An Early Onset Progressive Motor Neuron Disorder inScyl1-Deficient Mice Is Associated with Mislocalization of TDP-43

Abstract

The molecular and cellular bases of motor neuron diseases (MNDs) are still poorly understood. The diseases are mostly sporadic, with ϳ10% of cases being familial. In most cases of familial motor neuronopathy, the disease is caused by either gain-of-adverse-effect mutations or partial loss-of-function mutations in ubiquitously expressed genes that serve essential cellular functions. Here we show that deletion of Scyl1, an evolutionarily conserved and ubiquitously expressed gene encoding the COPI-associated protein pseudokinase SCYL1, causes an early onset progressive MND with characteristic features of amyotrophic lateral sclerosis (ALS). Skeletal muscles of Scyl1 Ϫ/Ϫ mice displayed neurogenic atrophy, fiber type switching, and disuse atrophy. Peripheral nerves showed axonal degeneration. Loss of lower motor neurons (LMNs) and large-caliber axons was conspicuous in Scyl1Ϫ/Ϫ animals. Signs of neuroinflammation were seen throughout the CNS, most notably in the ventral horn of the spinal cord. Neural-specific, but not skeletal muscle-specific, deletion of Scyl1 was sufficient to cause motor dysfunction, indicating that SCYL1 acts in a neural cell-autonomous manner to prevent LMN degeneration and motor functions. Remarkably, deletion of Scyl1 resulted in the mislocalization and accumulation of TDP-43 (TAR DNA-binding protein of 43 kDa) and ubiquilin 2 into cytoplasmic inclusions within LMNs, features characteristic of most familial and sporadic forms of ALS. Together, our results identify SCYL1 as a key regulator of motor neuron survival, and Scyl1 Ϫ/Ϫ mice share pathological features with many human neurodegenerative conditions.

SCYL2 Protects CA3 Pyramidal Neurons from Excitotoxicity during Functional Maturation of the Mouse Hippocampus

“…All oligonucleotides used in this study were produced by the Hartwell Center for Bioinformatics and Biotechnology (St. Jude Children's Research Hospital). Generation of the plasmid pBR322-DTA (diphtheria toxin A) has been described previously (Pelletier et al, 2012). Plasmids PL452 and PL451 were obtained from Dr. Neil A. Copeland (National Cancer Institute) (Liu et al, 2003).…”

“…The inverted-grid test was used to assess neuromuscular function and muscular strength (Pelletier et al, 2012). Mice were placed on top of an elevated cage grid.…”

“…HEK293T cells and SV40-transformed mouse embryonic fibroblasts (MEFs) were cultured in DMEM, high glucose, supplemented with FBS, L-glutamine, penicillin, and streptomycin as described previously (Pelletier et al, 2006). Hippocampal neurons were isolated from E18.5-P0 mouse brains and grown as described previously (Beaudoin et al, 2012).…”

“…Mouse tissues were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40 [Igepal-CA-630], 0.5% sodium deoxycholate, 0.1% SDS, and complete protease inhibitors and phosSTOP phosphatase inhibitors) (Roche). Western blotting was performed as described previously (Pelletier et al, 2006).…”

Scyl1 scaffolds class II Arfs to selective subcomplexes of coatomer via the γ-COP appendage domain

“…Homozygous recessive mdf mice develop a waddling gait, tremor and hind-limb paralysis at 4-8 weeks of age, with neurogenic atrophy (Blot et al, 1995). Moreover, a recently developed Scyl1-knockout mouse displays motor neuron degeneration similar to that observed in mdf mice, and Scyl1 2/2 pathology is associated with TDP-43-positive cytoplasmic inclusions in motor neurons (Pelletier et al, 2012), a hallmark of motor neuron diseases such as amyotrophic lateral sclerosis (ALS) (Neumann et al, 2006). Thus, it is vital to develop a better understanding of the role of Scyl1 in membrane trafficking.…”