Alcohol-Mediated Missplicing of Mcl-1 Pre-mRNA is Involved in Neurotoxicity

Abstract

Background Heavy and chronic ethanol (EtOH) exposure can cause significant structural and functional damage to the adult brain. The most devastating consequence of EtOH exposure is the neurotoxicity associated with the depletion of neurons. Regulation of splice variants in the brain can modulate protein functions, which may ultimately affect behaviors associated with alcohol dependence and EtOH-mediated neurotoxicity. Since alcohol consumption is associated with neurotoxicity, it is possible that altered splicing of survival and pro-survival factors during the development of alcoholism may contribute to the neurotoxicity. Methods Primary human neurons and a neuroblastoma cell line were exposed to different concentrations of EtOH for various time periods. Cell viability and neuronal marker expression were analyzed by MTT assay and immunoblotting, respectively. Effect of EtOH exposure on splicing regulatory protein expression and alternative splicing of candidate genes were analyzed by a biochemical approach. Transcriptional activity of SRSF1 gene was determined by reporter gene analysis. Results Our results suggest that EtOH exposure to neuronal cells at 25 mM and higher concentrations are detrimental. In addition, EtOH exposure caused a dramatic reduction in serine-arginine rich splicing factor 1 (SRSF1) expression levels. Furthermore, EtOH exposure led to pre-mRNA missplicing of Mcl-1, a pro-survival member of the Bcl-2 family, by downregulating the expression levels of serine/arginine rich splicing factor 1 (SRSF1). Moreover, ectopic expression of both SRSF1 and MCL-1L isoform was able to recover EtOH-mediated neurotoxicity. Conclusions Our results suggest that ethanol exposure can lead to pre-mRNA missplicing of Mcl-1 in neuronal cells. Our results indicate that ethanol exposure of neurons leads to a decrease in the ratio of Mcl-1L/Mcl-1S by favoring pro-apoptotic Mcl-1S splicing over anti-apoptotic Mcl-1L isoform suggesting that Mcl-1S may play a crucial role in neurotoxicity associated with alcohol consumption.

Alcohol exposure alters pre-mRNA splicing of antiapoptotic Mcl-1L isoform and induces apoptosis in neural progenitors and immature neurons

“…These differences in Mcl-1L expression could be related to the differences in its stability or half-life across the cell types. In line with our previous report 24 , EtOH exposure caused a dramatic reduction in SRSF1 protein levels in hNSPs, hNPCs, and immature neurons. Interestingly, SRSF1 levels in mature neuron cultures were slightly reduced but maintained in cells exposed to EtOH suggesting that its expression levels were sufficient to maintain Mcl-1L splicing over Mcl-1S.…”

“…However, little is known regarding the potential impact of alcohol on alternative splicing of genes during the fetal development and its involvement in the development of fetal alcohol spectrum disorder (FASD). We have recently shown that EtOH exposure of fetal neurons suppresses expression levels of serine/arginine rich splicing factor 1 (SRSF1) and causes missplicing of myeloid cell leukemia 1 (Mcl-1) by favoring the Mcl-1S splicing over Mcl-1L 24 . Several studies suggested that while the longer gene product Mcl-1L enhances cell survival by inhibiting apoptosis in response to various stress conditions in different cell types, the alternatively spliced shorter gene product Mcl-1S may promote apoptosis 25,26 .…”

Mitochondrial fusion and Bid-mediated mitochondrial apoptosis are perturbed by alcohol with distinct dependence on its metabolism

“…Furthermore, EtOH has been proposed to affect the post-translational modification of proteins in non-metabolizing cells 53 , 54 that might also contribute to tBid-induced OMMP. Recently, Sariyer et al 55 showed that in neuronal cells chrEtOH exposure causes Mcl-1 missplicing and reduces the anti-apoptotic isoform of Mcl-1 but our data did not show this in HepG2 cells. The most relevant mechanistic finding here was that chrEtOH facilitated the tBid-induced Bak oligomerization that alone can explain the increased OMMP in HepG2 cells.…”

SRSF3 Is a Critical Requirement for Inclusion of Exon 3 of BIS Pre-mRNA

“…Since the inclusion of exon 3 of BIS mRNA is critically determined by SRSF3 levels, the maintenance of SRSF3 at the appropriate physiological levels is indispensable for the synthesis of intact BIS protein. Therefore, the change in BIS mRNA in linearity and subsequent outcome on cellular fate should be investigated for the conditions in which SRSF3 is reduced, or, alternatively, the conditions in which the spliced form of SRSF3 is increased, such as exposure to alcohol or oxidative stress [ 39 , 40 , 41 ].…”