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Abstract: Spinal muscular atrophy (SMA), a neurodegenerative disorder primarily affecting motor neurons, is the most common genetic cause of infant death. This incurable disease is caused by the absence of a functional SMN1 gene and a reduction in full length survival of motor neuron (SMN) protein. In this study, a neuroprotective function of SMN was investigated in differentiated human SH-SY5Y cells using an adenoviral vector to over-express SMN protein. The pro-survival capacity of SMN was assessed in an Akt/PI3-kinase inhibition (LY294002) model, as well as an oxidative stress (hydrogen peroxide) and excitotoxic (glutamate) model. SMN over-expression in SH-SY5Y cells protected against Akt/phosphatidylinositol 3-kinase (PI3-kinase) inhibition, but not oxidative stress, nor against excitotoxicity in rat cortical neurons. Western analysis of cell homogenates from SH-SY5Y cultures over-expressing SMN harvested pre- and post-Akt/PI3-kinase inhibition indicated that SMN protein inhibited caspase-3 activation via blockade of calpain-mediated procaspase-3 cleavage. This study has revealed a novel anti-apoptotic function for the SMN protein in differentiated SH-SY5Y cells. Finally, the cell death model described herein will allow the assessment of future therapeutic agents or strategies aimed at increasing SMN protein levels. [Copyright &y& Elsevier]

Preconditioning describes a variety of treatments that induce neurons to become more resistant to a subsequent ischemic insult. How preconditioned neurons adapt to subsequent ischemic stress is not fully understood, but is likely to involve multiple protective mechanisms. We hypothesized hypoxic preconditioning induces protection by a coordinated up-regulation of antioxidant enzyme activity. To test this hypothesis, we developed two in vitro models of ischemia/reperfusion, involving oxygen–glucose deprivation (OGD) where neuronal cell death was predominantly by necrosis (necrotic model) or programmed cell death (PCD model). Hypoxic preconditioning 24 h prior to OGD significantly reduced cell death from 83% to 22% in the necrotic model and 68% to 11% in the PCD model. Consistent with the hypothesis, the activity of the antioxidant enzymes glutathione peroxidase, glutathione reductase, and Mn superoxide dismutase were significantly increased by 54%, 73% and 32%, respectively, in neuronal cultures subjected to hypoxic preconditioning. Furthermore, superoxide and hydrogen peroxide concentrations following OGD were significantly lower in the PCD model that had been subjected to hypoxic preconditioning. [Copyright &y& Elsevier]

Abstract: Spinal muscular atrophy (SMA), a fatal genetic motor disorder of infants, is caused by diminished full-length survival of motor neuron (SMN) protein levels. Normally involved in small nuclear ribonucleoprotein (snRNP) assembly and pre-mRNA splicing, recent studies suggest that SMN plays a critical role in regulating apoptosis. Interestingly, the anti-apoptotic Bcl-x isoform, Bcl-xL, is reduced in SMA. In a related finding, Sam68, an RNA-binding protein, was found to modulate splicing of SMN and Bcl-xL transcripts, promoting SMNΔ7 and pro-apoptotic Bcl-xS transcripts. Here we demonstrate that Bcl-xL expression increases SMN protein by ∼2-fold in SH-SY5Y cells. Conversely, SMN expression increases Bcl-xL protein levels by ∼6-fold in SH-SY5Y cells, and ∼2.5-fold in the brains of transgenic mice over-expressing SMN (PrP-SMN). Moreover, Sam68 protein levels were markedly reduced following SMN and Bcl-xL expression in SH-SY5Y cells, suggesting a feedback mechanism co-regulating levels of both proteins. We also found that exogenous SMN expression increased full-length SMN transcripts, possibly by promoting exon 7 inclusion. Finally, co-expression of SMN and Bcl-xL produced an additive anti-apoptotic effect following PI3-kinase inhibition in SH-SY5Y cells. Our findings implicate Bcl-xL as another potential target in SMA therapeutics, and indicate that therapeutic increases in SMN may arise from modest increases in total SMN. [Copyright &y& Elsevier]