SOD1 as a molecular switch for initiating the homeostatic ER stress response under zinc deficiency.

Zinc is an essential trace element, and impaired zinc homeostasis is implicated in the pathogenesis of various human diseases. However, the mechanisms cells use to respond to zinc deficiency are poorly understood. We previously reported that amyotrophic lateral sclerosis (ALS)-linked pathogenic mutants of SOD1 cause chronic endoplasmic reticulum (ER) stress through specific interactions with Derlin-1, which is a component of the ER-associated degradation machinery. Moreover, we recently demonstrated that this interaction is common to ALS-linked SOD1 mutants, and wild-type SOD1 (SOD1(WT)) comprises a masked Derlin-1 binding region (DBR). Here, we found that, under zinc-deficient conditions, SOD1(WT) adopts a mutant-like conformation that exposes the DBR and induces the homeostatic ER stress response, including the inhibition of protein synthesis and induction of a zinc transporter. We conclude that SOD1 has a function as a molecular switch that activates the ER stress response, which plays an important role in cellular homeostasis under zinc-deficient conditions.

The ASK1-specific inhibitors K811 and K812 prolong survival in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure. To develop effective treatments for this devastating disease, an appropriate strategy for targeting the molecule responsible for the pathogenesis of ALS is needed. We previously reported that mutant SOD1 protein causes motor neuron death through activation of ASK1, a mitogen-activated protein kinase kinase kinase. Additionally, we recently developed K811 and K812, which are selective inhibitors for ASK1. Here, we report the effect of K811 and K812 in a mouse model of ALS (SOD1(G93A) transgenic mice). Oral administration of K811 or K812 significantly extended the life span of SOD1(G93A) transgenic mice (1.06 and 1.08% improvement in survival). Moreover, ASK1 activation observed in the lumbar spinal cord of mice at the disease progression stage was markedly decreased in the K811- and K812-treated groups. In parallel, immunohistochemical analysis revealed that K811 and K812 treatment inhibited glial activation in the lumbar spinal cord of SOD1(G93A) transgenic mice. These results reinforce the importance of ASK1 as a therapeutic target for ALS treatment.

A systematic immunoprecipitation approach reinforces the concept of common conformational alterations in amyotrophic lateral sclerosis-linked SOD1 mutants.

Mutations in the Cu, Zn superoxide dismutase (SOD1) gene are one of the causative agents of amyotrophic lateral sclerosis (ALS). Although more than 100 different mutations in SOD1 have been identified, it is unclear whether all the mutations are pathogenic or just single nucleotide polymorphisms (SNPs) unrelated to the disease. Our previous systematic analysis found that all pathogenic SOD1 mutants (SOD1(mut)) have a common property, namely, an association with Derlin-1, a component of the endoplasmic reticulum-associated degradation machinery. For the proposed mechanism, we found that most pathogenic SOD1(mut) have a constitutively exposed Derlin-1-binding region (DBR), which is concealed in wild-type SOD1 (SOD1(WT)). Moreover, we generated MS785, a monoclonal antibody against DBR. MS785 distinguished most ALS-causative SOD1(mut) from both SOD1(WT) and non-toxic SOD1(mut). However, MS785 could not recognize SOD1(mut) that has mutations in the MS785 epitope region. Here, we developed a new diagnostic antibody, which could compensate for this shortcoming of MS785. We hypothesized that in ALS-causative SOD1(mut), the DBR-neighboring region [SOD1(30-40)] may also be exposed. We then generated MS27, a monoclonal antibody against SOD1(30-40). We found that MS27 could distinguish SOD1(WT) from the pathogenic SOD1(mut), which has mutations in the MS785 epitope region. Moreover, all pathogenic SOD1(mut), without exception, were immunoprecipitated with a combination of MS785 and MS27. The MS785-MS27 combination could be developed as a novel mechanism-based biomarker for the diagnosis of ALS.

A novel monoclonal antibody reveals a conformational alteration shared by amyotrophic lateral sclerosis-linked SOD1 mutants.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by the selective loss of upper and lower motoneurons. Although >100 different Cu, Zn superoxide dismutase (SOD1) mutations have been identified in ALS patients, it remains controversial whether all of them are disease-causative mutations. Therefore, it is necessary to develop molecular mechanism-based diagnosis and treatment of ALS caused by SOD1 mutations. METHODS: We previously reported that 3 pathogenic mutations of SOD1 cause chronic endoplasmic reticulum (ER) stress by inducing the binding of SOD1 to Derlin-1, a component of the ER homeostatic machinery. Here, we systematically analyzed 132 SOD1 mutants and found that most have a constitutively exposed Derlin-1-binding region (DBR) that is occluded in the wild-type protein. To develop the novel molecular mechanism-based antibody that can specifically recognize the aberrant structure of toxic SOD1 mutants, we generated the monoclonal antibody against the DBR. RESULTS: MS785, a monoclonal antibody generated against the DBR, distinguished most ALS-causative SOD1 mutants from both wild-type and nontoxic mutants. Moreover, MS785 recognized endogenous SOD1 in B lymphocytes derived from 14 ALS patients carrying SOD1 mutations but not from 11 healthy controls. INTERPRETATION: This is the first study to address the common property of all ALS-causative SOD1 mutants. MS785 is the first molecular mechanism-based antibody that was shown to be able to distinguish ALS-linked toxic SOD1 mutants from both wild-type and nontoxic mutants. MS785 may thus become an innovative tool for the diagnosis of ALS.

KLHDC10 Deficiency Protects Mice against TNFα-Induced Systemic Inflammation.

Systemic inflammatory response syndrome (SIRS) is a form of fatal acute inflammation for which there is no effective treatment. Here, we revealed that the ablation of Kelch domain containing 10 (KLHDC10), which we had originally identified as an activator of Apoptosis Signal-regulating Kinase 1 (ASK1), protects mice against TNFα-induced SIRS. The disease development of SIRS is mainly divided into two stages. The early stage is characterized by TNFα-induced systemic necroptosis, a regulated form of necrosis mediated by Receptor-interacting protein (RIP) 1/3 kinases. The later stage presents with an over-production of inflammatory cytokines induced by damage-associated molecular patterns (DAMPs), which are immunogenic cellular contents released from cells that underwent necroptosis. Analysis of TNFα-challenged mice revealed that KLHDC10-deficient mice show a reduction in the inflammatory response, but not in early systemic necroptosis. In vitro analysis suggested that the reduced inflammatory response observed in KLHDC10-deficient mice might be caused, in part, by enhanced necroptosis of inflammatory cells encountering DAMPs. Interestingly, the enhancement of necroptosis induced by KLHDC10 deficiency was selectively observed in inflammatory cells. Our results suggest that KLHDC10 is a cell-type specific regulator of necroptosis that ultimately contributes to the development of TNFα-induced SIRS.