Hidefumi Fukumitsu

Simultaneous expression of brain-derived neurotrophic factor and neurotrophin-3 in Cajal–Retzius, subplate and ventricular progenitor cells during early development stages of the rat cerebral cortex

To identify production sites and action targets of neurotrophins during neurogenesis, we investigated immunoreactivities of neurotrophins and their tyrosine kinase receptors in the cerebral cortex of rat embryos. Two sets of ligand-receptor systems, brain-derived neurotrophic factor/TrkB and neurotrophin-3/TrkC, were expressed simultaneously in Cajal-Retzius, subplate neurons and ventricular multipotent stem cells at embryonic days 13 and 15. Intraventricular administration of brain-derived neurotrophic factor or neurotrophin-3 at embryonic day 16 markedly modulated microtubule-associated protein II and/or Hu protein expression in different ways in the cortical plate cells by embryonic day 20. These observations indicate the involvement of autocrine and/or local paracrine action of brain-derived neurotrophic factor and/or neurotrophin-3 during formation of the cerebral cortex.

Brain-Derived Neurotrophic Factor Participates in Determination of Neuronal Laminar Fate in the Developing Mouse Cerebral Cortex

Lamina formation in the developing cerebral cortex requires precisely regulated generation and migration of the cortical progenitor cells. To test the possible involvement of brain-derived neurotrophic factor (BDNF) in the formation of the cortical lamina, we investigated the effects of BDNF protein and anti-BDNF antibody separately administered into the telencephalic ventricular space of 13.5-d-old mouse embryos. BDNF altered the position, gene-expression properties, and projections of neurons otherwise destined for layer IV to those of neurons for the deeper layers, V and VI, of the cerebral cortex, whereas anti-BDNF antibody changed some of those of neurons of upper layers II/III. Additional analysis revealed that BDNF altered the laminar fate of neurons only if their parent progenitor cells were exposed to it at approximately S-phase and that it hastened the timing of the withdrawal of their daughter neurons from the ventricular proliferating pool by accelerating the completion of S-phase, downregulation of the Pax6 (paired box gene 6) expression, an essential transcription factor for generation of the upper layer neurons, and interkinetic nuclear migration of cortical progenitors in the ventricular zone. These observations suggest that BDNF participates in the processes forming the neuronal laminas in the developing cerebral cortex. BDNF can therefore be counted as one of the key extrinsic factors that regulate the laminar fate of cortical neurons.

Inflammation-induced GDNF improves locomotor function after spinal cord injury.

Activation of microglia/macrophages after injury occurs limitedly in the CNS, which finding may explain unsuccessful axonal regeneration. Therefore, the relationship between lipopolysaccharide (LPS)-induced inflammation and recovery of locomotor function of rats after spinal cord injury was examined. High-dose LPS improved locomotor function greater than low-dose LPS, being consistent with the expression of neurotrophic factor (GDNF) in microglia/macrophages. Experiments using GDNF gene mutant mice confirmed that the increase in the GDNF mRNA level, rather than the reduction in the mRNA level of inducible NO synthase, could be correlated with the restoration activity of locomotor function. These results suggest that a higher degree of inflammation leads to a higher degree of repair of CNS injuries through GDNF produced by activated microglia/macrophages.

Brain-derived neurotrophic factor-like immunoreactivity in the adult rat central nervous system predominantly distributed in neurons with substantial amounts of brain-derived neurotrophic factor messenger RNA or responsiveness to brain-derived neurotrophic factor

Distribution of brain-derived neurotrophic factor-like immunoreactivity was investigated in the adult rat brain using two types of antibodies against peptides, V2 and V4, unique to the brain-derived neurotrophic factor. Western blot analysis showed that both antibodies specifically bound brain-derived neurotrophic factor, but not other neurotrophins, and that they recognized identical molecules of 18,000 mol. wt, but not the 14,500 mol. wt mass of mature form, in extracts from the rat hippocampus. Both antibodies recognized an identical precursor form (30,000 mol. wt) in lysates of COS7 cells transfected with brain-derived neurotrophic factor gene. These indicated that both antibodies predominantly recognized identical precursor protein(s) or its derivative(s) probably because of their much higher amounts than the amount of mature protein. Immunochemical studies showed that anti-V2 predominantly stained the cytoplasm of cells; whereas the anti-V4 bound to the nucleus, suggesting that the tertiary structure of immunoreactive molecules changed depending on their location. Cell populations with the immunoreactivity were similar in most brain sections stained with either anti-V2 or anti-V4 antibodies. These results suggest that brain-derived neurotrophic factor-like immunoreactivity distributes, in most cases, in neurons responding to brain-derived neurotrophic factor and in neurons expressing abundant brain-derived neurotrophic factor messenger RNA. These, taken together with other results concerning distributions of messenger RNAs of brain-derived neurotrophic factor and TrkB, provide additional information to elucidate the function of brain-derived neurotrophic factor in the rat central nervous system.

Involvement of glial cell line-derived neurotrophic factor in activation processes of rodent macrophages

The physiological roles of glial cell line‐derived neurotrophic factor (GDNF) expressed in the microglia/macrophages of the injured spinal cord have not yet been clarified. mRNA expression of chemokines, including monocyte chemoattractant protein (MCP)‐1, was evoked within 1 hr after transection of the spinal cord, and GDNF mRNA expression was similarly up‐regulated. Immunohistochemical analysis showed that GDNF was coexpressed with MCP‐1 in the CD11b‐positive cells. Therefore, we examined further the effects of GDNF on cultured rat peritoneal macrophages. GDNF enhanced the phagocytic activity of the macrophages via GFRα‐1, glycosylphosphatidylinositol‐anchored specific binding site of GDNF, in a c‐Ret‐independent manner. The influence of autocrine and/or paracrine GDNF synthesis was evaluated by performing activation experiments using macrophages cultured from heterozygous (+/−) GDNF gene‐deficient mice or wild‐type (+/+) mice. There were no morphological differences dependent on genetic types or stimulators. However, the GDNF mRNA level, but not the MCP‐1 or GFRα‐1 mRNA level, was substantially lower in the mutant macrophages than in the +/+ cells irrespective of stimulation with MCP‐1 or lipopolysaccharide (LPS). The phagocytic activity enhanced by MCP‐1 or LPS was significantly lower in the mutant cells (+/−) than in the +/+ ones, demonstrating the involvement of endogenous GDNF in the activation processes of macrophages in vitro and suggesting that not only neuroprotective function but also activation of macrophages is effected by the GDNF produced after a spinal cord injury.

Transforming growth factor-β1 enhances expression of brain-derived neurotrophic factor and its receptor, TrkB, in neurons cultured from rat cerebral cortex

The effects of transforming growth factor (TGF)‐β1 on expression of brain‐derived neurotrophic factor (BDNF) and its high‐affinity receptor, TrkB, in neurons cultured from the cerebral cortex of 18‐day‐old embryonic rats were examined. BDNF mRNA was significantly increased from 24–48 hr after the TGF‐β1 treatment over 20 ng/ml. Accumulation of BDNF protein in the culture medium was also potentiated by TGF‐β1, although the intracellular content of BDNF was nearly unchanged. The enhancement of BDNF mRNA expression was suppressed by the co‐presence of decorin, a small TGF‐β‐binding proteoglycan that inhibits the biological activities of TGF‐βs. mRNA expression of full‐length TrkB, the bioactive high‐affinity receptor for BDNF, was also upregulated after treatment with TGF‐β1. These observations suggest that: 1) TGF‐β1 potentiates BDNF/TrkB autocrine or local paracrine system; and 2) the neurotrophic activity of TGF‐β1 is partly responsible for the BDNF induced by TGF‐β1 itself. To test this latter possibility, we examined the neuronal survival activity of TGF‐β1 with or without K252a, a selective inhibitor of Trk family tyrosine kinases. TGF‐β1 significantly enhanced neuronal survival, but the co‐presence of K252a completely suppressed the activity, demonstrating the involvement of Trk receptor signaling in TGF‐β1‐mediated neuronal survival in cultured rat cortical neurons. These results seem to be in line with recent findings by other investigators that some neurotrophic factors including BDNF require TGF‐βs as a cofactor to exert their neurotrophic activities. J. Neurosci. Res. 66:369–376, 2001.

Hydrophobic dipeptide Leu-Ile protects against neuronal death by inducing brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor synthesis

We investigated whether certain hydrophobic dipeptides, Leu‐Ile, Leu‐Pro, and Pro‐Ile, which partially resemble the site on FK506 that binds to immunophilin, could stimulate glial cell line‐derived neurotrophic factor (GDNF) and brain‐derived neurotrophic factor (BDNF) synthesis in cultured neurons and found only Leu‐Ile to be an active dipeptide. Leu‐Ile protected against the death of mesencephalic neurons from wild‐type mice but not from mice lacking the BDNF or GDNF gene. Next, we examined the effects of i.p. or i.c.v. administration of Leu‐Ile on BDNF and GDNF contents. Both types of administration increased the contents of BDNF and GDNF in the striatum of mice. Also, peripheral administration of Leu‐Ile inhibited dopaminergic (DA) denervation caused by unilateral injection of 6‐hydroxydopamine (6‐OHDA) into the striatum of mice. The number of rotations following a methamphetamine challenge was lower in the Leu‐Ile‐treated group than in the nontreated group. Next, we compared the calcineurin activity and immunosuppressant activity of Leu‐Ile with those of FK506. Leu‐Ile was not inhibitory toward calcineurin cellular activity in cultured neuronal cells. Furthermore, Leu‐Ile did not suppress concanavalin A (ConA)‐induced synthesis/secretion of interleukin‐2 by cultured spleen cells, suggesting that the immunosuppressant activity of Leu‐Ile may be negligible when used as a therapeutic tool for neurodegenerative diseases.

Degenerative alterations in the visual pathway after NMDA-induced retinal damage in mice.

In the present study, intravitreal injection of N-methyl-d-aspartate (NMDA) into the left eye induced retinal damage (decreases in the number of retinal ganglion cells) at 1 day after the injection. At 7 days after the injection, atrophy of the optic tract was observed on the contralateral side, but not on the ipsilateral side. Number of neuronal nuclear specific protein (NeuN)-immunostained neurons were decreased in the contralateral dorsal LGN (dLGN) and contralateral ventral LGN-lateral (vLGN-l) at 90 and 180 days, respectively, after the injection. Furthermore, expressions of glial fibrillary acid protein (GFAP) were increased in the contralateral dLGN and contralateral vLGN-l at 7 and 30 days, respectively, and those of brain-derived neurotrophic factor (BDNF) were increased in the contralateral dLGN at 30 and 90 days and in the contralateral vLGN-l at 7 and 30 days. All NeuN-positive neuronal cells exhibited BDNF, whereas only some GFAP-positive astroglial cells exhibited BDNF. However, the contralateral ventral LGN-medial (vLGN-m) and ipsilateral LGN displayed no significant differences related to NeuN, GFAP, or BDNF immunohistochemistry. Taken together, these results indicate that time-dependent alterations occurred after the NMDA injection along the retinogeniculate pathway (from retina to LGN), and that the degree of damage in the LGN was region-dependent. In addition, the increased activated astroglial cells and expressions of BDNF in the damaged regions may play some roles in the cell-survival process of the LGN.

Neurotrophin-3 stimulates neurogenetic proliferation via the extracellular signal-regulated kinase pathway.

The effects of neurotrophin‐3 (NT3) administered into the ventricular space of 13.5‐day‐old mouse embryos on neurogenesis in the developing cerebral cortex were examined. 5‐Bromo‐2′‐deoxyuridine (BrdU) was injected into pregnant mice 3 hr after the NT3 administration to label the neural progenitor cells. NT3 increased the number of BrdU‐positive cells without altering their distribution. The increment in BrdU‐positive cells 24 hr after the BrdU injection was attributed to Pax6‐/BrdU‐positive cells (neural stem cells), which was followed by a significant elevation of the number of Tuj1‐/BrdU‐positive cells (neurons) 36 or 48 hr after the BrdU injection, suggesting that NT3 facilitated neurogenesis by acting in two sequential steps, i.e., causing proliferation of neural stem cells and generation of neurons from these progenitors. NT3 stimulated phosphorylation of extracellular signal‐regulated kinase (ERK) 1/2 and ERK5 in the cortical progenitors, and the effects of NT3 on the increase in total BrdU‐positive cells and Pax6‐/BrdU‐positive cells were diminished by an MEK inhibitor, suggesting the involvement of MEK‐mediated ERK signal transduction in the NT3 actions.

Pyrroloquinoline quinone attenuates iNOS gene expression in the injured spinal cord.

Pyrroloquinoline quinone (PQQ) is a naturally occurring redox cofactor that acts as an essential nutrient, antioxidant, and redox modulator. PQQ has been demonstrated to oxidize the redox modulatory site of N-methyl-d-aspartic acid (NMDA) receptors. Such agents are known to be neuroprotective in experimental stroke models. Therefore, we examined the possible ameliorating effect of PQQ on spinal cord injury (SCI) in adult rats. Intraperitoneal administration of PQQ effectively promoted the functional recovery of SCI rats after hemi-transection, which was preceded by the attenuation of the expression of inducible nitric oxide (NO) synthase (iNOS) mRNA in the injury site. NO is involved in the secondary detrimental mechanisms and has been implicated in NMDA receptor-mediated neurotoxicity. In fact, administration of PQQ induced significantly decreased lesion size and increased axon density adjoining the lesion area. These observations suggest that PQQ protects against the secondary damage by reducing iNOS expression following primary physical injury to the spinal cord.