Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology

For the majority of Duchenne muscular dystrophy (DMD) mutations, antisense oligonucleotide (AON)-mediated exon skipping has the potential to restore a functional protein. Here we show that weekly intravenous injections of morpholino phosphorodiamidate (morpholino) AONs induce expression of functional levels of dystrophin in body-wide skeletal muscles of the dystrophic mdx mouse, with resulting improvement in muscle function. Although the level of dystrophin expression achieved varies considerably between muscles, antisense therapy may provide a realistic hope for the treatment of a majority of individuals with DMD.     

Retinal asymmetry in birds

Vertebrate sensory systems are generally based on bilaterally symmetrical sense organs. It is evident, nevertheless, that birds preferentially use either their left or right eye for viewing novel or familiar stimuli [1], and perform visual discrimination tasks under monocular viewing conditions better with one eye than with the other [2] [3]. Because of the nearly complete contralateral decussation of the optic nerves in birds [4], it has been assumed that this division of labour is due solely to cerebral hemispheric specialisation, generated as a result of uneven photostimulation of the eyes of the developing embryo during the last three or four days before hatching [5] [6]. Here, however, we present evidence that in the European starling, Sturnus vulgaris, even the retinae are morphologically asymmetrical in terms of photoreceptor distribution. This is the first evidence for such asymmetry in any bird and suggests that retinal photoreceptor composition should be assessed during studies involving the lateralisation of visually mediated behaviours.     

Vascular endothelial insulin/IGF-1 signaling controls skin wound vascularization

Type 2 diabetes mellitus affects 6% of western populations and represents a major risk factor for the development of skin complications, of which impaired wound healing, manifested in e.g. "diabetic foot ulcer", is most prominent. Impaired angiogenesis is considered a major contributing factor to these non-healing wounds. At present it is still unclear whether diabetes-associated wound healing and skin vascular dysfunction are direct consequences of impaired insulin/IGF-1 signaling, or secondary due to e.g. hyperglycemia. To directly test the role of vascular endothelial insulin signaling in the development of diabetes-associated skin complications and vascular function, we inactivated the insulin receptor and its highly related receptor, the IGF-1 receptor, specifically in the endothelial compartment of postnatal mice, using the inducible Tie-2CreERT (DKO(IVE)) deleter. Impaired endothelial insulin/IGF-1 signaling did not have a significant impact on endothelial homeostasis in the skin, as judged by number of vessels, vessel basement membrane staining intensity and barrier function. In contrast, challenging the skin through wounding strongly reduced neo-angiogenesis in DKO(IVE) mice, accompanied by reduced granulation tissue formation reduced. These results show that endothelial insulin/IGF signaling is essential for neo-angiogenesis upon wounding, and imply that reduced endothelial insulin/IGF signaling directly contributes to diabetes-associated impaired healing.     

Social laughter is correlated with an elevated pain threshold

Although laughter forms an important part of human non-verbal communication, it has received rather less attention than it deserves in both the experimental and the observational literatures. Relaxed social (Duchenne) laughter is associated with feelings of wellbeing and heightened affect, a proximate explanation for which might be the release of endorphins. We tested this hypothesis in a series of six experimental studies in both the laboratory (watching videos) and naturalistic contexts (watching stage performances), using change in pain threshold as an assay for endorphin release. The results show that pain thresholds are significantly higher after laughter than in the control condition. This pain-tolerance effect is due to laughter itself and not simply due to a change in positive affect. We suggest that laughter, through an endorphin-mediated opiate effect, may play a crucial role in social bonding.     

Extensive and prolonged restoration of dystrophin expression with vivo-morpholino-mediated multiple exon skipping in dystrophic dogs

Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6-8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD.     

Ageing increases vulnerability to aβ42 toxicity in Drosophila

Age is the major risk factor for many neurodegenerative diseases, including Alzheimer's Disease (AD), for reasons that are not clear. The association could indicate that the duration or degree of exposure to toxic proteins is important for pathology, or that age itself increases susceptibility to protein toxicity. Using an inducible Drosophila model of AD, we investigated these possibilities by varying the expression of an Aβ42 transgene in neurons at different adult ages and measuring the effects on Aβ42 levels and associated pathological phenotypes. Acute induction of Arctic Aβ42 in young adult flies resulted in rapid expression and clearance of mRNA and soluble Arctic Aβ42 protein, but in irreversible expression of insoluble Arctic Aβ42 peptide. Arctic Aβ42 peptide levels accumulated with longer durations of induction, and this led to a dose-dependent reduction in negative geotaxis and lifespan. For a standardised level of mRNA expression, older flies had higher levels of Arctic Aβ42 peptide and associated toxicity, and this correlated with an age-dependent reduction in proteasome activity. Equalising Aβ42 protein at different ages shortened lifespan in correlation with the duration of exposure to the peptide, suggesting that Aβ42 expression accumulates damage over time. However, the relative reduction in lifespan compared to controls was greater in flies first exposed to the peptide at older ages, suggesting that ageing itself also increases susceptibility to Aβ42 toxicity. Indeed older flies were more vulnerable to chronic Aβ42 toxicity even with a much lower lifetime exposure to the peptide. Finally, the persistence of insoluble Aβ42 in both young and old induced flies suggests that aggregated forms of the peptide cause toxicity in later life. Our results suggest that reduced protein turnover, increased duration of exposure and increased vulnerability to protein toxicity at later ages in combination could explain the late age-of-onset of neurodegenerative phenotypes.     

Multiple paternity in Littorina obtusata (Gastropoda, Littorinidae) revealed by microsatellite analyses.

Parental identity for juvenile Littorina obtusata was determined from three egg masses by means of microsatellite DNA markers. Results confirm that the attendant adult female in each case was the dam of the offspring and that at least 4-6 males contributed to each brood. This correlates with our behavioral observations that indicated multiple copulations between the female and several males in each experimental aquarium. A significant number of offspring from each brood were sired by non-sampled males (males that had copulated with females before capture) whose sperm had been stored by the female. This is the first direct evidence of multiple paternity in the Littorinidae. Results are discussed in reference to current theories of sperm competition, male precedence, and cryptic female choice.