Heterozygous Igf1r deletion does not ameliorate pathological features associated with polyglutamine-containing huntingtin fragment.

The evolutionarily conserved insulin/IGF-1 signaling pathway has pleiotropic effects on various cellular processes. Hypomorphic alleles of the insulin/IGF-1 receptor enhance catabolic processes as well as stress resistance, which ultimately lead to lifespan extension in invertebrates. Moreover, decreased insulin/IGF-1 signaling promotes the maintenance of protein quality control and suppresses the onset of cellular toxicity caused by aggregate-prone proteins. As loss of protein homeostasis is a feature of many sporadic and inherited forms of neurodegenerative disorders, the pharmacological inhibition of the IGF-1 receptor represents a promising potential therapeutic strategy for currently untreatable neurodegenerative disorders. However, additional studies are required to determine whether this approach is suitable to delay pathology in clinically relevant models of neurodegenerative disorders. Here we show that, in a mouse model of Huntington's disease, heterozygous knockout of the Igf1r does not prevent premature lethality of mice expressing a short fragment of the mutant human huntingtin. Moreover, Igf1r haploinsufficiency does not suppress the formation of huntingtin-containing aggregates. Thus, partial genetic manipulation of the insulin/IGF-1 signaling pathway does not seem sufficient to counteract protein toxicity and extend animal survival.

Plastin 3 ameliorates spinal muscular atrophy via delayed axon pruning and improves neuromuscular junction functionality.

F-actin bundling plastin 3 (PLS3) is a fully protective modifier of the neuromuscular disease spinal muscular atrophy (SMA), the most common genetic cause of infant death. The generation of a conditional PLS3-over-expressing mouse and its breeding into an SMA background allowed us to decipher the exact biological mechanism underlying PLS3-mediated SMA protection. We show that PLS3 is a key regulator that restores main processes depending on actin dynamics in SMA motor neurons (MNs). MN soma size significantly increased and a higher number of afferent proprioceptive inputs were counted in SMAPLS3 compared with SMA mice. PLS3 increased presynaptic F-actin amount, rescued synaptic vesicle and active zones content, restored the organization of readily releasable pool of vesicles and increased the quantal content of the neuromuscular junctions (NMJs). Most remarkably, PLS3 over-expression led to a stabilization of axons which, in turn, resulted in a significant delay of axon pruning, counteracting poor axonal connectivity at SMA NMJs. These findings together with the observation of increased endplate and muscle fiber size upon MN-specific PLS3 over-expression suggest that PLS3 significantly improves neuromuscular transmission. Indeed, ubiquitous over-expression moderately improved survival and motor function in SMA mice. As PLS3 seems to act independently of Smn, PLS3 might be a potential therapeutic target not only in SMA but also in other MN diseases.

Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2ß in development.

Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2ß (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2ß is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2ß binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2ß regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2ß binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2ß protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2ß. Versions of Tra2ß lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2ß protein.

Deficiency of the splicing factor Sfrs10 results in early embryonic lethality in mice and has no impact on full-length SMN/Smn splicing.

The SR-like splicing factor SFRS10 (Htra2-beta1) is well known to influence various alternatively spliced exons without being an essential splicing factor. We have shown earlier that SFRS10 binds SMN1/SMN2 RNA and restores full-length (FL)-SMN2 mRNA levels in vitro. As SMN1 is absent in patients with spinal muscular atrophy (SMA), the level of FL-SMN2 determines the disease severity. Correct splicing of SMN2 can be facilitated by histone deacetylase inhibitors (HDACis) via upregulation of SFRS10. As HDACis are already used in SMA clinical trials, it is crucial to identify the spectrum of alternatively spliced transcripts modulated by SFRS10, because elevated SFRS10 levels may influence or misregulate also other biological processes. To address this issue, we generated a conditional Sfrs10 allele in mice using the Cre/loxP system. The ubiquitous homozygous deletion of Sfrs10, however, resulted in early embryonic lethality around E7.5, indicating an essential role of Sfrs10 during mouse embryogenesis. Deletion of Sfrs10 with recombinant Cre in murine embryonic fibroblasts (MEFs) derived from Sfrs10(fl/fl) embryos increased the low levels of SmnDelta7 3-4-fold, without affecting FL-Smn levels. The weak influence of Sfrs10 on Smn splicing was further proven by a Hb9-Cre driven motor neuron-specific deletion of Sfrs10 in mice, which developed normally without revealing any SMA phenotype. To assess the role of Sfrs10 on FL-SMN2 splicing, we established MEFs from Smn(-/-);SMN2(tg/tg);Sfrs10(fl/fl) embryos. Surprisingly, deletion of Sfrs10 by recombinant Cre showed no impact on SMN2 splicing but increased SMN levels. Our findings highlight the complexity by which alternatively spliced exons are regulated in vivo.

SAHA ameliorates the SMA phenotype in two mouse models for spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a common autosomal recessively inherited neuromuscular disorder determined by functional impairment of alpha-motor neurons within the spinal cord. SMA is caused by functional loss of the survival motor neuron gene 1 (SMN1), whereas disease severity is mainly influenced by the number of SMN2 copies. SMN2, which produces only low levels of full-length mRNA/protein, can be modulated by small molecules and drugs, thus offering a unique possibility for SMA therapy. Here, we analysed suberoylanilide hydroxamic acid (SAHA), a FDA-approved histone deacetylase inhibitor, as potential drug in two severe SMA mouse models each carrying two SMN2 transgenes: US-SMA mice with one SMN2 per allele (Smn(-/-);SMN2(tg/tg)) and Taiwanese-SMA mice with two SMN2 per allele (Smn(-/-);SMN2(tg/wt)), both on pure FVB/N background. The US-SMA mice were embryonically lethal with heterozygous males showing significantly reduced fertility. SAHA treatment of pregnant mothers rescued the embryonic lethality giving rise to SMA offspring. By using a novel breeding strategy for the Taiwanese model (Smn(-/-);SMN2(tg/tg) x Smn(-/+) mice), we obtained 50% SMA offspring that survive approximately 10 days and 50% control carriers in each litter. Treatment with 25 mg/kg twice daily SAHA increased lifespan of SMA mice by 30%, significantly improved motor function abilities, reduced degeneration of motor neurons within the spinal cord and increased the size of neuromuscular junctions and muscle fibers compared with vehicle-treated SMA mice. SMN RNA and protein levels were significantly elevated in various tissues including spinal cord and muscle. Hence, SAHA, which lessens the progression of SMA, might be suitable for SMA therapy.