The formin DAAM1 regulates the deubiquitinase activity of USP10 and integrin homeostasis.

The differentiation of fibroblasts into pathological myofibroblasts during wound healing is characterized by increased cell surface expression of αv-integrins. Our previous studies found that the deubiquitinase (DUB) USP10 removes ubiquitin from αv-integrins, leading to cell surface integrin accumulation, subsequent TGFß1 activation, and pathological myofibroblast differentiation. In this study, a yeast two-hybrid screen revealed a novel binding partner for USP10, the formin, DAAM1. We found that DAAM1 binds to and inhibits USP10's DUB activity through the FH2 domain of DAAM1 independent of its actin functions. The USP10/DAAM1 interaction was also supported by proximity ligation assay (PLA) in primary human corneal fibroblasts. Treatment with TGFß1 significantly increased USP10 and DAAM1 protein expression, PLA signal, and co-localization to actin stress fibers. DAAM1 siRNA knockdown significantly reduced co-precipitation of USP10 and DAAM1 on purified actin stress fibers, and ß1- and ß5-integrin ubiquitination. This resulted in increased αv-, ß1-, and ß5-integrin total protein levels, αv-integrin recycling, and extracellular fibronectin (FN) deposition. Together, our data demonstrate that DAAM1 inhibits USP10's DUB activity on integrins subsequently regulating cell surface αv-integrin localization and FN accumulation.

USP10 Promotes Fibronectin Recycling, Secretion, and Organization.

Purpose: Integrins play a central role in myofibroblast pathological adhesion, over-contraction, and TGFß activation. Previously, we demonstrated that after corneal wounding, αv integrins are protected from intracellular degradation by upregulation of the deubiquitinase USP10, leading to cell-surface integrin accumulation. Because integrins bind to and internalize extracellular matrix (ECM), we tested whether extracellular fibronectin (FN) accumulation can result from an increase in integrin and matrix recycling in primary human corneal fibroblasts (HCFs). Methods: Primary HCFs were isolated from cadaver eyes. HCFs were transfected with either USP10 cDNA or control cDNA by nucleofection. Internalized FN was quantified with a FN ELISA. Recycled extracellular integrin and FN were detected with streptavidin-488 by live cell confocal microscopy (Zeiss LSM 780). Endogenous FN extra domain A was detected by immunocytochemistry. Cell size and removal of FN from the cell surface was determined by flow cytometry. Results: USP10 overexpression increased α5ß1 (1.9-fold; P < 0.001) and αv (1.7-fold; P < 0.05) integrin recycling, with a concomitant increase in biotinylated FN internalization (2.1-fold; P < 0.05) and recycling over 4 days (1.7-2.2-fold; P < 0.05). The dependence of FN recycling on integrins was demonstrated by α5ß1 and αv integrin blocking antibodies, which, compared with control IgG, decreased biotinylated FN recycling (62% and 84%, respectively; P < 0.05). Overall, we established that extracellular FN was composed of approximately 1/3 recycled biotinylated FN and 2/3 endogenously secreted FN. Conclusions: Our data suggest that reduced integrin degradation with a subsequent increase in integrin/FN recycling after wounding may be a newly identified mechanism for the characteristic accumulation of ECM in corneal scar tissue.

Tau interferes with axonal neurite stabilization and cytoskeletal composition independently of its ability to associate with microtubules.

Tau impacts overall axonal transport particularly when overexpressed by interfering with translocation of kinesin along microtubules (MTs) and/or as a cargo of kinesin by outcompeting other kinesin cargo. To discern between which of these mechanisms was more robust during axonal outgrowth, we overexpressed phosphomimetic (E18; which is incapable of MT binding), phospho-null (A18) or wild-type (WT) full-length human tau conjugated to EGFP, the latter two of which bind MTs. Expression of WT and A18 displayed increased acetylated MTs and resistance to colchicine, while expression of E18 did not, indicating that E18 did not contribute to MT stabilization. Expression of all tau constructs reduced overall levels of neurofilaments (NFs) within axonal neurites, and distribution of NFs along neurite lengths. Since NFs are another prominent cargo of kinesin during axonal neurite outgrowth, this finding is consistent with WT, A18 and E18 inhibiting NF transport to the same extent by competing as cargo of kinesin. These findings indicate that tau can impair axonal transport independently of association with MTs in growing axonal neurites.

USP10 Targeted Self-Deliverable siRNA to Prevent Scarring in the Cornea.

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) (self-deliverable 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. αv integrins play a central role in myofibroblast pathological adhesion, overcontraction, and transforming growth factor ß (TGF-ß) activation. Previously, we demonstrated that αv 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.

Assembly and turnover of neurofilaments in growing axonal neurites.

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.

Influence of a GSK3ß phosphorylation site within the proximal C-terminus of Neurofilament-H on neurofilament dynamics.

Phosphorylation of the C-terminal tail of the heavy neurofilament subunit (NF-H) impacts neurofilament (NF) axonal transport and residence within axons by fostering NF-NF associations that compete with transport. We tested the role of phosphorylation of a GSK-3ß consensus site (S493) located in the proximal portion of the NF-H tail in NF dynamics by transfection of NB2a/d1 cells with NF-H, where S493 was mutated to aspartic acid (S493D) or to alanine (S493A) to mimic constitutive phosphorylation and non-phosphorylation. S493D underwent increased transport into axonal neurites, while S493A displayed increased perikaryal NF aggregates that were decorated by anti-kinesin. Increased levels of S493A co-precipitated with anti-kinesin indicating that reduced transport of S493A was not due to reduced kinesin association but due to premature NF-NF interactions within perikarya. S493D displayed increased phospho-immunoreactivity within axonal neurites at downstream C-terminal sites attributable to mitogen-activated protein kinase and cyclin-dependent kinase 5. However, S493D was more prone to proteolysis following kinase inhibition, suggesting that S493 phosphorylation is an early event that alters sidearm configuration in a manner that promotes appropriate NF distribution. We propose a novel model for sidearm configuration.

Omega-3 Hastens and Omega-6 Delays the Progression of Neuropathology in a Murine Model of Familial ALS.

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.