Yasutake Mori

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.

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.

CARM1 Regulates Proliferation of PC12 Cells by Methylating HuD

ABSTRACT HuD is an RNA-binding protein that has been shown to induce neuronal differentiation by stabilizing labile mRNAs carrying AU-rich instability elements. Here, we show a novel mechanism of arginine methylation of HuD by coactivator-associated arginine methyltransferase 1 (CARM1) that affected mRNA turnover of p21cip1/waf1 mRNA in PC12 cells. CARM1 specifically methylated HuD in vitro and in vivo and colocalized with HuD in the cytoplasm. Inhibition of HuD methylation by CARM1 knockdown elongated the p21cip1/waf1 mRNA half-life and resulted in a slow growth rate and robust neuritogenesis in response to nerve growth factor (NGF). Methylation-resistant HuD bound more p21cip1/waf1 mRNA than did the wild type, and its overexpression upregulated p21cip1/waf1 protein expression. These results suggested that CARM1-methylated HuD maintains PC12 cells in the proliferative state by committing p21cip1/waf1 mRNA to its decay system. Since the methylated population of HuD was reduced in NGF-treated PC12 cells, downregulation of HuD methylation is a possible pathway through which NGF induces differentiation of PC12 cells.

Induced HMGA1a expression causes aberrant splicing of Presenilin-2 pre-mRNA in sporadic Alzheimer's disease.

AbstractThe aberrant splicing isoform (PS2V), generated by exon 5 skipping of the Presenilin-2 (PS2) gene transcript, is a diagnostic feature of sporadic Alzheimers disease (AD). We found PS2V is hypoxia-inducible in human neuroblastoma SK-N-SH cells. We purified a responsible trans-acting factor based on its binding to an exon 5 fragment. The factor was identified as the high mobility group A1a protein (HMGA1a; formerly HMG-I). HMGA1a bound to a specific sequence on exon 5, located upstream of the 5′ splice site. HMGA1a expression was induced by hypoxia and the protein was accumulated in the nuclear speckles with the endogenous splicing factor SC35. Overexpression of HMGA1a generated PS2V, but PS2V was repressed by cotransfection with the U1 snRNP 70K protein that has a strong affinity to HMGA1a. HMGA1a could interfere with U1 snRNP binding to the 5′ splice site and caused exon 5 skipping. HMGA1a levels were significantly increased in the brain tissue from sporadic AD patients. We propose a novel mechanism of sporadic AD that involves HMGA1a-induced aberrant splicing of PS2 pre-mRNA in the absence of any mutations.

Mouse Prickle1 and Prickle2 are expressed in postmitotic neurons and promote neurite outgrowth

The Drosophila planar cell polarity (PCP) gene prickle has been previously indicated as one of the regulators of gastrulation in the early embryonic stage. However, the functional role of prickle in the brain in particular is not known. We first indicated that mouse Prickle1 and Prickle2 are continually expressed in the brain throughout the embryonic stages and are observed to be specifically expressed in the postmitotic neurons. Furthermore, Prickle1 or Prickle2 depletion effectively decreases the neurite outgrowth levels of mouse neuroblastoma Neuro2a cells. These results indicate that mouse Prickle1 and Prickle2 possibly regulate positive neurite formation during brain development.

Specific regional distribution of protein arginine methyltransferase 8 (PRMT8) in the mouse brain

The regional distribution of PRMT8 transcript was examined in mouse brain using in situ hybridization (ISH) histochemistry. The PRMT8 cRNA probe was specifically hybridized with CNS and the signals were observed only in the neurons. The distribution of the neurons expressing PRMT8 mRNA was not even throughout the brain. All of the regions related to general somatosensory system expressed PRMT8 mRNA strongly. Most of the relay nuclei intervening the special somatosensory system, such as the auditory, visual, and vestibular systems, were packed with PRMT8 mRNA expressing neurons. Forebrain limbic areas and thalamic nuclei relevant to limbic areas were also strongly labeled with the probe. Some areas related to the motor system, such as the caudate putamen, Purkinje cells, inferior olivary nucleus and cerebellar nuclei expressed PRMT8 mRNA strongly. These findings suggest that PRMT8 is chiefly involved in the somatosensory and limbic systems, and a part of motor system.

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.

PRMT1 and Btg2 regulates neurite outgrowth of Neuro2a cells.

Neurite outgrowth is one of the crucial events in the formation of neural circuits. The majority of studies on neurite outgrowth have focused on signal transduction processes based on phosphorylation and acetylation; a few studies have suggested the involvement of other molecular mechanisms. Recent progress in understanding the nature of protein arginine N-methyltransferases (PRMTs) raises the possibility of the involvement of protein methylation accompanied by cell shape changes during neuronal differentiation. Here, we show that PRMT1 play a pivotal role in the neurite outgrowth of Neuro2a cells. Our results revealed that PRMT1 depletion specifically affected neurite outgrowth but not the physiological processes involved in cell growth and differentiation. Furthermore, we demonstrated that Btg2, one of the PRMT1 binding partner, depletion down-regulated arginine methylation in the nucleus and inhibited neurite outgrowth. These results indicate that protein arginine methylation by PRMT1 in the nucleus is an important step in neuritogenesis.

Expression of an ADP-ribosylation factor like gene, ARF4L, is induced after transient forebrain ischemia in the gerbil

To elucidate the molecular mechanisms underlying post-ischemic phenomena including delayed neuronal death, we screened for genes which were induced in the hippocampus after transient global ischemia in the Mongolian gerbil by a differential display method, and cloned a gerbil homologue of human ADP-ribosylation factor 4L (ARF4L). Although the physiological roles of ARF4L are unknown, it is likely that ARF4L participates in vesicle transport between the endoplasmic reticulum (ER) and Golgi complex as it contains a GTP binding site, myristoylation site and coatmer binding motif (KKXX). In situ hybridization analysis indicated that the expression of ARF4L mRNA was elevated in neurons of the dentate gyrus (DG) and CA1 regions. In DG, the signals were detected 3 h after ischemia and peaked at 6 h with subsequent gradual reduction. On the other hand, in the CA1 region where cell death occurs in this model, ARF4L mRNA was slightly detected from 1 to 2 days after ischemia but was absent after 3 days. Other vesicle transport-related genes such as ARF1, ARL4 and beta-COP were also induced after 5-min ischemia, suggesting that vesicle transport was activated in hippocampal neurons after ischemic stress. To determine the cause of the induction of ARF4L gene expression after transient ischemia, we examined the changes in ARF4L mRNA expression in HEK 293 cells under hypoxic conditions compared with HSP70. The expression of ARF4L mRNA was elevated at 12 h after hypoxia exposure, similarly to HSP70. These results will help to elucidate the association of upregulation of vesicle transport systems including ARF4L and stress responses of neurons after transient ischemia.