Takayuki Manabe

Double-strand RNA dependent protein kinase (PKR) is involved in the extrastriatal degeneration in Parkinson's disease and Huntington's disease.

Double-strand RNA dependent protein kinase (PKR) plays an important role in control of cell death. We previously reported that activation of PKR is associated with hippocampal neuronal loss in Alzheimers disease (AD). Recent studies have reported that Parkinsons (PD) and Huntingtons (HD) disease brains displayed progressive hippocampal neuronal loss in extrastriatal degeneration. However, association between PKR and hippocampal neuronal loss in PD and HD brains is not known. In this report, brain tissues from patients with PD and HD displayed strong induction of phosphorylated-PKR (p-PKR) in hippocampal neurons. Immunoblotting analysis also demonstrated that levels of nuclear p-PKR in the hippocampus affected by these diseases were increased compared with age-matched disease controls. These results suggest that a close association exists between PKR and extrastriatal degeneration in PD and HD pathology.

JAB1 participates in unfolded protein responses by association and dissociation with IRE1

Recent papers have reported that neuronal death in patients with Alzheimers disease, Parkinsons disease, and cerebral ischemia has its origin in the endoplasmic reticulum (ER). IRE1alpha is one of the ER stress transducers that detect the accumulation of unfolded proteins in the ER. IRE1alpha mediates two major cellular responses, which are the unfolded protein response (UPR), a defensive response, and apoptosis that leads to cell death. However, little is known about the regulatory mechanisms that select between the UPR and apoptosis. We identified Jun activation domain-binding protein-1 (JAB1) as a molecule that interacts with IRE1alpha using a yeast two-hybrid system. We demonstrated that JAB1 binds to IRE1alpha in the absence of stress, but that binding is decreased by ER stress inducers. Moreover, mutant JAB1 down-regulates the UPR signaling pathway through tight binding with IRE1alpha. These results suggested that JAB1 may act as a key molecule in selecting the UPR or cell death by association and dissociation with IRE1alpha.

Metals accelerate production of the aberrant splicing isoform of the presenilin-2

Oxidative stress is a major risk factor for Alzheimers disease (AD) and other neurodegenerative disorders. Metals are known to be one of the factors that contribute to oxidative stress. Recently, we reported that the aberrant splicing isoform (PS2V) generated by skipping exon5 of the presenilin‐2 (PS2) gene is a diagnostic feature of sporadic AD (SAD). PS2V is inducible by exposure of human neuroblastoma to hypoxia. We examined whether this aberrant splicing was caused by metal‐induced oxidative stress, such as exposure to aluminum. As a result, we demonstrated that exposure to aluminum accelerated PS2V production induced by hypoxia. This acceleration of the production of PS2V to hypoxia was caused by chronic aluminum exposure, but was not related to the intracellular content of aluminum. HMGA1a is a mediator of PS2V production, and it was induced by aluminum as well as by hypoxia. Induction of HMGA1a was increased by chronic exposure to aluminum, and a nuclear extract containing HMGA1a bound to a specific sequence on exon5 of PS2 pre‐mRNA, as reported previously. Finally, the acceleration of PS2V production induced by aluminum under hypoxic conditions reflected, but has not yet been directly shown to cause, vulnerability to endoplasmic reticulum stress. These results suggest that exposure to some metals can accelerate and enhance PS2V generation, and that hypoxia plus chronic exposure to metals may promote the development of AD.

GRP94 reduces cell death in SH-SY5Y cells perturbated calcium homeostasis.

The endoplasmic reticulum (ER) resident—94 kDa glucose-regulated protein (GRP94), plays a pivotal role in cell death due to ER stress. In our study expression of GRP94 was increased in human neuroblastoma SH-SY5Y cells due to exposure to calcium ionophore A23187. A23187-mediated cell death was associated with activation of the major cysteine proteases, caspase-3 and calpain. Pretreatment with adenovirus-mediated antisense GRP94 (AdGRP94AS) reduced viability of SH-SY5Y cells subjected to A23187 treatment compared with wild type cells or cells with adenovirus-mediated overexpression of GRP94 (AdGRP94S). These results indicated that suppression of GRP94 is associated with accelerated cell death. Moreover, expression of GRP94 suppressed A23187-induced cell death and stabilized calcium homeostasis.

HMGA1a: sequence-specific RNA-binding factor causing sporadic Alzheimer's disease-linked exon skipping of presenilin-2 pre-mRNA.

Aberrant exon 5 skipping of presenilin‐2 (PS2) pre‐mRNA produces a deleterious protein isoform PS2V, which is almost exclusively observed in the brains of sporadic Alzheimers disease patients. PS2V over‐expression in vivo enhances susceptibility to various endoplasmic reticulum (ER) stresses and increases production of amyloid‐β peptides. We previously purified and identified high mobility group A protein 1a (HMGA1a) as a trans‐acting factor responsible for aberrant exon 5 skipping. Using heterologous pre‐mRNAs, here we demonstrate that a specific HMGA1a‐binding sequence in exon 5 adjacent to the 5′ splice site is necessary for HMGA1a to inactivate the 5′ splice site. An aberrant HMGA1a–U1 snRNP complex was detected on the HMGA1a‐binding site adjacent to the 5′ splice site during the early splicing reaction. A competitor 2′‐O‐methyl RNA (2′‐O‐Me RNA) consisting of the HMGA1a‐binding sequence markedly repressed exon 5 skipping of PS2 pre‐mRNA in vitro and in vivo. Finally, HMGA1a‐induced cell death under ER stress was prevented by transfection of the competitor 2′‐O‐Me RNA. These results provide insights into the molecular basis for PS2V‐associated neurodegenerative diseases that are initiated by specific RNA binding of HMGA1a.

Characterization of mouse Ire1α: cloning, mRNA localization in the brain and functional analysis in a neural cell line

In yeast, an endoplasmic reticulum (ER)-associated protein, Ire1p, is believed to initiate the unfolded protein response (UPR), that is responsible for protein folding in the ER under stressed conditions. Two mammalian homologs of Ire1p have been identified, Ire1 alpha and Ire1 beta. We have previously reported that familial Alzheimers disease linked presenilin-1 variants downregulate the signaling pathway of the UPR by affecting the phosphorylation of Ire1 alpha. In the present study, we cloned the mouse homolog of Ire1 alpha for generating genetically modified mice. Ire1 alpha was ubiquitously expressed in all mouse tissues examined, and was expressed preferentially in neuronal cells in mouse brain. This led us to investigate the effects of the downregulation of the UPR on the survival of neuronal cells under conditions of ER stress. Morphological and biochemical studies using a dominant-negative form of mouse Ire1 alpha have revealed that cell death caused by ER stress can be attributed to apoptosis, and that the downregulation of the UPR enhances the apoptotic process in the mouse neuroblastoma cell line, Neuro2a. Our results indicate that genetically modified mice such as transgenic mice with a dominant-negative form of Ire1 alpha might provide further understanding of the pathogenic mechanisms of Alzheimers disease and other neurodegenerative disorders.

Absence of endoproteolysis but no effects on amyloid β production by alternative splicing forms of presenilin-1, which lack exon 8 and replace D257A

It is well known that presenilin-1 (PS1) is involved in cleavage of amyloid precursor protein (APP) at the gamma-secretase site, and that the amino acids residues of D257 and D385 in PS1 are critical for this cleavage of APP and the endoproteolysis of itself. An alternatively spliced form of PS1 skipping exon 8 (PS1d8), which has D257A at the splice junction of exon 7/9, is expressed in human brain and in some cell lines. In this study, we examined production of Amyloid beta (A beta) and the endoproteolysis of the holoproteins in PS1d8-expressing neuroblastoma cells. Western blotting showed an absence of endoproteolysis in PS1d8. However, PS1d8 did not affect the production of A beta, which is different from the artificial point mutant PS1D257A. These results suggest that endoproteolysis of PS1 and gamma-secretase activity could be independent.

Role of ARF4L in Recycling Between Endosomes and the Plasma Membrane

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.

Possible involvement of the expression and phosphorylation of N-Myc in the induction of HMGA1a by hypoxia in the human neuroblastoma cell line

Increased expression of N-Myc and expression of the high mobility group protein A1a (HMGA1a) were observed in the nuclei of SK-N-SH cells following exposure to hypoxia. These observations were accompanied by the appearance of additional high molecular weight bands, which were eliminated by pretreatment with alkaline phosphatase. Immunoprecipitation showed phosphorylation of serine, threonine and tyrosine residues of N-Myc in the nucleus. These results suggest that hypoxia-induced signals in SK-N-SH cells lead to persistent expression of HMGA1a, which may induce expression of the transcription factor N-Myc, and that phosphorylation at serine, threonine and tyrosine residues of N-Myc occurs at an early stage after stimulation. Such signal consolidation processes could play a role in neuronal survival after hypoxia in neurons.