Transplantation of iPS-derived vascular endothelial cells improves white matter ischemic damage.

White matter infarct induces demyelination and brain dysfunction. We previously reported that transplantation of brain microvascular endothelial cells improved the behavioral outcome and promoted remyelination by increasing the number of oligodendrocyte precursor cells in the rat model of white matter infarct. In this study, we investigated the effects of transplantation of vascular endothelial cells generated from human induced pluripotent stem cells (iPSCs) on the rat model of white matter infarct. Seven days after induction of ischemic demyelinating lesion by injection of endothelin-1 into the internal capsule of a rat brain, iPSC-derived vascular endothelial cells (iVECs) were transplanted into the site of demyelination. The majority of iVECs transplanted into the internal capsule survived for 14 days after transplantation when traced by immunohistochemistry for a human cytoplasmic protein. iVEC transplantation significantly recovered hind limb rotation angle as compared to human iPSC or rat meningeal cell transplantation when evaluated using footprint test. Fourteen days after iVEC transplantation, the infarct area remarkably decreased as compared to that just before the transplantation when evaluated using magnetic resonance imaging or luxol fast blue staining, and remyelination was promoted dramatically in the infarct when assessed using luxol fast blue staining. Transplantation of iVECs increased the number of oligodendrocyte lineage cells and suppressed the inflammatory response and reactive astrocytogenesis. These results suggest that iVEC transplantation may prove useful in treatment for white matter infarct.

The dynamics of revascularization after white matter infarction monitored in Flt1-tdsRed and Flk1-GFP mice.

Subcortical white matter infarction causes ischemic demyelination and loss of brain functions, as the result of disturbances of the blood flow. Although angiogenesis is one of the recovery processes after cerebral infarction, the dynamics of revascularization after white matter infarction still remains unclear. We induced white matter infarction in the internal capsule of Flk1-GFP::Flt1-tdsRed double transgenic mice by injection of endothelin-1 (ET-1), a vasoconstrictor peptide, together with N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, and followed the changes in Flk1 and Flt1 expression in the vascular system in the infarct area. Reduction of Flt1-tdsRed-positive blood vessels 1 day after the injection and increase of Flk1-GFP-strongly-positive blood vessels 3 days after the injection were apparent. PDGFRß-strongly-positive (PDGFRß+) cells appeared in the infarct area 3 days after the injection and increased their number thereafter. Three days after the injection, most of these cells were in close contact with Flk1-GFP-positive endothelial cells, indicating these cells are bona fide pericytes. Seven days after the injection, the number of PDGFRß+ cells increased dramatically, and the vast majority of these cells were not in close contact with Flk1-GFP-positive endothelial cells. Taken together, our results suggest revascularization begins early after the ischemic insult, and the emerging pericytes first ensheath blood vessels and then produce fibroblast-like cells not directly associated with blood vessels.

X-ray irradiation induces disruption of the blood-brain barrier with localized changes in claudin-5 and activation of microglia in the mouse brain.

X-ray irradiation (X-irradiation) induces disruption of the blood-brain barrier (BBB). However, the mechanisms underlying the permeability changes are unclear. Therefore, in the present study, we examined the cellular and molecular changes produced by X-irradiation of the brain. Male ICR mice were irradiated locally on their head, posterior to the bregma, except for the eyes, with a single dose of 60 Gy. BBB permeability was assessed using Evans blue dye. We also examined vascular endothelial growth factor (VEGF) expression, microglial morphology, and the expression of the tight junction protein claudin-5 from 0.5 to 7 days after irradiation. An increase in BBB permeability and a decrease in the expression of VEGF protein occurred in a time-dependent manner. In addition, the number of activated microglia (CD68+/Iba-1+ double-positive cells), the amount of tumor necrosis factor-α protein and immunoreactivity of nuclear factor-kappaB increased by irradiation, while the expression of claudin-5 on vascular endothelial cells diminished markedly in the cerebral cortex starting 0.5 days after irradiation. These results suggest that the downregulation of claudin-5 expression mediated by activated microglia may contribute to the BBB disruption induced by X-irradiation.

BMP4 signaling in NPCs upregulates Bcl-xL to promote their survival in the presence of FGF-2.

We previously reported that BMP4 does not promote proliferation or differentiation of CD44-positive astrocyte precursor cells (APCs) but greatly promotes their survival in the presence of fibroblast growth factor-2 (FGF-2). In this study, we examined if BMP4 acts as a survival factor also for neural stem/progenitor cells (NPCs) isolated from ganglionic eminence of neonatal mouse brain. We found BMP4 promotes survival but not proliferation or differentiation of these cells, just as in the case for CD44-positive APCs. Microarray analysis revealed some candidate molecules in the signaling pathway downstream of BMP4. Among them, we focused on Id1 (inhibitor of DNA-binding 1) and Bcl-xL in this study. Expression of both genes was promoted in the presence of BMP4, and this promotion was reduced by dorsomorphin, an inhibitor of BMP4 signaling. Furthermore, cytochrome c release from mitochondria was significantly reduced in the presence of BMP4, suggesting up-regulation of Bcl-xL activity by BMP4. Id1 siRNA reduced the expression of Bcl-xL, and negated survival promoting effect of BMP4. These data suggest that BMP4 promotes survival of NPCs by enhancing the anti-apoptotic function of Bcl-xL via BMP4-Smad1/5/8-Id1 signaling.

Fibronectin on extracellular vesicles from microvascular endothelial cells is involved in the vesicle uptake into oligodendrocyte precursor cells.

We previously reported transplantation of brain microvascular endothelial cells (MVECs) into cerebral white matter infarction model improved the animal's behavioral outcome by increasing the number of oligodendrocyte precursor cells (OPCs). We also revealed extracellular vesicles (EVs) derived from MVECs promoted survival and proliferation of OPCs in vitro. In this study, we investigated the mechanism how EVs derived from MVECs contribute to OPC survival and proliferation. Protein mass spectrometry and enzyme-linked immunosorbent assay revealed fibronectin was abundant on the surface of EVs from MVECs. As fibronectin has been reported to promote OPC survival and proliferation via integrin signaling pathway, we blocked the binding between fibronectin and integrins using RGD sequence mimics. Blocking the binding, however, did not attenuate the survival and proliferation promoting effect of EVs on OPCs. Flow cytometric and imaging analyses revealed fibronectin on EVs mediates their internalization into OPCs by its binding to heparan sulfate proteoglycan on OPCs. OPC survival and proliferation promoted by EVs were attenuated by blocking the internalization of EVs into OPCs. These lines of evidence suggest that fibronectin on EVs mediates their internalization into OPCs, and the cargo of EVs promotes survival and proliferation of OPCs, independent of integrin signaling pathway.

Extracellular Vesicles from Vascular Endothelial Cells Promote Survival, Proliferation and Motility of Oligodendrocyte Precursor Cells.

We previously examined the effect of brain microvascular endothelial cell (MVEC) transplantation on rat white matter infarction, and found that MVEC transplantation promoted remyelination of demyelinated axons in the infarct region and reduced apoptotic death of oligodendrocyte precursor cells (OPCs). We also found that the conditioned medium (CM) from cultured MVECs inhibited apoptosis of cultured OPCs. In this study, we examined contribution of extracellular vesicles (EVs) contained in the CM to its inhibitory effect on OPC apoptosis. Removal of EVs from the CM by ultracentrifugation reduced its inhibitory effect on OPC apoptosis. To confirm whether EVs derived from MVECs are taken up by cultured OPCs, we labeled EVs with PKH67, a fluorescent dye, and added them to OPC cultures. Many vesicular structures labeled with PKH67 were found within OPCs immediately after their addition. Next we examined the effect of MVEC-derived EVs on OPC behaviors. After 2 days in culture with EVs, there was significantly less pyknotic and more BrdU-positive OPCs when compared to control. We also examined the effect of EVs on motility of OPCs. OPCs migrated longer in the presence of EVs when compared to control. To examine whether these effects on cultured OPCs are shared by EVs from endothelial cells, we prepared EVs from conditioned media of several types of endothelial cells, and tested their effects on cultured OPCs. EVs from all types of endothelial cells we examined reduced apoptosis of OPCs and promoted their motility. Identification of the molecules contained in EVs from endothelial cells may prove helpful for establishment of effective therapies for demyelinating diseases.

Blood Transcriptomic Markers in Patients with Late-Onset Major Depressive Disorder.

We investigated transcriptomic markers of late-onset major depressive disorder (LOD; onset age of first depressive episode ≥ 50 years) from the genes expressed in blood cells and identified state-dependent transcriptomic markers in these patients. We assessed the genes expressed in blood cells by microarray and found that the expression levels of 3,066 probes were state-dependently changed in the blood cells of patients with LOD. To select potential candidates from those probes, we assessed the genes expressed in the blood of an animal model of depression, ovariectomized female mice exposed to chronic ultra-mild stress, by microarray and cross-matched the differentially expressed genes between the patients and the model mice. We identified 14 differentially expressed genes that were similarly changed in both patients and the model mice. By assessing statistical significance using real-time quantitative PCR (RT-qPCR), the following 4 genes were selected as candidates: cell death-inducing DFFA-like effector c (CIDEC), ribonuclease 1 (RNASE1), solute carrier family 36 member-1 (SLC36A1), and serine/threonine/tyrosine interacting-like 1 (STYXL1). The discriminating ability of these 4 candidate genes was evaluated in an independent cohort that was validated. Among them, CIDEC showed the greatest discriminant validity (sensitivity 91.3% and specificity 87.5%). Thus, these 4 biomarkers should be helpful for properly diagnosing LOD.

Gene expression alterations in the medial prefrontal cortex and blood cells in a mouse model of depression during menopause.

AIMS: The prevalence of major depressive disorder (MDD) is higher in women than in men, and this may be due to the decline in estrogen levels that occurs during the menopausal transition. We studied the biological alterations in the medial prefrontal cortex (mPFC), which is a region that is highly implicated in the neurobiology of MDD, and the blood cells (BCs) of ovariectomized (OVX) mice subjected to chronic mild stress (CMS), which represents a mouse model of depression during menopause. MAIN METHODS: The mPFC and the BCs were obtained from the same individuals. Gene expression levels were analyzed by microarray. The data were used for the Ingenuity Pathway Analysis and the Gene Ontology analysis. KEY FINDINGS: The gene expression alterations (GEAs) induced by OVX were mainly associated with ribosomal and mitochondrial functions in both the mPFC and the BCs. Rapamycin-insensitive companion of mTOR (RICTOR) was identified as a possible upstream regulator of the OVX-induced GEAs in both tissues. The CMS-induced GEAs were associated with retinoic acid receptor signaling, inflammatory cytokines and post-synaptic density in the mPFC, but not in the BCs. SIGNIFICANCE: OVX and CMS independently affect biological pathways in the mPFC, which is involved in the development of the depression-like phenotype. Because a subset of the OVX-induced GEAs in the mPFC also occurred in the BCs, the GEAs in the BCs might be a useful probe to predict biological pathways in the corresponding brain tissue under specific conditions such as OVX in females.

Transplanted microvascular endothelial cells promote oligodendrocyte precursor cell survival in ischemic demyelinating lesions.

We previously showed that transplantation of brain microvascular endothelial cells (MVECs) greatly stimulated remyelination in the white matter infarct of the internal capsule (IC) induced by endothelin-1 injection and improved the behavioral outcome. In the present study, we examined the effect of MVEC transplantation on the infarct volume using intermittent magnetic resonance image and on the behavior of oligodendrocyte lineage cells histochemically. Our results in vivo show that MVEC transplantation reduced the infarct volume in IC and apoptotic death of oligodendrocyte precursor cells (OPCs). These results indicate that MVECs have a survival effect on OPCs, and this effect might contribute to the recovery of the white matter infarct. The conditioned-medium from cultured MVECs reduced apoptosis of cultured OPCs, while the conditioned medium from cultured fibroblasts did not show such effect. These results suggest a possibility that transplanted MVECs increased the number of OPCs through the release of humoral factors that prevent their apoptotic death. Identification of such humoral factors may lead to the new therapeutic strategy against ischemic demyelinating diseases.

Dynamic changes of CD44 expression from progenitors to subpopulations of astrocytes and neurons in developing cerebellum.

We previously reported that CD44-positive cells were candidates for astrocyte precursor cells in the developing cerebellum, because cells expressing high levels of CD44 selected by fluorescence-activated cell sorting (FACS) gave rise only to astrocytes in vitro. However, whether CD44 is a specific cell marker for cerebellar astrocyte precursor cells in vivo is unknown. In this study, we used immunohistochemistry, in situ hybridization, and FACS to analyze the spatial and temporal expression of CD44 and characterize the CD44-positive cells in the mouse cerebellum during development. CD44 expression was observed not only in astrocyte precursor cells but also in neural stem cells and oligodendrocyte precursor cells (OPCs) at early postnatal stages. CD44 expression in OPCs was shut off during oligodendrocyte differentiation. Interestingly, during development, CD44 expression was limited specifically to Bergmann glia and fibrous astrocytes among three types of astrocytes in cerebellum, and expression in astrocytes was shut off during postnatal development. CD44 expression was also detected in developing Purkinje and granule neurons but was limited to granule neurons in the adult cerebellum. Thus, at early developmental stages of the cerebellum, CD44 was widely expressed in several types of precursor cells, and over the course of development, the expression of CD44 became restricted to granule neurons in the adult.

Localization of acetylcholine-related molecules in the retina: implication of the communication from photoreceptor to retinal pigment epithelium.

It has been long speculated that specific signals are transmitted from photoreceptors to the retinal pigment epithelium (RPE). However, such signals have not been identified. In this study, we examined the retinal expression and localization of acetylcholine-related molecules as putative candidates for these signals. Previous reports revealed that α7 nicotinic acetylcholine receptors (nAChRs) are present in the microvilli of RPE cells that envelope the tips of photoreceptor outer segments (OS). Secreted mammalian leukocyte antigen 6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) is a positive allosteric modulator of the α7 nAChR. Therefore, we first focused on the expression of SLURP-1. SLURP-1 mRNA was expressed in the outer nuclear layer, which is comprised of photoreceptor cell bodies. SLURP-1 immunoreactivity co-localized with rhodopsin and S-opsin in photoreceptor OS, while choline acetyltransferase (ChAT) and high affinity choline transporter (CHT-1) were also expressed in photoreceptor OS. Immunoelectron microscopy identified that the majority of SLURP-1 was localized to the plasma membranes of photoreceptor OS. These results provide evidence that SLURP-1 is synthesized in photoreceptor cell bodies and transported to photoreceptor OS, where SLURP-1 may also be secreted. Our findings suggest that photoreceptor OS communicate via neurotransmitters such as ACh and SLURP-1, while RPE cells might receive these signals through α7 nAChRs in their microvilli.

Brain microvascular endothelial cell transplantation ameliorates ischemic white matter damage.

Ischemic insults affecting the internal capsule result in sensory-motor disabilities which adversely affect the patient's life. Cerebral endothelial cells have been reported to exert a protective effect against brain damage, so the transplantation of healthy endothelial cells might have a beneficial effect on the outcome of ischemic brain damage. In this study, endothelin-1 (ET-1) was injected into the rat internal capsule to induce lacunar infarction. Seven days after ET-1 injection, microvascular endothelial cells (MVECs) were transplanted into the internal capsule. Meningeal cells or 0.2% bovine serum albumin-Hank's balanced salt solution were injected as controls. Two weeks later, the footprint test and histochemical analysis were performed. We found that MVEC transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (P<0.01) and induced remyelination (P<0.01) compared with the control groups. Also the inflammatory response was repressed by MVEC transplantation, judging from fewer ED-1-positive activated microglial cells in the MVEC-transplanted group than in the other groups. Elucidation of the mechanisms by which MVECs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia.

CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum.

Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in preparing APC candidates from developing mouse cerebellum, characterized them in vitro, and found that BMPs are survival factors for these cells.

Cerebral capillary endothelial cells are covered by the VEGF-expressing foot processes of astrocytes.

Molecules that have crucial functions in both nervous and vascular systems have attracted keen attention recently, and the name "angioneurins" has been proposed. The most striking example of angioneurins is vascular endothelial growth factor A (VEGF), which was originally identified as a key regulator of angiogenesis and has only recently been found to have important functions in the nervous system. In this study, we compared VEGF expression in the vasculature in the brain with that in the aorta and the vasculature in the kidney in mice. In larger vessels containing smooth muscle cells, VEGF was expressed by smooth muscle cells covering the lining of endothelial cells, both in and outside the brain. In cerebral capillaries lacking smooth muscle cells, endothelial cells were closely covered by VEGF-expressing foot processes of astrocytes, whereas capillaries were surrounded by VEGF-expressing processes of podocytes in the renal glomeruli. We also found that cultured cerebral microvessel endothelial cells do not express VEGF, whereas cultured cortical astrocytes do express VEGF.

Flexural rigidity of individual microtubules measured by a buckling force with optical traps.

We used direct buckling force measurements with optical traps to determine the flexural rigidity of individual microtubules bound to polystyrene beads. To optimize the accuracy of the measurement, we used two optical traps and antibody-coated beads to manipulate each microtubule. We then applied a new analytical model assuming nonaxial buckling. Paclitaxel-stabilized microtubules were polymerized from purified tubulin, and the average microtubule rigidity was calculated as 2.0 x 10(-24) Nm2 using this novel microtubule buckling system. This value was not dependent on microtubule length. We also measured the rigidity of paclitaxel-free microtubules, and obtained the value of 7.9 x 10(-24) Nm2, which is nearly four times that measured for paclitaxel-stabilized microtubules.

Role of Ser50 phosphorylation in SCG10 regulation of microtubule depolymerization.

Members of the stathmin-like protein family depolymerize microtubules (MTs), probably due to the ability of each stathmin monomer to bind two tubulin heterodimers in a complex (T(2)S complex). SCG10, a member of this family, is localized in the growth cone of neurons. It has four identified sites of serine phosphorylation (S50, S63, S73, and S97). Of these, S50 and S97 are phosphorylated by cAMP-dependent protein kinase, an enzyme involved in growth cone guidance. When the equivalent sites in stathmins are phosphorylated, they lose their ability to depolymerize MTs. We investigated the specific role of the two cAMP-dependent protein kinase (PKA) phosphorylation sites in SCG10. A mutant of SCG10 phosphorylated only on S50 retained the ability to depolymerize MTs, but SCG10 phosphorylated on S97 or on both S50 and S97 lost MT-depolymerizing activity. Surface plasmon resonance studies revealed that the phosphorylation of SCG10 at these sites reduced the tubulin heterodimer binding, mainly due to a reduced rate of association. In particular, compared to the two other phosphorylated forms, SCG10 phosphorylated at S50 had a significantly smaller dissociation constant for the binding of the first tubulin heterodimer and larger association and dissociation rate constants for the binding of the second heterodimer. This indicates that the phosphorylation of S50 compensates for the effect of phosphorylation at other sites by modulating T2S complex formation. Furthermore, these results suggest that S50-P maintains MT-depolymerizing activity, which indicates that the biological functions of phosphorylation at S50 and S97 are different.

The origin of extensive colour polymorphism in Plateumaris sericea (Chrysomelidae, Coleoptera).

Evidence is presented to demonstrate that colour polymorphism in a beetle arises from structural colours produced by a five-layered reflector in the elytron. The colour of leaf beetles, Plateumaris sericea, ranges across the visible spectrum from blackish-blue to red. The elytra have two distinct layers: epicuticle and exocuticle. Morphological observations reveal that the multilayer structure within the exocuticle differs little among the different colour morphs but the layers within the epicuticle have characteristic thicknesses corresponding to the observed colour. The reflectors, consisting of five layers within the epicuticle, are responsible for all the different colours observed in P. sericea, as shown by theoretical analyses for a multilayer stack, and by showing that removal of the elytral surface, including epicuticle, results in the disappearance of the iridescent colour.