Caffeine induces in vivo premature appearance of telencephalic vesicles.

Caffeine administered to pregnant mice during germinative neuroepithelium preparation (embryonic days 8-10) dramatically accelerated primitive neuroepithelium evagination into telencephalic vesicles, versus age-matched controls. This histologically-documented, dose-dependent effect seemed reversible during subsequent neuronal migration if caffeine exposure was discontinued. Our in vivo model provides a new tool for studying telencephalic symmetry acquisition and for identifying genes potentially involved in holoprosencephaly, a developmental disorder characterized by defective telencephalic vesicle formation.

Caffeine does not affect excitotoxic brain lesions in newborn mice.

Caffeine is frequently administered to human pre-term newborns although its neurological impact has not been fully evaluated. In the present study performed in mice, we examined the effects of caffeine administration on neonatal excitotoxic lesions of the periventricular white matter, which mimics several aspects of human periventricular leukomalacia. In this model, caffeine exposure did not worsen white matter lesions. These data suggest that neonatal caffeine administration might not affect clastic lesions in pre-term infants.

Survival motor neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro.

Spinal muscular atrophy (SMA) is a genetic disorder characterized by degeneration of spinal cord motoneurons (MNs), resulting in muscular atrophy and weakness. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene and decreased SMN protein. SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoproteins and pre-mRNA splicing requirements. SMN reduction causes neurite degeneration and cell death without classical apoptotic features, but the direct events leading to SMN degeneration in SMA are still unknown. Autophagy is a conserved lysosomal protein degradation pathway whose precise roles in neurodegenerative diseases remain largely unknown. In particular, it is unclear whether autophagosome accumulation is protective or destructive, but the accumulation of autophagosomes in the neuritic beadings observed in several neurite degeneration models suggests a close relationship between the autophagic process and neurite collapse. In the present work, we describe an increase in the levels of the autophagy markers including autophagosomes, Beclin1 and light chain (LC)3-II proteins in cultured mouse spinal cord MNs from two SMA cellular models, suggesting an upregulation of the autophagy process in Smn (murine survival motor neuron protein)-reduced MNs. Overexpression of Bcl-xL counteracts LC3-II increase, contributing to the hypothesis that the protective role of Bcl-xL observed in some SMA models may be mediated by its role in autophagy inhibition. Our in vitro experimental data indicate an upregulation in the autophagy process and autophagosome accumulation in the pathogenesis of SMA, thus providing a valuable clue in understanding the mechanisms of axonal degeneration and a possible therapeutic target in the treatment of SMA.

Functional expression of voltage-gated calcium channels in human melanoma.

The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression.

Polypyrimidine tract binding proteins (PTB) regulate the expression of apoptotic genes and susceptibility to caspase-dependent apoptosis in differentiating cardiomyocytes.

Cardiac morphologic abnormalities in mice deficient for key regulators of the caspase-dependent signaling underscored its role in heart development. However, the mechanisms regulating apoptotic gene expression in the developing heart are unknown. As polypyrimidine tract binding proteins (PTB) determine gene isoform expression during myoblast differentiation and contribute to Apaf-1 translation in cell lines, we investigated whether PTB regulate apoptotic gene expression in differentiating cardiomyocytes. Our results show that PTB are expressed in the embryonic heart and are silenced during development, coinciding with a reduction in the expression of apoptotic genes. Overexpression of PTB in postnatal cardiomyocytes, which express low levels of PTB and apoptotic genes, induced an increase in the amount of pro-apoptotic proteins without affecting abundance of their respective transcripts. Translation of the reporter gene Firefly Luciferase preceded by the 5'-untranslated region of Apaf-1 or Caspase-3 was enhanced by PTB in cardiomyocytes. PTB silencing in fibroblasts induced a decrease of apoptotic protein levels. PTB overexpression in cardiomyocytes induced caspase activity and caspase-dependent DNA fragmentation during ischemia, which is otherwise caspase-independent in differentiated cardiomyocytes. Our results show that PTB contribute to apoptotic gene expression and modulate the susceptibility to caspase activation in differentiating rat cardiomyocytes.