The nebulin SH3 domain is dispensable for normal skeletal muscle structure but is required for effective active load bearing in mouse.

Nemaline myopathy (NM) is a congenital myopathy with an estimated incidence of 150,000 live births. It is caused by mutations in thin filament components, including nebulin, which accounts for about 50% of the cases. The identification of NM cases with nonsense mutations resulting in loss of the extreme C-terminal SH3 domain of nebulin suggests an important role of the nebulin SH3 domain, which is further supported by the recent demonstration of its role in IGF-1-induced sarcomeric actin filament formation through targeting of N-WASP to the Z-line. To provide further insights into the functional significance of the nebulin SH3 domain in the Z-disk and to understand the mechanisms by which truncations of nebulin lead to NM, we took two approaches: (1) an affinity-based proteomic screening to identify novel interaction partners of the nebulin SH3 domain; and (2) generation and characterization of a novel knockin mouse model with a premature stop codon in the nebulin gene, eliminating its C-terminal SH3 domain (NebΔSH3 mouse). Surprisingly, detailed analyses of NebΔSH3 mice revealed no structural or histological skeletal muscle abnormalities and no changes in gene expression or localization of interaction partners of the nebulin SH3 domain, including myopalladin, palladin, zyxin and N-WASP. Also, no significant effect on peak isometric stress production, passive tensile stress or Young's modulus was found. However, NebΔSH3 muscle displayed a slightly altered force-frequency relationship and was significantly more susceptible to eccentric contraction-induced injury, suggesting that the nebulin SH3 domain protects against eccentric contraction-induced injury and possibly plays a role in fine-tuning the excitation-contraction coupling mechanism.

Worsening of cardiomyopathy using deflazacort in an animal model rescued by gene therapy.

We have previously demonstrated that gene therapy can rescue the phenotype and extend lifespan in the delta-sarcoglycan deficient cardiomyopathic hamster. In patients with similar genetic defects, steroids have been largely used to slow down disease progression. Aim of our study was to evaluate the combined effects of steroid treatment and gene therapy on cardiac function. We injected the human delta-sarcoglycan cDNA by adeno-associated virus (AAV) 2/8 by a single intraperitoneal injection into BIO14.6 Syrian hamsters at ten days of age to rescue the phenotype. We then treated the hamsters with deflazacort. Treatment was administered to half of the hamsters that had received the AAV and the other hamsters without AAV, as well as to normal hamsters. Both horizontal and vertical activities were greatly enhanced by deflazacort in all groups. As in previous experiments, the AAV treatment alone was able to preserve the ejection fraction (70±7% EF). However, the EF value declined (52±14%) with a combination of AAV and deflazacort. This was similar with all the other groups of affected animals. We confirm that gene therapy improves cardiac function in the BIO14.6 hamsters. Our results suggest that deflazacort is ineffective and may also have a negative impact on the cardiomyopathy rescue, possibly by boosting motor activity. This is unexpected and may have significance in terms of the lifestyle recommendations for patients.

Perioral skin biopsy to study skeletal muscle protein expression.

Clinical trials for muscular dystrophy molecular treatment require multiple sampling of skeletal muscle to monitor protein rescue. This practice is invasive and could raise ethical problems. A less invasive tool to obtain sequential muscle sampling is necessary. Using indirect immunofluorescence, we evaluated muscle protein expression in myofiber bundles included in 2-2.5-mm punch skin biopsies from the perioral region from 6 healthy subjects and 6 patients with genetically defined forms of muscular dystrophy. Large intradermal bundles of orbicularis oris muscle were constantly present in skin biopsies. They showed a typical muscular antigenic pattern in controls and the expected protein defect in muscular dystrophy patients. These results demonstrate the feasibility of muscular protein expression analysis using skin biopsy. We propose this minimally invasive technique to follow-up the response to genetic or conventional therapies in muscular dystrophies and to confirm the diagnosis in some special clinical conditions.

Disease rescue and increased lifespan in a model of cardiomyopathy and muscular dystrophy by combined AAV treatments.

BACKGROUND: The BIO14.6 hamster is an excellent animal model for inherited cardiomyopathy, because of its lethal and well-documented course, due to a spontaneous deletion of delta-sarcoglycan gene promoter and first exon. The muscle disease is progressive and average lifespan is 11 months, because heart slowly dilates towards heart failure. METHODOLOGY/PRINCIPAL FINDINGS: Based on the ability of adeno-associated viral (AAV) vectors to transduce heart together with skeletal muscle following systemic administration, we delivered human delta-sarcoglycan cDNA into male BIO14.6 hamsters by testing different ages of injection, routes of administration and AAV serotypes. Body-wide restoration of delta-SG expression was associated with functional reconstitution of the sarcoglycan complex and with significant lowering of centralized nuclei and fibrosis in skeletal muscle. Motor ability and cardiac functions were completely rescued. However, BIO14.6 hamsters having less than 70% of fibers recovering sarcoglycan developed cardiomyopathy, even if the total rescued protein was normal. When we used serotype 2/8 in combination with serotype 2/1, lifespan was extended up to 22 months with sustained heart function improvement. CONCLUSIONS/SIGNIFICANCE: Our data support multiple systemic administrations of AAV as a general therapeutic strategy for clinical trials in cardiomyopathies and muscle disorders.

Natural antisense transcripts associated with genes involved in eye development.

Natural antisense transcripts (NATs) are a class of genes whose role in controlling gene expression is becoming more and more relevant. We describe the identification of eight novel mouse NATs associated with transcription factors (Pax6, Pax2, Six3, Six6, Otx2, Crx, Rax and Vax2) that play an important role in eye development and function. These newly identified NATs overlap with the mature processed mRNAs or with the primary unprocessed transcript of their corresponding sense genes, are predicted to represent either protein coding or non-coding RNAs and undergo extensive alternative splicing. Expression studies, by both RT-PCR and RNA in situ hybridization, demonstrate that most of these NATs, similarly to their sense counterparts, display a specific or predominant expression in the retina, particularly at postnatal stages. We found a significant reduction of the expression levels of one of these NATs, Vax2OS (Vax2 opposite strand) in a mouse mutant carrying the inactivation of Vax2, the corresponding sense gene. In addition, we overexpressed another NAT, CrxOS, in mouse adult retina using adeno-associated viral vectors and we observed a significant decrease in the expression levels of the corresponding sense gene, Crx. These results suggest that these transcripts are functionally related to their sense counterparts and may play an important role in regulating the molecular mechanisms that underlie eye development and function in both physiological and pathological conditions.

MBDin, a novel MBD2-interacting protein, relieves MBD2 repression potential and reactivates transcription from methylated promoters.

We have identified a human gene encoding a novel MBD2-interacting protein (MBDin) that contains an N-terminal GTP-binding site, a putative nuclear export signal (NES), and a C-terminal acidic region. MBDin cDNA was isolated through a two-hybrid interaction screening using the methyl-CpG-binding protein MBD2 as bait. The presence of the C-terminal 46-amino-acid region of MBD2 and both the presence of the acidic C-terminal 128-amino-acid region and the integrity of the GTP-binding site of MBDin were required for the interaction. Interaction between MBD2 and MBDin in mammalian cells was confirmed by immunoprecipitation experiments. Fluorescence imaging experiments demonstrated that MBDin mainly localizes in the cytoplasm but accumulates in the nucleus upon disruption of the NES or treatment with leptomycin B, an inhibitor of NES-mediated transport. We also found that MBDin partially colocalizes with MBD2 at foci of heavily methylated satellite DNA. An MBD2 deletion mutant lacking the C-terminal region maintained its subnuclear localization but failed to recruit MBDin at hypermethylated foci. Functional analyses demonstrated that MBDin relieves MBD2-mediated transcriptional repression both when Gal4 chimeric constructs and when in vitro-methylated promoter-reporter plasmids were used in transcriptional assays. Southern blotting and bisulfite analysis showed that transcriptional reactivation occurred without changes of the promoter methylation pattern. Our findings suggest the existence of factors that could be targeted on methylated DNA by methyl-CpG-binding proteins reactivating transcription even prior to demethylation.

PATZ attenuates the RNF4-mediated enhancement of androgen receptor-dependent transcription.

PATZ is a transcriptional repressor affecting the basal activity of different promoters, whereas RNF4 is a transcriptional activator. The association of PATZ with RNF4 switches the activation to repression of selected basal promoters. Because RNF4 interacts also with the androgen receptor (AR) functioning as a coactivator and, in turn, RNF4 associates with PATZ, we investigated whether PATZ functions as an AR coregulator. We demonstrate that PATZ does not influence directly the AR response but acts as an AR corepressor in the presence of RNF4. Such repression is not dependent on histone deacetylases. A mutant RNF4 that does not bind PATZ but enhances AR-dependent transcription is not influenced by PATZ, demonstrating that the repression by PATZ occurs only upon binding to RNF4. We also demonstrate that RNF4, AR, and PATZ belong to the same complex in vivo also in the presence of androgen, suggesting that repression is not mediated by the displacement of RNF4 from AR. Finally, we show that the repression of endogenous PATZ expression by antisense expression plasmids in LNCaP cells results in a stronger androgen response. Our findings demonstrate that PATZ is a novel AR coregulator that acts by modulating the effect of a coactivator. This could represent a novel and more general mechanism to finely tune the androgen response.