Identification of Rap1 as a target for the Crk SH3 domain-binding guanine nucleotide-releasing factor C3G.

C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide-releasing factor, shows sequence similarity to CDC25 and Sos family proteins (S. Tanaka, T. Morishita, Y. Hashimoto, S. Hattori, S. Nakamura, M. Shibuya, K. Matuoka, T. Takenawa, T. Kurata, K. Nagashima, and M. Matsuda, Proc. Natl. Acad. Sci. USA 91:3443-3447, 1994). The substrate specificity of C3G was examined by in vitro and in vivo experiments. C3G markedly stimulated dissociation of bound GDP from Rap1B but marginally affected the same reaction of other Ras family proteins (Ha-Ras, N-Ras, and RalA). C3G also stimulated binding of GTP-gamma S [guanosine 5'-3-O-(thio)triphosphate] to Rap1B. When C3G and Rap1A were expressed in COS7 cells, marked accumulation of the active GTP-bound form of Rap1A was observed, while Sos was not effective in the activation of Rap1A. These results clearly show that C3G is an activator for Rap1. Furthermore, expression of C3G with a membrane localization signal in a v-Ki-ras transformant, DT, induced a reversion of the cells to the flat form, possibly through the activation of endogenous Rap1.

The reversible change of GluR2 RNA editing in gerbil hippocampus in course of ischemic tolerance.

The ischemic tolerance is known to show protective effects on the neurons and the restricted Ca2+ influx through Ca2+ channels might be involved. In alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, ribonucleic acid (RNA) editing of the GluR2 subunit determines receptor desensitization and Ca2+ permeability. The authors investigated the effect of ischemic tolerance on the messenger RNA editing of Q/R and R/G sites of GluR2 subunit in hippocampus. It was found that the rate of RNA editing in Q/R site showed no change (100% edited), whereas that in R/G site decreased significantly (83.3% normal editing level to 60.4%) at day 3 (preconditioning period) and returned to normal level at day 14 (after preconditioning period). Further investigation revealed that the decrease of editing rate in ischemic tolerance resulted mainly from the decrease of editing in CA1 area.

Regulation of neuronal plasticity in the central nervous system by phosphorylation and dephosphorylation.

Neuronal plasticity can be defined as adaptive changes in structure and function of the nervous system, an obvious example of which is the capacity to remember and learn. Long-term potentiation and long-term depression are the experimental models of memory in the central nervous system (CNS), and have been frequently utilized for the analysis of the molecular mechanisms of memory formation. Extensive studies have demonstrated that various kinases and phosphatases regulate neuronal plasticity by phosphorylating and dephosphorylating proteins essential to the basic processes of adaptive changes in the CNS. These proteins include receptors, ion channels, synaptic vesicle proteins, and nuclear proteins. Multifunctional kinases (cAMP-dependent protein kinase, Ca2+/phospholipid-dependent protein kinase, and Ca2+/calmodulin-dependent protein kinases) and phosphatases (calcineurin, protein phosphatases 1, and 2A) that specifically modulate the phosphorylation status of neuronal-signaling proteins have been shown to be required for neuronal plasticity. In general, kinases are involved in upregulation of the activity of target substrates, and phosphatases downregulate them. Although this rule is applicable in most of the cases studied, there are also a number of exceptions. A variety of regulation mechanisms via phosphorylation and dephosphorylation mediated by multiple kinases and phosphatases are discussed.

Localization and developmental changes in the neuron-specific cyclin-dependent kinase 5 activator (p35nck5a) in the rat brain.

Mammalian brains contain a cde2-like protein kinase which is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a brain-specific regulatory subunit with a molecular weight of 35,000. In this study, we examined the temporal and spatial expression patterns of p35nck5a in the developing rat brain. Northern blot analysis showed that p35nck5a messenger RNA expression was low in the brain of 12-day postcoitum rats, and increased to a much higher level from 18 days postcoitum to two weeks after birth, and then declined at three weeks after birth. These developmental changes in p35nck5a expression correlated with the changes in Cdk5-associated kinase activity during brain development. These data suggest that p35nck5a is the specific activator for Cdk5 in the brain. Immunohistochemical and in situ hybridization studies demonstrated the presence of p35nck5a protein in postmitotic neurons but not in glial cells at all stages of brain development, indicating that p35nck5a is a neuron-specific protein. In the adult brain, the protein was rich in cell bodies and dendrites, and only very low amounts were detected in axons. In fetal and neonatal brains, however, axonal pathways such as the corpus callosum and external capsule were also stained with anti-p35nck5a antibody. Our findings suggest that p35nck5a is neuron specific, and a specific activator for Cdk5, and the subcellular localization of the two is strictly regulated depending on brain development. Neuronal Cdc2-like kinase may play key roles in neuronal maturation, synaptic formation, and neuronal plasticity.

Developmental alteration and neuron-specific expression of bone morphogenetic protein-6 (BMP-6) mRNA in rodent brain.

Bone morphogenetic proteins (BMPs) are a group of proteins which induce bone formation from mesenchymal cells. The existence of BMPs in the nervous system as well as in bone tissue has recently been reported. In this study, we show that BMP-6 is neuron-specific, and describe the temporal and spatial expression patterns of BMP-6 mRNA in the developing rat and gerbil brain. Northern blot analysis showed that the BMP-6 transcript level was specifically high from newborn to 3 weeks after birth compared with those in fetal and adult rats. In situ hybridization showed that most of the neurons possessed high levels of BMP-6 mRNA in the neonatal brain, while in the adult brain, BMP-6 mRNA level was significantly decreased in most of the neurons except those in hippocampus which retained high levels. Furthermore, to show that the BMP-6 expression was specific to neurons, we induced delayed neuronal cell death and compensative glial cell proliferation in the gerbil hippocampus by transient ischemia. Our findings collectively suggest that BMP-6 is neuron-specific and may play important roles in neuronal maturation and synapse formation.

Mitochondrial sulfhydryl groups. A possible endogenous probe of conformational changes in the mitochondrial membrane.

The protein-bound sulfhydryl (SH) groups of the mitochondrial membrane were determined with Ellman's reagent in energized and non-energized configurational states of mitochondria and submitochondrial particles. When beef heart mitochondria were energized by respiration, there was a decrease in titratable protein-bound SH groups which varied according to substrate: NADH-linked substrates induced a decrease of about 10 nmol per mg of protein,succinate about 7, and ascorbate-tetramethyl-p-phenylene-diamine about 3. Similar changes occurred in phosphorylating submitochondrial particles. A decrease in SH titer was also observed in non-energized conditions, induced by hypotonic treatment and by some reagents inhibiting electron transport and oxidative phosphorylation and inducing orthodox configuration. These changes in protein-bound SH groups might be useful in analyzing the conformational states of mitochondrial membranes.

Mutual interaction between host and graft tissues in embryonic neural transplantation.

The differentiation of AChE-positive cells in embryonic neural grafts from various brain regions into the hippocampus after fimbria-fornix transection was investigated. Rat fetal cell suspensions of basal forebrain, hippocampus, mesencephalon or cortex were prepared from fetuses of either embryonic day 14 (E14) or day 19 (E19) gestational age, and these were transplanted into the rat brain after unilateral fimbria-fornix transection. Dense acetylcholinesterase (AChE) positive fibres were observed in both E14 and E19 basal forebrain grafts. These fibres were also found in E14 hippocampal grafts. However E19 hippocampal grafts did not have any AChE-positive fibres. These results suggest that E14 hippocampal grafts might be affected by the host brain tissues and differentiate into cholinergic neurones.

Protective effect of vagus nerve stimulation on forebrain ischaemia in gerbil hippocampus.

The left vagus nerve was stimulated during transient forebrain ischaemia in gerbils. The animals were exposed to 3 min of forebrain ischaemic insult at 37.5 degrees C. On day 5 post-ischaemia, the animals were perfusion-fixed for qualitative and quantitative histopathological analyses. High current stimulation of the vagus nerve inhibited ischaemic neuronal damage in the hippocampal CA1 sector (p < 0.01), but low current stimulation did not (p < 0.01). These effects might have been due to inhibition of the effects of excitatory amino acids during ischaemia. These results indicate that vagus nerve stimulation might be protective to neurones subjected to ischaemic insult.

Developmental changes of cyclin dependent kinase 5 subcellular localization in the rat cerebellum.

Cyclin dependent kinase 5 (Cdk5) phosphorylates tau protein, a microtubule-associated protein, at pathological sites in vitro as well as in Alzheimer's disease brain. The enzyme is therefore regarded as an important candidate responsible for the progression of Alzheimer's disease. We and others have suggested that the enzyme has physiological roles in brain development and maturation. In this study, we investigated the exact distribution and developmental changes of the enzyme in cerebellum by immunohistochemistry and non-radioactive in situ hybridization. Immunohistochemistry demonstrated that Cdk5 was consistently expressed in the cerebellum at all developing stages. However, the subcellular localization of Cdk5 dramatically changed during maturation of the cerebellum. In the early neonatal stage, Cdk5 was strongly expressed in the cell bodies of neurones. With neuronal maturation Cdk5 immunoreactivity changed its subcellular localization from the cell body to axon. In terminally differentiated neurons, the immunoreactivity was only detected in the axon. These results suggest that subcellular localization of Cdk5 is strictly regulated and may play an important role in neuronal maturation.

Expression of calbindin-D28K by reactive astrocytes in gerbil hippocampus after ischaemia.

Calbindin-D28K (Calbindin) is a member of the superfamily of calcium-binding proteins that is implicated in the regulation of intracellular calcium. In the adult mammalian brain, calbindin was thought to be present only in neurones, where it is believed to serve a neuroprotective role. We now report the expression of calbindin after ischaemia in reactive astrocytes in the CA1 subfield of the hippocampus. Since other calcium-binding proteins, such as S-100 and calmodulin, which induce transformation or proliferation of glia, occur in astrocytes, it is conceivable that the expression of calbindin after ischaemia might be an important part of the process of gliosis.

Embryonic transplantation and ischemic memory deficit.

Transient forebrain ischemia is associated with selective neuronal vulnerability and persistent memory deficit. This study compares functional outcome and morphological changes in rats subjected to post-ischemic CA1 or hilus/dentate gyrus region hippocampal fetal transplantation. Ischemia was produced by bilateral common carotid artery occlusion with hypotension. Fetal hippocampal neurons were transplanted into both sides of the CA1 or hilus/dentate gyrus region of the dorsal hippocampus, 1 week post-ischemia. Four weeks post transplantation, the rats underwent behavioral testing for 5 consecutive days using the water maze trial. All animals were perfusion fixed for morphological studies. Transplants in the CA1 region of the dorsal hippocampus were associated with memory and morphological recovery, while grafts placed into the hilus/dentate gyrus region of the dorsal hippocampus were not. Similarly, neurons transplanted in the CA1 region of the dorsal hippocampus were morphologically similar to CA1 pyramidal cell neurons and stained positive with calbindin D(28k). In contrast the grafts transplanted into the hilus/dentate gyrus region of the dorsal hippocampus were morphologically heterogeneous and staining with calbindin D(28k) was not as robust. Post-ischemic transplantation in the CA1 region of the dorsal hippocampus is effective in improving memory and morphological function.

A calcineurin inhibitor, FK506, blocks voltage-gated calcium channel-dependent LTP in the hippocampus.

The effects of FK506, an immunosuppressant and protein phosphatase 2B (calcineurin) inhibitor, on the voltage-gated calcium channel (VGCC)-dependent long-term potentiation (LTP) were investigated in the CA1 region of mice hippocampal slices. VGCC-dependent LTP was induced either by a brief application of a potassium channel blocker tetraethyleneanmonium (TEA), or by a strong tetanic stimulation under the blockade of NMDA-receptors. FK506 (1-50 microM) produced dose-dependent inhibition on TEA-induced LTP. Cyclosporin A (CysA 50 microM), another calcineurin inhibitor, showed a similar inhibitory effect on TEA-induced LTP. FK506 (10 microM) also blocked the strong tetanus-induced LTP, but had no effect on the post-tetanic potentiation. By using a subthreshold weak tetanic stimulation protocol, we also found that low concentration of FK506 (1 microM) produced neither inhibition nor potentiation on VGCC-dependent LTP. These results showed FK506 and CysA exerted inhibitory effects on VGCC-dependent LTP, and suggest that calcineurin is involved in the processes of this kind of synaptic plasticity.

Changes in the expression of novel Cdk5 activator messenger RNA (p39nck5ai mRNA) during rat brain development.

We previously reported that a neuron-specific Cdk5 activator, p35nck5ai, was most prominent in the newborn rat brain. In the adult brain, the expression decreased in most regions except hippocampus and primary olfactory cortex. A novel neuron-specific Cdk5 activator, p39nck5ai, has been recently cloned. To clarify whether two activators were differentially distributed throughout brain development, in this study, we examined the spatial and temporal expression of p39nck5ai in the development rat brain. Northern blot analysis showed that p39nck5ai expression was low in 15-day old fetuses and newborn, and was most prominent in the 1-3 week-old rat brains. In the adult rat brain, expression declined to the same level as in newborn rat brain. In situ hybridization showed that p39nck5ai mRNA was weakly expressed in all neurons of all regions in the newborn rat brain and the transcriptional level was highest in all regions in the 3 week-old rat brain. In the adult, expression was decreased in most neurons except Purkinje and granule cells in the cerebellum which retained high levels. These results suggest that p35nck5a and p39nck5ai may have different functional roles in distinct brain regions during different states of the rat brain development.

Immunosuppressant FK506 prevents mossy fiber sprouting induced by kindling stimulation.

Kindling stimulation induces expansive growth of the axons of the dentate granule cells, the mossy fiber, into several areas of the hippocampus. An intraperitoneal injection of the immunosuppressant drug FK506, which is a specific inhibitor of Ca(2+)-calmodulin dependent phosphatase, calcineurin, prevented the full development of kindling as well as mossy fiber sprouting. The results show a correlation between mossy fiber sprouting and the development of kindling. The results suggest also that calcineurin may have a promoting role in mossy fiber sprouting and subsequent synaptogenesis.

A chronological study of the expression of glial fibrillary acidic protein and calbindin-D28 k by reactive astrocytes in the electrically lesioned rat brain.

Immunoreactivity of neuronal and glial marker proteins of reactive astrocytes around the electrically damaged pyramidal layer and stratum radiatum of the hippocampal CA1 region and corpus callosum was chronologically studied in electrically lesioned rat brains. A monoclonal antibody against calbindin-D28 k (CD28-Ab) and a polyclonal antibody against glial fibrillary acidic protein (GFAP-Ab) were used for immunostaining. Immunoreactivity of CD28 and GFAP in the reactive astrocytes was detected in brains 1-6 weeks post-lesion but not in non-lesioned brains. The number of immunohistochemically stained reactive astrocytes around the electrically damaged areas were counted and then compared with the number of those in the same areas of non-lesioned brains. The number of CD28- and GFAP-immunoreactive astrocytes began to increase around the lesion from 1-3 weeks following lesion in the pyramidal layer of the hippocampal CA1 region and from 1-4 weeks following lesion in the stratum radiatum of the hippocampal CA1 region and corpus callosum. These immunoreactive astrocytes could be observed for 6 weeks (the maximum survival time studies) in all areas of the lesioned brains studied. The increase in the number of reactive astrocytes might have been induced by the stimulatory effects of neurotrophic factors, or growth factors, produced around the lesioned site. The constancy in the number of reactive astrocytes after 3 and 4 weeks in the lesioned areas may have been due to the termination of the initial phase of the repair process, i.e. space-filling. Reactive astrocytes which were stained by GFAP-Ab were separated into two groups, based on the presence of CD28, i.e. CD28-positive and CD28-negative reactive astrocytes. The presence of CD28 might confer certain functions via calcium-mediated mechanisms on CD28-positive astrocytes in addition to the constructive role mediated by GFAP.

Immunohistochemical localization of calcineurin, calmodulin-stimulated phosphatase, in the rat hippocampus using a monoclonal antibody.

Immunohistochemical localization of calcineurin, a calmodulin-stimulated phosphatase, was examined in the rat hippocampus by using a monoclonal antibody VD3 which is specific for the A subunit (61 kDa) of calcineurin. The stratum lucidum, where the mossy fiber terminal forms giant synaptic boutons, showed strong immunoreactivity.

Involvement of calmodulin-dependent protein kinases-I and -IV in long-term potentiation.

Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) are thought to be involved in the induction of long-term potentiation (LTP). In the present study, LTP was induced by theta burst stimulation in the Schaffer collateral area of the stratum radiatum in the hippocampal CA1 region of the rat hippocampus. LTP-induced and control hippocampal slices were studied by Western blot and immunohistochemical analyses using CaMK-I, -II and -IV antibodies. Increased amounts of all three CaMKs were found in LTP-induced hippocampal slices as indicated by Western blot as well as by the density of their immunoreactivity. Our data clearly shows that not only CaMK-II but also CaMK-I and -IV contribute to synaptic plasticity formed in LTP.

Immunosuppressants and calcineurin inhibitors, cyclosporin A and FK506, reversibly inhibit epileptogenesis in amygdaloid kindled rat.

Calcineurin (CaN) immunoreactivity and content increased markedly in kindled rat brain, and this increment was due to CaN in the membrane fraction. Investigation of the effects of cyclosporin A and FK506 (immunosuppressants which inhibit CaN activity in T lymphocytes) in the kindling phenomena showed that the kindling stage progression was reversibly blocked by these drugs. These findings suggest that calcineurin may play an essential role in acquiring epileptogenesis in kindling.

Distinct cellular compartment of cyclin-dependent kinase 5 (Cdk5) and neuron-specific Cdk5 activator protein (p35nck5a) in the developing rat cerebellum.

We have elucidated the spatial and temporal localization of Cdk5 and p35nck5a in the developing rat postnatal cerebellum. Both proteins were highly expressed in cell bodies of post mitotic and immature neurons. The localization of Cdk5 in cellular compartment was changed from cell body to the axon in development. On the other hand, p35nck5a was always expressed in the cell body throughout cerebellum development. The Cdk5 kinase activity was correlated with the expression of p35nck5a rather than that of Cdk5. These results indicate that p35nck5a is a physiological activator of Cdk5 in immature neurons and further suggest that Cdk5 has another function in mature neurons.

Identification of a src family protein specifically expressed in rat astrocytes by immunohistochemistry and double immunofluorescent study.

Identification of a src-related tyrosine kinase and its regional and cellular distributions were studied in rat brain. The study was performed using a specific antibody raised against a synthetic peptide corresponding to the conserved autophosphorylation site of src-family tyrosine kinases. The antibody (alpha-src antibody) recognized a 43 kDa polypeptide in plasma membrane enriched fraction. The immunohistochemical data revealed that the polypeptide is localized in astrocytes of corpus callosum and fimbria hippocampus. The presence of this src-related protein in astrocytes suggests that it may have some control in their proliferation and differentiation.