Here, we show that the biochemical and cell biological properties of SCYL3 are similar to those of SCYL1 and both proteins work in conjunction to maintain motor neuron viability. of SCYL3 are similar to those of SCYL1 and both proteins work in conjunction to maintain motor neuron viability. Specifically, although lack of in mice has no apparent effect on embryogenesis and postnatal life, it accelerates the onset of the motor neuron disorder caused by deficiency. Dicloxacillin Sodium hydrate Growth abnormalities, motor dysfunction, hindlimb paralysis, muscle wasting, neurogenic atrophy, motor neuron degeneration, and loss of large-caliber axons in peripheral nerves Dicloxacillin Sodium hydrate occurred at an earlier age in double-deficient mice than in and highlight the importance the SCYL family of proteins in regulating neuronal function and survival. Only male mice were used in this study. SIGNIFICANCE STATEMENT SCYL1 and SCYL2, members of the SCY1-like family of pseudokinases, have well established roles in neuronal function. Herein, we uncover the role of SCYL3 in maintaining motor neuron viability. Although targeted disruption of in mice had little or no effect on embryonic development and postnatal life, it accelerated disease onset associated with the loss of Dicloxacillin Sodium hydrate and double-deficient mice had neuronal defects characteristic of amyotrophic lateral sclerosis, including TDP-43 pathology, at an earlier age than did and confirm that SCYL family members are critical regulators of neuronal function and survival. are associated with a rare syndrome characterized by liver failure, cerebellar atrophy, ataxia and, most importantly peripheral neuropathy (Schmidt et al., 2015). In mice, spontaneous or targeted deletion of causes an early onset motor neuron disorder characterized by progressive loss of motor function, muscle wasting, loss of large-caliber axons in the periphery, and loss of lower motor neurons in the ventral horn of the spinal cord (Blot et al., 1995; Schmidt et al., 2007; Pelletier et al., 2012). The disease is neural cell-autonomous and involves molecular pathways associated with other motor neuron disorders such as amyotrophic lateral sclerosis (ALS; Pelletier et al., 2012). Mislocalization of the RNA-binding protein (RBP) TDP-43 from the nucleus to cytoplasmic inclusions, a hallmark of ALS and frontotemporal lobar degeneration, occurs in lower motor neurons of in mice causes severe neurological disorders, including impaired suckling behavior, which result in premature death of most newborn mice (Gingras et al., 2015). In surviving mice, the absence of results in the degeneration of several neuronal populations, most notably CA3 pyramidal neurons of the hippocampus (Gingras et al., 2015). The degenerative process associated with the loss of SCYL2 is also neural cell-autonomous and caused by excessive excitatory signaling (Gingras et al., 2015). Although these studies clearly established SCYL1 and SCYL2 as critical regulators of neuronal function and survival, the biological function of SCYL3 has remained elusive. In this study, we use a combination of biochemical, cell biological and genetic approaches to elucidate the function of SCYL3 in mammalian development and physiology. We show that SCYL3 is a widely expressed Golgi membrane-associated protein with biochemical and cell biological properties similar to those of SCYL1 and both proteins play an overlapping role in maintaining motor neuron viability in mice. Although targeted disruption of in mice did not produce overt abnormalities, absence of accelerated the onset of the motor phenotype associated with loss of and double-deficient mice also correlated with TDP-43 pathology in spinal motor neurons suggesting a link between SCYL1, SCYL3, and TDP-43 proteostasis. Materials and Methods SCYL3 antibody. A peptide corresponding to amino acids 7C27 [ALKSYTLRESPFTLPSGLAVY (serum 7688)] of SCYL3 was synthesized by the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children’s Research Hospital (St. Jude) and conjugated to glutaraldehyde-activated keyhole limpet hemocyanin (KLH). The KLH-conjugated peptide was used to immunize rabbits (Rockland Immunochemicals). The serum was enriched for the peptide of interest by affinity chromatography on a matrix coupled to the corresponding peptide. Selectivity of the antibody was tested against other SCY1-like family members by RNA interference (RNAi)-mediated knockdown of SCYL1, SCYL2, and SCYL3. No cross-reactivity with other SCYL proteins was found. Reactivity of the peptide-purified antibody was also tested by Western blot, using protein extracts from was subcloned by gap repair into pBR322-DTA. The first cassette containing the neomycin-resistance gene flanked by 2 loxP sites (PL452) was inserted in intron 4. After Mcam excision of the neomycin cassette by CRE recombinase, a second cassette containing the neomycin-resistance and thymidine kinase cDNAs flanked by 2 Flp.