Kazunori Imaizumi

Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response.

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.

Two cis-acting elements in the 3′ untranslated region of α-CaMKIIregulate its dendritic targeting

Dendritic localization of the α subunit of Ca2+/calmodulin-dependent protein kinase II (αCaMKII) mRNA in CNS neurons requires its 3′ untranslated region (3′UTR). We investigated this targeting mechanism by identifying two cis-acting elements in the 3′UTR. One is a 30-nucleotide element that mediated dendritic translocation. A homologous sequence in the 3′UTR of neurogranin, transcripts of which also reside in dendrites, also funtioned in cis to promote its dendritic transport. Other putative elements in the αCaMKII mRNA inhibit its transport in a resting state. This inhibition was removed in depolarized neurons, and such activity-dependent derepression was a primary requirement for their dendritic targeting.

Molecular Cloning of a Novel Polypeptide, DP5, Induced during Programmed Neuronal Death

To study the molecular mechanisms underlying neuronal programmed cell death (PCD), we performed differential display screening for genes, the expression of which was induced during PCD in the sympathetic neuron culture model deprived of NGF. We cloned a gene encoding a novel polypeptide (DP5) which consisted of 92 amino acids. DP5 polypeptide had no homology with any other known protein and contained no motif that would indicate its putative biochemical functions. DP5 mRNA levels peaked at 15 h after nerve growth factor withdrawal, concurrent with the time at which neurons were committed to die. The induction of DP5 gene expression was blocked when cell death was rescued by treatment with cycloheximide, KCl, or the cyclic AMP analogue CPTcAMP. Overexpression of the full-length DP5 in cultured sympathetic neurons was in itself sufficient to induce apoptosis. These results suggest that DP5 plays a role in programmed neuronal death.

DIFFERENTIAL EXPRESSION OF SGK MRNA, A MEMBER OF THE SER/THR PROTEIN KINASE GENE FAMILY, IN RAT BRAIN AFTER CNS INJURY

We cloned genes the expression of which were induced 3 days after cortical injury of rat brain by a differential display technique, and four novel and known sequences were isolated. Among these sequences, the sgk gene which was recently identified as a novel member of the serine/threonine protein kinase gene family, was selected for analysis of its expression patterns in rat brain by northern blotting and in situ hybridization, because hybridization signals were strong at the lesion sites. Expression of sgk mRNA was induced within 3 days after injury, and was maintained at a high level for at least 14 days. The cells which strongly expressed the sgk gene were in the deep layers of the cortex and in the corpus callosum. In situ hybridization analysis for sgk and myelin proteolipid protein mRNA using serial sections showed that the distribution of both signals was very similar at the damaged regions. Therefore, it is likely that the sgk transcript is expressed by oligodendrocytes after brain injury. Investigation of the developmental expression of the sgk gene showed that neurons in layers I and II of the cortex, lateroposterior and laterodorsal thalamic nucleus, and ventral posterolateral and posteromedial thalamic nucleus strongly expressed sgk mRNA at postnatal day 1 and day 7, but these neurons showed no expression in fetal or adult brain. These results suggest that the induction of sgk gene may be associated with a series of axonal regenerations after brain injury, and in addition, the sgk gene may also play important roles in the development of particular groups of neurons in the postnatal brain.

The Cell Death-promoting Gene DP5, Which Interacts with the BCL2 Family, Is Induced during Neuronal Apoptosis Following Exposure to Amyloid β Protein

DP5, which contains a BH3 domain, was cloned as a neuronal apoptosis-inducing gene. To confirm that DP5 interacts with members of the Bcl-2 family, 293T cells were transiently co-transfected with DP5 and Bcl-xl cDNA constructs, and immunoprecipitation was carried out. The 30-kDa Bcl-xl was co-immunoprecipitated with Myc-tagged DP5, suggesting that DP5 physically interacts with Bcl-xl in mammalian cells. Previously, we reported that DP5 is induced during neuronal apoptosis in cultured sympathetic neurons. Here, we analyzed DP5 gene expression and the specific interaction of DP5 with Bcl-xl during neuronal death induced by amyloid-β protein (A β). DP5 mRNA was induced 6 h after treatment with A β in cultured rat cortical neurons. The protein encoded by DP5 mRNA showed a specific interaction with Bcl-xl. Induction of DP5 gene expression was blocked by nifedipine, an inhibitor of l-type voltage-dependent calcium channels, and dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. These results suggested that the induction of DP5 mRNA occurs downstream of the increase in cytosolic calcium concentration caused by A β. Moreover, DP5 specifically interacts with Bcl-xl during neuronal apoptosis following exposure to A β, and its binding could impair the survival-promoting activities of Bcl-xl. Thus, the induction of DP5 mRNA and the interaction of DP5 and Bcl-xl could play significant roles in neuronal degeneration following exposure to A β.

Accumulation of murine amyloidbeta42 in a gene-dosage-dependent manner in PS1 'knock-in' mice.

The establishment of an animal model with a missense mutation of presenilin‐1 (PS1) is an initial step toward understanding the molecular pathogenesis of familial Alzheimer’s disease (FAD) and developing therapeutic strategies for the disease. We previously described a Japanese family with FAD caused by the I213T mutation of PS1, in which typical signs and symptoms of Alzheimer’s disease were observed at the age of 45u2003±u20034.2u2003years [Hardy, J. (1997) Trends. Neurosci., 20, 154–159; Kamino, K etu2003al. (1996) Neurosci. Lett., 208, 195–198]. Here, we report the establishment of ‘knock‐in’ mice with the I213T PS1 missense mutation. Northern blot and reverse transcription polymerase chain reaction (RT‐PCR) analyses showed that the mutated PS1 allele was expressed at the same level as the endogenous PS1 allele, demonstrating that the PS1 missense mutation was successfully introduced into the mouse PS1 locus, and therefore that the situation mimics that in FAD patients bearing PS1 missense mutations. Amyloid beta (Aβ) 42(43) peptide, but not Aβ40 peptide, accumulated in ‘knock‐in’ mice at the age of 16–20u2003weeks. A clear gene‐dosage effect on the increase of Aβ42(43) was observed in ‘knock‐in’ mice: the percentage increase of Aβ42(43) in mice with mutations in both alleles was twice as high as that in mice with a single allele. These results indicate that the level of the mutated PS1 gene expression is likely to be critically involved in the production of highly amyloidogenic Aβ42(43), and confirm that PS1 mutation has an important effect on amyloid precursor protein (APP) processing, in proportion to the level of the expression of the mutant gene.

Are cerebrovascular factors involved in Alzheimer's disease?

Recent epidemiological studies have shown that vascular risk factors may be involved in Alzheimers disease (AD) as well as dementia in general. To investigate the relation between a vascular disorder and AD pathology, current criteria are defective because most depend on exclusion of a cerebrovascular disorder. Epidemiological studies have indicated the possibilities that arteriosclerosis, abnormal blood pressure, diabetes mellitus and smoking may be related to the pathogenesis of AD. As for the mechanism that vascular disorders influence AD, it is presumed that amyloid deposition may be caused by a vascular disorder. Alternatively, a vascular event may cause progression of subclinical AD to a clinical stage. Insulin resistance and apolipoprotein E may also be involved in these mechanisms. Our studies show that ischemia-induced the Alzheimer-associated gene presenilin 1 (PS1) and endoplasmic reticulum-stress, generated from a vascular disorder, may unmask clinical AD symptoms caused by presenilin mutation, suggesting that a vascular factor might be involved in the onset of familial AD.

Alzheimer-associated presenilin-1 gene is induced in gerbil hippocampus after transient ischemia

To investigate the biological roles of the presenilin-1 (PS-1) gene after neuronal injury, the changes of PS-1 mRNA expression in the gerbil hippocampus after transient ischemia were examined. From 1 day to 3 day-reperfusion after 5 min-ischemia, PS-1 mRNA was induced in the hippocampus compared with the sham-operated control. The cells which induced the PS-1 genes were neurons of CA3 and dentate gyrus, the region relatively resistant to ischemic stress. These findings suggest that the induction of PS-1 genes may be associated with some responses of neurons damaged by transient ischemia.

Induction of SPI-3 mRNA, encoding a serine protease inhibitor, in gerbil hippocampus after transient forebrain ischemia.

We cloned genes the expression of which is induced in the Mongolian gerbil (Meriones unguiculatus) hippocampus after transient forebrain ischemia by a differential display technique. Among these genes, a rat serine protease inhibitor SPI-3 homologue was isolated. Present analyses suggested that the expression of gerbil SPI-3 mRNA was closely associated with delayed neuronal death and may block activities of proteases leaking from degenerating neurons or may support neuronal survival.

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.