Yoshio Hata

A novel cell transplantation protocol and its application to an ALS mouse model

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease, which selectively affects motor neurons throughout the central nervous system. The extensive distribution of motor neurons is an obstacle to applying cell transplantation therapy for the treatment of ALS. To overcome this problem, we developed a cell transplantation method via the fourth cerebral ventricle in mice. We used mouse olfactory ensheathing cells (OECs) and rat mesenchymal stem cells (MSCs) as donor cells. OECs are reported to promote regeneration and remyelination in the spinal cord, while MSCs have a capability to differentiate into several types of specific cells including neural cells. Furthermore both types of cells can be relatively easily obtained by biopsy in human. Initially, we confirmed the safety of the operative procedure and broad distribution of grafted cells in the spinal cord using wild-type mice. After transplantation, OECs distributed widely and survived as long as 100 days after transplantation, with a time-dependent depletion of cell number. In ALS model mice, OEC transplantation revealed no adverse effects but no significant differences in clinical evaluation were found between OEC-treated and non-transplanted animals. After MSC transplantation into the ALS model mice, females, but not males, showed a statistically longer disease duration than the non-transplanted controls. We conclude that intrathecal transplantation could be a promising way to deliver donor cells to the central nervous system. Further experiments to elucidate relevant conditions for optimal outcomes are required.

Latent TGF-β binding protein-2 is essential for the development of ciliary zonule microfibrils

Latent TGF-β-binding protein-2 (LTBP-2) is an extracellular matrix protein associated with microfibrils. Homozygous mutations in LTBP2 have been found in humans with genetic eye diseases such as congenital glaucoma and microspherophakia, indicating a critical role of the protein in eye development, although the function of LTBP-2 in vivo has not been well understood. In this study, we explore the in vivo function of LTBP-2 by generating Ltbp2(-/-) mice. Ltbp2(-/-) mice survived to adulthood but developed lens luxation caused by compromised ciliary zonule formation without a typical phenotype related to glaucoma, suggesting that LTBP-2 deficiency primarily causes lens dislocation but not glaucoma. The suppression of LTBP2 expression in cultured human ciliary epithelial cells by siRNA disrupted the formation of the microfibril meshwork by the cells. Supplementation of recombinant LTBP-2 in culture medium not only rescued the microfibril meshwork formation in LTBP2-suppressed ciliary epithelial cells but also restored unfragmented and bundled ciliary zonules in Ltbp2(-/-) mouse eyes under organ culture. Although several reported human mutant LTBP-2 proteins retain normal domain structure and keep the fibrillin-1-binding site intact, none of these mutant proteins were secreted from their producing cells, suggesting secretion arrest occurred to the LTBP-2 mutants owing to conformational alteration. The findings of this study suggest that LTBP-2 is an essential component for the formation of microfibril bundles in ciliary zonules.

Developmental and Visual Input-Dependent Regulation of the CB1 Cannabinoid Receptor in the Mouse Visual Cortex

The mammalian visual system exhibits significant experience-induced plasticity in the early postnatal period. While physiological studies have revealed the contribution of the CB1 cannabinoid receptor (CB1) to developmental plasticity in the primary visual cortex (V1), it remains unknown whether the expression and localization of CB1 is regulated during development or by visual experience. To explore a possible role of the endocannabinoid system in visual cortical plasticity, we examined the expression of CB1 in the visual cortex of mice. We found intense CB1 immunoreactivity in layers II/III and VI. CB1 mainly localized at vesicular GABA transporter-positive inhibitory nerve terminals. The amount of CB1 protein increased throughout development, and the specific laminar pattern of CB1 appeared at P20 and remained until adulthood. Dark rearing from birth to P30 decreased the amount of CB1 protein in V1 and altered the synaptic localization of CB1 in the deep layer. Dark rearing until P50, however, did not influence the expression of CB1. Brief monocular deprivation for 2 days upregulated the localization of CB1 at inhibitory nerve terminals in the deep layer. Taken together, the expression and the localization of CB1 are developmentally regulated, and both parameters are influenced by visual experience.

Activation of p38 mitogen-activated protein kinase is required for in vivo brain-derived neurotrophic factor production in the rat hippocampus.

Several lines of evidence strongly suggest that brain-derived neurotrophic factor (BDNF) is associated with the formation, storage and recall of memory in the hippocampus and that it is important to maintain a considerable level of hippocampal BDNF in order to keep normal functions. BDNF can be synthesized in an activity-dependent manner. In fact, kainic acid or AMPA enhances BDNF levels in hippocampal granule neurons. However, the mechanisms of BDNF production are largely unclear. Recently, we have found that riluzole, which blocks voltage-gated sodium channels and thereby reduces glutamate release, actually strengthens immunoreactivity of BDNF in hippocampal granule neurons of rats. Therefore, we examined the riluzole-activated signaling pathways for BDNF production. Riluzole increased levels of phospho-p38 mitogen-activated protein kinase (p38 MAPK), as well as BDNF levels. Inhibition of p38 MAPK by SB203580 reduced riluzole effects, while activation of p38 MAPK by anisomycin increased levels of BDNF, suggesting that p38 MAPK can mediate BDNF production. Riluzole-induced elevation of phospho-activating transcription factor-2, a transcription factor downstream of p38 MAPK, was also observed. A blocker of N-type voltage-gated calcium channels reduced the effects of riluzole on BDNF production and p38 MAPK activation. We also examined a possible involvement of the adenosine A1 receptor in BDNF production because riluzole can influence ecto-nucleotide levels. An A1 receptor agonist inhibited riluzole-induced elevation of BDNF levels, whereas an antagonist not only increased levels of BDNF and active p38 MAPK but also augmented riluzole effects. These results indicate that, in the rat hippocampus, there is an in vivo signaling pathway for BDNF synthesis mediated by p38 MAPK, and that N-type voltage-gated calcium channels and/or adenosine A1 receptors contribute to p38 MAPK activation.

A phase advance of the light-dark cycle stimulates production of BDNF, but not of other neurotrophins, in the adult rat cerebral cortex: association with the activation of CREB.

Circadian variation in the expression of brain‐derived neurotrophic factor (BDNF) indicates that BDNF is involved in the regulation of diurnal rhythms in a variety of biological processes. However, it is still unclear which brain regions alter their BDNF levels in response to external light input. Therefore, in selected brain regions of adult male rats, we investigated diurnal variation, as well as the effects of a single eight‐hour phase advance of the light‐dark cycle, on the levels of BDNF and of other neurotrophins. The cerebellum, hippocampus and cerebral cortex containing visual cortex (VCX) showed diurnal variation in BDNF protein levels and the VCX also in NT‐3 levels. In the VCX and the region containing the entorhinal cortex and amygdala (ECX), BDNF protein levels were increased 12 h after the phase advance, while BDNF mRNA levels were increased significantly in the VCX and slightly in the ECX after 4 h. After one week, however, BDNF protein levels were reduced in eight brain regions out of 13 examined. BDNF levels in the ECX and VCX were significantly different between light rearing and dark rearing, while a hypothyroid status did not produce an effect. Cyclic AMP responsive element‐binding protein (CREB), a transcription factor for BDNF, was greatly activated by the phase advance in the ECX and VCX, suggesting the existence of CREB‐mediated pathways of BDNF synthesis that are responsive to external light input.

Netrin-4 regulates thalamocortical axon branching in an activity-dependent fashion

Significance The environmental control of neuronal wiring is one of the most intriguing issues in neuroscience. However, the molecular mechanisms are largely unknown. Here, we demonstrate that the expression of the netrin family member netrin-4 (NTN4) is activity-dependent in the developing cortex and promotes terminal branching of thalamocortical axons. Evidence further shows that unc-5 homolog B (Unc5B), a putative receptor of NTN4, is expressed in the developing thalamus and mediates NTN4 signaling. These results suggest that NTN4 is the key molecule that underlies activity-dependent axon branch formation in neocortical circuits. Axon branching is remodeled by sensory-evoked and spontaneous neuronal activity. However, the underlying molecular mechanism is largely unknown. Here, we demonstrate that the netrin family member netrin-4 (NTN4) contributes to activity-dependent thalamocortical (TC) axon branching. In the postnatal developmental stages of rodents, ntn4 expression was abundant in and around the TC recipient layers of sensory cortices. Neuronal activity dramatically altered the ntn4 expression level in the cortex in vitro and in vivo. TC axon branching was promoted by exogenous NTN4 and suppressed by depletion of the endogenous protein. Moreover, unc-5 homolog B (Unc5B), which strongly bound to NTN4, was expressed in the sensory thalamus, and knockdown of Unc5B in thalamic cells markedly reduced TC axon branching. These results suggest that NTN4 acts as a positive regulator for TC axon branching through activity-dependent expression.

Experience-Driven Axon Retraction without Binocular Imbalance in Developing Visual Cortex

Refinement of the neural circuit during brain maturation is regulated by experience-driven neural activity. In the mammalian visual cortex, monocular visual deprivation (MD) in the early postnatal life causes a significant loss of cortical responses to a deprived eye and the retraction of input axons serving the deprived eye. A competitive interaction between inputs serving both eyes has been supposed to underlie the effects of MD because the loss of cortical response is much weaker when both eyes are deprived of vision. Also, the input axons do not retract after binocular deprivation. Here, we report that uncorrelated activity between presynaptic and postsynaptic neurons can solely lead to the retraction of geniculocortical axons in the absence of activity imbalance between two inputs. We analyzed the morphology of geniculocortical axons in a pharmacologically inhibited visual cortex of animals with normal vision and of binocularly deprived animals. In the normal vision animals, the axonal arbors in the inhibited cortex showed robust retraction. On the other hand, the arbors in binocularly deprived animals remained mostly intact. These results suggest that a homosynaptic associative mechanism, rather than a heterosynaptic competition between inputs, may play an important role in experience-driven axon retraction.

Effects of anesthesia on immunohistochemical detection of phosphorylated extracellular signal-regulated kinase in cerebral cortex

During attempts to examine the phosphorylation status of extracellular signal-regulated kinase (ERK) in cerebral cortex immunohistochemically, we determined whether deep anesthesia for euthanasia disturbs the phosphorylation status of ERK, because the anesthesia might influence activity-dependent phosphorylation of ERK. We compared effects of short (2 and 5 min) and long (>10 min) anesthesia by pentobarbital on the immunoreactivity for phosphorylated ERK in the visual and entorhinal cortices of rat. The long anesthesia drastically reduced the density of phosphorylated ERK immunopositive cells to about 15% of the short anesthesia condition. The reduction was observed in all cortical regions. We found no significant difference in pERK immunoreactivity between 2 and 5 min groups. A reduction of similar degree was induced by long anesthesia with isoflurane. Even if a similar duration of anesthesia is given, the immunohistochemical results possibly contain a variation due to the individual difference in the sensitivity to the anesthetics. We demonstrated that the variation of pERK immunopositive cell density in the visual cortex was significantly reduced by normalizing the values to the density in the nonvisual area in the entorhinal cortex, thus enabling us to detect differences between animals under different visual conditions with higher sensitivity. Therefore, the variation could be reduced by calculating the ratio of immunoreactivity in the area of interest to that in other cortical area as reference.

NT-4 protein is localized in neuronal cells in the brain stem as well as the dorsal root ganglion of embryonic and adult rats

We have newly established a sensitive, two‐site enzyme immunoassay system for neurotrophin‐4 (NT‐4) and investigated its tissue distribution in the rat nervous system. The minimal limit of detection of the assay is 0.3 pg/0.2 mL of assay mixture. Concentrations of NT‐4 were found to be extremely low in all brain regions, irrespective of the animal age, the highest level being found in the brain stem of 40‐day‐old rats, at 0.12 ng/g wet weight. NT‐4 levels in young adult rats were significantly lower in the thalamus and higher in the olfactory bulb, neocortex, hypothalamus and brain stem than respective levels in 1‐week‐old rats. NT‐4 immunoreactivity was strong in large neurons of the red nucleus and pontine reticular nucleus as well as the locus coeruleus, and moderate in cells in the mesencephalic trigeminal nucleus and interstitial nucleus of the medial longitudinal fasciculus. In the rat embryo, stong staining of NT‐4 was detected in cells of regions corresponding to the midbrain/pons from E11.5 through E15.5. The intensity was decreased after E13.5 when the cytoplasm of cells in the medulla oblongata, fibers of the cerebellar primordium, and both cells and fibers of the dorsal root ganglion were also stained. Concentrations of NT‐4 were detected in regions including the hindbrain and the dorsal root ganglion. Immunoblotting of NT‐4‐immunoreactive proteins extracted from these two regions revealed a band corresponding to mature NT‐4 with a molecular mass of ∼14 kDa. Kainic acid and another glutamte agonist, (+/–)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid did not affect NT‐4 levels in the hippocampus. The present results show NT‐4 to be localized in very limited brain cells and fibers from the embyonic period through to the young adult, suggesting specific roles in brain functions.

Cortical activity regulates corticothalamic synapses in dorsal lateral geniculate nucleus of rats.

In the visual system, the afferent axons from the dorsal lateral geniculate nucleus (dLGN) to the primary visual cortex (V1) show significant activity-dependent plasticity in early postnatal life. To determine whether activity-dependent plasticity operates also in feedback projections from V1 to dLGN, we inactivated cortical inputs pharmacologically and examined possible changes in the density of synaptic proteins, vesicular glutamate transporter 1 (VGluT1) and type 1 metabotropic glutamate receptor alpha (mGluR1alpha), which locate pre- and postsynaptically at feedback projections, respectively in dLGN of rats. The intensity of the immunohistochemical signal of mGluR1alpha in dLGN significantly decreased following the cortical inactivation for at least 2 days, and the decrease was maintained under cortical inactivation until 28 days. On the other hand, the signal intensity of VGluT1 showed a significant increase following 14 or 28 days of cortical inactivation. In adult rats, however, we found no significant change in VGluT1 signal intensity and only a small and transient downregulation of mGluR1alpha following 7-day inactivation. Thus, the decrease in presynaptic activity induces a rapid downregulation of postsynaptic mGluR1alpha followed by a delayed upregulation of presynaptic VGluT1 in young rats. These results suggest that feedback synapses are regulated by neural activity during development.