Kohsuke Kanekura

Characterization of amyotrophic lateral sclerosis- linked P56S mutation of vesicle- associated membrane protein- associated protein b (VAPB/ALS8)

The P56S mutation in VAPB (vesicle-associated membrane protein-associated protein B) causes autosomal dominant motoneuronal diseases. Although it was reported that the P56S mutation induces localization shift of VAPB from endoplasmic reticulum (ER) to non-ER compartments, it remains unclear what the physiological function of VAPB is and how the P56S mutation in VAPB causes motoneuronal diseases. Here we demonstrate that overexpression of wild type VAPB (wt-VAPB) promotes unfolded protein response (UPR), which is an ER reaction to suppress accumulation of misfolded proteins, and that small interfering RNA for VAPB attenuates UPR to chemically induced ER stresses, indicating that VAPB is physiologically involved in UPR. The P56S mutation nullifies the function of VAPB to mediate UPR by inhibiting folding of VAPB that results in insolubility and aggregate formation of VAPB in non-ER fractions. Furthermore, we have found that expression of P56S-VAPB inhibits UPR, mediated by endogenous wt-VAPB, by inducing aggregate formation and mislocalization into non-ER fractions of wt-VAPB. Consequently, the P56S mutation in a single allele of the VAPB gene may diminish the activity of VAPB to mediate UPR to less than half the normal level. We thus speculate that the malfunction of VAPB to mediate UPR, caused by the P56S mutation, may contribute to the development of motoneuronal degeneration linked to VAPB/ALS8.

ER stress and unfolded protein response in amyotrophic lateral sclerosis.

Several theories on the pathomechanism of amyotrophic lateral sclerosis (ALS) have been proposed: misfolded protein aggregates, mitochondrial dysfunction, increased glutamate toxicity, increased oxidative stress, disturbance of intracellular trafficking, and so on. In parallel, a number of drugs that have been developed to alleviate the putative key pathomechanism of ALS have been under clinical trials. Unfortunately, however, almost all studies have finished unsuccessfully. This fact indicates that the key ALS pathomechanism still remains a tough enigma. Recent studies with autopsied ALS patients and studies using mutant SOD1 (mSOD1) transgenic mice have suggested that endoplasmic reticulum (ER) stress-related toxicity may be a relevant ALS pathomechanism. Levels of ER stress-related proteins were upregulated in motor neurons in the spinal cords of ALS patients. It was also shown that mSOD1, translocated to the ER, caused ER stress in neurons in the spinal cord of mSOD1 transgenic mice. We recently reported that the newly identified ALS-causative gene, vesicle-associated membrane protein-associated protein B (VAPB), plays a pivotal role in unfolded protein response (UPR), a physiological reaction against ER stress. The ALS-linked P56S mutation in VAPB nullifies the function of VAPB, resulting in motoneuronal vulnerability to ER stress. In this review, we summarize recent advances in research on the ALS pathomechanism especially addressing the putative involvement of ER stress and UPR dysfunction.

Abnormal expression of TIP30 and arrested nucleocytoplasmic transport within oligodendrocyte precursor cells in multiple sclerosis

Oligodendrocyte precursor cells (OPCs) persist near the demyelinated axons arising in MS but inefficiently differentiate into oligodendrocytes and remyelinate these axons. The pathogenesis of differentiation failure remains elusive. We initially hypothesized that injured axons fail to present Contactin, a positive ligand for the oligodendroglial Notch1 receptor to induce myelination, and thus tracked axoglial Contactin/Notch1 signaling in situ, using immunohistochemistry in brain tissue from MS patients containing chronic demyelinated lesions. Instead, we found that Contactin was saturated on demyelinated axons, Notch1-positive OPCs accumulated in Contactin-positive lesions, and the receptor was engaged, as demonstrated by cleavage to Notch1-intracellular domain (NICD). However, nuclear translocalization of NICD, required for myelinogenesis, was virtually absent in these cells. NICD and related proteins carrying nuclear localization signals were associated with the nuclear transporter Importin but were trapped in the cytoplasm. Abnormal expression of TIP30, a direct inhibitor of Importin, was observed in these OPCs. Overexpression of TIP30 in a rat OPC cell line resulted in cytoplasmic entrapment of NICD and arrest of differentiation upon stimulation with Contactin-Fc. Our results suggest that extracellular inhibitory factors as well as an intrinsic nucleocytoplasmic transport blockade within OPCs may be involved in the pathogenesis of remyelination failure in MS.

ALS-linked P56S-VAPB, an aggregated loss-of-function mutant of VAPB, predisposes motor neurons to ER stress-related death by inducing aggregation of co-expressed wild-type VAPB

A point mutation (P56S) in the vapb gene encoding an endoplasmic reticulum (ER)‐integrated membrane protein [vesicle‐associated membrane protein‐associated protein B (VAPB)] causes autosomal‐dominant amyotrophic lateral sclerosis. In our earlier study, we showed that VAPB may be involved in the IRE1/XBP1 signaling of the unfolded protein response, an ER reaction to inhibit accumulation of unfolded/misfolded proteins, while P56S‐VAPB formed insoluble aggregates and lost the ability to mediate the pathway (loss‐of‐function), and suggested that P56S‐VAPB promoted the aggregation of co‐expressed wild‐type (wt)‐VAPB. In this study, a yeast inositol‐auxotrophy assay has confirmed that P56S‐VAPB is functionally a null mutant in vivo. The interaction between P56S‐VAPB and wt‐VAPB takes place with a high affinity through the major sperm protein domain in addition to the interaction through the C‐terminal transmembrane domain. Consequently, wt‐VAPB is speculated to preferentially interact with co‐expressed P56S‐VAPB, leading to the recruitment of wt‐VAPB into cytosolic aggregates and the attenuation of its normal function. We have also found that expression of P56S‐VAPB increases the vulnerability of NSC34 motoneuronal cells to ER stress‐induced death. These results lead us to hypothesize that the total loss of VAPB function in unfolded protein response, induced by one P56S mutant allele, may contribute to the development of P56S‐VAPB‐induced amyotrophic lateral sclerosis.

Involvement of c-Jun N-terminal kinase in amyloid precursor protein-mediated neuronal cell death

Amyloid precursor protein (APP), the precursor of Aβ, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti‐APP antibody. The c‐Jun N‐terminal kinase (JNK) can mediate various neurotoxic signals, including Aβ neurotoxicity. However, the relationship of APP‐mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Aβ independent. Here we examined whether JNK is involved in APP‐mediated neuronal cell death and found that: (i) neuronal cell death by antibody‐bound APP was inhibited by dominant‐negative JNK, JIP‐1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK‐mediated cell death closely coincided with that of APP‐mediated cell death. Pertussis toxin (PTX) suppressed APP‐mediated cell death but not caJNK‐induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP‐mediated cytotoxicity. In the presence of PTX, the PTX‐resistant mutant of Gαo, but not that of Gαi, recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP‐mediated neuronal cell death as a downstream signal transducer of Go.

Characterization of the toxic mechanism triggered by Alzheimer's amyloid‐β peptides via p75 neurotrophin receptor in neuronal hybrid cells

Neuronal pathology of the brain with Alzheimers disease (AD) is characterized by numerous depositions of amyloid‐β peptides (Aβ). Aβ binding to the 75‐kDa neurotrophin receptor (p75NTR) causes neuronal cell death. Here we report that Aβ causes cell death in neuronal hybrid cells transfected with p75NTR, but not in nontransfected cells, and that p75NTRL401K cannot mediate Aβ neurotoxicity. We analyzed the cytotoxic pathway by transfecting pertussis toxin (PTX)‐resistant G protein α subunits in the presence of PTX and identified that Gαo, but not Gαi, proteins are involved in p75NTR‐mediated Aβ neurotoxicity. Further investigation suggested that Aβ neurotoxicity via p75NTR involved JNK, NADPH oxidase, and caspases‐9/3 and was inhibited by activity‐dependent neurotrophic factor, insulin‐like growth factor‐I, basic fibroblast growth factor, and Humanin, as observed in primary neuron cultures. Understanding the Aβ neurotoxic mechanism would contribute significantly to the development of anti‐AD therapies.

Two serine residues distinctly regulate the rescue function of Humanin, an inhibiting factor of Alzheimer's disease-related neurotoxicity: functional potentiation by isomerization and dimerization

The 24‐residue peptide Humanin (HN), containing two Ser residues at positions 7 and 14, protects neuronal cells from insults of various Alzheimers disease (AD) genes and Aβ. It was not known why the rescue function of (S14G)HN is more potent than HN by two to three orders of magnitude. Investigating the possibility that the post‐translational modification of Ser14 might play a role, we found that HN with d‐Ser at position 14 exerts neuroprotection more potently than HN by two to three orders of magnitude, whereas d‐Ser7 substitution does not affect the rescue function of HN. On the other hand, S7A substitution nullified the HN function. Multiple series of experiments indicated that Ser7 is necessary for self‐dimerization of HN, which is essential for neuroprotection by this factor. These findings indicate that the rescue function of HN is quantitatively modulated by d‐isomerization of Ser14 and Ser7‐relevant dimerization, allowing for the construction of a very potent HN derivative that was fully neuroprotective at 10 pm against 25 µm Aβ1–43. This study provides important clues to the understanding of the neuroprotective mechanism of HN, as well as to the development of novel AD therapeutics.

Molecular Mechanisms for Neuronal Cell Death by Alzheimer’s Amyloid Precursor Protein-Relevant Insults

To develop a therapeutic intervention for Alzheimer’s disease (AD), it is necessary to clarify the mechanisms underlying the pathogenesis of AD, in which senile plaques, neurofibrillary tangles and neuronal loss in the cerebrum are the central abnormalities. A number of studies have focused on the major component of the senile plaques, which is amyloid-β (Aβ) and its precursor protein APP, and have investigated the roles of these molecules in the onset, progression and inhibition of AD. For multiple reasons, however, their roles in AD, especially in neuronal death, remain elusive and a unified concept for their roles has not yet been established. Recently, it has been found that APP functions normally as a neuronal surface transmembrane protein. In this article, we review the molecular mechanisms of neuronal cell death by these APP-relevant insults and discuss the functions of APP in regard to intracellular signal transducers, including c-Jun N-terminal kinase. We also revise the roles of Aβ in neuronal death and survival.

Transforming Growth Factor β2 Is a Neuronal Death-Inducing Ligand for Amyloid-β Precursor Protein

ABSTRACT APP, amyloid β precursor protein, is linked to the onset of Alzheimers disease (AD). We have here found that transforming growth factor β2 (TGFβ2), but not TGFβ1, binds to APP. The binding affinity of TGFβ2 to APP is lower than the binding affinity of TGFβ2 to the TGFβ receptor. On binding to APP, TGFβ2 activates an APP-mediated death pathway via heterotrimeric G protein Go, c-Jun N-terminal kinase, NADPH oxidase, and caspase 3 and/or related caspases. Overall degrees of TGFβ2-induced death are larger in cells expressing a familial AD-related mutant APP than in those expressing wild-type APP. Consequently, superphysiological concentrations of TGFβ2 induce neuronal death in primary cortical neurons, whose one allele of the APP gene is knocked in with the V642I mutation. Combined with the finding indicated by several earlier reports that both neural and glial expression of TGFβ2 was upregulated in AD brains, it is speculated that TGFβ2 may contribute to the development of AD-related neuronal cell death.

Humanin antagonists: mutants that interfere with dimerization inhibit neuroprotection by Humanin

The 24‐residue peptide Humanin (HN) protects neuronal cells from insults of various Alzheimers disease (AD) genes and Aβ by forming a homodimer. We have previously shown that P3A, S7A, C8A, L9A, L12A, T13A, S14A and P19A mutations nullify the neuroprotective function of HN [Yamagishi, Y., Hashimoto, Y., Niikura, T. & Nishimoto, I. (2003) Peptides, 24, 585–595]. Here we examined whether any of these ‘null’ mutants could function as dominant‐negative mutants. Homodimerization‐defective mutants, P3A‐, L12A‐, S14A‐ and P19A‐HN, specifically blocked neuroprotection by HN, but not by activity‐dependent neurotrophic factor. Furthermore, insertion of S7A, the mutation that blocks the homodimerization of HN, but not insertion of G5A abolished the antagonizing function of L12A‐HN. While L12A‐HN and G5A/L12A‐HN actually inhibited HN homodimerization, S7A/L12A‐HN had no effect. These data indicate that P3A‐, L12A‐, S14A‐ and P19A‐HN function as HN antagonists by forming an inactive dimer with HN. This study provides a novel insight into the understanding of the in vivo function of HN, as well as into the development of clinically applicable HN neutralizers.