Derlin-1 overexpression ameliorates mutant SOD1-induced endoplasmic reticulum stress by reducing mutant SOD1 accumulation

https://doi.org/10.1016/j.neuint.2010.12.010Get rights and content

Abstract

Unfolded protein responses, including induction of stress sensor kinases, chaperones, and apoptotic mediators, are involved in the familial amyotrophic lateral sclerosis (ALS) model related to mutant Cu/Zn superoxide dismutase (SOD1) and sporadic ALS. We hypothesized that the endoplasmic reticulum-resident factor Derlin-1 plays a pivotal role in the regulation of misfolded proteins evoked by mutant SOD1. We show that Derlin-1 overexpression reduced mutant SOD1-induced cell toxicity and increased cell viability by suppressing the activation of the ER stress pathway factors: immunoglobulin-binding protein, activating transcription factor 6 p50, and C/EBP homologous protein. Interestingly, exogenous Derlin-1 resulted in a decrease in the amount of mutant SOD1, and a lesser decrease in that of wild-type SOD1, in transfected cells. Reduced SOD1 protein expression was observed in the microsomal fraction of wild-type and mutant SOD1 cells. Our results indicate that Derlin-1 regulates the turn over of SOD1 by promoting the proteasomal and autophagosomal degradation of SOD1 protein, but not by decreasing mutant SOD1 mRNA levels. Insights into the effects of Derlin-1 on mutant SOD1 may facilitate advancements in the treatment of motor neuron degeneration associated with ALS.

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

References (19)

There are more references available in the full text version of this article.

Cited by (33)

  • Proteotoxic stress and the ubiquitin proteasome system

    2024, Seminars in Cell and Developmental Biology
  • SERS “hot spot” enhance-array assay for misfolded SOD1 correlated with white matter lesions and aging

    2023, Analytica Chimica Acta
    Citation Excerpt :

    Regarding the conformational misfolding of SOD1, oxidative stress is considered to be the most common way. Experimental evidence has demonstrated that the production of misfolded SOD1 is closely related to the endoplasmic reticulum (ER) and mitochondria [17,18], particularly in the nervous system. The ER and mitochondria are the most significant sites for oxidative stress and misfolded SOD1 is usually enriched in the aggregates region for mitochondria [17].

  • Chemical stresses fail to mimic the unfolded protein response resulting from luminal load with unfolded polypeptides

    2018, Journal of Biological Chemistry
    Citation Excerpt :

    These compounds, however, nonspecifically activate all three mammalian UPR branches and their use has pleiotropic consequences resulting in lethal multiorganelle and multicompartmental failures (18). A handful of studies analyzed responses to expression of unfolded proteins in the mammalian ER (e.g. Refs. 19–24). Strikingly however, given the meaning of the acronym UPR (i.e. responses to unfolded proteins), comparative high throughput studies at the transcriptome and proteome level aiming to distinguish chemical- versus unfolded protein–induced ER stresses of the same magnitude and to molecularly characterize these latter, remain to be performed.

  • Proteostasis impairment in ALS

    2016, Brain Research
    Citation Excerpt :

    Another mechanism by which mutant SOD1 can impact ER homeostasis, is via its aberrant association with Derlin-1, a component of the ER Associated Degradation (ERAD) machinery, which leads to its inhibition, resulting in ER stress and proteostasis impairment (Nishitoh et al., 2008). Here, Derlin-1 overexpression was shown to be efficient in reducing the amount of accumulated mutant SOD1, leading to improved cell survival due to the inhibition of ER stress and concomitant activation of proteasomal and autophagy-associated degradation of mutant SOD1 (Mori et al., 2011). These findings further strengthen the notion that these pathways are complex modulators of cellular proteostasis involving active cross-talks with different homeostasis-associated processes.

  • Proteasome activation: An innovative promising approach for delaying aging and retarding age-related diseases

    2015, Ageing Research Reviews
    Citation Excerpt :

    Moreover, strategies aiming to enhance the E3-ubiquitin ligases have shown promising results (Sone et al., 2010; Yonashiro et al., 2009). The proteasomal function can be also enhanced through the up-regulation of proteins relevant to the ER stress such as Derlin-1, overexpresssion of which reduced the protein aggregation of mutant SOD1 in cell cultures (Mori et al., 2011), or TorsinA that restored the normal neuronal function in the C. elegans model of ALS (Thompson et al., 2014). Regarding chemical activation, melittin treatment of ALS model mice restored the proteasomal activity followed by reduced neuronal death (Yang et al., 2011).

  • Calcium-dependent protein folding in amyotrophic lateral sclerosis

    2013, Cell Calcium
    Citation Excerpt :

    However, it is not yet clear exactly how mutated SOD1 toxicity is linked to ER stress and apoptosis. Several reports have described that Derlin-1, which is involved in retrotranslocation of misfolded proteins across the ER membrane for purposes of degradation, can trigger ER stress owing to its interaction with mutant SOD1 [53]. Furthermore, the BH3-only protein, Bim has been directly linked to ER stress and the ensuing mitochondrial apoptosis in the murine neuroblastoma cell line, Neuro2a [54].

View all citing articles on Scopus
View full text