Mutations in the optineurin (OPTN) gene have been implicated in both familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of this protein in the central nervous system (CNS) and how it may contribute to ALS pathology are unclear. Here, we found that optineurin actively suppressed receptor-interacting kinase 1 (RIPK1)-dependent signaling by regulating its turnover. Loss of OPTN led to progressive dysmyelination and axonal degeneration through engagement of necroptotic machinery in the CNS, including RIPK1, RIPK3, and mixed lineage kinase domain-like protein (MLKL). Furthermore, RIPK1- and RIPK3-mediated axonal pathology was commonly observed in SOD1(G93A) transgenic mice and pathological samples from human ALS patients. Thus, RIPK1 and RIPK3 play a critical role in mediating progressive axonal degeneration. Furthermore, inhibiting RIPK1 kinase may provide an axonal protective strategy for the treatment of ALS and other human degenerative diseases characterized by axonal degeneration.

In the previous reports, we showed that the familial Alzheimer's disease (AD)-linked presenilin-1 (PS1) mutation induced the fragility to the endoplasmic reticulum (ER) stress and that caspase-4 mediates ER stress-induced- and beta-amyloid induced-apoptotic signaling in human cells. These results suggest the involvement of ER stress and caspase-4 in the cell death observed in AD. In this report, we studied the activation of caspase-4 in the familial AD-linked PS I mutation (Delta E9). Cleavage of caspase-4 under ER stress was enhanced by the overexpression of the familial AD-linked mutation (Delta E9), showing that caspase-4 is a key caspase involved in the apoptotic signaling of AD. We also showed that the overexpression of caspase-4 induced cleavage of caspase-9 and caspase-3 without releasing cytochrome-c from the mitochondria. Thus, caspase-4 activates downstream caspases independently of mitochondrial apoptotic signaling and this might contribute to the pathogenesis of AD.To sum up our data, the familial AD-linked PS1 mutation accelerates the cleavage of caspase-4 under the ER stress and results in the activation of caspase-9 and caspase-3, apoptosis signal, without releasing cytochrome-c. (C) 2007 Elsevier Ltd. All rights reserved.

Stimulation of death receptors by agonists such as FasL and TNFalpha activates apoptotic cell death in apoptotic-competent conditions or a type of necrotic cell death dependent on RIP1 kinase, termed necroptosis, in apoptotic-deficient conditions. In a genome-wide siRNA screen for regulators of necroptosis, we identify a set of 432 genes that regulate necroptosis, a subset of 32 genes that act downstream and/or as regulators of RIP1 kinase, 32 genes required for death-receptor-mediated apoptosis, and 7 genes involved in both necroptosis and apoptosis. We show that the expression of subsets of the 432 genes is enriched in the immune and nervous systems, and cellular sensitivity to necroptosis is regulated by an extensive signaling network mediating innate immunity. Interestingly, Bmf, a BH3-only Bcl-2 family member, is required for death-receptor-induced necroptosis. Our study defines a cellular signaling network that regulates necroptosis and the molecular bifurcation that controls apoptosis and necroptosis.

Necroptosis is a cellular mechanism of necrotic cell death induced by apoptotic stimuli in the form of death domain receptor engagement by their respective ligands under conditions where apoptotic execution is prevented. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphological features as unregulated necrotic death. Here we report that necrostatin-1, a previously identified small-molecule inhibitor of necroptosis, is a selective allosteric inhibitor of the death domain receptor-associated adaptor kinase RIP1 in vitro. We show that RIP1 is the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. In addition, we show that two other necrostatins, necrostatin-3 and necrostatin-5, also target the RIP1 kinase step in the necroptosis pathway, but through mechanisms distinct from that of necrostatin-1. Overall, our data establish necrostatins as the first-in-class inhibitors of RIP1 kinase, the key upstream kinase involved in the activation of necroptosis.

Neuronal Lewy body-like hyaline inclusions (LBHI) and astrocytic hyaline inclusions (Ast-HI) containing mutant Cu/Zn superoxide dismutase 1 (SOD1) are morphological hallmarks of familial amyotrophic lateral sclerosis (FALS) associated with mutant SOD1. However, the mechanisms by which mutant SOD1 contributes to formation of LBHI/Ast-HI in FALS remain poorly defined. Here, we report induction of LBHI/Ast-HI-like hyaline inclusions (LHIs) in vitro by ER stress in neuroblastoma cells. These LHI closely resemble LBHI/Ast-HI in patients with SOD1-linked FALS. LHI and LBHI/Ast-HI share the following features: 1) eosinophilic staining with a pale core, 2) SOD1, ubiquitin and ER resident protein (KDEL) positivity and 3) the presence of approximately 15-25 nm granule-coated fibrils, which are morphological hallmark of mutant SOD1-linked FALS. Moreover, in spinal cord neurons of L84V SOD1 transgenic mice at presymptomatic stage, we observed aberrant aggregation of ER and numerous free ribosomes associated with abnormal inclusion-like structures, presumably early stage neuronal LBHI. We conclude that the LBHI/Ast-HI seen in human patients with mutant SOD1-linked FALS may arise from ER dysfunction.

The human ADP-ribosylation factor-like protein, ARF4L is a member of the ARF family, which are small GTP-binding proteins that play significant roles in vesicle transport and protein secretion. However, little is known about the physiological roles of ARF4L. In this study, to understand the biological functions of ARF4L, we carried out immunocytochemical analysis of ARF4L molecules with mutations in the functional domains. ARF4L was shown to be distributed to the plasma membrane following binding to GTP (Q80L), and into endosomes following binding to GDP (T35N). Moreover, the inactive-form of ARF4L (T35N) causes localization of transferrin receptors to the endosomal compartment, while the active form (Q80L) causes transport to the plasma membrane. These findings indicate that ARF4L drive the transport of cargo protein and subsequent fusion of recycling vesicles with the plasma membrane for maintenance of the cell surface.

Missense mutations in the human presenilin-1 ( PS1 ) gene, which is found on chromosome 14, cause early-onset familial Alzheimer’s disease (FAD). FAD-linked PS1 variants alter proteolytic processing of the amyloid precursor protein and cause an increase in vulnerability to apoptosis induced by various cell stresses. However, the mechanisms responsible for these phenomena are not clear. Here we report that mutations in PS1 affect the unfolded-protein response (UPR), which responds to the increased amount of unfolded proteins that accumulate in the endoplasmic reticulum (ER) under conditions that cause ER stress. PS1 mutations also lead to decreased expression of GRP78/Bip, a molecular chaperone, present in the ER, that can enable protein folding. Interestingly, GRP78 levels are reduced in the brains of Alzheimer’s disease patients. The downregulation of UPR signalling by PS1 mutations is caused by disturbed function of IRE1, which is the proximal sensor of conditions in the ER lumen. Overexpression of GRP78 in neuroblastoma cells bearing PS1 mutants almost completely restores resistance to ER stress to the level of cells expressing wild-type PS1. These results show that mutations in PS1 may increase vulnerability to ER stress by altering the UPR signalling pathway.