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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., 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. Transplantation of iPSC‐derived vascular endothelial cells (iVECs) ameliorated ischemic demyelinating (DM) lesion in endothelin‐1 (ET‐1)‐treated internal capsule (IC) of a rat brain. iVEC transplantation improved locomotor function when evaluated using footprint (FP) test, and reduced infarct area when monitored using magnetic resonance imaging (MRI). In addition, transplanted iVECs promoted remyelination in the infarct area when examined using luxol fast blue (LFB) staining. Taken together, our results suggest that transplantation of human iPSC‐derived vascular endothelial cells may lead to development of effective therapies for white matter infarct.

GABA is a major neurotransmitter in the mammalian central nervous system. Glutamate decarboxylase (GAD) synthesizes GABA from glutamate, and two isoforms of GAD, GAD65, and GAD67, are separately encoded by the Gad2 and Gad1 genes, respectively. The phenotypes differ in severity between GAD single isoform-deficient mice and rats. For example, GAD67 deficiency causes cleft palate and/or omphalocele in mice but not in rats. In this study, to further investigate the functional roles of GAD65 and/or GAD67 and to determine the contribution of these isoforms to GABA synthesis during development, we generated various kinds of GAD isoform(s)-deficient rats and characterized their phenotypes. The age of death was different among Gad mutant rat genotypes. In particular, all Gad1

ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4(+/−)/ARF5(−/−) mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4(+/−)/ARF5(−/−) mice of both sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4(+/−)/ARF5(−/−) mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of pore-forming voltage-dependent Na(+) channel subunit Nav1.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein localized in the AIS and the consequent tremors in ARF4(+/−)/ARF5(−/−) mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class II ARF proteins in ARF4(+/−)/ARF5(−/−) mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Nav1.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Nav1.6, to the AIS. SIGNIFICANCE STATEMENT We found that decreasing the expression of class II ARF proteins, through the generation of ARF4(+/−)/ARF5(−/−) mice, impairs Nav1.6 distribution to the axon initial segment (AIS) of cerebellar Purkinje cells (PCs), thereby resulting in the impairment of action potential firing of PCs. The ARF4(+/−)/ARF5(−/−) mutant mice exhibited movement-associated essential tremor (ET)-like behavior with pharmacological profiles similar to those in ET patients. The exogenous expression of ARF5 reduced the tremor phenotype and restored the localization of Nav1.6 immunoreactivity to the AIS in ARF4(+/−)/ARF5(−/−) mice. Thus, our results suggest that class II ARFs are involved in the localization of Nav1.6 to the AISs in cerebellar PCs and that the reduction of class II ARF activity leads to ET-like movement disorder.

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. PDGFRβ-strongly-positive (PDGFRβ+) 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 PDGFRβ+ 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.

The HapMap Project is a major international research effort to construct a resource to facilitate the discovery of relationships between human genetic variations and health and disease. The Ser19Stop single nucleotide polymorphism (SNP) of human phytanoyl-CoA hydroxylase-interacting protein-like (PHYHIPL) gene was detected in HapMap project and registered in the dbSNP. PHYHIPL gene expression is altered in global ischemia and glioblastoma multiforme. However, the function of PHYHIPL is unknown. We generated PHYHIPL Ser19Stop knock-in mice and found that PHYHIPL impacts the morphology of cerebellar Purkinje cells (PCs), the innervation of climbing fibers to PCs, the inhibitory inputs to PCs from molecular layer interneurons, and motor learning ability. Thus, the Ser19Stop SNP of the PHYHIPL gene may be associated with cerebellum-related diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00766-x.

Pachychoroid spectrum diseases have been described as a new clinical entity within the spectrum of macular disorders. “Pachychoroid” is defined as choroidal thickening associated with dilated outer choroidal vessels often showing retinal pigment epithelium (RPE) degeneration. Although various clinical studies on the pachychoroid spectrum diseases have been conducted, the pathophysiology of pachychoroid has yet to be fully elucidated. In this study, we attempted to establish a mouse model of pachychoroid. We sutured vortex veins in eyes of wild type mice to imitate the vortex vein congestion in pachychoroid spectrum diseases. Fundus photography and ultra-widefield indocyanine green angiography showed dilated vortex veins from the posterior pole to the ampulla in eyes after induction of choroidal congestion. Optical coherence tomography and tissue sections presented choroidal thickening with dilatation of choroidal vessels. The RPE-choroid/retina thickness ratios on the tissue sections in the treated day 1 and day 7 groups were significantly greater than that in the control group (0.19±0.03 and 0.16±0.01 vs. 0.12±0.02, P

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.

Neuroblasts derived from neural stem cells (NSCs) in the subventricular zone (SVZ) migrate along the rostral migratory stream into the olfactory bulb to generate interneurons under normal physiological conditions. When demyelination occurs, NSCs or neural progenitor cells (NPCs) in the SVZ provide newly formed oligodendrocytes to demyelinated lesions. The plasticity of NSC/NPC lineages may tend to oligodendrogenesis under the influence of demyelinated lesions. The mechanisms, however, still remain unknown. This study revealed that focal demyelination in the corpus callosum caused activation of the microglia, not only at the site of demyelination but also in the SVZ, and dramatically increased the generation of oligodendrocyte progenitor cells (OPCs) in the SVZ. Furthermore, the inhibition of microglial activation by minocycline treatment decreased OPC generation in the SVZ, suggesting that microglial activation in the SVZ, induced by the focal demyelination in the corpus callosum, regulates NSC/NPC lineage plasticity in situ. In contrast to the findings regarding demyelination in the corpus callosum, inducing focal demyelination in the internal capsule did not induce either microglial activation or OPC generation in the SVZ. These results suggest that the mechanism of OPC generation in the SVZ after inducing demyelinating lesions could be different across the demyelinated regions.

We have previously reported that transient receptor potential vanilloid 2 (TRPV2) can be activated by mechanical stimulation, which enhances axonal outgrowth in developing neurons; however, the molecular mechanisms that govern the contribution of TRPV2 activation to axonal outgrowth remain unclear. In the present study, we examined this mechanism by using PC12 cells as a neuronal model. Overexpression of TRPV2 enhanced axonal outgrowth in a mechanical stimulus-dependent manner. Accumulation of TRPV2 at the cell surface was 4-fold greater in the growth cone compared with the soma. In the growth cone, TRPV2 is not static, but dynamically accumulates (within ∼100 ms) to the site of mechanical stimulation. The dynamic and acute clustering of TRPV2 can enhance very weak mechanical stimuli

Physiological brain temperature is an important determinant of brain function, and it is well established that changes in brain temperature dynamically influence hippocampal neuronal activity. We previously demonstrated that the thermosensor TRPV4 is activated at physiological brain temperature in hippocampal neurons thereby controlling neuronal excitability in vitro. Here, we examined whether TRPV4 regulates neuronal excitability through its activation by brain temperature in vivo. We locally cooled the hippocampus using our novel electrical device and demonstrated constitutive TRPV4 activation in normal mouse brain. We generated a model of partial epilepsy by utilizing kindling stimuli in the ventral hippocampus of wild type (WT) or TRPV4-deficient (TRPV4KO) mice and obtained electroencephalograms (EEG). The frequencies of epileptic EEG in WT mice were significantly larger than those in TRPV4KO mice. These results indicate that TRPV4 activation is involved in disease progression of epilepsy. We expected that disease progression would enhance hyperexcitability and lead to hyperthermia in the epileptogenic foci. To confirm this hypothesis, we developed a new device to measure exact brain temperature only in a restricted local area. From the recording results by the new device, we found that the brain temperatures in epileptogenic zones were dramatically elevated compared with normal regions. Furthermore, we demonstrated that the temperature elevation was critical for disease progression. Based on these results, we speculate that brain cooling treatment at epileptogenic foci would effectively suppress epileptic discharges through inhibition of TRPV4. Notably, the cooling treatment drastically suppressed neuronal discharges dependent on the inactivation of TRPV4.

The Ca(2+)-dependent activator protein for secretion 1 (CAPS1) protein plays a regulatory role in the dense-core vesicle exocytosis pathway. To clarify the functions of this protein in the brain, we searched for novel interaction partners of CAPS1 by mass spectrometry. We identified a specific interaction of CAPS1 with septin family proteins. We also demonstrated that the C-terminal region of the CAPS1 protein binds to part of the deduced GTP-binding domain of septin proteins. It is possible that a tertiary complex of septin, CAPS, and syntaxin contributes to dense-core vesicle trafficking and exocytosis in neurons.

Using region-specific injection of hyaluronic acid, we developed a mouse model of acute retinal detachment (RD) to investigate molecular mechanisms of photoreceptor cell death triggered by RD. We focused on the transient receptor potential vanilloid 4 (TRPV4) ion channel, which functions as a thermosensor, osmosensor, and/or mechanosensor. After RD, the number of apoptotic photoreceptors was reduced by ∼50% in TRPV4KO mice relative to wild-type mice, indicating the possible involvement of TRPV4 activation in RD-induced photoreceptor cell death. Furthermore, TRPV4 expressed in Müller glial cells can be activated by mechanical stimuli caused by RD-induced swelling of these cells, resulting in release of the cytokine MCP-1, which is reported as a mediator of Müller glia-derived strong mediator for RD-induced photoreceptor death. We also found that the TRPV4 activation by the Müller glial swelling was potentiated by body temperature. Together, our results suggest that RD adversely impacts photoreceptor viability via TRPV4-dependent cytokine release from Müller glial cells and that TRPV4 is part of a novel molecular pathway that could exacerbate the effects of hypoxia on photoreceptor survival after RD.SIGNIFICANCE STATEMENTIdentification of the mechanisms of photoreceptor death in retinal detachment is required for establishment of therapeutic targets for preventing loss of visual acuity. In this study, we found that TRPV4 expressed in Müller glial cells can be activated by mechanical stimuli caused by RD-induced swelling of these cells, resulting in release of the cytokine MCP-1, which is reported as a mediator of Müller glia-derived strong mediator for RD-induced photoreceptor death. We also found that the TRPV4 activation by the Müller glial swelling was potentiated by body temperature. Hence, TRPV4 inhibition could suppress cell death in RD pathological conditions and suggests that TRPV4 in Müller glial cells might be a novel therapeutic target for preventing photoreceptor cell death after RD.

Compartmentalization of neuronal function is achieved via specifically localized clustering of ion channels in discrete subcellular membrane domains. Transient receptor potential (TRP) channels exhibit highly variable cellular and subcellular patterns of expression. We previously revealed that the thermo-sensor TRPV4 (activated above 34 °C) is gated by physiological brain temperatures in hippocampal neurons and thereby controls their excitability. Here, we examined synaptic clustering of TRPV4 in developing hippocampal neurons. We found that TRPV4 accumulated in the soma of immature hippocampal neurons, and did not localize to post-synaptic locations although PSD-95-labeled post-synaptic structures were evident. During the maturation of neurons, TRPV4 was targeted to dendrites and also clustered at post-synaptic locations. Taken together, we reveal that TRPV4 localizes to post-synaptic sites and the post-synaptic targeting is strictly regulated in a neuronal maturation-dependent manner.

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.

During central nervous development, multi-potent neural stem/progenitor cells located in the ventricular/subventricular zones are temporally regulated to mostly produce neurons during early developmental stages and to produce glia during later developmental stages. After birth, the rodent cerebellum undergoes further dramatic development. It is also known that neural stem/progenitor cells are present in the white matter (WM) of the postnatal cerebellum until around P10, although the fate of these cells has yet to be determined. In the present study, it was revealed that primary neurospheres generated from cerebellar neural stem/progenitor cells at postnatal day 3 (P3) mainly differentiated into astrocytes and oligodendrocytes. In contrast, primary neurospheres generated from cerebellar neural stem/progenitor cells at P8 almost exclusively differentiated into astrocytes, but not oligodendrocytes. These results suggest that the differentiation potential of primary neurospheres changes depending on the timing of neural stem/progenitor cell isolation from the cerebellum. To identify the candidate transcription factors involved in regulating this temporal change, we utilized DNA microarray analysis to compare global gene-expression profiles of primary neurospheres generated from neural stem/progenitor cells isolated from either P3 or P8 cerebellum. The expression of zfp711, zfp618, barx1 and hoxb3 was higher in neurospheres generated from P3 cerebellum than from P8 by real-time quantitative PCR. Several precursor cells were found to express zfp618, barx1 or hoxb3 in the WM of the cerebellum at P3, but these transcription factors were absent from the WM of the P8 cerebellum.

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.

The kidney is one of the major loci for the expression of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). While CBS-deficient ( Cbs−/−) mice display homocysteinemia/methioninemia and severe growth retardation, and rarely survive beyond the first 4 wk, CTH-deficient ( Cth−/−) mice show homocysteinemia/cystathioninemia but develop with no apparent abnormality. This study examined renal amino acid reabsorption in those mice. Although both 2-wk-old Cbs−/−and Cth−/−mice had normal renal architecture, their serum/urinary amino acid profiles largely differed from wild-type mice. The most striking feature was marked accumulation of Met and cystathionine in serum/urine/kidney samples of Cbs−/−and Cth−/−mice, respectively. Levels of some neutral amino acids (Val, Leu, Ile, and Tyr) that were not elevated in Cbs−/−serum were highly elevated in Cbs−/−urine, and urinary excretion of other neutral amino acids (except Met) was much higher than expected from their serum levels, demonstrating neutral aminoaciduria in Cbs−/−(not Cth−/−) mice. Because the bulk of neutral amino acids is absorbed via a B0AT1 transporter and Met has the highest substrate affinity for B0AT1 than other neutral amino acids, hypermethioninemia may cause hyperexcretion of neutral amino acids.

Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca(2+) transients in astrocytes, and these Ca(2+) transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4(+) astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4(+) astrocytes. After activation, both TRPV4(+) and TRPV4(-) astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4(+) astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4(+) astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands.

In the mouse cerebellum, Ca

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.

Physiological roles of the transsulfuration pathway have been recognized by its contribution to the synthesis of cytoprotective cysteine metabolites, such as glutathione, taurine/hypotaurine, and hydrogen sulfide (H(2)S), whereas its roles in protecting against methionine toxicity remained to be clarified. This study aimed at revealing these roles by analyzing high-methionine diet-fed transsulfuration-defective cystathionine γ-lyase-deficient (Cth(-/-)) mice. Wild-type and Cth(-/-) mice were fed a standard diet (1 × Met: 0.44%) or a high-methionine diet (3 × Met or 6 × Met), and hepatic conditions were monitored by serum biochemistry and histology. Metabolome analysis was performed for methionine derivatives using capillary electrophoresis- or liquid chromatography-mass spectrometry and sulfur-detecting gas chromatography. The 6 × Met-fed Cth(-/-) (not 1 × Met-fed Cth(-/-) or 6 × Met-fed wild type) mice displayed acute hepatitis, which was characterized by markedly elevated levels of serum alanine/aspartate aminotransferases and serum/hepatic lipid peroxidation, inflammatory cell infiltration, and hepatocyte ballooning; thereafter, they died of gastrointestinal bleeding due to coagulation factor deficiency. After 1 week on 6 × Met, blood levels of ammonia/homocysteine and hepatic levels of methanethiol/3-methylthiopropionate (a methionine transamination product/methanethiol precursor) became significantly higher in Cth(-/-) mice than in wild-type mice. Although hepatic levels of methionine sulfoxide became higher in 6 × Met-fed wild-type mice and Cth(-/-) mice, those of glutathione, taurine/hypotaurine, and H(2)S became lower and serum levels of homocysteine became much higher in 6 × Met-fed Cth(-/-) mice than in wild-type mice. Thus, transsulfuration plays a critical role in the detoxification of excessive methionine by circumventing aberrant accumulation of its toxic transamination metabolites, including ammonia, methanethiol, and 3-methylthiopropionate, in addition to synthesizing cysteine-derived antioxidants to counteract accumulated pro-oxidants such as methionine sulfoxide and homocysteine.

The diverse physiological effects of sphingosine 1-phosphate (S1P) are mostly mediated by its five cognate G protein-coupled receptors, S1P(1)-S1P(5), which have attracted much attention as future drug targets. To gain insight into S1P(2)-mediated signaling, we analyzed frequent spontaneous seizures in S1P(2)-deficient (S1P(2)(-/-)) mice obtained after several backcrosses onto a C57BL/6N background. Full-time video recording of 120 S1P(2)(-/-) mice identified 420 seizures both day and night between postnatal days 25 and 45, which were accompanied by high-voltage synchronized cortical discharges and a series of typical episodes: wild run, tonic-clonic convulsion, freezing, and, occasionally, death. Nearly 40% of 224 S1P(2)(-/-) mice died after such seizures, while the remaining 60% of the mice survived to adulthood; however, approximately half of the deliveries from S1P(2)(-/-) pregnant mice resulted in neonatal death. In situ hybridization revealed exclusive s1p(2) expression in the hippocampal pyramidal/granular neurons of wild-type mice, and immunohistochemistry/microarray analyses identified enhanced gliosis in the whole hippocampus and its neighboring neocortex in seizure-prone adult S1P(2)(-/-) mice. Seizure-prone adult S1P(2)(-/-) mice displayed impaired spatial working memory in the eight-arm radial maze test and increased anxiety in the elevated plus maze test, whereas their passive avoidance learning memory performance in the step-through test and hippocampal long-term potentiation was indistinguishable from that of wild-type mice. Our findings suggest that blockade of S1P(2) signaling may cause seizures/hippocampal insults and impair some specific central nervous system functions.

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.

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.

To examine lesions caused by seizures in the developing brain, seizures were induced by the intraperitoneal injection of kainate and nicotine into juvenile mice. After a week, whole brain sections were examined using histochemistry and the gene expression profiles in the neocortices and hippocampi were analyzed using a DNA microarray. Propidium iodide and Fluoro-Jade C staining revealed that kainate but not nicotine-induced degeneration of the hippocampal pyramidal neurons. Comparative analyses of 12,488 probe sets on the microarray chip revealed the differential expression of 208 and 1243 probe sets in the neocortices and hippocampi of kainate-injected mice, respectively, as well as that of 535 and 436 probe sets in the neocortices and hippocampi of nicotine-injected mice, respectively, the patterns of change were largely drug-specific and region-specific. Among a variety of kainate-modified genes including those representing neurodegeneration and astrogliosis, we identified an increased gene expression of the lysosomal cysteine protease cathepsin S in the hippocampi of kainate-injected mice. Western blot analysis of the hippocampal homogenates revealed that kainate induced a 3.3-fold increase in cathepsin S expression. Immunohistochemistry using cell type-specific markers showed that cathepsin S was induced in microglia, especially those surrounding degenerating pyramidal neurons, but not in neurons themselves or astroglia, in the hippocampal CA1 region of kainate-injected mice. These results indicate that seizures induced by kainate elicit neurodegeneration, astrogliosis, and microglial activation accompanied by the expression of cathepsin S while those induced by nicotine do not.

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.

The origins and developmental regulation of cerebellar oligodendrocytes are largely unknown, although some hypotheses of embryonic origins have been suggested. Neural stem cells exist in the white matter of postnatal cerebellum, but it is unclear whether these neural stem cells generate oligodendrocytes at postnatal stages. We previously showed that cerebellar progenitor cells, including neural stem cells, widely express CD44 at around postnatal day 3. In the present study, we showed that CD44-positive cells prepared from the postnatal day 3 cerebellum gave rise to neurospheres, while CD44-negative cells prepared from the same cerebellum did not. These neurospheres differentiated mainly into oligodendrocytes and astrocytes, suggesting that CD44-positive neural stem/progenitor cells might generate oligodendrocytes in postnatal cerebellum. We cultured CD44-positive cells from the postnatal day 3 cerebellum in the presence of signaling molecules known as mitogens or inductive differentiation factors for oligodendrocyte progenitor cells. Of these, only FGF-2 promoted survival and proliferation of CD44-positive cells, and these cells differentiated into O4+ oligodendrocytes. Furthermore, we examined the effect of FGF-2 on cerebellar oligodendrocyte development ex vivo. FGF-2 enhanced proliferation of oligodendrocyte progenitor cells and increased the number of O4+ and CC1+ oligodendrocytes in slice cultures. These results suggest that CD44-positive cells might be a source of cerebellar oligodendrocytes and that FGF-2 plays important roles in their development at an early postnatal stage.

For homeothermic animals, constant body temperature is an important determinant of brain function. It is well established that changes in brain temperature dynamically influence hippocampal activity. We previously reported that the thermosensor TRPV4 (activated above 34 °C) is activated at the physiological temperature in hippocampal neurons and controls neuronal excitability in vitro. Here, we examined if TRPV4 regulates neuronal excitability through its activation at the physiological temperature in vivo. We found that TRPV4-deficient (TRPV4KO) mice exhibit reduced depression-like and social behaviors compared to wild-type (WT) mice, and the number of c-fos positive cells in the dentate gyrus was significantly reduced upon the depression-like behaviors. We measured resting membrane potentials (RMPs) in the hippocampal granule cells from slice preparations at 35 °C and found that TRPV4-positive neurons significantly depolarized the RMPs through TRPV4 activation at the physiological temperature. The depolarization increased the spike numbers depending on the enhancement of TRPV4 activation. We also found that theta-frequency electroencephalogram (EEG) activities in TRPV4KO mice during wake periods were significantly reduced compared with those in WT mice. Taken together, we report for the first time that TRPV4 activation at the physiological temperature is important to regulate neuronal excitability and behaviors in mammals.

During CNS development, multipotent neural stem cells give rise first to various kinds of specified precursor cells, which proliferate extensively before terminally differentiating into either neurons or glial cells. Control of proliferation of the precursor cells plays a crucial role in determining the number of cells in the CNS. Proliferation is driven by mitogens, but how it is terminated remains a mystery. We examined the role of p27/Kip1 (p27), a cyclin-dependent kinase inhibitor, in the control of proliferation of cerebellar granule cell precursors (GCPs). We found that there is an intracellular mechanism that stops GCP division and causes GCPs to differentiate and that p27 is part of this mechanism. It is still not clear either whether the specified precursor cells are irreversibly determined to differentiate into their particular cell types. We examined the developmental plasticity of GCPs in vitro and found that at least some GCPs are not irreversibly committed to neuronal development but can be induced to differentiate into astroglial cells by appropriate extracellular signals. Keywords:: neural stem cell, neural precursor cell, proliferation, differentiation, plasticity

During CNS development, multipotent neural stem cells give rise first to various kinds of specified precursor cells, which proliferate extensively before terminally differentiating into either neurons or glial cells. It is still not clear, however, whether the specified precursor cells are irreversibly determined to differentiate into their particular cell types. In this study, we show that isolated mouse cerebellar granule cell precursors from the outermost, proliferative zone of the external germinal layer can differentiate into astroglial cells when exposed to sonic hedgehog (Shh) and bone morphogenetic proteins. These induced cells initially expressed both glial fibrillary acidic protein and neuronal markers, but they then lost their neuronal markers and acquired S100-β, a marker of differentiated astroglial cells. These results indicate that at least some granule cell precursors are not irreversibly committed to neuronal development but can be induced to differentiate into astroglial cells by appropriate extracellular signals.

Ca2+-dependent activator protein for secretion 2 (CAPS2) is a protein that is essential for enhanced release of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from cerebellar granule cells. We previously identified dex3, a rare alternative splice variant of CAPS2, which is overrepresented in patients with autism and is missing an exon 3 critical for axonal localization. We recently reported that a mouse model CAPS2Δex3/Δex3 expressing dex3 showed autistic-like behavioral phenotypes including impaired social interaction and cognition and increased anxiety in an unfamiliar environment. Here, we verified impairment in axonal, but not somato-dendritic, localization of dex3 protein in cerebellar granule cells and demonstrated cellular and physiological phenotypes in postnatal cerebellum of CAPS2Δex3/Δex3 mice. Interestingly, both BDNF and NT-3 were markedly reduced in axons of cerebellar granule cells, resulting in a significant decrease in their release. As a result, dex3 mice showed developmental deficits in dendritic arborization of Purkinje cells, vermian lobulation and fissurization, and granule cell precursor proliferation. Paired-pulse facilitation at parallel fiber-Purkinje cell synapses was also impaired. Together, our results indicate that CAPS2 plays an important role in subcellular locality (axonal vs. somato-dendritic) of enhanced BDNF and NT-3 release, which is indispensable for proper development of postnatal cerebellum.

Ca2+-dependent activator protein for secretion 1 (CAPS1) plays a regulatory role in the dense-core vesicle (DCV) exocytosis pathway, but its functions at the cellular and synaptic levels in the brain are essentially unknown because of neonatal death soon after birth inCaps1knock-out mice. To clarify the functions of the protein in the brain, we generated two conditional knock-out (cKO) mouse lines: 1) one lackingCaps1in the forebrain; and 2) the other lackingCaps1in the cerebellum. Both cKO mouse lines were born normally and grew to adulthood, although they showed subcellular and synaptic abnormalities. Forebrain-specificCaps1cKO mice showed reduced immunoreactivity for the DCV marker secretogranin II (SgII) and thetrans-Golgi network (TGN) marker syntaxin 6, a reduced number of presynaptic DCVs, and dilatedtrans-Golgi cisternae in the CA3 region. Cerebellum-specificCaps1cKO mice had decreased immunoreactivity for SgII and brain-derived neurotrophic factor (BDNF) along the climbing fibers. At climbing fiber–Purkinje cell synapses, the number of DCVs was markedly lower and the number of synaptic vesicles was also reduced. Correspondingly, the mean amplitude of EPSCs was decreased, whereas paired-pulse depression was significantly increased. Our results suggest that loss of CAPS1 disrupts the TGN–DCV pathway, which possibly impairs synaptic transmission by reducing the presynaptic release probability.

During development, control of proliferation of neuronal precursor cells plays a crucial role in determining the number of neurons. Proliferation is driven by mitogens, but how it is terminated remains a mystery. In this study, we examined the role of cyclin-dependent kinase inhibitors in the control of proliferation of cerebellar granule cell precursors (GCPs). Among the inhibitors we examined, only p27/Kip1 (p27) was expressed at significant levels in cells of the granule cell lineage in the developing and adult cerebellum. In developing cerebella, p27 was expressed in the external germinal layer (the deeper regions), the molecular layer, and the granule layer. In adult cerebella, p27 was expressed in the cells of the granule layer. We isolated and purified GCPs from cerebella of developing mice and examined their bromodeoxyuridine (BrdU) uptake and p27 expression at various times. We found that there was an inverse correlation between BrdU uptake and p27 expression. Even in the presence of saturating amounts of Sonic hedgehog, a potent mitogen, the cells eventually stopped dividing and differentiated, expressing p27 strongly. We also examined mice in which one or both copies of the p27 gene have been inactivated by targeted gene disruption and found that their cerebella were larger than those of wild-type mice. In cell cultures, GCPs prepared from p27-deficient mice showed enhanced proliferation compared with GCPs from wild-type mice. Taken together, these results suggest that there is an intracellular mechanism that stops GCP division and causes GCPs to differentiate and that p27 is part of this mechanism.

A major hallmark of apoptosis is normotonic shrinkage of cells. Here, we studied the relation between apoptotic cell shrinkage and apoptotic cell death. Induction of the apoptotic volume decrease (AVD) under normotonic conditions was found to be coupled to facilitation of the regulatory volume decrease (RVD), which is known to be attained by parallel operation of Cl − and K + channels, under hypotonic conditions. Both the AVD induction and the RVD facilitation were found to precede cytochrome c release, caspase-3 activation, DNA laddering, and ultrastructural alterations in three cell types after apoptotic insults with two distinct apoptosis inducers. Also, the AVD was not prevented by a broad-spectrum caspase inhibitor. When the AVD induction and the RVD facilitation were prevented by blocking volume-regulatory Cl − or K + channels, these cells did not show succeeding apoptotic biochemical and morphological events and were rescued from death. Thus, it is concluded that the AVD, which is caused by disordered cell volume regulation, is an early prerequisite to apoptotic events leading to cell death.

We examined the role of ion movement in staurosporine-induced apoptosis of vascular endothelial cells. Cultured vascular endothelial cells from bovine carotid arteries were used. Apoptosis was determined by propidium iodide assay. Treatment of the endothelial cells with staurosporine (10 nmol/l-1 micromol/l) for 6 h induced nuclear fragmentation in a dose-dependent manner. Staurosporine (1 micromol/l) elicited apoptosis in 70.5+/-1.5% of cells. Concomitant treatment of endothelial cells with 1 mmol/l of 4, 4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), a chloride-bicarbonate exchange blocker, completely inhibited staurosporine-induced apoptosis. Other ion transporter inhibitors such as dimethyl amiloride and anthracene-9 carboxylic acid were less effective inhibitors of staurosporine-induced apoptosis of endothelial cells. DIDS prevented staurosporine-induced apoptosis of endothelial cells as well as cardiomyocytes. Next, we determined whether chloride ions or bicarbonate are involved in apoptosis. Incubation with a chloride ion removal buffer did not inhibit staurosporine-induced apoptosis of endothelial cells. However, endothelial cell apoptosis was completely suppressed by an inhibitor of caspase, benzyloxycarbonyl-Asp-CH(2)-O(C)O-dichlorobenzene (zD-dcb, 50 micromol/l). Staurosporine (1 micromol/l) increased the intracellular pH of endothelial cells, and DIDS (1 mmol/l), but not a caspase inhibitor, inhibited this increase in pH caused by staurosporine. Our findings suggest that endothelial cell apoptosis induced by staurosporine may be associated with the Cl(-)and bicarbonate (HCO-3) ions. Thus, Cl(-)efflux from cells or HCO-3 influx to cells (which increases pH) may play an important role in signal transduction leading events such as activation of caspase in staurosporine-induced apoptosis.

There is increasing evidence that programmed cell death (PCD) depends on a novel family of intracellular cysteine proteases, called caspases, that includes the Ced-3 protease in the nematode Caenorhabditis elegans and the interleukin-1β–converting enzyme (ICE)-like proteases in mammals. Some developing cells, including lens epithelial cells, erythroblasts, and keratinocytes, lose their nucleus and other organelles when they terminally differentiate, but it is not known whether the enzymatic machinery of PCD is involved in any of these normal differentiation events. We show here that at least one CPP32 (caspase-3)-like member of the caspase family becomes activated when rodent lens epithelial cells terminally differentiate into anucleate lens fibers in vivo, and that a peptide inhibitor of these proteases blocks the denucleation process in an in vitro model of lens fiber differentiation. These findings suggest that at least part of the machinery of PCD is involved in lens fiber differentiation.

Thermosensitive transient receptor potential (thermo TRP) channels are important for sensory transduction. Among them, TRPV2 has an interesting characteristic of being activated by very high temperature (>52 °C). In addition to the heat sensor function, TRPV2 also acts as a mechanosensor, an osomosensor and a lipid sensor. It has been reported that TRPV2 is expressed in heart, intestine, pancreas and sensory nerves. In the central nervous system, neuronal TRPV2 expression was reported, however, glial expression and the precise roles of TRPV2 have not been determined. To explore the functional expression of TRPV2 in astrocytes, the expression was determined by histological and physiological methods. Interestingly, TRPV2 expression was detected in plasma membrane of astrocytes, and the astrocytic TRPV2 was activated by very high temperature (>50 °C) consistent with the reported characteristic. We revealed that the astrocytic TRPV2 was also activated by lysophosphatidylcholine, a known endogenous lipid ligand for TRPV2, suggesting that astrocytic TRPV2 might regulate neuronal activities in response to lipid metabolism. Thus, for the first time we revealed that TRPV2 is functionally expressed in astrocytes in addition to neurons.

Neural stem cells (NSCs) are present in postnatal murine cerebellum. The detailed characteristics of these NSCs have never been reported. This study isolated NSC-like cells from postnatal mouse cerebellum. These cells proliferated in response to epidermal growth factor, expressed various NSC markers, and had the ability to self-renew. Neurosphere assays revealed that these cells could differentiate into neurons, astrocytes, and oligodendrocytes, indicating multipotency as NSCs. Although possessing multipotency, most of these cells differentiated into astrocytes spontaneously in vitro. Both ciliary neurotrophic factor (CNTF) and bone morphogenetic protein 2 (BMP2) facilitated expression of glial fibrillary acidic protein (GFAP) and some other characteristics of mature astrocytes by these cells. However, the shape and expression of glutamine transporter GLT-1 of GFAP(+) cells generated in the presence of CNTF or BMP2 differed significantly, suggesting that CNTF and BMP2 induced differentiation of these NSCs into two distinct types of astrocytes.

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.

We recently proposed that most mammalian cells other than blastomeres may be programmed to kill themselves unless continuously signaled by other cells not to. Many observations indicate that some mammalian cells are programmed in this way, but is it the case for most mammalian cells? As it is impractical to test all of the hundreds of types of mammalian cells, we have focused on two tissues--lens and cartilage--which each contain only a single cell type: if there are cells that do not require signals from other cells to avoid programmed cell death (PCD), lens epithelial cells and cartilage cells (chondrocytes) might be expected to be among them. We have previously shown that rat lens epithelial cells can survive in serum-free culture without signals from other cell types but seem to require signals from other lens epithelial cells to survive: without such signals they undergo PCD. We show here that the same is true for rat (and chick) chondrocytes. They can survive for weeks in culture at high cell density in the absence of other cell types, serum, or exogenous proteins or signaling molecules, but they die with the morphological features of apoptosis in these conditions at low cell density. Medium from high density cultures, FCS, or a combination of known growth factors, all support prolonged chondrocyte survival in low density cultures, as long as antioxidants are also present. Moreover, medium from high density chondrocyte cultures promotes the survival of lens epithelial cells in low density cultures and vice versa. Chondrocytes isolated from adult rats behave similarly to those isolated from developing rats. These findings support the hypothesis that most mammalian cells require signals from other cells to avoid PCD, although the signals can sometimes be provided by cells of the same type, at least in tissues that contain only one cell type.

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

During retinogenesis, the basic helix-loop-helix proneural gene math5 (atoh7) initiates the generation of the first-born neurons, retinal ganglion cells (RGCs), by activating a network of RGC transcription factors, including Brn-3b (POU4F2). Herein, we show that the expression of DLX1 and DLX2 is significantly down-regulated in math5-null retina but is markedly increased in Brn-3b-null retina. Interestingly, Brn-3b interacts with DLX1 through its homeodomain, and this interaction represses DLX1 activity. Retrovirus-mediated mis-expression of DLX1 or DLX2 dramatically increases the number of amacrine/bipolar cells and concurrently reduces rod photoreceptors. Conversely, combined ectopic expression of Brn-3b with DLX1 or DLX2 promotes the production of RGCs and inhibits amacrine cell differentiation. Thus, DLX1/2 play an essential role in cell fate selection between amacrine and RGCs. Brn-3b suppresses the role of DLX1/2 through physical interaction and biases the competent precursors toward RGC fates.

We have studied the survival requirements of developing lens epithelial cells to test the hypothesis that most cells are programmed to kill themselves unless they are continuously signaled by other cells not to do so. The lens cells survived for weeks in both explant cultures and high-density dissociated cell cultures in the absence of other cells or added serum or protein, suggesting that they do not require signals from other cell types to survive. When cultured at low density, however, they died by apoptosis, suggesting that they depend on other lens epithelial cells for their survival. Lens epithelial cells cultured at high density in agarose gels also survived for weeks, even though they were not in direct contact with one another, suggesting that they can promote one another's survival in the absence of cell-cell contact. Conditioned medium from high density cultures promoted the survival of cells cultured at low density, suggesting that lens epithelial cells support one another's survival by secreting survival factors. We show for the first time that normal cell death occurs within the anterior epithelium in the mature lens, but this death is strictly confined to the region of the anterior suture.

Polybrominated biphenyls (PBBs) are polyhalogenated, bioaccumulative flame retardant chemicals, which have been used in a variety of consumer and household products. They were accidentally introduced into the food chain in Michigan in 1973 and have remained a source of health concern. Studies have shown that exposure to PBB may cause adverse neurotoxic effects. We therefore examined the effects of BP-6, a PBB mixture, on thyroid hormone (TH) receptor (TR)-mediated transcription, on TH-induced Purkinje cell dendritogenesis, and on TH-induced cerebellar granule cell neurite extension. Our study shows that BP-6 suppressed TR-mediated transcription in CV-1 cells. Mammalian two-hybrid studies revealed that BP-6 did not inhibit coactivator binding to TR nor did it recruit corepressors to TR. Further examination using the liquid chemiluminescent DNA pull down assay revealed partial dissociation of TR from TH response element (TRE). In primary rat cerebellar culture, BP-6 significantly suppressed TH-induced dendrite arborization of Purkinje cells, and in reaggregate rat granule cell culture, impaired TH-induced neurite extension of granule cells. Taken together, our results indicate that BP-6 may disrupt TH homeostasis and consequently impair normal neuronal development.

Thermosensitive TRP (thermo TRP) channels are well recognized for their contributions to sensory transduction, responding to a wide variety of stimuli including temperature, nociceptive stimuli, touch, and osmolarity. However, the precise roles for the thermo TRP channels during development have not been determined. To explore the functional importance of thermo TRP channels during neural development, the temporal expression was determined in embryonic mice. Interestingly, TRPV2 expression was detected in spinal motor neurons in addition to the dorsal root ganglia from embryonic day 10.5 and was localized in axon shafts and growth cones, suggesting that the channel is important for axon outgrowth regulation. We revealed that endogenous TRPV2 was activated in a membrane stretch-dependent manner in developing neurons by knocking down the TRPV2 function with dominant-negative TRPV2 and TRPV2-specific shRNA and significantly promoted axon outgrowth. Thus, for the first time we revealed that TRPV2 is an important regulator for axon outgrowth through its activation by membrane stretch during development.

The effect of interleukin 6 (IL-6) upon arachidonic acid (AA) metabolism was studied in cultured bovine vascular endothelial cells (BVECs). Although, in those BVECs pretreated with IL-6 for 48 h, there was no effect upon cell proliferation, it was discovered that IL-6 suppressed the conversion of AA to prostaglandin I2 (PGI2) in cellular homogenates. First signs of the suppression occurred 3 h after incubation, and thereafter the suppression increased with time until it leveled off at 12 h. This effect of IL-6 was concentration-dependent, ranging from 20 ng/ml to a maximum of 100 ng/ml with IL-6 at 47% of control. IL-6 had no effect upon AA conversion when exogenously added to the BVEC homogenates. To investigate the mechanism of the suppression induced by IL-6, we studied its effect upon PGI2 synthesizing enzymes. We found that IL-6 had no effect upon the release of AA from phospholipids, but inhibited cyclooxygenase, the key enzyme for PGI2 production from AA. To amplify PGI2 production, AA was added exogenously to both control and IL-6-treated BVECs, and in this case also, the conversion activity in IL-6-treated BVEC's was suppressed. Through immunoblot analysis with an antibody to cyclooxygenase, it was determined that the level of cyclooxygenase protein was lowered in IL-6-treated cells. This is the first report to confirm down expression of cyclooxygenase in addition to the first observation as to the effect of IL-6 on endothelial cells.

In our previous study, it was revealed that the exogenous arachidonic acid is mainly metabolized by the lipoxygenase pathway in the cell-free homogenate of cultured astrocytes. This is apparently in contrast with other studies reporting production and release of the cyclooxygenase products (prostaglandins and thromboxanes) by cultured astrocytes. To help specify the reason for this discrepancy, the metabolism of endogenous arachidonic acid in the intact monolayer of cultured astrocytes was examined. When the astrocytes were stimulated with calcium ionophore A23187, a peak coeluted with authentic 12-hydroxyeicosatetraenoic acid (12-HETE) on reverse-phase high-performance liquid chromatography (RP-HPLC) was observed. Formation of this peak was not affected by indomethacin, a speciic inhibitor for cyclooxygenase, but completely inhibited by BW755C, an inhibitor for both cyclooxygenase and lipooxygenases. Furthermore, the ultraviolet spectrum of the substance giving this peak agreed well with that of authentic 12-HETE. The amount of 12-HETE formed and released by the astrocytes was estimated to be 293.1 ng/mg protein/1 h. Taken together, these results suggest that the endogenous arachidonic acid is mainly metabolized by 12-lipoxygenase in the intact monolayer of astrocytes.

Cystathionine beta-synthase-deficient mice (Cbs(-/-)) exhibit several pathophysiological features similar to hyperhomocysteinemic patients, including endothelial dysfunction and hepatic steatosis. Heterozygous mutants (Cbs(+/-)) on the C57BL/6J background are extensively analyzed in laboratories worldwide; however, detailed analyses of Cbs(-/-) have been hampered by the fact that they rarely survive past the weaning age probably due to severe hepatic dysfunction. We backcrossed the mutants with four inbred strains (C57BL/6J(Jcl), BALB/cA, C3H/HeJ and DBA/2J) for seven generations, and compared Cbs(-/-) phenotypes among the different genetic backgrounds. Although Cbs(-/-) on all backgrounds were hyperhomocysteinemic/hypermethioninemic and suffered from lipidosis/hepatic steatosis at 2 weeks of age, >30% of C3H/HeJ-Cbs(-/-) survived over 8 weeks whereas none of DBA/2J-Cbs(-/-) survived beyond 5 weeks. At 2 weeks, serum levels of total homocysteine and triglyceride were lowest in C3H/HeJ-Cbs(-/-). Adult C3H/HeJ-Cbs(-/-) survivors showed hyperhomocysteinemia but escaped hypermethioninemia, lipidosis and hepatic steatosis. They appeared normal in general behavioral tests but showed cerebellar malformation and impaired learning ability in the passive avoidance step-through test, and required sufficient dietary supplementation of cyst(e)ine for survival, demonstrating the essential roles of cystathionine beta-synthase in the central nervous system function and cysteine biosynthesis. Our C3H/HeJ-Cbs(-/-) mice could be useful tools for investigating clinical symptoms such as mental retardation and thromboembolism that are found in homocysteinemic patients.

Cystathionine gamma-lyase (CSE) is the last key enzyme in the transsulfuration pathway for the biosynthesis of cysteine from methionine in mammals, and catalyzes the hydrolysis of cystathionine into cysteine. Cysteine can be provided through diet; however, several investigators have suggested that infants may require dietary supplements of cysteine because of very low or undetectable CSE activity in their livers. We have previously shown that CSE levels are much lower in the livers and kidneys of fetal and infant mice than in those of adult mice, suggesting that the maternal supply of cysteine is important for the early development of mice. Here we examined changes of CSE expression in the livers and kidneys of dams during gestation and lactation. Hepatic enlargement was observed as early as gestational day 12.5 (G12.5) and thereafter became more prominent, whereas expression of CSE in the livers was found after postpartum day 1 (P1) and reached a peak at P14. The maintenance of lactation was essential for both hepatic enlargement and CSE expression. In contrast, kidneys gained weight only slightly during lactation while CSE expression in kidneys was markedly induced at G15.5 and then gradually declined through to P28. Serum concentrations of homocysteine (the precursor of cystathionine) were significantly lower in G18.5 dams than in virgins or G15.5 dams, suggesting that the expression of CSE in the kidneys contributes to the effective clearance of homocysteine during the late gestational stage.

We examined the effects of 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS), an inhibitor of the chloride-bicarbonate exchangers and chloride channels, on death in cultured cerebellar granule neurons. Various stimuli, such as reduction of extracellular K + concentration, removal of growth factors, and staurosporine treatment, induced cell death. This death was blocked by DIDS in a dose dependent manner. In the presence of DIDS, the cells exposed to such stimuli did not show DNA fragmentation, but retained the ability to exclude trypan blue and to metabolize MTT to formazan. On the other hand, pretreatment of the cells with DIDS did not show any protective effects. The neuroprotective effect of DIDS was not influenced by extracellular Na + , Cl − , HCO 3 − or Ca 2+ concentrations, although reduction of extracellular Cl − or Ca 2+ concentrations per se induced neuronal death. Other chloride-bicarbonate exchange blockers like 4-acetamido-4′-isothiocyanatostilmene-2,2′-disulfonic acid (SITS) or 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS) showed no significant effects on neuronal survival under these death-inducing stimuli. Dimethylamiloride, an inhibitor of the Na + /H + exchanger, did not influence neuronal death induced by these stimuli. Cells undergoing death showed gradual intracellular acidification, and DIDS did not inhibit this response, although DIDS (2 mM) per se induced transitory acidification followed by recovery within 10 min. DIDS did not influence intracellular Ca 2+ or Cl − levels during the lethal process. DIDS suppressed the cleavage of caspase-3 in the cells exposed to the death-inducing stimuli. These findings suggest that the neuroprotective effect of DIDS is mediated by a novel mechanism other than by nonselective inhibition of transporters or channels, and that DIDS blocks the death program upstream of caspases and downstream of all of the activation processes triggered by various stimuli.