Alternative splicing of competing 5′ splice sites is regulated by enhancers and silencers in the spliced exon. We have characterized sequences and splicing factors that regulate alternative splicing of PLP and DM20, myelin proteins produced by oligodendrocytes (OLs) by selection of 5′ splice sites in exon 3. We identify a G-rich enhancer (M2) of DM20 5′ splice site in exon 3B and show that individual G triplets forming M2 are functionally distinct and the distal group plays a dominant role. G-rich M2 and a G-rich splicing enhancer (ISE) in intron 3 share similarities in function and protein binding. The G-rich sequences are necessary for binding of hnRNPs to both enhancers. Reduction in hnRNPH and F expression in differentiated OLs correlates temporally with increased PLP/DM20 ratio. Knock down of hnRNPH increased PLP/DM20 ratio, while hnRNPF did not. Silencing hnRNPH and F increased the PLP/DM20 ratio more than hnRNPH alone, demonstrating a novel synergistic effect. Mutation of M2, but not ISE reduced the synergistic effect. Replacement of M2 and all G runs in exon 3B abolished it almost completely. We conclude that developmental changes in hnRNPH/F associated with OLs differentiation synergistically regulate PLP alternative splicing and a G-rich enhancer participates in the regulation.

Gliosis is a biological process that occurs during injury repair in the central nervous system and is characterized by the overexpression of the intermediate filaments (IFs) glial fibrillary acidic protein (GFAP) and vimentin. A common thread in manyThe overexpression of glial fibrillary acidic protein (GFAP) 2 with vimentin is a hallmark of reactive gliosis in the central nervous system (CNS) (1, 2). These intermediate filaments (IFs) are expressed by reactive astrocytes and macro-and microglia during traumatic and inflammatory injury and in a range of CNS degenerative diseases (2). In fact, an enigma of major retinal diseases, including age-related macular degeneration, glaucoma, diabetic retinopathy, and retinopathy of prematurity, is retinal gliosis, for which there is no available clinical treatment (3-5).Important fundamental insights on the structural and mechanical functions of IFs (6, 7) have now been validated in mouse lines deficient in type III IFs (2). These studies have illuminated that, whereas overexpression of vimentin and GFAP during CNS stress response and injury repair contributes to scar formation (8), their deficiency can be protective of tissue functions in certain contexts. For instance, pathogenic angiogenesis is impaired in vimentin-deficient (Vim KO) mice due to the decreased ability of newly formed blood vessels to cross the retinal inner limiting membrane in the model of hypoxia-induced retinal neovascularization (9). Interestingly, that study also identified in vimentin and GFAP double deficient (Vim GFAP dKO) mice, and to a lesser extent in Vim KO mice, that the retinal ganglion layer is highly sensitive to mechanical stress, which was not observed in GFAP KO mice. Pathological neovascularization was also reduced in Vim KO mice in the corneal alkali injury model (10) and delayed vascularization in skin injury model (11), which is attributed to defective vascular endothelial cell integrity (12), because vimentin is the sole type III IF expressed in endothelial cells (13). On the other hand, Vim GFAP dKO mice subjected to spinal cord or brain injury recover favorably with improvement of glial scars (14). In fact, the complete absence of type III IFs in Vim GFAP dKO mice helps promote axonal regeneration and regain ambulatory function after spinal cord injury (15). These Vim GFAP dKO

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Sirtuin 2 (SIRT2), a NAD-dependent deacetylase expressed by oligodendrocytes (OLs), the myelin-producing cells of the central nervous system (CNS), is markedly up-regulated during active myelination (Li et al. 2007; Southwood et al. 2007; Werner et al. 2007). SIRT2 is a component of the myelin proteome and is severely reduced in the Plp1 knockout mouse brain, in which both PLP and DM20 are absent (Werner et al. 2007). The mechanisms that regulate SIRT2 expression in OLs and myelin remain to be investigated. We report for the first time that the expression of SIRT2 is regulated by the QKI-dependent pathway and this effect is mediated through selective regulation of PLP. In the homozygous quakingviable (qkv/qkv) mutant mouse that harbors QKI deficiency in OLs (Bockbrader and Feng 2008; Ebersole et al. 1996; Hardy et al. 1996), PLP, but not DM20 mRNA, was selectively down-regulated and SIRT2 protein was severely reduced while SIRT2 mRNA expression was unaffected. Expression of the cytoplasmic isoform QKI6 in OLs (Zhao et al. 2006) rescued SIRT2 expression in the qkv/qkv mutant concomitantly with restoration of PLP expression. Moreover, SIRT2 protein is diminished in myelin tracts and compact myelin of the PLP-ISEdel mutant brain, in which PLP protein but not DM20 is selectively reduced (Wang et al. 2008). In contrast, SIRT2 expression and its cellular function in regulating process complexity are not affected by the absence of PLP in PLP-ISEdel non-myelinating oligodendrocytes. Collectively, our results indicate that the abundance of SIRT2 in myelin is dependent on PLP, but not DM20.

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