Viral delivery of antioxidant genes as a therapeutic strategy in experimental models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder with no effective treatment to date. Despite its multi-factorial aetiology, oxidative stress is hypothesized to be one of the key pathogenic mechanisms. It is thus proposed that manipulation of the expression of antioxidant genes that are downregulated in the presence of mutant SOD1 may serve as a therapeutic strategy for motor neuronal protection. Lentiviral vectors expressing either PRDX3 or NRF2 genes were tested in the motor neuronal-like NSC34 cell line, and in the ALS tissue culture model, NSC34 cells expressing the human SOD1(G93A) mutation. The NSC34 SOD1(G93A) cells overexpressing either PRDX3 or NRF2 showed a significant decrease in endogenous oxidation stress levels by 40 and 50% respectively compared with controls, whereas cell survival was increased by 30% in both cases. The neuroprotective potential of those two genes was further investigated in vivo in the SOD1(G93A) ALS mouse model, by administering intramuscular injections of adenoassociated virus serotype 6 (AAV6) expressing either of the target genes at a presymptomatic stage. Despite the absence of a significant effect in survival, disease onset or progression, which can be explained by the inefficient viral delivery, the promising in vitro data suggest that a more widespread CNS delivery is needed

Site Specific Modification of Adeno-Associated Virus Enables Both Fluorescent Imaging of Viral Particles and Characterization of the Capsid Interactome

Adeno-associated viruses (AAVs) are attractive gene therapy vectors due to their low toxicity, high stability, and rare integration into the host genome. Expressing ligands on the viral capsid can re-target AAVs to new cell types, but limited sites have been identified on the capsid that tolerate a peptide insertion. Here, we incorporated a site-specific tetracysteine sequence into the AAV serotype 9 (AAV9) capsid, to permit labelling of viral particles with either a fluorescent dye or biotin. We demonstrate that fluorescently labelled particles are detectable in vitro, and explore the utility of the method in vivo in mice with time-lapse imaging. We exploit the biotinylated viral particles to generate two distinct AAV interactomes, and identify several functional classes of proteins that are highly represented: actin/cytoskeletal protein binding, RNA binding, RNA splicing/processing, chromatin modifying, intracellular trafficking and RNA transport proteins. To examine the biological relevance of the capsid interactome, we modulated the expression of two proteins from the interactomes prior to AAV transduction. Blocking integrin αVβ6 receptor function reduced AAV9 transduction, while reducing histone deacetylase 4 (HDAC4) expression enhanced AAV transduction. Our method demonstrates a strategy for inserting motifs into the AAV capsid without compromising viral titer or infectivity

Candidate Proteins, Metabolites and Transcripts in the Biomarkers for Spinal Muscular Atrophy (BforSMA) Clinical Study

Spinal Muscular Atrophy (SMA) is a neurodegenerative motor neuron disorder resulting from a homozygous mutation of the survival of motor neuron 1 (SMN1) gene. The gene product, SMN protein, functions in RNA biosynthesis in all tissues. In humans, a nearly identical gene, SMN2, rescues an otherwise lethal phenotype by producing a small amount of full-length SMN protein. SMN2 copy number inversely correlates with disease severity. Identifying other novel biomarkers could inform clinical trial design and identify novel therapeutic targets.To identify novel candidate biomarkers associated with disease severity in SMA using unbiased proteomic, metabolomic and transcriptomic approaches.A cross-sectional single evaluation was performed in 108 children with genetically confirmed SMA, aged 2-12 years, manifesting a broad range of disease severity and selected to distinguish factors associated with SMA type and present functional ability independent of age. Blood and urine specimens from these and 22 age-matched healthy controls were interrogated using proteomic, metabolomic and transcriptomic discovery platforms. Analyte associations were evaluated against a primary measure of disease severity, the Modified Hammersmith Functional Motor Scale (MHFMS) and to a number of secondary clinical measures.A total of 200 candidate biomarkers correlate with MHFMS scores: 97 plasma proteins, 59 plasma metabolites (9 amino acids, 10 free fatty acids, 12 lipids and 28 GC/MS metabolites) and 44 urine metabolites. No transcripts correlated with MHFMS.In this cross-sectional study, "BforSMA" (Biomarkers for SMA), candidate protein and metabolite markers were identified. No transcript biomarker candidates were identified. Additional mining of this rich dataset may yield important insights into relevant SMA-related pathophysiology and biological network associations. Additional prospective studies are needed to confirm these findings, demonstrate sensitivity to change with disease progression, and assess potential impact on clinical trial design.Clinicaltrials.gov NCT00756821

Overview of Current Drugs and Molecules in Development for Spinal Muscular Atrophy Therapy

The full text of this article can be found here: https://link.springer.com/article/10.1007/s40265-018-0868-

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCF^Slmb degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

Pathogenic Huntingtin Repeat Expansions in Patients with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis.

We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40-64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington's disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered

Primary Care Consultations About Medically Unexplained Symptoms: How Do Patients Indicate What They Want?

BACKGROUND: Patients with medically unexplained physical symptoms (MUS) are often thought to deny psychological needs when they consult general practitioners (GPs) and to request somatic intervention instead. We tested predictions from the contrasting theory that they are transparent in communicating their psychological and other needs. OBJECTIVE: To test predictions that what patients tell GPs when they consult about MUS is related transparently to their desire for (1) emotional support, (2) symptom explanation and (3) somatic intervention. DESIGN: Prospective naturalistic study. Before consultation, patients indicated what they wanted from it using a self-report questionnaire measuring patients' desire for: emotional support, explanation and reassurance, and physical investigation and treatment. Their speech during consultation was audio-recorded, transcribed and coded utterance-by-utterance. Multilevel regression analysis tested relationships between what patients sought and what they said. PARTICIPANTS: Patients (N = 326) consulting 33 GPs about symptoms that the GPs designated as MUS. RESULTS: Patients who wanted emotional support spoke more about psychosocial problems, including psychosocial causes of symptoms and their need for psychosocial help. Patients who wanted explanation and reassurance suggested more physical explanations, including diseases, but did not overtly request explanation. Patients' wish for somatic intervention was associated only with their talk about details of such interventions and not with their requests for them. CONCLUSIONS: In general, patients with medically unexplained symptoms provide many cues to their desire for emotional support. They are more indirect or guarded in communicating their desire for explanation and somatic intervention