Semaphorin 3C preserves survival and induces neuritogenesis of cerebellar granule neurons in culture.

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.

[Molecular basis of Huntington's disease and possible pathogenic mechanisms]. / Bases moleculares de la enfermedad de Huntington y posibles mecanismos patogénicos.

INTRODUCTION: Huntington s disease is one of the, at least, nine neurological disorders caused by a CAG triplet expansion coding for a poly glutamine sequence in the corresponding protein. Huntington s disease affects 3 7 in 100.000 individuals in Western Europe descendent population and the symptomatology comprises motor (including chorea and rigidity), cognitive (subcortical dementia), and psychological (including irritability and depression) manifestations until death. DEVELOPMENT: Neuropathology is extremely restricted, with atrophy occurring in the striatum and, to a lesser extent, in the cerebral cortex. Microscopically, the neuropathology is characterized by neuronal loss, reactive gliosis, and intraneuronal protein aggregates. Since the initial description of this disease by George Huntington in 1872, substantial advance has been achieved in the understanding of this pathology. The pathogenic gene and mutation were identified in 1993. This allowed the generation of multiple in vitro, cellular, and animal models of Huntington s disease. These studies have originated multiple hypotheses regarding the mechanism by which huntingtin with an expanded poly glutamine tract exerts its toxicity. CONCLUSION: We try to summarize the current knowledge about this disease from the clinical manifestations to the molecular basis, in an attempt to offer a global view of this pathology.

Bases moleculares de la enfermedad de Huntington y posibles mecanismos patogénicos / Molecular basis of huntington's disease and possible pathogenic mechanisms

Introducción. La enfermedad de Huntington (EH) es una de las nueve enfermedades neurológicas autosómicas dominantes causadas por una mutación de expansión de tripletes de CAG codificantes para secuencias de poliglutamina. Afecta a 3-7 casos por 100.000 de la población descendiente de Europa occidental, y su sintomatología abarca trastornos de tipo motor (corea y rigidez, entre otros), cognitivo (demencia subcortical) y psicológico (como irritabilidad y depresión), que terminan con la muerte del individuo. Desarrollo. La neuropatología de la EH es extremadamente restringida, con una marcada atrofia del estriado y en menor medida de la corteza cerebral. Microscópicamente, la patología estriatal se caracteriza por la pérdida neuronal, gliosis y presencia de agregados proteicos. Desde que, en el año 1872, el doctor George Huntington describiera por primera vez la enfermedad, ha habido múltiples avances en su conocimiento. El descubrimiento del gen responsable de la enfermedad en el año 1993 ha dado lugar a la generación de múltiples modelos in vitro, celulares y animales que han permitido ahondar en los fundamentos moleculares de esta neuropatología. Basándose en estos estudios, han surgido varias líneas de investigación que intentan explicar el mecanismo por el cual la huntingtina con la poliglutamina expandida provoca la neuropatología. Conclusión. En este trabajo hemos pretendido dar una visión completa de la enfermedad avanzando desde los aspectos puramente sintomatológicos hasta los moleculares, para entender de un modo global la neuropatología de la EH (AU)

Loss of mRNA levels, binding and activation of GTP-binding proteins for cannabinoid CB1 receptors in the basal ganglia of a transgenic model of Huntington's disease.

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.

Fibronectin modulation by A beta amyloid peptide (25-35) in cultured astrocytes of newborn rat cortex.

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.

Proteasomal-dependent aggregate reversal and absence of cell death in a conditional mouse model of Huntington's disease.

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.