Hisae Kadowaki

ALS-linked mutant SOD1 induces ER stress- and ASK1-dependent motor neuron death by targeting Derlin-1

Mutation in Cu/Zn-superoxide dismutase (SOD1) is a cause of familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 protein (SOD1(mut)) induces motor neuron death, although the molecular mechanism of SOD1(mut)-induced cell death remains controversial. Here we show that SOD1(mut) specifically interacted with Derlin-1, a component of endoplasmic reticulum (ER)-associated degradation (ERAD) machinery and triggered ER stress through dysfunction of ERAD. SOD1(mut)-induced ER stress activated the apoptosis signal-regulating kinase 1 (ASK1)-dependent cell death pathway. Perturbation of binding between SOD1(mut) and Derlin-1 by Derlin-1-derived oligopeptide suppressed SOD1(mut)-induced ER stress, ASK1 activation, and motor neuron death. Moreover, deletion of ASK1 mitigated the motor neuron loss and extended the life span of SOD1(mut) transgenic mice. These findings demonstrate that ER stress-induced ASK1 activation, which is triggered by the specific interaction of Derlin-1 with SOD1(mut), is crucial for disease progression of familial ALS.

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation

Amyloid β (Aβ) is a main component of senile plaques in Alzheimers disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Aβ-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Aβ-induced neuronal cell death. Aβ activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1−/− neurons were defective in Aβ-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Aβ-induced neurotoxicity, which plays a central role in Alzheimers disease.

Survival and apoptosis signals in ER stress: the role of protein kinases

The endoplasmic reticulum (ER) is the organelle in which newly synthesized secretory and transmembrane proteins form their proper tertiary structure by post-translational modification, folding, and oligomerization. However, many of these proteins are unfolded or misfolded by extracellular or intracellular stimuli. The accumulation of misfolded proteins constitutes a risk for living cells. Eukaryotic cells possess at least three different mechanisms to adapt to ER stress and thereby survive: (1) translational attenuation to limit further accumulation of misfolded proteins; (2) transcriptional activation of genes encoding ER-resident chaperones; and (3) the ER-associated degradation (ERAD) pathway to restore the folding capacity. If the cells are exposed to prolonged or strong ER stress, the cells are destroyed by apoptosis. Recent evidence indicates that ER stress signaling pathways are mediated in part by several protein kinases and play an important role in the pathogenesis of neurodegenerative disorders. The main purpose of this review is to summarize current knowledge about the protein kinases involved in ER stress, and their involvement in the pathogenesis of neurodegenerative disorders.

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.

Detection of mutations in the insulin receptor gene in patients with insulin resistance by analysis of single-stranded conformational polymorphisms

SummaryWe analyzed single-stranded conformational poly morphisms to screen for mutations and polymorphisms in the insulin receptor gene in subjects with or without insulin resistance. Using this new technique, we demonstrated the existence of mutations in the insulin receptor gene which we had identified previously. In addition, a new mutation was found in exon 20 of the insulin receptor gene in a patient with moderate insulin resistance associated with morbid obesity, acanthosis nigricans, and polycystic ovary syndrome. The patient was heterozygous for a mutation substituting Leu (CTG) for Pro (CCG) at codon 1178. Pro1178 is a part of a characteristic sequence motif (D1150 F1151 G1152-A1177 P1178 E1179) common to many protein kinases. Analysis of single-stranded conformational polymorphisms was also used to estimate the frequency of a polymorphism at codon 1058. The two codons CAC (1058 His) and CAT (1058 His) both had a prevalence of 50% in 30 Japanese subjects. These data demonstrate that analysis of single-stranded conformational polymorphisms is a simple and sensitive screening method for mutations and polymorphisms in the insulin receptor gene in subjects with or without insulin resistance. Identification of a mutation in the insulin receptor gene in a patient with a moderate degree of insulin resistance associated with morbid obesity suggests that insulin receptor mutations may exist in patients with Type 2 (non-insulin-dependent) diabetes mellitus associated with a moderate degree of insulin resistance.

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

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.

Signaling Pathways from the Endoplasmic Reticulum and Their Roles in Disease

The endoplasmic reticulum (ER) is an organelle in which newly synthesized secretory and transmembrane proteins are assembled and folded into their correct tertiary structures. However, many of these ER proteins are misfolded as a result of various stimuli and gene mutations. The accumulation of misfolded proteins disrupts the function of the ER and induces ER stress. Eukaryotic cells possess a highly conserved signaling pathway, termed the unfolded protein response (UPR), to adapt and respond to ER stress conditions, thereby promoting cell survival. However, in the case of prolonged ER stress or UPR malfunction, apoptosis signaling is activated. Dysfunction of the UPR causes numerous conformational diseases, including neurodegenerative disease, metabolic disease, inflammatory disease, diabetes mellitus, cancer, and cardiovascular disease. Thus, ER stress-induced signaling pathways may serve as potent therapeutic targets of ER stress-related diseases. In this review, we will discuss the molecular mechanisms of the UPR and ER stress-induced apoptosis, as well as the possible roles of ER stress in several diseases.

CHIP-dependent termination of MEKK2 regulates temporal ERK activation required for proper hyperosmotic response.

The extracellular signal‐regulated kinase (ERK) pathway is an important signalling pathway that regulates a large number of cellular processes, including proliferation, differentiation and gene expression. Hyperosmotic stress activates the ERK pathway, whereas little is known about the regulatory mechanisms and physiological functions of ERK activation in hyperosmotic response. Here, we show that MAPK/ERK kinase kinase 2 (MEKK2), a member of the MAPKKK family, mediated the specific and transient activation of ERK, which was required for the induction of aquaporin 1 (AQP1) and AQP5 gene expression in response to hyperosmotic stress. Moreover, we identified the E3 ubiquitin ligase carboxyl terminus of Hsc70‐interacting protein (CHIP) as a binding partner of MEKK2. Depletion of CHIP by small‐interference RNA or gene targeting attenuated the degradation of MEKK2 and prolonged the ERK activity. Interestingly, hyperosmolality‐induced gene expression of AQP1 and AQP5 was suppressed by CHIP depletion and was reversed by inhibition of the prolonged phase of ERK activity. These findings show that transient activation of the ERK pathway, which depends not only on MEKK2 activation, but also on CHIP‐dependent MEKK2 degradation, is crucial for proper gene expression in hyperosmotic stress response.

USP14 inhibits ER-associated degradation via interaction with IRE1α

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) lumen induces ER stress. Eukaryotic cells possess the ER quality control systems, the unfolded protein response (UPR), to adapt to ER stress. IRE1alpha is one of the ER stress receptors and mediates the UPR. Here, we identified ubiquitin specific protease (USP) 14 as a binding partner of IRE1alpha. USP14 interacted with the cytoplasmic region of IRE1alpha, and the endogenous interaction between USP14 and IRE1alpha was inhibited by ER stress. Overexpression of USP14 inhibited the ER-associated degradation (ERAD) pathway, and USP14 depletion by small interfering RNA effectively activated ERAD. These findings suggest that USP14 is a novel player in the UPR by serving as a physiological inhibitor of ERAD under the non-stressed condition.

Involvement of ASK1–p38 pathway in the pathogenesis of diabetes triggered by pancreatic ß cell exhaustion

BACKGROUND Diabetes mellitus is characterized by high blood glucose levels. Pancreatic ß cell death contributes to type 1 and type 2 diabetes. Akita mice, which harbor a human permanent neonatal diabetes-linked mutation (Cys96Tyr) in the insulin gene, are well established as an animal model of diabetes caused by pancreatic ß cell exhaustion. Mutant Insulin 2 protein (Ins2(C96Y)) induces endoplasmic reticulum (ER) stress and pancreatic ß cell death in Akita mice, although the molecular mechanism of Ins(C96Y)-induced cell death remains unclear. METHODS We investigate the mechanisms of Ins2(C96Y)-induced pancreatic ß cell death in vitro and in vivo, using p38 inhibitor (SB203580), MIN6 cell (pancreatic ß cell line), Akita mice and apoptosis signal-regulating kinase 1 (ASK1) knockout mice. RESULTS The expression of Ins(C96Y) activated the ASK1-p38 pathway. Deletion of ASK1 mitigated Ins(C96Y)-induced pancreatic ß cell death and delayed the onset of diabetes in Akita mice. Moreover, p38 inhibitor suppressed Ins(C96Y)-induced MIN6 cell death. CONCLUSIONS These findings suggest that ER stress-induced ASK1-p38 activation, which is triggered by the accumulation of Ins(C96Y), plays an important role in the pathogenesis of diabetes. GENERAL SIGNIFICANCE Pancreatic ß cell death caused by insulin overload appears to be involved in the pathogenesis of type 1 and type 2 diabetes. Inhibition of the ASK1-p38 pathway may be an effective therapy for various types of diabetes.