Co-regulation of survival of motor neuron and Bcl-xL expression: Implications for neuroprotection in spinal muscular atrophy

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.

Introduction

Spinal muscular atrophy (SMA) is an often fatal autosomal recessive genetic disease typified by the degeneration of anterior horn cells of the spinal cord, muscle weakness and atrophy. With an incidence of one in 6,000 live births, SMA is the most common genetic cause of infant mortality. Clinically characterized by profound muscle weakness, hypotonia and trunk paralysis, SMA is classified into 3 main subtypes (I–III) based on disease severity and age of onset (Munsat and Davies, 1992).

SMA is a monogenic disorder whereby the survival of motor neuron (SMN1) gene is mutated or absent in more than 95% of cases (Lefebvre et al., 1995). During human evolution, a 500 kb inverted duplication of the SMN1 locus has given rise to a second copy of the gene, termed SMN2. The SMN2 gene is essentially identical to SMN1 except for a single translationally silent nucleotide change (C→T) at position 6 of exon 7, which causes exclusion of exon 7 in SMN2 transcripts (Lorson et al., 1999, Monani et al., 1999). Consequently, only about 10% of SMN2 protein is functional, while the other 90% is truncated and unstable. The occurrence of multiple SMN2 gene copies in patient groups correlates with increased full-length SMN transcripts, greater amounts of functional protein, and reduced disease severity (Lefebvre et al., 1997, Feldkotter et al., 2002).

The SMN protein is highly expressed during embryonic development and ubiquitously expressed within the cytoplasm and nucleus of cells (La Bella et al., 1998). SMN plays a role in the assembly of small nuclear ribonucleoproteins (snRNPs), a component essential for pre-mRNA splicing (Liu et al., 1997, Pellizzoni et al., 1999). Among other functions, SMN also interacts with key regulators of cell survival, such as Bcl-2, ZPR1 and p53 (Iwahashi et al., 1997, Gangwani et al., 2001, Young et al., 2002), and appears to possess an important anti-apoptotic function (Kerr et al., 2000, Vyas et al., 2002, Anderton et al., 2011).

Curiously, like SMN, expression of the anti-apoptotic Bcl-2 family member, Bcl-xL, is also reduced in SMA patients and SMA mouse models (Soler-Botija et al., 2003, Tsai et al., 2008). Bcl-xL is highly expressed in the CNS and regulates cell survival by inhibiting the pro-apoptotic Bax and Bak proteins (Merry and Korsmeyer, 1997). Importantly, Bcl-xL expression rescues SMA-like motor defects (Garcera et al., 2011) and increases the life-span of transgenic type III SMA mice (Tsai et al., 2008). The co-occurrence of reduced Bcl-xL and SMN in SMA suggests co-regulated expression of these two proteins. Interestingly, phosphorylation of the RNA-binding protein, Sam68, promotes exon 7 inclusion in SMN2 transcripts and splicing of the pro-survival Bcl-xL transcript (Paronetto et al., 2007, Pedrotti et al., 2010).

We used recombinant adenoviral vectors and transgenic mice to investigate the potentially important relationship between SMN and Bcl-xL expression. Expression of SMN strongly increased Bcl-xL expression in SH-SY5Y cells, and in PrP-SMN mouse brain tissue. Remarkably, expression of both SMN and Bcl-xL reduced total Sam68 levels, supporting the notion that SMN positively regulates its own expression, possibly by modulating Sam68 levels. Finally, we demonstrate that co-expression of Bcl-xL and SMN has an additive neuroprotective effect against phosphatidylinositol-3 kinase (PI3-kinase) inhibition-induced apoptosis, thereby identifying Bcl-xL as a valid therapeutic target in SMA treatment.