山口 淳

The precuneus is an association area in the posteromedial cortex (PMC) that is involved in high-order cognitive functions through integrating multi-modal information. Previous studies have shown that the precuneus is functionally heterogeneous and subdivided into several subfields organized by the anterior-posterior and ventral-dorsal axes. Further, the precuneus forms the structural core of brain connectivity as a rich-club hub and overlaps with the default mode network (DMN) as the functional core. This review summarizes recent research on the connectivity and cognitive functions of the precuneus. We then present our recent tractography-based studies of the precuneus and contextual these results here with respect to possible cognitive functions and resting-state networks.

Z-DNA binding protein 1 (ZBP1) is a cytosolic nucleic acid sensor, functioning as a critical mediator of inflammation and cell death pathways. Since neuroinflammation could occur in response to damage-associated molecular patterns (DAMPs), ZBP1 might be involved in neuroinflammation after stroke. However, the spatiotemporal expression profile of ZBP1 in the post-stroke brain remains to be elucidated. The aim of this study is to demonstrate the spatiotemporal expression patterns of ZBP1 in the post-stroke brain using a mouse photothrombotic stroke model. Real-time PCR assays showed that ZBP1 is induced on days 3-14 post stroke. ZBP1 immunoreactivity was observed in Iba1-positive microglia/macrophages in peri-infarct regions by immunohistochemistry. ZBP1-positive cells were spread in layers surrounding the infarct core by 7-14 days post stroke. Interestingly, ZBP1 immunoreactivity was also detected in CD206-positive border-associated macrophages (BAMs) in the meninges. Furthermore, ZBP1-expressing cells were positive for antibodies against inflammatory mediators such as Toll-like receptor 4 (TLR4), Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF), and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Morphological analysis with confocal microscopy showed that the co-localization signals of ZBP1 and its adaptor, TRIF, are increased by glucose oxidase (GOx) treatment, which has been reported to induce mitochondrial DNA (mtDNA) release. These results suggest that ZBP1 is induced in peri-infarct microglia/macrophages and may be involved in DAMPs-mediated neuroinflammation involving mtDNA in the post-infarct brain.

It is generally hypothesized that functional connectivity (FC) reflects the underlying structural connectivity (SC). The precuneus is associated with highly integrated cognitive functions. However, our understanding of the structural connections that could underlie them is limited. This study aimed to characterize the cortico-cortical connections by probabilistic tractography. The precuneus corresponds to the five cortical areas (7Am, PCV, 7Pm, 7m, POS2) on the HCP MMP atlas. We first conducted the atlas-based probabilistic tractography. The anterior part (7Am) was strongly connected to the sensorimotor region. The dorsal part (7Am, 7Pm) was highly connected with the adjacent parietal and temporal cortex, while the ventral part (PCV, 7m) showed strong connections with the adjacent posterior cingulate and medial prefrontal cortex. The most posterior part (POS2) was explicitly connected to the visual cortex. In addition, there was a correlation between SC and resting-state fMRI connectivity (Spearman's rank correlation coefficient = 0.322 ± 0.019, p < 0.05 corrected at subject level). Collectively, the current study revealed the characteristic connectional profile of precuneus, which could shed light on the structural heterogeneity for the future functional analyses.

The intervention at the stage of mild cognitive impairment (MCI) is promising for preventing Alzheimer's disease (AD). This study aims to search for the optimal machine learning (ML) model to classify early and late MCI (EMCI and LMCI) subtypes using multimodal MRI data. First, the tract-based spatial statistics (TBSS) analyses showed LMCI-related white matter changes in the Corpus Callosum. The ROI-based tractography addressed the connected cortical areas by affected callosal fibers. We then prepared two feature subsets for ML by measuring resting-state functional connectivity (TBSS-RSFC method) and graph theory metrics (TBSS-Graph method) in these cortical areas, respectively. We also prepared feature subsets of diffusion parameters in the regions of LMCI-related white matter alterations detected by TBSS analyses. Using these feature subsets, we trained and tested multiple ML models for EMCI/LMCI classification with cross-validation. Our results showed the ensemble ML model (AdaBoost) with feature subset of diffusion parameters achieved better performance of mean accuracy 70%. The useful brain regions for classification were those, including frontal, parietal lobe, Corpus Callosum, cingulate regions, insula, and thalamus regions. Our findings indicated the optimal ML model using diffusion parameters might be effective to distinguish LMCI from EMCI subjects at the prodromal stage of AD.

The precuneus, involved in various cognitive processes, is considered to form the midline core of the default mode network (DMN), while the medial temporal lobe (MTL) is a subsystem of the DMN. Until now, the anatomical study of the precuneus-MTL connection is limited in humans. One possible reason is the precuneus' territory of the posteromedial cortex (PMC) is inconsistent across studies. The primary purpose of this study is to investigate the structural connectivity (SC) of precuneus-MTL, focusing on its anatomical organization using the Human Connectome Project Multi-modal Parcellation (HCP MMP) atlas. We first conducted the quantitative tractography analyses using the HCP dataset. The major streamlines originated from the posterior precuneus and were projected to the MTL extensively. Next, to complement the tractography data, we conducted the white matter dissection in the post-mortem human brain. We observed the major fiber bundles arise from the posterior precuneus extending to the anterior parahippocampal gyrus, which could support our tractography results. Then we analyzed the relationship between SC and resting-state functional connectivity (rsFC) of the precuneus-MTL. Although the SC-rsFC correlation was scarce on the whole, the posterior precuneus (POS2, 7Pm. 7m) showed a relatively high correlation (r = 0.38349, p < 0.05) with the posterior MTL (PreS, H, ProS, PHA1, PHA2). Our findings suggest the posterior precuneus is highly connected to MTL structurally, which could have an effect on the resting-state functional connectivity. In addition, the precuneus might consist of the heterogeneous connectivity-based subdivisions.

BACKGROUND: Chronic pain is a highly refractory and complicated condition that persists even without nociception. Several genome-wide gene expression analyses have shown that the immune response and inflammatory cytokines affect chronic pain establishment in the acute pain phase. However, compared with the acute phase, the chronic phase has a poorly elucidated gene expression profile. This study aimed to determine the gene expression profile in the spinal cord of a neuropathic pain mouse model in the chronic phase to elucidate the chronic pain characteristics. METHODS: We established a sciatic nerve cuff mouse model as a neuropathic pain model by placing a 2-mm section of a split PE-20 polyethylene tube around the sciatic nerve. The spinal cord was harvested at the L4-6 level at 28 postoperative days. Next, we examined differentially expressed genes (DEGs) through RNA sequencing (RNA-seq) compared with the sham group; moreover, we conducted enrichment analyses of the expressed genes. To reveal the chronic pain characteristics, we compared the gene expression profiles of the spinal cord between the acute and chronic phases in the neuropathic pain model. Among the chronic pain-related genes categorized in the dendrites, we focused on cyclin-dependent kinase-like 5 (CDKL5). We analyzed CDKL5 expression and function using real-time polymerase chain reaction (PCR), immunohistochemistry, and neurite extension assay in Neuro 2a (N2a) cells. We used three types of CDKL5 plasmids: wild type, nuclear localization signal-attached, and K42R kinase-dead CDKL5. RESULTS: We identified 403 DEGs, including 104 upregulated and 43 downregulated genes (false discovery rate < 0.01). Rather than inflammation or immune response, the most enriched terms in the chronic phase were "regulation of plasma membrane-bounded cell projection organization" and "dendrite." Real-time PCR assay confirmed increased CDKL5 expression in the ipsilateral dorsal horn. CDKL5 was broadly expressed in the ipsilateral dorsal horn across all layers. The neurite extension assay revealed that the cytoplasmic kinase function of CDKL5 was necessary for neurite outgrowth in N2a cells. CONCLUSION: RNA-seq of the spinal cord revealed that the most enriched genes during the chronic pain phase were involved in regulating axon and dendrite morphogenesis, including CDKL5. Our findings suggest that neural remodeling affects chronic pain establishment. Since patients with CDKL5 mutations have shown reduced pain perception, our findings suggest that CDKL5 in the spinal cord could result in neural remodeling during the chronic pain phase through cytoplasmic kinase activity.

<title>Abstract</title> The intraparietal sulcus (IPS) in the posterior parietal cortex (PPC) is well-known as an interface for sensorimotor integration in visually guided actions. However, our understanding of the human neural network between the IPS and the cortical visual areas has been devoid of anatomical specificity. We here identified a distinctive association fiber tract “IPS-FG” to connect the IPS areas and the fusiform gyrus (FG), a high-level visual region, by white matter dissection and tractography. The major fiber bundles of this tract appeared to arise from the medial bank of IPS, in the superior parietal lobule (SPL), and project to the FG on the ventral temporal cortex (VTC) in post-mortem brains. This tract courses vertically at the temporo-parieto-occipital (TPO) junction where several fiber tracts intersect to connect the dorsal-to-ventral cortical regions, including the vertical occipital fasciculus (VOF). We then analyzed the structural connectivity of this tract with diffusion-MRI (magnetic resonance imaging) tractography. The quantitative tractography analysis revealed the major streamlines of IPS-FG interconnect the posterior IPS areas (e.g., IP1, IPS1) with FG (e.g., TF, FFC, VVC, PHA2, PIT) on the Human Connectome Project multimodal parcellation atlas (HCP MMP 1.0). Since the fronto-parietal network, including the posterior IPS areas, is recruited by multiple cognitive demands, the IPS-FG could play a role in the visuomotor integration as well as the top-down modulation of various cognitive functions reciprocally.

The neuroinflammation in the ischemic brain could occur as sterile inflammation in response to damage-associated molecular patterns (DAMPs). However, its long-term dynamic transcriptional changes remain poorly understood. It is also unknown whether this neuroinflammation contributes to the recovery or just deteriorates the outcome. The purpose of this study is to characterize the temporal transcriptional changes in the post-stroke brain focusing on DAMPs-related genes by RNA-sequencing during the period of 28 days. We conducted the RNA-sequencing on day 1, 3, 7, 14, 28 post-stroke in the mouse photothrombosis model. The gross morphological observation showed the ischemic lesion on the ipsilateral cortex turned into a scar with the clearance of cellular debris by day 28. The transcriptome analyses indicated that post-stroke period of 28 days was classified into four categories (I Baseline, II Acute, III Sub-acute-#1, IV Sub-acute-#2 phase). During this period, the well-known genes for DAMPs, receptors, downstream cascades, pro-inflammatory cytokines, and phagocytosis were transcriptionally increased. The gene ontology (GO) analysis of biological process indicated that differentially expressed genes (DEGs) are genetically programmed to achieve immune and inflammatory pathways. Interestingly, we found the biphasic induction of various genes, including DAMPs and pro-inflammatory factors, peaking at acute and sub-acute phases. At the sub-acute phase, we also observed the induction of genes for phagocytosis as well as regulatory and growth factors. Further, we found the activation of CREB (cAMP-response element binding protein), one of the key players for neuronal plasticity, in peri-ischemic neurons by immunohistochemistry at this phase. Taken together, these findings raise the possibility the recurrent inflammation occurs at the sub-acute phase in the post-stroke brain, which could be involved in the debris clearance as well as neural reorganization.

The vertical occipital fasciculus (VOF) is an association fiber tract coursing vertically at the posterolateral corner of the brain. It is re-evaluated as a major fiber tract to link the dorsal and ventral visual stream. Although previous tractography studies showed the VOF's cortical projections fall in the dorsal and ventral visual areas, the post-mortem dissection study for the validation remains limited. First, to validate the previous tractography data, we here performed the white matter dissection in post-mortem brains and demonstrated the VOF's fiber bundles coursing between the V3A/B areas and the posterior fusiform gyrus. Secondly, we analyzed the VOF's structural connectivity with diffusion tractography to link vision-associated cortical areas of the HCP MMP1.0 atlas, an updated map of the human cerebral cortex. Based on the criteria the VOF courses laterally to the inferior longitudinal fasciculus (ILF) and craniocaudally at the posterolateral corner of the brain, we reconstructed the VOF's fiber tracts and found the widespread projections to the visual cortex. These findings could suggest a crucial role of VOF in integrating visual information to link the broad visual cortex as well as in connecting the dual visual stream.

FUS (Fused-in-Sarcoma) is a multifunctional DNA/RNA binding protein linked to familial amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). Since FUS is localized mainly in the nucleus with nucleo-cytoplasmic shuttling, it is critical to understand physiological functions in the nucleus to clarify pathogenesis. Here we report a yeast two-hybrid screening identified FUS interaction with nuclear matrix-associated protein SAFB1 (scaffold attachment factor B1). FUS and SAFB1, abundant in chromatin-bound fraction, interact in a DNA-dependent manner. N-terminal SAP domain of SAFB1, a DNA-binding motif, was required for its localization to chromatin-bound fraction and splicing regulation. In addition, depletion of SAFB1 reduced FUS's localization to chromatin-bound fraction and splicing activity, suggesting SAFB1 could tether FUS to chromatin compartment thorough N-terminal DNA-binding motif. FUS and SAFB1 also interact with Androgen Receptor (AR) regulating ligand-dependent transcription. Moreover, FUS interacts with another nuclear matrix-associated protein Matrin3, which is muted in a subset of familial ALS cases and reportedly interacts with TDP43. Interestingly, ectopic ALS-linked FUS mutant sequestered endogenous Matrin3 and SAFB1 in the cytoplasmic aggregates. These findings indicate SAFB1 could be a FUS's functional platform in chromatin compartment to regulate RNA splicing and ligand-dependent transcription and shed light on the etiological significance of nuclear matrix-associated proteins in ALS pathogenesis.

FUS/TLS (fused in sarcoma/translocated in liposarcoma) encodes a multifunctional DNA/RNA binding protein with non-classical carboxy (C)-terminal nuclear localization signal (NLS). A variety of ALS-linked mutations are clustered in the C-terminal NLS, resulting in the cytoplasmic mislocalization and aggregation. Since the arginine methylations are implicated in the nuclear-cytoplasmic shuttling of FUS, a methylation inhibitor could be one of therapeutic targets for FUS-linked ALS. We here examined effects of methylation inhibitors on the cytoplasmic mislocalization and aggregates of ALS-linked C-terminal FUS mutant in a cell culture system. Treatment with adenosine dialdehyde (AdOx), a representative global methyltransferase inhibitor, remarkably mitigated the cytoplasmic mislocalization and aggregation of FUS mutant, which is consistent with previous reports. However, AdOx treatment of higher concentration and longer time period evoked the intranuclear aggregation of the ectopic expressed FUS protein. The pull down assay and the morphological analysis indicated the binding between FUS and Transportin could be potentiated by AdOx treatment through modulating methylation status in RGG domains of FUS. These findings indicated the treatment with a methylation inhibitor at the appropriate levels could alleviate the cytoplasmic mislocalization but in excess this could cause the intranuclear aggregation of FUS C-terminal mutant.

Ischemic stroke is a devastating neural event as currently no therapies other than physical rehabilitation are available to enhance recovery after stroke. To identify endogenous mediators to repair stroke brain, we performed the expression profiling analysis of transcripts in the mouse photothrombotic stroke brain. Based on real-time PCR analysis, we found VGF, identified as a nerve growth factor (NGF)-regulated transcript, was induced transcriptionally in stroke brain at 1-7 days after insult. The immunoreactivites of VGF were observed in the neurons around the ischemic core of stroke brain. Experiments with various inhibitors and plasmid transfections indicated that cAMP response element binding protein-mediated complex signaling pathways are possibly implicated in the NGF-mediated VGF expressions in vitro. Furthermore, the over-expression of VGF promoted neurite extensions and conferred protections from ischemic stress in vitro. These findings raise the possibility the application of VGF could be one of the promising therapeutic strategies to enhance recovery after stroke.

Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked PUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in similar to 10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as PUS wild type were assembled into stress granules under stress conditions, and these were also recruited to PUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis. (C) 2014 Elsevier Inc. All rights reserved.

Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). In order to identify binding partners for FUS/TLS, we performed a yeast two-hybrid screening and found that protein arginine methyltransferase 1 (PRMT1) is one of binding partners primarily in the nucleus. In vitro and in vivo methylation assays showed that FUS/TLS could be methylated by PRMT1. The modulation of arginine methylation levels by a general methyltransferase inhibitor or conditional over-expression of PRMT1 altered slightly the nucleus-cytoplasmic ratio of FUS/TLS in cell fractionation assays. Although co-localized primarily in the nucleus in normal condition, FUS/TLS and PRMT1 were partially recruited to the cytoplasmic granules under oxidative stress, which were merged with stress granules (SGs) markers in SH-SY5Y cell. C-terminal truncated form of FUS/TLS (FUS-dC), which lacks C-terminal nuclear localization signal (NLS), formed cytoplasmic inclusions like ALS-linked FUS mutants and was partially co-localized with PRMT1. Furthermore, conditional over-expression of PRMT1 reduced the FUS-dC-mediated SGs formation and the detergent-insoluble aggregates in HEK293 cells. These findings indicate that PRMT1-mediated arginine methylation could be implicated in the nucleus-cytoplasmic shuttling of FUS/TLS and in the SGs formation and the detergent-insoluble inclusions of ALS-linked FUS/TLS mutants.

A pivotal role of c-jun N-terminal kinase (INK) on neuronal apoptosis has been demonstrated in a rodent stroke model. MAP kinase phosphatase 1 (MKP-1) is an archetypal member of the dual-specificity protein phosphatase (DUSP) family, which inactivates mitogen-activated protein kinase (MAPK) including JNK through dephosphorylation. MKP-1, one of immediate early genes in stress conditions, was induced at transcriptional level in hypoxia/re-oxygenation (H/R) in neuroblastoma N1E115 cells, however the activation of JNK was not suppressed in the acute phase of re-oxygenation. Small interference RNA-mediated knock-down of MKP-1 enhanced phospho-JNK and neuronal death that is rescued by INK inhibitor in H/R. Conversely, conditional over-expression of MKP-1 suppressed phospho-JNK, the expression of proapoptotic genes, and neuronal death in H/R. Further the immunoreactivity of MKP-1 was detected in the neurons and partially co-localized with that of phospho-JNK in the surrounding zone of ischemia in rat MCA-O (middle cerebral artery occlusion) reperfusion model. These findings indicate that over-expression of MKP-1 could suppress neuronal death possibly through regulating JNK signaling in vitro and be a prominent neuroprotective target for the treatment of acute cerebral infarction. (C) 2011 Elsevier B.V. All rights reserved.

Balancing intake of diverse nutrients is important for organismal growth, reproduction, and survival. A shift in an organism's optimal diet due to changes in nutritional requirements after developmental or environmental changes is referred to as dietary switch and has been observed in several species [1]. Here we demonstrate that female Drosophila melanogaster also undergo a dietary switch following mating that leads to an increased preference for yeast, the major source of protein in their diet. We also demonstrate that S6 kinase (S6K) and serotonin production are involved in the postmating dietary switch. To further investigate the ability of D. melanogaster to balance nutrient intake, we examined the dietary preferences of adult flies following deprivation of yeast or sucrose. We observe that following conditioning on a diet deficient in either carbohydrates or yeast, D. melanogaster show a strong preference for the deficient nutrient. Furthermore, flies with activated dS6K or flies fed a serotonin precursor exhibit enhanced preference for yeast in this assay. Our results suggest that TOR signaling and serotonin may play an important role in maintaining nutrient balance in D. melanogaster. These studies may contribute to our understanding of metabolic disorders such as obesity and diabetes [2].

Abstract Background SIRT1 is a mammalian homologue of NAD+-dependent deacetylase sirtuin family. It regulates longevity in several model organisms and is involved with cell survival, differentiation, metabolism among other processes in mammalian cells. SIRT1 modulates functions of various key targets via deacetylation. Recent studies have revealed SIRT1 protects neurons from axonal degeneration or neurodegeneration. Further, SIRT1 null mice exhibit growth retardation and developmental defects, suggesting its critical roles in neurons and development. Results To identify novel binding partners for SIRT1 in the central nervous system, we performed yeast two-hybrid screening on human fetal brain cDNA library and found that zyxin is a possible binding partner. SIRT1 and zyxin transcript were both preferentially expressed in developmental mouse brain. Zyxin accumulates in the nucleus where it is co-localized with SIRT1 after treatment with leptomycin B in COS-7 cells. Furthermore, SIRT1 deacetylates zyxin, suggesting SIRT1 could interact with nuclear-accumulated zyxin and modulate its function through deacetylation. Conclusion Zyxin could be a novel interacting partner of SIRT1. Zyxin is an adaptor protein at focal adhesion plaque, regulating cytoskeletal dynamics and signal transduction to convey signal from the ECM (extracellular matrix) to the nucleus. Our results raise the possibility that SIRT1 regulates signal transmission from ECM to the nucleus by modulating the functions of zyxin via deacetylation.

The critical role of protein synthesis in regulating lifespan has been evidenced. This study shows that adult-onset RNAi inactivation of eukaryotic initiation factor 2B delta (eIF2B delta/F11A3.2), a subunit of eIF2B, extends the mean lifespan of Caenorhabditis elegans. eIF2B is a GDP-GTP exchange factor for eIF2-a rate-limiting factor for protein synthesis initiation. (35)S-methionine incorporation assay showed that global protein synthesis is reduced by eIF2B delta/F11A3.2 RNAi. Inhibition of eIF2B delta/F11A3.2 during adulthood conferred thermal and oxidative stress resistance and reduced the fecundity and fat storage, suggesting the possible trade-offs of resources between reproduction and somatic maintenance. Lifespan extension by adult-onset eIF2B delta/F11A3.2 RNAi is suppressed in FOXO transcription factor daf-16 deletion mutants. Adult-onset eIF2B delta/F11A3.2 RNAi increases the expression of stress-resistant genes, including hsp-16.2, hsp-70, hsp90, and sod-3, some of which are reported to be targets of DAF-16. Adult-onset eIF2B delta/F11A3.2 RNAi in daf-16 mutants reduced fecundity, but did not extend lifespan. Furthermore, adult-onset eIF2B delta/F11A3.2 RNAi did not extend the lifespan of germline-defective glp-4 organisms. Thus, it is possible that eIF2B delta/F11A3.2 RNAi during adulthood prolongs lifespan via daf-16, which induces stress resistance in organisms. This might be the mechanism, at least in part, for trade-offs of resources between reproduction and somatic maintenance.-Tohyama, D., Yamaguchi, A., Yamashita, T. Inhibition of a eukaryotic initiation factor (eIF2B delta/F11A3.2) during adulthood extends lifespan in Caenorhabditis elegans. FASEB J. 22, 4327-4337 ( 2008)

Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system [T. Yamashita, BK Mueller, K. Hata, Neogenin and RGM signaling in the central nervous system, Curt. Opin. Neurobiol. 17 (2007) 29-34]. Functional studies in Xenopus and chick embryos have revealed the roles of RGM in axon guidance and laminar patterning, while those in mouse embryos have demonstrated its function in regulating the cephalic neural tube closure. Importantly, RGM inhibition enhanced the growth of injured axons and promoted functional recovery after spinal cord injury in rats. Here, we identified two RGMa-derived peptides that functioned as antagonists against RGMa. The peptides studied in vitro dose-dependently suppressed the neurite growth inhibition and growth cone collapse induced by RGMa. Thus, these peptides are promising reagents to treat injuries of the central nervous system. (C) 2008 Elsevier Inc. All rights reserved.

Abstract Although myelin‐associated neurite outgrowth inhibitors express their effects through RhoA/Rho‐kinase, the downstream targets of Rho‐kinase remain unknown. We examined the involvement of myosin II, which is one of the downstream targets of Rho‐kinase, by using blebbistatin – a specific myosin II inhibitor – and small interfering RNA targeting two myosin II isoforms, namely, MIIA and MIIB. We found that neurite outgrowth inhibition by repulsive guidance molecule (RGMa) was mediated via myosin II, particularly MIIA, in cerebellar granule neurons. RGMa induced myosin light chain (MLC) phosphorylation by a Rho‐kinase‐dependent mechanism. After spinal cord injury in rats, phosphorylated MLC in axons around the lesion site was up‐regulated, and this effect depends on Rho‐kinase activity. Further, RGMa‐induced F‐actin reduction in growth cones and growth cone collapse were mediated by MIIA. We conclude that Rho‐kinase‐dependent activation of MIIA via MLC phosphorylation induces F‐actin reduction and growth cone collapse and the subsequent neurite retraction/outgrowth inhibition triggered by RGMa.

Disrupted-in-schizophrenia 1 (DISC1) is a gene disrupted by a ( 1; 11) (q42.1; q14.3) translocation that segregates with major psychiatric disorders in a Scottish family. To investigate how DISC1 confers susceptibility to psychiatric disorders, we previously identified fasciculation and elongation protein zeta-1 and Kendrin as DISC1-interacting molecules in a yeast twohybrid screen of a human brain complementary DNA library. Here, we have further identified a novel DISC1-interacting protein, termed DISC1-Binding Zinc-finger protein (DBZ), which has a predicted C2H2-type zinc-finger motif and coiled-coil domains. DBZ was co-immunoprecipitated with DISC1 in lysates of PC12 cells and rat brain tissue. The domain of DISC1 interacting with DBZ was close to the translocation breakpoint in the DISC1 gene. DBZ messenger RNA (mRNA) was expressed in human brains, but not in peripheral tissues. In situ hybridization revealed high expression of DBZ mRNA in the hippocampus, olfactory tubercle, cerebral cortex and striatum in rats. Because this pattern of localization was similar to that of the pituitary adenylate cyclase (PAC1) receptor for pituitary adenylate cyclase-activating polypeptide (PACAP), which has recently been implicated in neuropsychological functions, we examined whether DISC1/DBZ interaction was involved in the PACAP signaling pathway. PACAP upregulated DISC1 expression and markedly reduced the association between DISC1 and DBZ in PC12 cells. A DISC1-binding domain of DBZ reduced the neurite length in PC12 cells after PACAP stimulation and in primary cultured hippocampal neurons. The present results provide some new molecular insights into the mechanisms of neuronal development and neuropsychiatric disorders.

Generally, the axons in the adult mammalian central nervous system (CNS) do not regenerate after injuries. This lack of regeneration results in partial disability or complete paralysis. To date, several axon growth inhibitors that are known to inhibit axon regeneration in the CNS have been identified in the myelin. Recent research has revealed the molecular mechanisms underlying the action of these inhibitors. RhoA and Rho-kinase-a downstream effector of RhoA-are key signals that elicit axon inhibition. These findings provide a number of promising strategies to promote axon regeneration after CNS injuries. Some strategies such as Rho/Rho-kinase inhibition have proved effective in animal models. Elucidation of the complete mechanism of the signal transduction will be useful for the development of efficient therapeutic strategies against injuries of the CNS. (C) 2006 Prous Science. All rights reserved.

Spontaneous regeneration of lesioned fibers is limited in the adult central nervous system due to an unfavorable environment. Several myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. These proteins induce activation of Rho in some neurons. Inhibition of Rho or Rho-kinase, downstream effector of Rho, promotes axon regeneration in vivo. Recent reports suggest that several other proteins are axon growth inhibitors. Therapeutic strategy to block the multiple axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. In addition, it is noted that synaptic plasticity in pre-existing pathways and the formation of new circuits through collateral sprouting of lesioned and unlesioned fibers are important components of the recovery process.

Application of differential display to cultured rat astrocytes allowed cloning of Herp cDNA. Although Herp was strongly induced by endoplasmic reticulum (ER) stress, it decayed rapidly consequent to proteasome-mediated degradation. To investigate the role of this molecule in terms of the stress response, Herp knockout cells were developed using F9 embryonic carcinoma cells. F9 Herp null cells were more vulnerable to ER stress compared with F9 wild-type cells. In the early period of ER stress (0-8 h after tunicamycin treatment), Herp null cells displayed enhanced ER stress signalling and stabilization of an endogenous ERAD substrate, compared with wild-type cells. In the intermediate period (8-20 h after tunicamycin treatment), Herp null cells displayed reduced ER stress signalling, whereas in the late period (20-40 h after tunicamycin treatment), Herp null cells manifested irreversible cellular changes that lead to apoptotic cell death. Transfection analysis revealed that the N-terminal region, including the ubiquitin-like domain of Herp, was required for the survival of F9 cells under ER stress. These results indicate that Herp is a short-lived Ub-like protein improving the balance of folding capacity and protein loads in the ER and plays crucial roles for the ER stress resistance in F9 cells.

博士学位:博士(医学)/論第18946号/平成16年6月23日授与Abstract:The Rho family of small GTPases, key regulators of the actin cytoskeleton in eukaryotic cells from yeast to human, is implicated in the control of neuronal morphology. Guanine nucleotide exchange factors (GEFs) are upstream positive regulators of Rho GTPases and integrate extracellular signaling for appropriate activation of Rho GTPases at specific subcellular regions. Here we describe the identification of a novel Dbl family GEF for Rho GTPases in Homo sapiens and Mus musculus. It contains a tandem Dbl homology-pleckstrin homology domain and FERM domain, characteristic of the plasma membrane proteins linker. This gene, termed FERM domain including RhoGEF (FIR), was abundantly expressed in brain, lung, and testis, as well as embryonic hippocampal and cortical neurons. FIR was found to activate the biochemical pathway specific for Rac1 but not for RhoA or Cdc42. Ectopic expression of FIR in the cortical neurons resulted in significantly shortened neurites and excessive growth cones, presumably mediated by Rac1. These results suggest that FIR may regulate neurite remodeling by mediating the signaling pathways from membrane proteins to Rac.

The rat homologue of a mitochondrial ATP-dependent protease Lon was cloned from cultured astrocytes exposed to hypoxia. Expression of Lon was enhanced in vitro by hypoxia or ER stress, and in vivo by brain ischemia. These observations suggested that changes in nuclear gene expression (Lon) triggered by ER stress had the potential to impact important mitochondrial processes such as assembly and/or degradation of cytochrome c oxidase (COX). In fact, steady-state levels of nuclear-encoded COX IV and V were reduced, and mitochondrial-encoded subunit II was rapidly degraded under ER stress. Treatment of cells with cycloheximide caused a similar imbalance in the accumulation of COX subunits, and enhanced mRNA for Lon and Yme1, the latter another mitochondrial ATP-dependent protease. Furthermore, induction of Lon or GRP75/mtHSP70 by ER stress was inhibited in PERK (−/−) cells. Transfection studies revealed that overexpression of wild-type or proteolytically inactive Lon promoted assembly of COX II into a COX I–containing complex, and partially prevented mitochondrial dysfunction caused by brefeldin A or hypoxia. These observations demonstrated that suppression of protein synthesis due to ER stress has a complex effect on the synthesis of mitochondrial-associated proteins, both COX subunits and ATP-dependent proteases and/or chaperones contributing to assembly of the COX complex.

Mild hypothermia is a well-known method of reducing brain damage caused by traumatic, hypoxic, and ischemic injury. To elucidate the neuroprotective mechanism induced by hypothermic treatment, we compared gene expression profiles in the hippocampus of gerbils rendered ischemic for 15 min and then reperfused for 3 h under conditions of normothermia (37 +/-0.5 degreesC) or hypothermic treatment (34 +/-0.5 degreesC). Using the differential display method, we observed significantly reduced expression of the 78 kDa glucose regulated protein (GRP78), in ischemic gerbil hippocampus that underwent normothermic reperfusion, but normal GRP78 expression in animals that underwent hypothermic reperfusion. In situ hybridization and Northern blot analysis showed GRP78 mRNA expression was reduced in the CAI region of the hippocampus under normothermic conditions, but was not reduced under hypothermic conditions. Western blot analysis also showed the levels of immunoreactive GRP78 protein decreased in neurons of the hippocampal CA-1 region under normothermia, but not under hypothermic treatments. Furthermore, adenovirus-mediated overexpression of GRP78 protects rat hippocampal neurons from cell death and inhibits the rise in intracellular calcium concentration normally induced by hydrogen peroxide. These results suggest that reduction in GRP78 expression contributes to cell damage in the ischemic brain and that hypothermia-mediated restoration of GRP78 expression is one mechanism that enhances neuronal survival. (C) 2001 Published by Elsevier Science B.V.

Upon binding to the cAMP-response element of a gene's promoter, the transcription factor known as cAMP-response element-binding protein (CREB) facilitates transcription of many different neuronal genes including those involved with synaptic function. Based on our previous reports of gene structure (GenBank™ accession number AF029701), we now demonstrate that activated CREB binds to the proximal promoter of the human presenilin-1 (PS-1) gene to activate PS-1 transcription in rat and in human neuronal cells. Specific stimulation of the N-methyl-D-aspartate subtype of neuronal glutamate receptors activates CREB and results in increased PS-1 expression. Similarly, treatment with brain-derived neurotrophic factor activates CREB and increases PS-1 expression in a dose-dependent fashion. By using adenovirus vectors expressing dominant negative forms of CREB, we were able to show that induction of PS-1 expression requires the activation of CREB. Conversely, constitutive expression of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) results in activation of CREB and increased PS-1 expression that can be blocked by the addition of selective MEK inhibitors. Our findings suggest a hypothesis where stimulation of N-methyl-D-aspartate receptors signals CREB activation to enhance PS-1 gene product expression that contributes to normal neuronal functions.

Oxygen-regulated protein 150 kD (ORP150) is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. Although ORP150 was sparingly upregulated in neurons from human brain undergoing ischemic stress, there was robust induction in astrocytes. Cultured neurons overexpressing ORP150 were resistant to hypoxemic stress, whereas astrocytes with inhibited ORP150 expression were more vulnerable. Mice with targeted neuronal overexpression of ORP150 had smaller strokes compared with controls. Neurons with increased ORP150 demonstrated suppressed caspase-3-like activity and enhanced brain-derived neurotrophic factor (BDNF) under hypoxia signaling. These data indicate that ORP150 is an integral participant in ischemic cytoprotective pathways.

Inheritance of the ε-4 allele of the apolipoprotein E gene (APOE4) is a major risk factor for the development of Alzheimer's disease (AD). Although the association between APOE4 and AD is well documented, the mechanism by which apolipoprotein E exerts an isoform-specific effect on neurons in disease is unknown. In this report, we demonstrate that apoE4 stimulates the transcriptional activity of cAMP-response element-binding protein (CREB) by activating the extracellular signal-regulated kinase (ERK) cascade in rat primary hippocampal neurons. In contrast, apoE3 was unable to stimulate CREB transcriptional activity and unable to activate the ERK pathway. Elevation of intracellular Ca2+ levels are also involved because treatment with receptor-associated protein, nifedipine, MK801, removal of Ca2+ from the medium and dantrolene all served to inhibit calcium elevation and attenuate the activation of CREB. Treatment with an apoE peptide was also found to facilitate transcription of the CREB-dependent genes, c-fos and Bcl-2. In contrast to treatment with apoE3, our findings suggest apoE4 and apoE-peptide induce a novel signaling pathway.

Survival factors suppress apoptosis by activating the serine/threonine kinase Akt. To investigate the molecular mechanism underlying activated Akt's ability to protect neurons from hypoxia or nitric oxide (NO) toxicity, we focused on the apoptosis-related functions of p53 and caspases, We eliminated p53 by employing p53-deficient neurons and increased p53 by infection with recombinant adenovirus capable of transducing p53 expression, and we now show that p53 is implicated in the apoptosis induced by hypoxia or NO treatments of primary cultured hippocampal neurons. Although hypoxia and NO induced p53, treatment with insulin-like growth factor-1 significantly inhibited caspase-3-like activation, neuronal death and transcriptional activity of p53. These insulin-like growth factor-1 effects are prevented by wortmannin, a phosphatidylinositol 3-kinase inhibitor, Adenovirus-mediated expression of activated-Akt kinase suppressed p53-dependent transcriptional activation of responsive genes such as Bax, suppressed caspase-3-like protease activity and suppressed neuronal cell death with no effect on the cellular accumulation and nuclear translocation of p53. In contrast, overexpression of kinase-defective Akt failed to suppress these same activities. These results suggest a mechanism where Akt kinase activation reduces p53's transcriptional activity that ultimately rescues neurons from hypoxia- or NO-mediated cell death.

As a model of the reperfusion injury found in stroke, we have exposed neurons to hypoxia followed by reoxygenation. Neurons treated with hypoxia/reoxygenation (H/R) respond by activating nuclear factor-κB (NFκB), releasing cytochrome c from their mitochondria, and ultimately dying. Further supporting an apoptotic mechanism, expression of the antiapoptotic Bcl-2 and Bcl-x proteins was increased following H/R. In this model, adenoviral- mediated transduction of IκB expression inhibited NFκB activation and significantly accelerated cytochrome c release and caspase-dependent neuronal death. At the same time, expression of mutated IκB prevented the increased expression of endogenous Bcl-2 and Bcl-x. In the presence of mutated IκB, singular overexpression of only Bcl-2 by adenoviral-mediated transduction significantly inhibited cytochrome c release, caspase-3-like activation, and cell death in response to H/R. These findings suggest a pathway where NFκB activation induces overexpression of Bcl-2 and Bcl-x, which function to prevent apoptotic cell death following H/R treatments.

Application of differential display to cultured rat astrocytes subjected to hypoxia allowed cloning of a novel cDNA, termed stress-associated endoplasmic reticulum protein 1 (SERP1). Expression of SERP1 was enhanced in vitro by hypoxia and/or reoxygenation or other forms of stress, causing accumulation of unfolded proteins in endoplasmic reticulum (ER) stress, and in vivo by middle cerebral artery occlusion in rats. The SERP1 cDNA encodes a 66–amino acid polypeptide which was found to be identical to ribosome-associated membrane protein 4 (RAMP4) and bearing 29% identity to yeast suppressor of SecY 6 protein (YSY6p), suggesting participation in pathways controlling membrane protein biogenesis at ER. In cultured 293 cells subjected to ER stress, overexpression of SERP1/RAMP4 suppressed aggregation and/or degradation of newly synthesized integral membrane proteins, and subsequently, facilitated their glycosylation when the stress was removed. SERP1/RAMP4 interacted with Sec61α and Sec61β, which are subunits of translocon, and a molecular chaperon calnexin. Furthermore, Sec61α and Sec61β, but not SERP1/RAMP4, were found to associate with newly synthesized integral membrane proteins under stress. These results suggest that stabilization of membrane proteins in response to stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, other components of translocon, and molecular chaperons in ER.

Based on the neurotrophic properties of astrocytes in response to ischemia, the current work focuses on the mechanism for cultured astrocytes to adapt to a hypoxic environment. Intracellular glucose levels in primary cultured rat astrocytes exposed to hypoxia fell by 30% within 24 h, in parallel with a decrease in glycogen stores. Glycolytic metabolism was crucial for cell survival during hypoxia, as 2-deoxyglucose resulted in rapid ATP depletion and cell death. The mechanism for maintaining glucose levels under these conditions appeared to be mobilization of glycogen stores, rather than increased extracellular uptake of glucose, as gluconolactone (an inhibitor of beta 1-4 amyloglucosidase) induced a rapid fall in cellular ATP in cultures subjected to hypoxia, whereas cytochalasin B was without affect. Addition of cycloheximide diminished the viability of astrocytes in hypoxia, suggesting an obligatory role of de-novo gene expression to respond to hypoxia. Consistently, the results of differential display suggested the induction of glycolytic enzymes, including aldolase 4 (EC 4.1.2.13), hexokinase II (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), and triosephosphate isomerase (EC 5.3.1.1) in the hypoxic culture. Marked induction of these glycolytic enzymes in hypoxic astrocytes was confirmed by Northern blot analysis. These data provide a theoretical basis to understand the ability of astrocytes to tolerate ischemic condition. (C) 1999 Published by Elsevier Science B.V. All rights reserved.