Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting in the selective death of motor neurons in the brain and spinal cord. Some familial cases of ALS are caused by dominant mutations in the gene encoding superoxide dismutase (SOD1). The emergence of interfering RNA (RNAi) for specific gene silencing could be therapeutically beneficial for the treatment of such dominantly inherited diseases. We generated a lentiviral vector to mediate expression of RNAi molecules specifically targeting the human SOD1 gene (SOD1). Injection of this vector into various muscle groups of mice engineered to overexpress a mutated form of human SOD1 (SOD1(G93A)) resulted in an efficient and specific reduction of SOD1 expression and improved survival of vulnerable motor neurons in the brainstem and spinal cord. Furthermore, SOD1 silencing mediated an improved motor performance in these animals, resulting in a considerable delay in the onset of ALS symptoms by more than 100% and an extension in survival by nearly 80% of their normal life span. These data are the first to show a substantial extension of survival in an animal model of a fatal, dominantly inherited neurodegenerative condition using RNAi and provide the highest therapeutic efficacy observed in this field to date.

Following injury, dorsal root ganglion (DRG) neurons undergo transcriptional changes so as to adopt phenotypic changes that promote cell survival and axonal regeneration. Here we used a microarray approach to profile changes in a population of small noncoding RNAs known as microRNAs (miRNAs) in the L4 and L5 DRG following sciatic nerve transection. Results showed that 20 miRNA transcripts displayed a significant change in expression levels, with 8 miRNAs transcripts being altered by more than 1.5-fold. Using quantitative reverse transcription PCR, we demonstrated that one of these miRNAs, miR-21, was upregulated by 7-fold in the DRG at 7 days post-axotomy. In dissociated adult rat DRG neurons lentiviral vector-mediated overexpression of miR-21 promoted neurite outgrowth on a reduced laminin substrate. miR-21 directly downregulated expression of Sprouty2 protein, as confirmed by Western blot analysis and 3′ untranslated region (UTR) luciferase assays. Our data show that miR-21 is an axotomy-induced miRNA that enhances axon growth, and suggest that miRNAs are important players in regulating growth pathways following peripheral nerve injury.

Angiotensin II (ANGII) acting on ANGII type 1 (AT1) receptors in the solitary tract nucleus (NTS) depresses the baroreflex. Since ANGII stimulates the release of nitric oxide (NO), we tested whether the ANGII‐mediated depression of the baroreflex in the NTS depended on NO release. In a working heart‐brainstem preparation (WHBP) of rat NTS microinjection of either ANGII (500 fmol) or a NO donor (diethylamine nonoate, 500 pmol) both depressed baroreflex gain by ‐56 and ‐67 %, respectively (P < 0.01). In contrast, whilst ANGII potentiated the peripheral chemoreflex, the NO donor was without effect. NTS microinjection of non‐selective NO synthase (NOS) inhibitors (l‐NAME; 50 pmol) or (l‐NMMA; 200 pmol) prevented the ANGII‐induced baroreflex attenuation (P > 0.1). In contrast, a neurone‐specific NOS inhibitor, TRIM (50 pmol), was without effect. Using an adenoviral vector, a dominant negative mutant of endothelial NOS (TeNOS) was expressed bilaterally in the NTS. Expression of TeNOS affected neither baseline cardiovascular parameters nor baroreflex sensitivity. However, ANGII microinjected into the transfected region failed to affect the baroreflex. Immunostaining revealed that eNOS‐positive neurones were more numerous than those labelled for AT1 receptors. Neurones double labelled for both AT1 receptors and eNOS comprised 23 ± 5.4 % of the eNOS‐positive cells and 57 ± 9.2 % of the AT1 receptor‐positive cells. Endothelial cells were also double labelled for eNOS and AT1 receptors. We suggest that ANGII activates eNOS located in either neurones and/or endothelial cells to release NO, which acts selectively to depress the baroreflex.

We have developed a non-primate-based lentiviral vector based on the equine infectious anemia virus (EIAV) for efficient gene transfer to the central and peripheral nervous systems. Previously we have demonstrated that pseudotyping lentiviral vectors with the rabies virus glycoprotein confers retrograde axonal transport to these vectors. In the present study we have successfully produced high-titer EIAV vectors pseudotyped with envelope glycoproteins from Rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Chandipura strains); rabies virus [various Evelyn-Rokitnicki-Abelseth ERA strains and challenge virus standard (CVS)]; Lyssavirus Mokola virus, a rabies-related virus; and Arenavirus lymphocytic choriomeningitis virus (LCMV). These vectors were delivered to the striatum or spinal cord of adult rats or muscle of neonatal mice by direct injection. We report that the lentiviral vectors pseudotyped with envelopes from the VSV Indiana strain, wild-type ERA, and CVS strains resulted in strong transduction in the striatum, while Mokola- and LCMV-pseudotyped vectors exhibited moderate and weak transduction, respectively. Furthermore ERA- and CVS-pseudotyped lentiviral vectors demonstrated retrograde transport and expression in distal neurons after injection in brain, spinal cord, and muscle. The differences in transduction efficiencies and retrograde transport conferred by these envelope glycoproteins present novel opportunities in designing therapeutic strategies for different neurological diseases.