Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS.

Many mutations confer one or more toxic function(s) on copper/zinc superoxide dismutase 1 (SOD1) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we found that oxidized wild-type SOD1 and mutant SOD1 share a conformational epitope that is not present in normal wild-type SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord were markedly C4F6 immunoreactive, indicating that an aberrant wild-type SOD1 species was present. Recombinant, oxidized wild-type SOD1 and wild-type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to that of FALS-linked mutant SOD1. Our findings suggest that wild-type SOD1 can be pathogenic in SALS and identify an SOD1-dependent pathogenic mechanism common to FALS and SALS

PP1 Forms an Active Complex with TLRR (lrrc67), a Putative PP1 Regulatory Subunit, during the Early Stages of Spermiogenesis in Mice

Mammalian spermatogenesis is a highly regulated developmental pathway that demands dramatic rearrangement of the cytoskeleton of the male germ cell. We have described previously a leucine rich repeat protein, TLRR (also known as lrrc67), which is associated with the spermatid cytoskeleton in mouse testis and is a binding partner of protein phosphatase-1 (PP1), an extremely well conserved signaling molecule. The activity of PP1 is modulated by numerous specific regulators of which TLRR is a candidate. In this study we measured the phosphatase activity of the TLRR-PP1 complex in the adult and the developing mouse testis, which contains varying populations of developing germ cell types, in order to determine whether TLRR acts as an activator or an inhibitor of PP1 and whether the phosphatase activity of this complex is developmentally regulated during spermatogenesis. Additionally, we assayed the ability of bacterially expressed TLRR to affect the enzymatic activity of PP1. Furthermore, we examined phosphorylation of TLRR, and elements of the spermatid cytoskeleton during the first wave of spermatogenesis in the developing testis. We demonstrate here that the TLRR complex is associated with a phosphatase activity in adult mouse testis. The relative phosphatase activity of this complex appears to reach a peak at about 21 days after birth, when pachytene spermatocytes and round spermatids are abundant in the seminiferous epithelium of the mouse testis. TLRR, in addition to tubulin and kinesin-1B, is phosphorylated during the first wave of spermatogenesis. These findings indicate that the TLRR-PP1 complex is active prior to translocation of TLRR toward the sperm flagella and that TLRR, and constituents of the spermatid cytoskeleton, may be subject to regulation by reversible phosphorylation during spermatogenesis in murine testis

Quantification of Retrograde Axonal Transport in the Rat Optic Nerve by Fluorogold Spectrometry

PURPOSE: Disturbed axonal transport is an important pathogenic factor in many neurodegenerative diseases, such as glaucoma, an eye disease characterised by progressive atrophy of the optic nerve. Quantification of retrograde axonal transport in the optic nerve usually requires labour intensive histochemical techniques or expensive equipment for in vivo imaging. Here, we report on a robust alternative method using Fluorogold (FG) as tracer, which is spectrometrically quantified in retinal tissue lysate. METHODS: To determine parameters reflecting the relative FG content of a sample FG was dissolved in retinal lysates at different concentrations and spectra were obtained. For validation in vivo FG was injected uni- or bilaterally into the superior colliculus (SC) of Sprague Dawley rats. The retinal lysate was analysed after 3, 5 and 7 days to determine the time course of FG accumulation in the retina (n = 15). In subsequent experiments axona transport was impaired by optic nerve crush (n = 3), laser-induced ocular hypertension (n = 5) or colchicine treatment to the SC (n = 10). RESULTS: Spectrometry at 370 nm excitation revealed two emission peaks at 430 and 610 nm. We devised a formula to calculate the relative FG content (c(FG)), from the emission spectrum. c(FG) is proportional to the real FG concentration as it corrects for variations of retinal protein concentration in the lysate. After SC injection, c(FG) monotonously increases with time (p = 0.002). Optic nerve axonal damage caused a significant decrease of c(FG) (crush p = 0.029; hypertension p = 0.025; colchicine p = 0.006). Lysates are amenable to subsequent protein analysis. CONCLUSIONS: Spectrometrical FG detection in retinal lysates allows for quantitative assessment of retrograde axonal transport using standard laboratory equipment. It is faster than histochemical techniques and may also complement morphological in vivo analyses