Cystathionine beta-synthase (CBS; EC 4.2.1.22) is a key enzyme in the generation of cysteine from methionine. A deficiency of CBS leads to homocystinuria, an inherited human disease characterized by mental retardation, seizures, psychiatric disturbances, skeletal abnormalities, and vascular disorders; however, the underlying mechanisms remain largely unknown. Here, we show the regional and cellular distribution of CBS in the adult and developing mouse brain. In the adult mouse brain, CBS was expressed ubiquitously, but it is expressed most intensely in the cerebellar molecular layer and hippocampal dentate gyrus. Immunohistochemical analysis revealed that CBS is preferentially expressed in cerebellar Bergmann glia and in astrocytes throughout the brain. At early developmental stages, CBS was expressed in neuroepithelial cells in the ventricular zone, but its expression changed to radial glial cells and then to astrocytes during the late embryonic and neonatal periods. CBS was most highly expressed in juvenile brain, and a striking induction was observed in cultured astrocytes in response to EGF, TGF-alpha, cAMP, and dexamethasone. Moreover, CBS was significantly accumulated in reactive astrocytes in the hippocampus after kainic acid-induced seizures, and cerebellar morphological abnormalities were observed in CBS-deficient mice. Taken together, these results suggest that CBS plays a crucial role in the development and maintenance of the CNS and that radial glia/astrocyte dysfunction might be involved in the complex neuropathological features associated with abnormal homocysteine metabolism.

Microglia are the primary immune surveillance cells in the brain, and when activated they play critical roles in inflammatory reactions and tissue repair in the damaged brain. Microglia rapidly extend their processes toward the damaged areas in response to stimulation of the metabotropic ATP receptor P2Y(12) by ATP released from damaged tissue. This chemotactic response is a highly important step that enables microglia to function properly at normal and pathological sites in the brain. To investigate the molecular pathways that underlie microglial process extension, we developed a novel method of modeling microglial process extension that uses transwell chambers in which the insert membrane is coated with collagen gel. In this study, we showed that ATP increased microglial adhesion to collagen gel, and that the ATP-induced process extension and increase in microglial adhesion were inhibited by integrin blocking peptides, RGD, and a functional blocking antibody against integrin-beta1. An immunoprecipitation analysis with an antibody against the active form of integrin-beta1 showed that P2Y(12) mediated the integrin-beta1 activation by ATP. In addition, time-lapse imaging of EGFP-labeled microglia in mice hippocampal slices showed that RGD inhibited the directional process extension toward the nucleotide source, and immunohistochemical staining showed that integrin-beta1 accumulated in the tips of the microglial processes in rat hippocampal slices stimulated with ADP. These findings indicate that ATP induces the integrin-beta1 activation in microglia through P2Y(12) and suggest that the integrin-beta1 activation is involved in the directional process extension by microglia in brain tissue.

A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons. Keywords: a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, development, GluR1 subunit, post-synaptic density-95/ Dlg/ ZO-1 domain, Shank3, synapse. Transmission at excitatory synapses is primarily mediated by glutamate acting on three classes of ligand-gated ion channels, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate and NMDA receptors (Wisden and Seeburg 1993;Hollmann and Heinemann 1994). In addition to their role in synaptic transmission, these glutamate receptors (GluRs) have been thought to play a crucial role in many brain functions, including activity-dependent synaptogenesis during development and synaptic plasticity (McDonald and Johnston 1990;Bliss and Collingridge 1993).Many excitatory synapses in young developing neurons have been found to express only NMDA receptors, which are continuously blocked by magnesium at resting membrane potentials. As no evoked transmission is observed even when glutamate is present, these synapses are referred to as 'silent synapses'. During later development, AMPA receptors are delivered and clustered on the synaptic membrane in an activity-dependent manner, and the synapses subsequently become functionally active (Durand et al. 1996;Wu et al. 1996;Pickard et al. 2000;Liao et al. 2001;Isaac 2003). Thus, the clustering of AMPA receptors on the synaptic membrane is an essential event during synaptogenesis. Address correspondence and reprint requests to S. Kohsaka, Department of Neurochemistry, National Institute of Neu...

J. Neurochem. (2012) 121, 217–227. Abstract The extension of microglial processes toward injured sites in the brain is triggered by the stimulation of the purinergic receptor P2Y12 by extracellular ATP. We recently showed that P2Y12 stimulation by ATP induces microglial process extension in collagen gels. In the present study, we found that a P2Y12 agonist, 2‐methylthio‐ADP (2MeSADP), failed to induce the process extension of microglia in collagen gels and that co‐stimulation with adenosine, a phosphohydrolytic derivative of ATP, and 2MeSADP restored the chemotactic process extension. An adenosine A3 receptor (A3R)‐selective agonist restored the chemotactic process extension, but other receptor subtype agonists did not. The removal of adenosine by adenosine deaminase and the blocking of A3R by an A3R‐selective antagonist inhibited ADP‐induced process extension. The A3R antagonist inhibited ADP‐induced microglial migration, and an A3R agonist promoted 2MeSADP‐stimulated migration. ADP and the A3R agonist activated Jun N‐terminal kinase in microglia, and a Jun N‐terminal kinase inhibitor inhibited the ADP‐induced process extension. An RT‐PCR analysis showed that A1R and A3R were expressed by microglia sorted from adult rat brains and that the A2AR expression level was very low. These results suggested that A3R signaling may be involved in the ADP‐induced process extension and migration of microglia.

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