance P-mRNA levels. Taken together, these results suggest that the loss of CB1 receptor might be preferential to the enkephalinergic CB1 receptor-containing striatopallidal neurons, and further implicate the CB1 receptor to the subsequent HD symptomatology and neuropathology.
Brain Research 04/2002; 929(2):236-42. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neuronal intranuclear inclusions are a histopathological hallmark of Huntington's disease. Nevertheless, the precise mechanism by which they are formed and their relevance to neuronal cell death and/or dysfunction remains unclear. We recently generated a conditional mouse model of Huntington's disease (HD94) in which silencing expression of mutated huntingtin led to the disappearance of intranuclear aggregates and amelioration of the behavioral phenotype. Here, we analyze primary striatal neuronal cultures from HD94 mice to explore the dynamics of aggregate formation and reversal, the possible mechanisms involved, and the correlation between aggregates and neuronal death. In parallel, we examine symptomatic adult HD94 mice in similar studies and explored the relationship between aggregate clearance and behavioral reversal. We report that, in culture, aggregate formation and reversal were rapid processes, such that 2 d of transgene expression led to aggregate formation, and 5 d of transgene suppression led to aggregate disappearance. In mice, full reversal of aggregates and intranuclear mutant huntingtin was more rapid than reported previously and preceded the motor recovery by several weeks. Furthermore, the proteasome inhibitor lactacystin inhibited the aggregate clearance observed in culture, thus indicating that aggregate formation is a balance between the rate of huntingtin synthesis and its degradation by the proteasome. Finally, neither expression of the mutant huntingtin nor aggregates compromised the viability of HD94 cultures. This correlated with the lack of cell death in symptomatic HD94 mice, thus demonstrating that neuronal dysfunction, and not cell loss, triggered by mutant huntingtin underlies symptomatology.
Journal of Neuroscience 12/2001; 21(22):8772-81. · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fibronectin appears to be present in Senile Plaques of Alzheimer's disease brains. These senile or neuritic plaques are surrounded by dystrophic neurites, activated microglia and reactive astrocytes. The purpose of this work was to establish if a direct correlation exists between the production of Fibronectin (FN) by astrocytes and the presence of amyloid, analysing the modification of this protein produced after the treatment of cultured astrocytes with amyloid peptide (25-35). Our data showed that the addition of previously polymerised A beta-peptide to cultured astrocytes induced a marked increase in FN immunoreactivity that is in part dependent on phosphatases 2A or phosphatase 1, since was partially inhibited by okadaic acid. The increased amount of FN did not appear to be associated to any specific single isoform of which are mainly present in the rat brain. Our data suggest that in vivo FN accumulated in senile plaques may be the result, at least in part, of the response of reactive astrocyte to the presence of amyloid peptide. The importance of FN up-regulation in vivo, as part of a 'positive' response of the astrocytes to produce molecules that favours neurite outgrowth, is discussed.