神保 恵理子

Many human proteins have homopolymeric amino acid (HPAA) tracts, but their physiological functions or cellular effects are not well understood. Previously, we expressed 20 HPAAs in mammalian cells and showed characteristic intracellular localization, in that hydrophobic HPAAs aggregated strongly and caused high cytotoxicity in proportion to their hydrophobicity. In the present study, we investigated the cytotoxicity of these aggregate-prone hydrophobic HPAAs, assuming that the ubiquitin proteasome system is impaired in the same manner as other well-known aggregate-prone polyglutamine-containing proteins. Some highly hydrophobic HPAAs caused a deficiency in the ubiquitin proteasome system and excess endoplasmic reticulum stress, leading to apoptosis. These results indicate that the property of causing excess endoplasmic reticulum stress by proteasome impairment may contribute to the strong cytotoxicity of highly hydrophobic HPAAs, and proteasome impairment and the resulting excess endoplasmic reticulum stress is not a common cytotoxic effect of aggregate-prone proteins such as polyglutamine.

Dysferlin is a type-II transmembrane protein and the causative gene of limb girdle muscular dystrophy type 2B and Miyoshi myopathy (LGMD2B/MM), in which specific loss of dysferlin labeling has been frequently observed. Recently, a novel mutant (L1341P) dysferlin has been shown to aggregate in the muscle of the patient. Little is known about the relationship between degradation of dysferlin and pathogenesis of LGMD2B/MM. Here, we examined the degradation of normal and mutant (L1341P) dysferlin. Wild-type (wt) dysferlin mainly localized to the ER/Golgi, associated with retrotranslocon, Sec61 alpha, and VCP(p97), and was degraded by endoplasmic reticulum (ER)-associated degradation system (ERAD) composed of ubiquitin/proteasome. In contrast, mutant dysferlin spontaneously aggregated in the ER and induced eukaryotic translation initiation factor 2 alpha (eIF2 alpha) phosphorylation and LC3 conversion, a key step for autophagosome formation, and finally, ER stress cell death. Unlike proteasome inhibitor, E64d/pepstatin A, inhibitors of lysosomal proteases did not stimulate the accumulation of the wt-dysferlin, but stimulated aggregation of mutant dysferlin in the ER. Furthermore, deficiency of Atg5 and dephosphorylation of eIF2 alpha, key molecules for LC3 conversion, also stimulated the mutant dysferlin aggregation in the ER. Rapamycin, which induces eIF2 alpha phosphorylation-mediated LC3 conversion, inhibited mutant dysferlin aggregation in the ER. Thus, mutant dysferlin aggregates in the ER-stimulated autophagosome formation to engulf them via activation of ER stress-eIF2 alpha phosphorylation pathway. We propose two ERAD models for dysferlin degradation, ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Mutant dysferlin aggregates on the ER are degraded by the autophagy/lysosome ERAD(II), as an alternative to ERAD(I), when retrotranslocon/ERAD(I) system is impaired by these mutant aggregates.

Although a mutation (R553H) in the forkhead box (FOX)P2 gene is associated with speech/language disorder, little is known about the function of FOXP2 or its relevance to this disorder. In the present study, we identify the forkhead nuclear localization domains that contribute to the cellular distribution of FOXP2. Nuclear localization of FOXP2 depended on two distally separated nuclear localization signals in the forkhead domain. A truncated version of FOXP2 lacking the leu-zip, Zn2+ finger, and forkhead domains that was observed in another patient with speech abnormalities demonstrated an aggregated cytoplasmic localization. Furthermore, FOXP2 (R553H) mainly exhibited a cytoplasmic localization despite retaining interactions with nuclear transport proteins (importin a and P). Interestingly, wild type FOXP2 promoted the transport of FOXP2 (R553H) into the nucleus. Mutant and wild type FOXP2 heterodimers in the nucleus or FOXP2 R553H in the cytoplasm may underlie the pathogenesis of the autosomal dominant speech/language disorder. (c) 2006 Elsevier Inc. All rights reserved.

RA175/TSLC1/SynCAM/IGSF4A (RA175), a member of the immunoglobulin superfamilly with Ca(2+)-independent homophilic trans-cell adhesion activity, participates in synaptic and epithelial cell junctions. To clarify the biological function of RA175, we disrupted the mouse Igsf4a (Ra175/Tslc1/SynCam/Igsf4a Ra175) gene. Male mice lacking both alleles of Ra175 (Ra175(-/-)) were infertile and showed oligo-astheno-teratozoospermia; almost no mature motile spermatozoa were found in the epididymis. Heterozygous males and females and homozygous null females were fertile and had no overt developmental defects. RA175 was mainly expressed on the cell junction of spermatocytes, elongating and elongated spermatids (steps 9 to 15) in wild-type testes; the RA175 expression was restricted to the distal site (tail side) but not to the proximal site (head side) in elongated spermatids. In Ra175(-/-) testes, elongated and mature spermatids (steps 13 to 16) were almost undetectable; round spermatids were morphologically normal, but elongating spermatids (steps 9 to 12) failed to mature further and to translocate to the adluminal surface. The remaining elongating spermatids at improper positions were finally phagocytosed by Sertoli cells. Furthermore, undifferentiated and abnormal spermatids exfoliated into the tubular lumen from adluminall surfaces. Thus, RA175-based cell junction is necessary for retaining elongating spermatids in the invagination of Sertoli cells for their maturation and translocation to the adluminall surface for timely release.

RA175 is a new member of the immunoglobulin superfamily with trans interaction activity, and it plays a role as a tumor suppressor in lung carcinoma (TSLC1) and as a cell adhesion molecule promoting the formation of functional synapses (SynCAM). Little is known about the biological function of R-A175/TSLC1/SynCAM neural network formation during neurogenesis. We examined the distribution and colocalization of the RA175/TSLC1/SynCAM protein with other members of the immunoglobulin superfamily such as NCAM, L1, and TAG-1 in the mouse developing nervous system. Consistent with the expression of RA175/TSLC1/SynCAM mRNA, the protein was localized in the brain neuroepithelium at embryonic day (E) 9.5, neural crest at E10.5, motor neurons at E10.5, and olfactory epithelium at E16.5. In contrast with its mRNA, the protein was intensely detected on the fasciculated axons in the floor plates, ventral root, and dorsal funiculus in the E10.5-11.5 spinal cord and colocalized with NCAM and L1 on the ventral root and dorsal funiculus and partly colocalized with TAG-I on the commissural axons and dorsal faniculus. In the E13.5-15.5 brain, RA175/TSLC1/SynCAM colocalized with NCAM and L1 on the developing thalamocortical fibers from the internal capsule (IC) and partly colocalized with TAG-1 on the cortical efferent axons in the intermediate zone (IZ). RA175/TSLC1/SynCAM was localized on the axons of some of the cortical neurons cultured in vitro. Thus, in addition to cell adhesion activity in the neuroepithelium and the synapses, RA175/TSLC1/SynCAM may be involved in neuronal migration, axon growth, pathfinding, and fasciculation on the axons of differentiating neurons. (C) 2004 Elsevier B.V. All rights reserved.

Caspase-3-deficient 129/Sv mice show hyperplasia of the brain at embryonic (E) day 10.5-12.5, but caspase-3-deficient C57L/136 mice do not. We examined the relationship between activation of caspase-3 and programmed cell death (PCD) during forebrain development of various mouse strains (129/Sv, ICR, C57L/136, and CBA) using terminal deoxytransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and immunostaining with antiserum against the caspase-3 (anti-m3D175) cleavage site. A number of anti-m3D175 positive cells and TUNEL positive cells were detected in the ventral side of the forebrain of 129/Sv and ICR mice at E8.5-9 but not in C57L/136 and CBA mice. Ac-DEVD-MCA cleavage activity, a caspase-3-like activity, also suggests the preferential activation of caspase-3 in the ventral forebrain of ICR mice but not in C57L/136 mice. Developmental changes of TUNEL and anti-m3D175 reactivities were essentially similar during brain morphogenesis of ICR and 129/Sv mice. The number of TUNEL/anti-m3D175 positive cells decreased in the neuroepithelium of the ventral forebrain at E9.5 before generation of the medial ganglionic eminence (MGE). TUNEL and/or anti-m3D175 reactivity was slightly detectable in the MGE at E10.5, from which neuroprogenitor cells follow a tangential migratory route to the cortex. Activation of caspase-9 was also immunohistochemically detected in the ventral forebrain at E8.5-9, suggesting that activation of caspase-3 and caspase-9 occurs in the PCD of this region. Thus, it is likely that decreased cell death in the ventral forebrain of caspase-3- and caspase-9-deficient 129/Sv mice increases the number of neuroprogenitor cells in the MGE, leading to hyperplasia of the forebrain. (C) 2003 Elsevier B.V. All rights reserved.

RA175, a new immunoglobulin superfamily member, is preferentially expressed during differentiation of P19 embryonic carcinoma (EC) cells induced by retinoic acid. In the present study, we isolated mouse RA175 cDNA in its entirety and showed that RA175 is the mouse ortholog of TSLC1, a tumor suppressor gene in human lung cancer. RA175/TSLC1 was localized in the adherent region of human lung squamous carcinoma cells and in the differentiated P19 EC cells. RA175/TSLC1 showed homophilic trans-interaction activity in a Ca2+-independent manner. RA175/TSLC1 was preferentially expressed in the polarized cells lining the lumen of developing mouse lung epithelium. This suggests that RA175/TSLC1 is a cell adhesion molecule that is acting as a tumor suppressor gene in the metastasis of lung tumors. RA175/TSLC1 may be necessary for cells to remain tightly associated in the epithelium, thereby suppressing metastasis. (C) 2003 Elsevier Science (USA). All rights reserved.

Caspase-3-deficient 129/Sv mice show hyperplasia of the forebrain at embryonic day (E) 10.5, which suggests that caspase-3-dependent programmed cell death (PCD) plays an essential role in brain morphogenesis prior to neurogenesis. However, little is known about region-specific caspase-3-dependent PCD in the developing forebrain. We examined the PCD region in the early developmental brain at E9.5 by whole mount terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL). In addition to hindbrain, TUNEL-reactivity was detected in the ventral forebrain and in the caudal portion of the front nasal region, just behind the regions expressing fgf-8 and otx-2. It has been shown recently that brain hyperplasia induced by caspase-3-deficiency is mouse strain-dependent; such that brain abnormalities were observed in caspase-3-deficient 129/Sv mice but not in caspase-3-deficient C57BL/6 mice. We examined the caspase-3-dependent PCD in the ventral forebrain of 129/Sv and C57BL/6 mouse embryos (E8.5-9 and E9.5) by double staining of TUNEL and antiserum against the active form of caspase-3 (anti-m3DI75). TUNEL/anti-m3DI75 reactivity in the ventral forebrain was mouse strain-dependent, such that many TUNEL/anti-m3DI75-positive cells were detected in the ventral forebrains of 129/Sv mice, but were not observed in C57BL/6 mice. Thus, it is likely that this region is the site of the strain-specific caspase-3-dependent PCD. A strain-dependent 'modulator' that regulates both caspase-3-dependent and -independent cell death pathways may control PCD in the ventral forebrain at E8.5-9.5.

Excess endoplasmic reticulum (ER) stress induces processing of caspase-12, which is located in the ER, and cell death. However, little is known about the relationship between caspase-12 processing and cell death. We prepared antisera against putative caspase-12 cleavage sites (anti-m12D318 and anti-m12D341) and showed that overexpression of caspase-12 induced autoprocessing at D-318 but did not induce cell death. Mutation analysis confirmed that D-318 was a unique autoprocessing site. In contrast, tunicamycin, one of the ER stress stimuli, induced caspase-12 processing at the N-terminal region and the C-terminal region (both at D-318 and D-341) and cell death, Anti-m12D318 and anti-m12D341 immunoreactivities were located in the ER of the tunicamycin-treated cells, and some immunoreactivities were located around and in the nuclei of the apoptotic cells. Thus, processing at the N-terminal region may be necessary for the translocation of processed caspase-12 into nuclei and cell death induced by ER stress, Some of the caspase-12 processed at the N-terminal and C-terminal regions may directly participate in the apoptotic events in nuclei.

Accumulation of unfolded and malfolded proteins causes endoplasmic reticulum (ER) stress, stimulating unfolded protein response (UPR) and c-Jun N-terminal kinase (JNK) activation and activating caspase-12 located on the ER. Little is known about the relationship between the ER stress and polyglutamine [poly(Q)] aggregates. Poly(Q)(72) repeats [poly(Q)(72)] induced the stimulation of ER stress signals such as JNK activation, upregulation of Grp78/Bip and caspase-12 activation in C2C5 cells. We prepared antiserum against the cleavage site of mouse caspase-12 at D(318) (anti-m12D318), and showed that poly(Q)(72) with perinuclear aggregates, cytoplasmic inclusions and nuclear inclusions stimulated JNK activation and anti-m12D318 immunoreactivity, but poly(Q)(72) with dispersed aggregates and small nuclear aggregates showed a significantly less effect. poly(Q)(72) and poly(Q)(11) dispersed in cytoplasm did not. Anti-m12D318-positive cells showed apoptotic features. Unlike anti-m8D387 immunoreactivity, the anti-m12D318 immunoreactivity was not coaggregated with poly(Q). Ac-IETD-fmk (caspase-8 inhibitor) and Ac-DEVD-CHO (caspase-3 inhibitor) did not prevent the anti-m12D318 immunoreactivity induced by poly(Q)(72) aggregates. Anti-m12D318 immunoreactivity was detected in caspase-8(-/-) and caspase-3(-/-) mouse embryonic fibroblasts expressing poly(Q)(72) aggregates. Thus, caspase-12 was activated by poly(0)72 aggregates via a pathway independent of caspase-8 and caspase-3 activation, and caspase-12 activation was closely associated with poly(Q) aggregate-mediated cell death. Stimulation of ER stress signals may be involved in the pathogenesis of neurodegenerative disorders with poly(Q) expansion.

Truncated polypeptides containing expanded polyglutamine (polyQ) stretches tend to form cytoplasmic or nuclear aggregates in cultured cells, leading to cell death. Although it has been shown recently that caspase-8 coaggregates with polyQ and is activated during polyQ-mediated cell death, little is known of the location and timing of caspase-8 activation by nuclear polyQ aggregates. Also, the relationship between nuclear polyQ aggregate-mediated cell death and activation of other caspases is unclear. In P19 embryonal carcinoma (EC) cells, which can be made to differentiate into neuronal cells, polyQ72 repeats preferentially aggregate in the nucleus. Nuclear aggregates of polyQ72 induced P19 EC cell death, with a high frequency of cells exhibiting morphology characteristic of apoptosis (i.e., roundness, cell shrinkage, chromatin condensation) and DNA fragmentation. In the present study, we used antisera that specifically recognized the active forms of caspase-8, -3, and -9 but not their proforms, and showed that only caspase-8 and -3 were activated during the generation of polyQ72 aggregates in P19 EC cell nuclei. Furthermore, we showed that the caspase inhibitor z-VAD-fmk inhibited DNA fragmentation, but only partially inhibited the appearance of apoptotic morphology. Thus, caspase activation, including caspase-8 and -3, is necessary for polyQ-mediated DNA fragmentation but not sufficient for polyQ-mediated cell death in P19 EC cells. (C) 2000 Wiley-Liss, Inc.

Caspases, which play crucial roles during apoptosis, are activated from their inactive proforms in a sequential cascade of cleavage by other members of the caspase family. Caspase-9 is autoprocessed by the Apaf-1/cytochrome c pathway and acts at an early point in this cascade, whereas Bcl-xL, an antiapoptotic member of the Bcl-2 family, prevents activation of caspases in vitro. Little is known, however, about the relation between caspase-9 and Bcl-xL during development of the mammalian nervous system. We used antisera against two cleavage sites in mouse caspase-9 that recognize only the activated form of mouse caspase-9, and we examined immunohistochemically the activation of mouse caspase-9 in the nervous system of Bcl-x-deficient mouse embryos. Mouse caspase-9 is processed at both D353 and D368, but it is processed preferentially at D368 during apoptosis of cultured cells induced by various stimuli and in the nervous system of Bcl-x-deficient mouse embryos. We show that Bcl-xL protects against caspase-9- and/or caspase-3- dependent apoptosis in the caudal portion of the ventral hindbrain, anterior horn cells, and dorsal root ganglia neurons of the normal mouse embryos and against caspase-9/caspase-3-independent apoptosis in the dorsal region of the nervous system including the dorsal spinal cord. Furthermore, we demonstrate that Bcl-xL blocks cytochrome c release from mitochondria, causing activation of caspase-9 in anterior horn cells and dorsal root ganglia neurons in mouse embryos at embryonic day 11.5. (C) 2000 Elsevier Science B.V.

The relation between activation of caspase-8 and polyglutamine aggregates has been focused. We prepared an antiserum (anti-m8D387) that recognizes the active form but not the preform of mouse caspase-8. We used immunostaining with anti-m8D387 antiserum to compare the localizations of activated mcaspase-8 in L929 (clone 1422) cells induced by TNF and polyglutamine aggregates. Anti-m8D387 was positive throughout cytoplasm of the TUNEL-positive cells induced by TNF treatment, whereas the anti-m8D387 reactivity was not positive throughout cytoplasm of the cells expressing polyglutamine but was restricted to polyglutamine aggregates. In contrast with TNF-treated cells, cells expressing anti-m8D387-positive cytoplasmic polyglutamine aggregates did not undergo TUNEL-positive apoptosis. Thus activated caspase-8 associated with polyglutamine aggregates alone was not sufficient to induce TUNEL-positive apoptosis of L929 (clone 1422) cells. The distribution of activated caspase-8 associated with polyglutamine aggregates may be essential for the polyglutamine-mediated cell death or downstream of caspase-8 may be different in the TNF-treated cells and cells expressing polyglutamine. (C) 2000 Academic Press.

Caspases and Bcl-xL, the mammalian homologues of the Caenorhabditis elegans (C. elegans) ced-3 and ced-9 genes, respectively, regulate apoptosis of various cells. Caspase-3 is processed into an active form (p20 or p17 and p12) during apoptosis. We investigated the relation between caspase-3 and Bcl-xL during development by examining activation of caspase-3 and apoptotic cells in Bcl-x-deficient (bcl-x(-/-)) mice at embryonic (E) day 11.5. We used a double-staining technique with a cleavage site-directed antibody against caspase-3 (anti-p20/17) and terminal-deoxytransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL). Bcl-xL-deficiency increased both numbers of p20/17-positive and -negative apoptotic cells in dorsal root ganglia (DRG); the numbers of p20/17-positive apoptotic cells in the caudal parts of the ventral hindbrain and ventral spinal cord; and the numbers of p20/17-negative apoptotic cells in the dorsal midbrain, dorsal hindbrain, and dorsal spinal cord. Thus, Bcl-xL blocks the caspase-3-dependent apoptotic pathway in the restricted regions of the nervous system during development. Furthermore, these observations suggest that Bcl-xL protects against activation of the caspase-3-independent apoptotic pathway. Other caspases or apoptotic mechanisms may also be activated in the nervous systems of bcl-x(-/-) mice. Copyright (C) 1999 Elsevier Science B.V.

RA70, which is expressed during neuronal differentiation of P19 EC, is highly homologous to human src kinase-associated phosphoprotein (SKAP55). Here we isolated human full-length RA70 cDNA. Unlike SKAP55, which is specifically expressed in thymus and T cells, RA70 was expressed ubiquitously in various tissues including lung, skeletal muscle, and spleen, and in various cell lines including human monocytic leukemia (U937) cells, but RA70 was undetectable in thymus and T cell lymphoma (Jurkat) cells. RA70 as well as SKAP55 proved to be a protein with molecular weight 55 kDa associated with SH2 domain of Fyn. Interaction between RA70 and src family kinases, Fyn, Hck and Lyn, was detected during monocytes/macrophage-differentiation of U937 cells. Thus, like SKAP55, RA70 is an adaptor protein of the src family kinases. RA70 may play an essential role in the src signaling pathway in various cells.

We previously demonstrated that Caspase-3 is highly expressed in dorsal root ganglia and trigeminal ganglia of mouse embryos [T. Mukasa, K. Urase, Y.M. Momoi, I. Kimura, T. Momoi, Specific expression of CPP32 in sensory neurons of mouse embryos and activation of CPP32 in the apoptosis induced by a withdrawal of NGF, Biochem. Biophys. Res. Commun., 231 (1997) 770-774.]. Since, however, Caspases are processed into active form during apoptosis, it is difficult to examine the involvement of activated Caspases in naturally occurring cell death during development by immunohistochemical staining or in situ hybridization method. We prepared a cleavage site-directed antiserum against Caspase-3 (anti-p20/17). This antiserum reacted with fragment (p20/17) of Caspase-3, but not proCaspase-3 (p32), proCaspase-7 (p34) and its cleaved fragment (p24). We examined the relationship between the activation of Caspase-3 and the appearance of the naturally occurring apoptotic cells in the nervous system during development. In the trigeminal ganglia and dorsal root ganglia, the expression of Caspase-3 mRNA was maximal before the appearance of p20/17-positive cells and apoptotic cells. In the mouse brain, many p20/17-positive cells and apoptotic cells were observed in the neuroepithelium in the early developmental stages, but very few p20/17- positive cells were detected in postmitotic neurons in the cerebral cortex although Caspase-3 mRNA was expressed highly. Caspase-3 is activated mainly during apoptosis of neuroepithelial cells in the early developmental stages but not of mature neurons at postnatal stages.

Free D-Ser, D-Asp and total B-amino acids were significantly higher (p < 0.05) in Alzheimer (AD) ventricular CSF than in normal CSF. There was no significant difference in the total L-amino acids between AD and normal CSF, but L-Gln and L-His were significantly higher (p < 0.05) in AD-CSF, The higher concentrations of these D- and L-amino acids in AD ventricular CSF could reflect the degenerative process that occurs in Alzheimer's brain since ventricular CSF is the repository of amino acids from the brain.