Derlin-1 overexpression ameliorates mutant SOD1-induced endoplasmic reticulum stress by reducing mutant SOD1 accumulation
Graphical abstract
Research highlights
▶ Exogenous Derlin-1 reduced mutant SOD1-induced cell toxicity by suppressing ER stress. ▶ Derlin-1 resulted in a decrease in the expression of mutant SOD1 in cells. ▶ Reduced SOD1 protein expression was observed in the microsomal fraction. ▶ Derlin-1 regulates the turn over of SOD1 by promoting degradation of SOD1 protein.
Introduction
Amyotrophic lateral sclerosis (ALS) is one of the most common adult-onset paralytic diseases and is characterized by loss of both upper and lower motor neurons (Cleveland and Rothstein, 2001). Approximately 5–10% of all ALS patients have the familial form of ALS (FALS), and in 15–20% cases of FALS, family members have mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) (Rosen et al., 1993). Several lines of transgenic mice that overexpress mutant human SOD1 develop phenotypes closely resembling those associated with human ALS and are useful for studies on the pathogenesis of ALS (Gurney et al., 1994, Nagai et al., 2001).
An increased load of misfolded proteins in the endoplasmic reticulum (ER) triggers ER stress signaling, also known as the unfolded protein response (UPR) (Harding, 1992, Kaufmann et al., 2004). UPRs include induction of stress sensor kinases, chaperones, and apoptotic mediators, and are observed not only in cell- or rodent-models of FALS but also in the spinal cord of human ALS patients (Atkin et al., 2008). This implies that ER stress plays a pivotal role in the generic pathophysiology of motor neuron death. The ER-associated degradation (ERAD) system eliminates misfolded proteins via degradation in the cytosol. Misfolded ER proteins are retrotranslocated across the ER membrane into the cytosol, where ubiquitin-conjugating enzymes target them for proteasomal degradation. ERAD requires a number of dedicated ER-resident factors, including Der1p, Der3p/Hrd1p, and Hrd3p (Travers et al., 2000). In support of the theory that mutant SOD1 specifically evokes UPRs, a recent study revealed that mutant SOD1 specifically interacts with the C-terminal cytoplasmic region of Derlin-1, a mammalian homologue of Der1p, and triggers ER stress by attenuating the retrotranslocation of substrates required for ERAD (Nishitoh et al., 2008). On the other hand, exogenously expressed Derlin-1 was found to be colocalized with the cystic fibrosis transmembrane conductance regulator (CFTR), a substrate for the ubiquitin-proteasome system, in the ER, where it reduced wild-type CFTR expression and efficiently degraded the disease-associated CFTR folding mutants (Sun et al., 2006). These findings suggest that Derlin-1 recognizes misfolded, non-ubiquitylated substrates and promotes their dislocation and degradation as an adaptation to ER stress.
We hypothesized that Derlin-1 plays a pivotal role in the regulation of misfolded proteins produced by mutant SOD1. We show that Derlin-1 overexpression reduced mutant SOD1-induced cell toxicity by suppressing the activation of ER stress pathway factors such as immunoglobulin-binding protein (BiP), activating transcription factor 6 (ATF6) p50, and C/EBP homologous protein (CHOP). Interestingly, exogenous Derlin-1 expression resulted in a decrease in mutant SOD1 expression, and a lesser decrease in wild-type SOD1 expression, in transfected neuro2a cells. SOD1 expression reduced in the microsomal fraction of both wild-type and mutant cells owing to accelerated proteasomal and autophagosomal degradation of SOD1 proteins. Our finding that Derlin-1 controls the expression of mutant SOD1 may facilitate advancements for the treatment of motor neuron degeneration in ALS.
Section snippets
Construction of expression vectors, cell culture, and transfection
The human SOD1 (wild-type, G93A, or G85R) expression vectors used have been previously described (Yamashita et al., 2010). Human Derlin-1 cDNAs were amplified by PCR using primers that included appropriate restriction enzyme sites. The enzyme-digested inserts were ligated into pcDNA5/FRT/TO (Invitrogen, Carlsbad, CA, USA), and the DNA sequences of the recombinant cDNAs thus obtained were confirmed by the dideoxynucleotide chain termination method. Derlin-2 and Derlin-3 expression vectors were
Wild-type SOD1 and mutant SOD1 partially colocalized with Derlin-1 in ER
Derlin-1 is involved in ERAD, and its dysfunction exacerbates mutant SOD1-induced ER stress (Nishitoh et al., 2008). We examined whether overexpressed Derlin-1 could colocalize with SOD1, as suggested by Nishitoh et al. (2008). We performed immunofluorescence analysis using neuro2a cells cotransfected with FLAG-tagged SOD1 (wild-type, G93A, or G85R), and Derlin-1 or its control vector, pcDNA5. Staining with anti-FLAG and anti-Derlin-1 antibodies revealed partial colocalization of both wild-type
Discussion
Recent reports have suggested that ER stress is related to the pathogenesis of FALS and SALS (Kikuchi et al., 2006, Ilieva et al., 2007, Atkin et al., 2008). These imply that ER stress plays a pivotal role in the generic pathophysiology of motor neuron death, although it remains unclear as to whether the overload of misfolded proteins in the ER followed by UPRs serves as a trigger or a consequence of motor neuron degeneration. In support of the theory that mutant SOD1 specifically elicited
Acknowledgments
This work was supported by a Grant-in-Aid for Young Scientists (B), the Ministry of Education, Culture, Sports, Science and Technology of Japan; Grants-in-Aid from the Research Committee of CNS Degenerative Diseases, the Ministry of Health, Labour and Welfare of Japan; The Nakabayashi Trust For ALS Research; ALS Foundation, Japan ALS Association; Kanae Foundation for the Promotion of Medical Science; and Advanced Education Program for Integrated Clinical, Basic and Social Medicine, Graduate
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