Dementia model mice exhibited improvements of neuropsychiatric symptoms as well as cognitive dysfunction with neural cell transplantation.

Elderly patients with dementia suffer from cognitive dysfunctions and neuropsychiatric symptoms (NPS) such as anxiety and depression. Alzheimer's disease (AD) is a form of age-related dementia, and loss of cholinergic neurons is intimately associated with development of AD symptoms. We and others have reported that neural cell transplantation ameliorated cognitive dysfunction in AD model mice. It remains largely unclear whether neural cell transplantation ameliorates the NPS of AD. It would be interesting to determine whether NPS correlates with cognitive dysfunctions before and after neural cell transplantation in AD model mice. Based on the revalidation of our previous data from a Morris water maze test, we found that neural cell transplantation improved anxiety and depression significantly and marginally affected locomotion activity in AD mice. A correlation analysis revealed that the spatial learning function of AD mice was correlated with their NPS scores both before and after cell transplantation in a similar manner. In contrast, in the mice subjected to cell transplantation, spatial reference memory function was not correlated with NPS scores. These results suggested the neural cell transplantation in the AD model mice significantly improved NPS to the same degree as cognitive dysfunctions, possibly via distinct mechanisms, such as the cholinergic and GABAergic systems.

Female dominance of both spatial cognitive dysfunction and neuropsychiatric symptoms in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a prevalent neurological disorder affecting memory function in elderly persons. Indeed, AD exhibits abnormality in cognitive behaviors and higher susceptibility to neuropsychiatric symptoms (NPS). Various factors including aging, sex difference and NPS severity, are implicated during in development of AD. In this study, we evaluated behavioral abnormalities of AD model, PDAPP transgenic mice at young age using the Morris Water Maze test, which was established to assess hippocampal-dependent learning and memory. We found that female AD model mice exhibited spatial learning dysfunction and highly susceptible to NPS such as anxiety and depression, whereas spatial reference memory function was comparable in female PDAPP Tg mice to female wild type (WT) mice. Spatial learning function was comparable in male AD model mice to male WT mice. Multiple regression analysis showed that spatial learning dysfunction was associated with NPS severity such as anxiety and depression. Furthermore, the analysis showed that spatial reference memory function was associated with status of depression, but not anxiety. Thus, these results suggest female dominance of spatial learning dysfunction in the AD model mice accompanying increased NPS severity. The understandings of AD model may be useful for the development of therapeutic agents and methods in human AD.

Roles of Reelin/Disabled1 pathway on functional recovery of hemiplegic mice after neural cell transplantation; Reelin promotes migration toward motor cortex and maturation to motoneurons of neural grafts.

Reelin is a large glycoprotein which regulates central nervous system (CNS) development. Dysfunctions of Reelin were reported on certain neuropsychiatric diseases. We examined involvement of Reelin pathway in functional recovery of hemiplegic mice after neural transplantation. Reelin was expressed 1 day after cryogenic injury of right motor cortex. We transplanted neural stem/progenitor cells (NSPCs) from wild-type mice into ipsilateral striatum of hemiplegic mice. The grafts migrated from the striatum and reached the injured cortex 14 days after transplantation. The transplantation significantly improved their motor functions (P < .05). The NSPCs migrating toward the cortex expressed Reelin receptors, Apoer and Vldlr, and phosphorylated Disabled1 (Dab1), a downstream signaling molecule of Reelin. The grafts expressed Ncadherin and active form of Integrin ß1, both of which were known to become active with Reelin stimulation. At day 28, the grafts expressed Ctip2, Crim1, Foxp2, and Fezf2, all of which were forebrain motoneuron associated markers, and Nfm and Synapsin1 on the damaged cortex. We then transplanted NSPCs of yotari mice (yot/yot genotype) having nonfunctional Dab1 by a mutation of its gene. Majority of the grafts from yotari mice (>80%) did not migrate and thus remained at the striatum. The grafts did not express the forebrain motoneuron associated markers nor the cell adhesion molecules including Ncadherin and active Integrin ß1. Reelin pathway was involved in graft migration by regulating certain adhesion molecules and in their differentiation to functional motoneurons accompanying synapse formation. We suggested involvement of Reelin pathway for neural regeneration and functional recovery of hemiplegic mice in adulthood after neural transplantation.

Relative abundance of Megamonas hypermegale and Butyrivibrio species decreased in the intestine and its possible association with the T cell aberration by metabolite alteration in patients with Behcet's disease (210 characters).

OBJECTIVES: We have previously demonstrated that the phylum Actinobacteria, the family Lactobacillaceae, and the genus Bifidobacterium increased in relative abundance of gut microbiota in patients with Behcet's disease (BD). The phylum Firmicutes and the class Clostridia were predominant in the feces of normal individuals. The class Clostridia includes short-chain fatty acid-producing bacteria, important for the balance between regulatory T cells and helper T type 17 (Th17) cells. It is possible that the bacterial compositional alteration causes low intestinal short-chain fatty acid concentrations, leading to skewed immune functions in patients with BD. METHODS: To test the hypothesis, we examined species composition and gene functions from the 16S rRNA data by utilizing PICRUSt software. RESULTS: We have shown that relative abundance of Eggerthella lenta, Acidaminococcus species, Lactobacillus mucosae, Bifidobacterium bifidum, Lactobacillus iners, Streptococcus species, and Lactobacillus salivarius increased significantly in patients with BD. Relative abundance of Megamonas hypermegale, Butyrivibrio species, Streptococcus infantis, and Filifactor species increased significantly in normal individuals compared with BD patients. In the functional annotation analysis by PICRUSt, we found prevalent gene functions of the pentose phosphate pathway and the inosine monophosphate biosynthesis in patients with BD. The data suggested that BD gut microbes altered nucleic acid and fatty acid synthesis. CONCLUSIONS: These compositional and functional alterations of gut microbes may accompany unfavorable molecular exchanges between intestinal immunocompetent cells and gut microbes, and these interactions may have an association with the immune aberration in patients with BD.

Propionate-producing bacteria in the intestine may associate with skewed responses of IL10-producing regulatory T cells in patients with relapsing polychondritis.

Relapsing polychondritis (RP) is an inflammatory disease of unknown causes, characterized by recurrent inflammation in cartilaginous tissues of the whole body. Recently, researchers have reported that, in mouse experiments, altered gut microbe-dependent T cell differentiation occurred in gut associated lymphoid tissues. Here, we investigated whether gut microbe alteration existed, and if so, the alteration affected peripheral T cell differentiation in patients with RP. In an analysis of gut microbiota, we found increased annotated species numbers in RP patients compared with normal individuals. In the RP gut microbiota, we observed several predominant species, namely Veillonella parvula, Bacteroides eggerthii, Bacteroides fragilis, Ruminococcus bromii, and Eubacterium dolichum, all species of which were reported to associate with propionate production in human intestine. Propionate is a short-chain fatty acid and is suggested to associate with interleukin (IL)10-producing regulatory T (Treg) cell differentiation in gut associated lymphoid tissues. IL10 gene expressions were moderately higher in freshly isolated peripheral blood mononuclear cells (PBMC) of RP patients than those of normal individuals. Six hours after the initiation of the cell culture, regardless of the presence and absence of mitogen stimulation, IL10 gene expressions were significantly lower in RP patients than those in normal individuals. It is well known that PBMC of patients with autoimmune and inflammatory diseases show hyporesponsiveness to mitogen stimulation. We suggest that, in RP patients, continuous stimulation of intestinal T cells by excessive propionate leads to the spontaneous IL10 production and a subsequent refractory period of T cells in patients with RP. The hyporesponsiveness of Treg cells upon activation may associate with inflammatory cytokine production of PBMC and subsequently relate to chondritis in RP patients.

Neuronal Cell Sheets of Cortical Motor Neuron Phenotype Derived from Human iPSCs.

Transplantation of stem cells that differentiate into more mature neural cells brings about functional improvement in preclinical studies of stroke. Previous transplant approaches in the diseased brain utilized injection of the cells in a cell suspension. In addition, neural stem cells were preferentially used for grafting. However, these cells had no specific relationship to the damaged tissue of stroke and brain injury patients. The injection of cells in a suspension destroyed the cell-cell interactions that are suggested to be important for promoting functional integrity of cortical motor neurons. In order to obtain suitable cell types for grafting in patients with stroke and brain damage, a protocol was modified for differentiating human induced pluripotent stem cells from cells phenotypically related to cortical motor neurons. Moreover, cell sheet technology was applied to neural cell transplantation, as maintaining the cell-cell communications is regarded important for the repair of host brain architecture. Accordingly, neuronal cell sheets that were positive Forebrain Embryonic Zinc Finger (Fez) family zinc finger 2 (FEZF2), COUP-TF-interacting protein 2, insulin-like growth factor-binding protein 4 (IGFBP4), cysteine-rich motor neuron 1 protein precursor (CRIM1), and forkhead box p2 (FOXP2) were developed. These markers are associated with cortical motoneurons that are appropriate for the transplant location in the lesions. The sheets allowed preservation of cell-cell interactions shown by synapsin1 staining after transplantation to damaged mouse brains. The sheet transplantation brought about partial structural restoration and the improvement of motor functions in hemiplegic mice. Collectively, the novel neuronal cell sheets were transplanted into damaged motor cortices; the cell sheets maintained cell-cell interactions and improved the motor functions in the hemiplegic model mice. The motoneuron cell sheets are possibly applicable for stroke patients and patients with brain damage by using patient-specific induced pluripotent stem cells.

Bifidobacteria Abundance-Featured Gut Microbiota Compositional Change in Patients with Behcet's Disease.

Gut microbiota compositional alteration may have an association with immune dysfunction in patients with Behcet's disease (BD). We conducted a fecal metagenomic analysis of BD patients. We analyzed fecal microbiota obtained from 12 patients with BD and 12 normal individuals by sequencing of 16S ribosomal RNA gene. We compared the relative abundance of bacterial taxa. Direct comparison of the relative abundance of bacterial taxa demonstrated that the genera Bifidobacterium and Eggerthella increased significantly and the genera Megamonas and Prevotella decreased significantly in BD patients compared with normal individuals. A linear discriminant analysis of bacterial taxa showed that the phylum Actinobacteria, including Bifidobacterium, and the family Lactobacillaceae exhibited larger positive effect sizes than other bacteria in patients with BD. The phylum Firmicutes and the class Clostridia had large effect sizes in normal individuals. There was no significant difference in annotated species numbers (as numbers of operational taxonomic unit; OTU) and bacterial diversity of each sample (alpha diversity) between BD patients and normal individuals. We next assigned each sample to a position using three axes by principal coordinates analysis of the OTU table. The two groups had a significant distance as beta diversity in the 3-axis space. Fecal sIgA concentrations increased significantly in BD patients but did not correlate with any bacterial taxonomic abundance. These data suggest that the compositional changes of gut microbes may be one type of dysbiosis (unfavorable microbiota alteration) in patients with BD. The dysbiosis may have an association with the pathophysiology of BD.

Possible association of proinflammatory cytokines including IL1ß and TNFα with enhanced Th17 cell differentiation in patients with Behcet's disease.

We have reported that helper T type 17 (Th17) cells increased in patients with Behcet's disease (BD). It remains obscure how Th17 cells increase in the patients. We here analyzed whether T cells preferentially differentiate into Th17 cells in response to various inflammatory cytokines in patients with BD. Exogenous interleukin (IL)23 sustained the higher Th17 cell frequencies of CD4+CD45RO+ T cells after a 2-day culture in vitro in patients with BD, whereas the T cell subpopulation of normal individuals did not respond to IL23 to sustain/increase Th17 cell frequencies. IL23 receptor positive cell frequencies in freshly isolated BD CD4+CD45RO+ T cells correlated with Th17 cell frequencies assessed by intracellular cytokine staining. After a 2-day culture with IL23, BD CD4+ T cells retained the correlation between IL23 receptor expression level and extent of IL17 secretion (as indicated by Th17 cell frequencies), whereas such correlation was not noted in normal individuals. IL23 signals with its receptor were thus suggested to induce IL17 secretion (Th17 cell frequencies) in a short-time culture in patients with BD. We cultured CD4+CD45RO- T cells for 11 days with various inflammatory cytokines to study which cytokine associated with the enhanced Th17 frequencies in the patients. IL17 production by CD4+CD45RO- T cells of BD patients increased significantly by the supplementation of IL1ß and tumor necrosis factor (TNF)α, in addition to IL23, compared with that of normal individuals. These results suggest that proinflammatory cytokines, such as IL1ß, TNFα, and IL23, may associate with the expansion of Th17 cells in patients with BD. This study was registered with the University Hospital Medical Information Network-Clinical Trials Registry (UMIN000003806).

Cellular and molecular mechanisms of the restoration of human APP transgenic mouse cognitive dysfunction after transplant of human iPS cell-derived neural cells.

Cholinergic neuronal loss is a common finding in patients with Alzheimer's disease (AD) and AD model mice. We previously transplanted neurons derived from human induced pluripotent stem (iPS) cells into the hippocampus of human amyloid precursor protein transgenic AD model mice. In the present study, we examined the cellular and molecular mechanisms involved in the alleviation of cognitive dysfunction in transplanted mice. After transplant, mice showed improvement in cognitive function, confirming our previous findings. Human choline acetyltransferase (ChAT)-positive cholinergic neurons were distributed throughout the cortex of the grafted mice. Human and mouse ChAT-positive neurons and alpha7 nicotinic acetylcholine receptor (α7nAChR)-positive neurons were significantly increased in the cortex and hippocampus of the grafted mice compared with the vehicle-injected mice. In addition, human and mouse vesicular GABA transporter (VGAT)-positive neurons were located mainly in the hippocampus and, though the number was small, human VGAT-positive neurons were observed in the cortex. In the grafted mouse cortex, the number of GABA receptor (GABAR)-positive neurons of both human origin and mouse origin were significantly increased compared with those in the vehicle-injected mouse cortex. The α7nAChR-positive and GABAR-positive neurons expressed phosphorylated Akt and c-fos in the cortex, suggesting that these receptor-expressing neurons were possibly activated by the neurotransmitters secreted from the grafted neurons. Collectively, the grafted and host neurons may form positive feedback loops via neurotransmitter secretion in both the cerebral cortex and hippocampus, leading to alleviation of cognitive dysfunction in dementia model mice.

Restoration of spatial memory dysfunction of human APP transgenic mice by transplantation of neuronal precursors derived from human iPS cells.

PDGF promoter driven amyloid precursor protein (PDAPP) transgenic mice were accompanied by age dependent amyloid ß deposition and progressive spatial memory dysfunction which emerges within a few months of age. We conducted transplantation of neuronal precursors of cholinergic neuron phenotype which were derived from human iPS (hiPS) cells into bilateral hippocampus of PDAPP mice. We first generated neuronal precursors with cholinergic neuron phenotype from hiPS cells by culturing them with retinoic acid (RA), sonic hedgehog (SHH) and noggin-Fc (NOG). Spatial memory function of PDAPP mice was significantly impaired compared to that of nontransgenic littermates at age 8 weeks. After neuronal precursor transplantation, subsequent memory dysfunction of PDAPP mice was significantly improved, compared to that of vehicle injected PDAPP mice. We observed choline acetyltransferase (ChAT) positive cholinergic human neurons and vesicle GABA transporter (VGAT) positive GABAergic human neurons in PDAPP mouse hippocampus 45 days after the transplantation. Neuronal precursors with cholinergic neuron phenotype derived from hiPS cells survived in PDAPP mouse hippocampus and their spatial memory loss was improved. hiPS cells may become applicable for the treatment of patients with dementia.

Role of SDF1/CXCR4 interaction in experimental hemiplegic models with neural cell transplantation.

Much attention has been focused on neural cell transplantation because of its promising clinical applications. We have reported that embryonic stem (ES) cell derived neural stem/progenitor cell transplantation significantly improved motor functions in a hemiplegic mouse model. It is important to understand the molecular mechanisms governing neural regeneration of the damaged motor cortex after the transplantation. Recent investigations disclosed that chemokines participated in the regulation of migration and maturation of neural cell grafts. In this review, we summarize the involvement of inflammatory chemokines including stromal cell derived factor 1 (SDF1) in neural regeneration after ES cell derived neural stem/progenitor cell transplantation in mouse stroke models.

Establishment of retinal progenitor cell clones by transfection with Pax6 gene of mouse induced pluripotent stem (iPS) cells.

We previously reported that transfection of Pax6 gene which regulated early events in eye development into mouse ES cells brought about their differentiation into retinal progenitors. Here, we attempted to establish cloned retinal progenitors which had ability to further differentiate into photoreceptor like cells by transfecting mouse induced pluripotent stem (iPS) cells with Pax6 gene. Undifferentiated iPS cells were transfected with Pax6 cDNA, followed by selection with G418. After limiting dilution culture, we selected cloned Pax6-transfected cells, which simultaneously expressed mRNAs of Nestin, Musashi1, Six3 and Chx10 for further characterization. We obtained totally 8 clonally expanding Pax6-transfected cells. They started to express mRNAs of Brn3b, Cone-rod homeobox (Crx), pkc, CD73, rhodopsin and the γ-subunit of rod cGMP phosphodiesterase (PDEγ). Flow cytometric analysis revealed that almost half of the cells were CD73+, a marker of photoreceptor precursors. Western blotting confirmed cytoplasmic protein expression of rhodopsin. High KCl stimulation increased free Ca influx into the cells on Ca(2+) imaging. iPS cells transfected with Pax6 gene, followed by subsequent limiting dilution culture became retinal progenitors including photoreceptor like cells. The cloned cell lines may be useful for analyzing differentiation requirement of retinal progenitors.

A novel avian homologue of CD72, chB1r, down modulates BCR-mediated activation signals.

The avian B cell differentiation antigen chB1 is a C-type lectin membrane protein most homologous to mammalian CD72. Here, we report a new chB1-related gene, chB1r, that is located 18 kb away the chB1 gene. The cytoplasmic domain of chB1r protein contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs: ITIM1 and 2), which are identical to those found in CD72, whereas chB1 lacks the second ITIM2. Although chB1 expression is restricted to the bursa and an immature B cell line, chB1r is highly expressed in the bursa, spleen and both immature and mature B cell lines, a pattern that parallels CD72 expression. SHP-1 and Grb2 interact with phosphorylated tyrosine residues within chB1r ITIM1 and ITIM2, respectively. By contrast, ITIM1 of chB1 does not interact with SHP-1. Functional characterization using chB1r/chB1 double-deficient DT40 B cells demonstrated that ITIM1 in chB1r transduces a negative signal for BCR-mediated nuclear factor of activated T cells (NF-AT) activation and that ITIM2 attenuates this negative signal. This study has established chB1r as the genuine avian homologue of mammalian CD72, and revealed an opposing role for the two ITIMs through binding with SHP-1 and Grb2 for regulation of BCR-mediated NF-AT activation.

CD72 stimulation modulates anti-IgM induced apoptotic signaling through the pathway of NF-kappaB, c-Myc and p27(Kip1).

Engagement of mIgM induces G1 arrest and apoptosis in immature B cells. The biochemical mechanism(s) regulating the cell death process are poorly understood. Cross-linking of CD72 (a B cell co-receptor) with anti-CD72 antibody was shown to protect B cells from apoptosis. We investigated the molecular mechanism involved in apoptosis preventing signaling mediated by CD72 ligation using a derivative (WEHIdelta) of the WEHI231 cell line which is representative of immature B cells. Apoptotic WEHIdelta cells following cross-linking of mIgM demonstrate a dramatic loss of c-Myc protein after transient up-regulation. In contrast, pre-ligation of CD72 was able to sustain c-Myc expression after transient up-regulation. Cross-linking of mIgM of WEHIdelta cells causes accumulation of the Cdk inhibitor, p27(Kip1). CD72 pre-ligation was shown to inhibit the accumulation of p27(Kip1) protein. Moreover, NF-kappaB activity was not suppressed in WEHIdelta cells after mIgM cross-linking when the cells were pre-treated with anti-CD72 antibody. These results strongly suggest that the apoptosis preventing signal evoked by CD72 ligation is delivered through the pathway of NF-kappaB, c-Myc, p27(Kip1) and cyclin.

Targeting of MIST to Src-family kinases via SKAP55-SLAP-130 adaptor complex in mast cells.

MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST-SLAP-130-SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells.