The Keio Journal of Medicine


Decreased Neurogenesis as a Risk Factor for Mental Diseases
Noriko Osumi and Team CREST

Among several endophenotypes (biological markers) of schizophrenia, deficits in prepulse inhibition (PPI) and the smaller hippocampus are reproducible ones that can be measured in patients and various animal models. From QTL analyses using two lines of mice that show different PPI performance, our group has identified FABP7 (B-FABP/BLBP), a gene encoding a fatty acid binding protein, as a responsible gene for schizophrenia. Fabp7 knockout mice also exhibit impaired neurogenesis and smaller hippocampi. Similar association is reported in knockout mice for NPAS3 and neuregulin1, and also found in Small eye rats (Pax6 -/+ rats) that have a mutation in Pax6 gene, an upstream to Fabp7 and a pivotal player in neurogenesis. We thus suspect that decrease in neurogenesis may serve as a vulnerable condition not only for depression but also schizophrenia. We have tried to establish well-grounded ways to increase neurogenesis in hope for preventing and treating schizophrenia and related mental diseases. We have focused on polyunsaturated fatty acids (PUFAs) that can be bound to FABPs. At the moment, we have succeeded to increase production of new neurons via administrating n-6 fatty acid, arachidonic acid (ARA), in the wild type rat pups for 4 weeks. We then raised Pax6 -/+ rats with ARA-diet, and were able to prevent PPI deficits.

Epigenetic Regulation of Neural Cell Differentiation Plasticity in the Adult Mammalian Brain
Kinichi Nakashima

Major CNS cell types, i.e., neurons, astrocytes and oligodendrocytes are generated from common neural progenitor cells (NPCs). It is becoming apparent that extracellular cues and intracellular epigenetic modification play critical roles in the regulation of NPC-lineage specification. However, the mechanism of how cell-type specificity in differentiated cells is regulated remains a open question. In this study, we show that a methyl-CpG binding protein, MeCP2 is a cell-intrinsic determinant regulating cellular competences to respond to astrogliogenic signals. Neurons expressing highly amount of MeCP2 did not express an astrocytic marker, GFAP, even when stimulated with astrocyte-inducing cytokine, LIF (Leukemia Inhibitory Factor). In contrast, oligodendrocytes, which do not express MeCP2, could differentiate into GFAP-positive astrocytes in response to LIF both in vivo and in vitro. Furthermore, ectopic expression of MeCP2 in oligodendrocyte inhibited LIF-induced astrocytic gene expression. A central challenge in the field of neuroscience is to understand the mechanisms whereby diverse functions of various cell-types are generated in the adult CNS. Our findings suggest a molecular basis for a part of cellular identity or plasticity and could be a first step toward unraveling the above-mentioned mechanisms.

Regeneration of the Damaged CNS Using Pluripotent Stem Cells
Hideyuki Okano, Yohei Okada, Gentaro Kumagai and Masaya Nakamura

Although transplanted neural stem/progenitor cells (NS/PCs) differentiated into neuron, astrocyte, and oligodendrocyte and promoted functional recovery after spinal cord injury (SCI), it remains to be resolved about the differentiation potential and survival rate play a role in functional recovery after transplantation. To address this, we took advantage of our original in vitro differentiation of embryonic stem (ES) cells. We newly developed a novel in vitro model which allows recapitulation of CNS development by taking advantage of ES cell-derived neurospheres. This method enables efficient derivation of highly neurogenic FGF-responsive NS/PCs with early temporal identities and high plasticity, which are subsequently specified to be EGF-responsive and more gliogenic during repeated passages. Here we show that Coup-tf I and II are required for the temporal specification of NS/PCs, including their acquisition of gliogenic competence. We cultured mouse ES cells-derived neurogenic primary neurosphere (ES-PNS) and gliogenic secondary neurosphere (ES-SNS) in vitro and transplanted them into mouse SCI models. PNS- and SNS-derived cells 10-20 % survived similarly without tumor formation and differentiated with the properties of in vitro. Surprisingly, transplantation of gliogenic ES-SNS, but not neurogenic ES-PNS, promoted axonal growth, remyelination, angiogenesis, and functional recovery after SCI. Thus, injured spinal cord required gliogenic ES-derived neurospheres for functional recovery and gave an important cue for the establishment of the regenerative medicine for SCI using ES or induced pluripotent stem cells (iPS cells) in the near future.

Function and Regulation of Adult Mammalian Neurogenesis
Fred H. Gage

While most neurons in the adult central nervous system (CNS) are terminally differentiated, evidence now exists that small populations of neurons are generated in the adult olfactory bulb and hippocampus. In the adult hippocampus, newly born neurons originate from putative stem cells that exist in the subgranular zone of the dentate gyrus. Progeny of these putative stem cells differentiate into neurons in the granular layer within a month of the cells' birth, and this late neurogenesis continues throughout the adult life of all mammals. Stem cells can be harvested from a variety of brain and spinal cord regions, genetically modified, and transplanted back to the brain and spinal cord where they can differentiate into mature glia and neurons depending on the local environment. In addition, environmental stimulation can differentially affect the proliferation, migration, and differentiation of these cells in vivo. These environmentally induced changes in the structural organization of the hippocampus, result in changes in electrophysiological responses in the hippocampus, as well as in hippocampal related behaviors. We are studying the cellular, molecular, as well as environmental influences that regulate neurogenesis in the adult brain and spinal cord. We have recently identified several novel mechanisms that regulate proliferation, survival and differentiation of these adult derived stem cells. The functional and practical significance of these findings will be discussed in light of their implications for alternative or expanded views of structural plasticity in the adult brain.

Cytokine-mediated Modulation of Inducible Regulatory
T Cells
Akihiko Yoshimura

Anti-inflammatory cytokines, TGF-β and IL-10 have been implicated in the suppression of autoimmune and inflammatory dieses. TGF-β is believed to be an effector molecule of natural occurring Foxp3-positive regulatory T cells (nTreg). In addition, Foxp3 is shown to be induced in CD4+Foxp3- T cells in vitro after stimulation with TGF-β, which is called inducible Treg (iTreg). Induction of iTregs could be therapeutic for autoimmune diseases and allergy. However, molecular mechanism of iTreg induction has been poorly understood. We analyzed Foxp3 promoter and identified an enhancer in the Foxp3 gene in which NF-AT and Smad2/3 bind and cooperatively induce Foxp3 expression. Furthermore, we found that STAT6, which is activated by IL-4, inhibited Smad-dependent Foxp3 promoter activation, while retinoic acid enhanced it by the binding of RARα/RXR complex to a specific Foxp3 promoter enhancer region. These data indicate that Foxp3 expression is tightly regulated by various extracellular factors. To clarify the significance of Foxp3 induction by TGF-β in vivo, we generated T cell specific Smad2-deficeint (cKO) mice. As expected, Foxp3 induction was severely impaired by TGF-β in vitro in T cells from Smad2-cKO mice. Although these mice rarely develop autoimmune diseases, they were sensitive to Th1 type diseases such as DSS-induced colitis and resistant to Th17 type diseases such as experimental autoimmune encephalomyelitis (EAE). These were mostly due to enhanced IFN-γ production in cKO mice. Furthermore, Smad2-deficient naive T cells adoptively transferred into Rag2-/- mice produced higher levels of IFNγ than wilt type T cells, resulting in the induction of severer colitis. Therefore, suppression of IFNγ production is one of the important immunoregualtory mechanisms of TGF-β. It has been shown that Foxp3 overexpression in T cells results in the suppression of various cytokines including IFNγ. Thus, induction of Foxp3 could be involved in the suppression of IFNγ production by TGF-β. However, we found that TGF-β still suppressed IFNγ production in T cells from scurfy (Foxp3-deficeint) mice. Taken together, we propose that there is Foxp3-dependent and independent mechanisms for the immunoregulatory effect of TGF-β.

The Role of Regulatory T Cells in Tumor Immunity and Immunotherapy
Yutaka Kawakami, Chie Kudo-Saito, Tomonori Yaguchi, Naoshi Kawamura and Hidetoshi Sumimoto

Systemic and local immunosuppression is a problem occurs in cancer patients. Cancer cells produce not only various immunosuppressive molecules, but also induce immunosuppressive cells, including regulatory T cells (Treg), myeloid derived suppressor cells (MDSC), and tolerogenic dendritic cells (tDC). To restore immunocompetence of cancer patients, it is important to understand the mechanisms for the cancer induced immunosuppression. Treg is thought to be one of the major immunosuppressive cells increased in tumor bearing animals and cancer patients. In an effective immunotherapy we have developed, intratumoral injection of DC following tumor cryoablation, depletion of FoxP3+ CD4+ T cells further enhanced it's anti-tumor effects, indicating that inhibition of Treg is important for effective immunothetrapy. In a combined immunization trial with CD4+ T cell epitope peptide of gp100 melanoma antigen along with CD8+ CTL epitope surprisingly decreased induction of gp100 specific CD8+ CTL possibly through induction of gp100 specific Treg. Administration of Ab specific for CTLA-4 which is highly expressed on Treg to melanoma patients resulted in tumor regression in some patients who showed autoimmune reactions possibly due to blockade of Treg suppressive effect. Adoptive T cell immunotherapy following lymphodepletive treatment using cyclophosphamide, fludarabine, or total body irradiation, which also decrease Treg, resulted in dramatic tumor reduction in more than half of patients with advanced melanoma. In human melanoma cells, we have identified signaling molecules such as BRAF, STAT3, β-catenin, and snail, responsible for production of Treg inducing molecules, including TGF-β and IL10. Snail is the major EMT (epithelial mesenchymal transition) inducing transcription factor. Snail transfected human cancer cells showing EMT features such as increased migration and metastatic ability, induced FoxP3+ CD4+ immunosuppressive Treg from PBMC. Intratumoral injection of siRNA into subcutaneously implanted snail transfected murine tumors resulted in tumor reduction accompanied by decrease of FoxP3+ CD4+ T cells and increase of CD8+T cells,, indicating that EMT enhances tumor metastasis through immunosuppression partly via Treg induction as well as increased invasive ability. These results indicated that Treg is the major immunosuppressive factor in cancer bearing hosts, and depletion of Treg or their suppressive activity by targeting molecules expressed in Treg or cancer cells may restore immunocompetence of cancer patients and enhance anti-tumor effects of current immunotherapy.

Roles of Thymic Epithelial Cells for the Establishment of Immunological Self-tolerance
Mitsuru Matsumoto

Thymic epithelial cells (TEC) play pivotal roles in the establishment of self-tolerance through critical dialogue with developing thymocytes. Unique actions of two transcriptional regulators within TECs, NF-κB-inducing kinase (NIK) and AIRE (an autoimmune regulator), for the establishment of self-tolerance have recently been highlighted by studies using a strain of mouse bearing a natural mutation of the NIK gene (aly mice) and gene-targeted mice, respectively. Previous studies have demonstrated essential roles of NIK downstream of the lymphotoxin-β receptor (LTβR), which is essential for the development of secondary lymphoid organs; aly mice lack all lymph nodes and Peyer's patches because of the defective LTβR signaling. Now additional roles of NIK in thymic organogenesis downstream of LTβR, mainly through the developmental regulation of TECs, have emerged, although the corresponding ligand(s) for LTβR participating in this action have not been fully characterized. In contrast, AIRE, a gene responsible for the development of an organ-specific autoimmune disease that demonstrates monogenic autosomal recessive inheritance, contributes to the establishment of self-tolerance probably by controlling the expression of self-antigens through yet undetermined molecular mechanisms. Thus, it is highly likely that a group of genes control self-tolerance within TECs through unique and coordinated actions, and that an understanding of this process would help to unravel the pathogenesis of autoimmune disease.

Regulatory T Cells for the Control of Immunological Diseases
Shimon Sakaguchi

Naturally arising CD25+CD4+ regulatory T cells (Tregs) are engaged in the maintenance of immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses, such as autoimmune disease and allergy. Tregs specifically express the transcription factor Foxp3, a key regulator of Treg development and function. Mutations of the Foxp3 gene are indeed the cause of the disease called IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome, in which thymic production of Tregs and their function is severely impaired. Ectopic expression of Foxp3 in conventional T cells is sufficient to confer suppressive activity to naive T cells, repress production of cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-γ), and up-regulate Treg-associated molecules such as CD25, and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). One of the key issues to understand the role of Foxp3+ natural Tregs in immune suppression is to determine how Foxp3 controls these molecular events and what is the molecular basis of Treg-mediated suppression. We have recently shown that the transcription factor AML1/Runx1, which is crucially required for normal hematopoiesis including thymic T cell development, activates IL-2 and IFN-γ gene expression in conventional CD4+ T cells through binding to their respective promoters. In natural Tregs, Foxp3 physically interacts with AML1/Runx1, thereby confer suppressive activity to them. Further, Treg-specific conditional knockout of AML1/Runx1, and also CTLA-4, which natural Tregs constitutively express at a high level, impairs Treg function and results in the development of autoimmune disease and allergy. Based on these and other findings on the molecular basis of Treg development and function, it will be discussed how Treg contribute to the maintenance of immunological self-tolerance and immune homeostasis, and how they can be exploited at the molecular level to control physiological and pathological immune responses.