Spinal muscular atrophy is a common autosomal recessive neuromuscular disorder and the leading hereditary cause of death in early childhood. No cure is available. The disease determining gene for SMA is the survival motor neuron gene 1. SMN1 produces full length transcripts only, whereas the majority of transcripts derived from the copy gene SMN2 lack exon 7 due to alternative splicing. Although the amount of fully-functional SMN2-derived FL-SMN protein is not sufficient to overcome the absence of SMN1 and to prevent disease onset, SMN2 has been shown to be the main disease modifying gene. SMN2 is present in all SMA patients, which all lack functional SMN1. Therefore, and due to its disease modifying property, SMN2 became the most interesting target for a potential SMA therapy. It has been proven that epigenetic modification by HDACi activates the transcription of SMN2. Additionally, HDACi treatment has been shown to modulate the splicing pattern of SMN2 by the up-regulation of the splicing factor SFRS10, resulting in increased FL-SMN levels. Elevation of the SMN protein level was proven to ameliorate SMA progression in vitro, ex vivo and in vivo. In the present work two newly identified second generation HDAC inhibitors of different chemical classes, namely M344 and FK228, were shown to be able to elevate SMN and SFRS10 protein levels in fibroblast cell lines derived from SMA patients. Moreover, it was proven by quantitative real-time PCR that both HDACis up-regulated the FL-SMN2 transcript amounts and revealed no change or even a decrease in the delta7-SMN2 level indicating a reversion of the splicing pattern. The ability to correct the SMN2 splicing was confirmed by the finding that both substances were able to elevate SFRS10 protein levels. Semi-quantitative Western blot analysis revealed that the treatment with M344 resulted in an up to 7-fold and with FK228 in an up to 4.4-fold augmentation of the SMN protein level. FK228 proved its efficacy to elevate SMN levels also in a murine fibroblast line derived from a SMA-like mouse, indicating potency to modulate SMN2 expression regardless of the genetic background. Both substances were able to increase the number of subnuclear gems, indicating the elevated production of fully-functional SMN proteins. However, the performed MTT-assays revealed for both substances an in vitro toxicity profile, which restrained further in vivo characterization. To identify the HDAC isoenzyme involved in SMN2 expression regulation, all 11 classical HDACs were knocked-down by siRNA. Only after the KD of HDAC8, the SMN protein level was significantly up-regulated. HDAC8 overexpression experiments were performed, which could prove that HDAC8 activity regulates the SMN protein expression. ChIP experiments revealed that HDAC8 binds to the promoter region of SMN2, resembling the involvement in the epigenetic regulation of SMN2 expression. This finding was confirmed by treating SMN1 deleted fibroblasts with HDAC8 specific inhibitor. qRT-PCR expression analysis and semi-quantitative Western blots revealed that the up-regulation of SMN by PCI-34051 resembled the elevation of SMN by the knock-down of HDAC8. Previously, our group has shown for the first time that the FDA-approved drug SAHA is able to elevate the SMN level in vitro and ex vivo. Thus, this work proved that SAHA is a potent and non-toxic candidate drug for SMA treatment, since the in vivo characterization of this compound revealed a beneficial effect on disease progression in two different SMA mouse models. Since the SMN2 copy number is crucial for the severity of SMA, a real-time PCR based assay was developed to precisely determine the SMN2 copy number of SMA-like mice. In the very severe SMA mouse model SAHA treatment of the pregnant mother mice could prevent embryonic lethality of the SMA-like mice. In another SMA mouse model, which was here characterized for the first time in detail, the direct application of SAHA to the SMA-like mice revealed a significant amelioration of the SMA progression. The mean lifespan was increased by ~30%, from 9.9 to 12.9 days. Moreover, the SAHA-treated SMA-like mice showed better motor function abilities. Additionally, in histochemical attempts it was shown that SAHA-treated SMA-like mice revealed less degeneration of the a-motor neurons in the anterior horns of the spinal cord and larger muscle fibers. Immunofluorescence stains proved that SAHA-treated SMA mice showed an increase in neuromuscular junction size. Furthermore, qRT-PCR approaches as well as quantitative Western blotting displayed that SAHA treatment elevated SMN protein and transcript levels in the brain, spinal cord, muscle and liver. Although SAHA was able to ameliorate the SMA phenotype, it was not able to fully rescue the mice from disease progression. Since SAHA is an FDA-approved drug and was shown to ameliorate the SMA progression, it can be considered as an attractive candidate for a potential SMA therapy
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