Neurofilaments (NFs) are thought to provide stability to the axon. We examined NF dynamics within axonal neurites of NB2a/d1 neuroblastoma by transient transfection with green fluorescent protein-tagged NF-heavy (GFP-H) under the control of a tetracycline-inducible promoter. Immunofluorescent and biochemical analyses demonstrated that GFP-H expressed early during neurite outgrowth associated with a population of centrally-situated, highly-phosphorylated crosslinked NFs along the length of axonal neurites (‘bundled NFs’). By contrast, GFP-H expressed after considerable neurite outgrowth displayed markedly reduced association with bundled NFs and was instead more evenly distributed throughout the axon. This differential localization was maintained for up to 2 weeks in culture. Once considerable neurite outgrowth had progressed, GFP that had previously associated with the NF bundle during early expression was irreversibly depleted by photobleaching. Cessation of expression allowed monitoring of NF turnover. GFP-H associated bundled NFs underwent slower decay than GFP-H associated with surrounding, less-phosphorylated NFs. Notably, GFP associated with bundled NFs underwent similar decay rates within the core and edges of this bundle. These results are consistent with previous demonstration of a resident NF population within axonal neurites, but suggest that this population is more dynamic than previously considered.

Background:Amyotrophic lateral sclerosis (ALS) is a progressive disease of motor neurons that has no cure or effective treatment. Any approach that could sustain minor motor function during terminal stages would improve quality of life.Objective:We examined the impact of omega-3 (Ω-3) and Ω-6, on motor neuron function in mice expressing mutant human superoxide dismutase-1 (SOD-1), which dominantly confers familial ALS and induces a similar sequence of motor neuron decline and eventual death when expressed in mice.Method:Mice received standard diets supplemented with equivalent amounts of Ω-3 and Ω-6 or a 10x increase in Ω-6 with no change in Ω-3 commencing at 4 weeks of age. Motor function and biochemical/histological parameters were assayed by standard methodologies.Results:Supplementation with equivalent Ω-3 and Ω-6 hastened motor neuron pathology and death, while 10x Ω-6 with no change in Ω-3 significantly delayed motor neuron pathology, including preservation of minor motor neuron function during the terminal stage.Conclusion:In the absence of a cure or treatment, affected individuals may resort to popular nutritional supplements such as Ω-3 as a form of “self-medication”. However, our findings and those of other laboratories indicate that such an approach could be harmful. Our findings suggest that a critical balance of Ω-6 and Ω-3 may temporarily preserve motor neuron function during the terminal stages of ALS, which could provide a substantial improvement in quality of life for affected individuals and their caregivers.

No abstract

Ocular scarring after surgery, trauma, or infection leads to vision loss. The transparent cornea is an excellent model system to test anti-scarring therapies. Cholesterol-conjugated fully modified asymmetric small interfering RNAs (siRNAs) (selfdeliverable siRNAs [sdRNAs]) are a novel modality for in vivo gene knockdown, transfecting cells and tissues without any additional formulations. Myofibroblasts are a main contributor to scarring and fibrosis. a v integrins play a central role in myofibroblast pathological adhesion, overcontraction, and transforming growth factor b (TGF-b) activation. Previously, we demonstrated that a v integrins are protected from intracellular degradation after wounding by upregulation of the deubiquitinase (DUB) ubiquitin-specific protease 10 (USP10), leading to integrin cell surface accumulation. In this study, we tested whether knockdown of USP10 with a USP10-targeting sdRNA (termed US09) will reduce scarring after wounding a rabbit cornea in vivo. The wounded corneal stroma was treated once with US09 or non-targeting control (NTC) sdRNA. At 6 weeks US09 treatment resulted in faster wound closure, limited scarring, and suppression of fibrotic markers and immune response. Specifically, fibronectin-extra domain A (EDA), collagen III, and a-smooth muscle actin (p < 0.05), CD45 + cell infiltration (p < 0.01), and apoptosis at 24 (p < 0.01) and 48 h (p < 0.05) were reduced post-wounding. Corneal thickness and cell proliferation were restored to unwounded parameters. Targeting the DUB, USP10 is a novel strategy to reduce scarring. This study indicates that ubiquitin-mediated pathways should be considered in the pathogenesis of fibrotic healing.