Shigeo Uchino

Visualization of microglia in living tissues using Iba1-EGFP transgenic mice

Microglia are thought to play important roles not only in repairing injured tissue but in regulating neuronal activity, and visualizing the cells is very useful as a means of further investigating the function of microglia in vivo. We previously cloned the ionized calcium‐binding adaptor molecule 1 (Iba1) gene, which is expressed selectively in microglia/microphages. To generate new transgenic mice to visualize microglia with enhanced green fluorescent protein (EGFP), we here constructed a plasmid carrying EGFP cDNA under control of the Iba1 promoter. This construct was injected into C57B/6 mouse zygotes, and the Iba1‐EGFP transgenic line was developed. Fluorescent in‐situ hybridization analysis revealed that the Iba1‐EGFP transgene was located on chromosome 11D. No obvious defects were observed during development or in adulthood, and the EGFP fluorescence remained invariant over the course of at least four generations. Judging from the immunoreactivity with anti‐Iba1 antibody, all EGFP‐positive cells in the adult brain were ramified microglia. In the developing transgenic embryos, EGFP signals were detected as early as embryonic Day 10.5. The most prominent EGFP signals were found in forebrain, spinal cord, eye, foreleg, yolk sac, liver, and vessel walls. At postnatal Day 6, clear EGFP signals were observed in the supraventricular corpus callosum, known as “fountain of microglia,” where ameboid microglia migrate into the brain parenchyma and mature into ramified microglia. Iba1‐EGFP transgenic mice thus permit observation of living microglia under a fluorescence microscope and provide a useful tool for studying the function of microglia in vivo.

Direct interaction of post-synaptic density-95/Dlg/ZO-1 domain-containing synaptic molecule Shank3 with GluR1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor.

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 α‐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.

SHANK3 as an autism spectrum disorder-associated gene

SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses, and plays important roles in the formation, maturation, and maintenance of synapses. Haploinsufficiency of the SHANK3 gene causes a developmental disorder, 22q13.3 deletion syndrome (known as Phelan-McDermid syndrome), that is characterized by severe expressive language and speech delay, hypotonia, global developmental delay, and autistic behavior. Since several SHANK3 mutations have been identified in a particular phenotypic group in patients with autism spectrum disorder (ASD), the SHANK3 is strongly suspected of being involved in the pathogenesis and neuropathology of ASD. Five CpG-islands have been identified in the SHANK3 gene, and tissue-specific expression of SHANK3 is regulated by DNA methylation in an epigenetic manner. Cumulative evidence has shown that several SHANK3 variants are expressed in the developing rodent brain and that their expression is regulated by DNA methylation of intragenic promoters. We identified novel SHANK3 transcripts whose transcription started at the vicinity of the CpG-island 2 in the mouse brain. Shank3 mutant mice exhibit autistic-like behaviors, including impaired social interaction and repetitive behaviors. In this article we review recent findings in regard to higher brain functions of SHANK3, epigenetic regulation of SHANK3 expression, and SHANK3-related ASD that were obtained from genetic analyses in ASD patients, molecular biological studies using developing mouse brains, and studies of Shank3 mutant mice.

DEVELOPMENTAL CHANGES IN DISTRIBUTION OF DEATH-ASSOCIATED PROTEIN KINASE MRNAS

Death‐associated protein kinase (DAP‐kinase) is Ca2+/calmodulin‐dependent serine/threonine kinase that contains ankyrin repeats and the death domain. It has been isolated as a positive mediator of interferon‐γ‐induced apoptotic cell death of HeLa cells. In order to reveal the physiological role of DAP‐kinase, the tissue distribution and developmental changes in mRNA expression of DAP‐kinase were investigated by Northern blot and in situ hybridization analyses. DAP‐kinase mRNA was predominantly expressed in brain and lung. In brain, DAP‐kinase mRNA had already appeared at embryonic day 13 (E13) and was, thereafter, detected throughout the entire embryonic period. High levels of expression were detected in proliferative and postmitotic regions within cerebral cortex, hippocampus, and cerebellar Purkinje cells. These findings suggest that DAP‐kinase may play an important role in neurogenesis where a physiological type of cell death takes place. The overall expression of DAP‐kinase mRNA in the brain gradually declined at postnatal stages, and the expression became restricted to hippocampus, in which different expression patterns were observed among rostral, central, and caudal coronal sections, suggesting that DAP‐kinase may be implicated in some neuronal functions. Furthermore, it was found that the expression of DAP‐kinase mRNA was increased prior to a certain cell death induced by transient forebrain ischemia, indicating a possible relationship between DAP‐kinase and neuronal cell death. J. Neurosci. Res. 58:674–683, 1999.

NMDA receptor antagonist memantine promotes cell proliferation and production of mature granule neurons in the adult hippocampus

Memantine, which is used clinically for the treatment of Alzheimers disease (AD), is classified as an N-methyl-d-aspartate (NMDA) receptor antagonist. Since previous studies have shown that NMDA receptor antagonists promote neurogenesis in the adult brain, we examined the effect of memantine on neurogenesis in the adult mouse hippocampus. We intraperitoneally injected 3-month-old mice with memantine (at 10 or 50 mg/kg body weight) followed by 5-bromo-2-deoxyuridine (BrdU) injections (3x) after 3 days. We then examined the number of BrdU+ cells in the dentate gyrus (DG) of the hippocampus at different time points. The number of BrdU+ cells in the 50 mg/kg memantine-injected group increased by 2.1-fold (1 day after BrdU-injection), 3.4-fold (after 7 days), and 6.8-fold (after 28 days), whereas the 10 mg/kg dose of memantine had little effect on labeling compared to the control group. Immunohistochemical staining at 28 days after BrdU-injection revealed that the newly generated cells in the 50 mg/kg memantine-group had differentiated into mature granule neurons. Moreover, when 12-month-old mice were injected with memantine, cell proliferation was promoted in the DG (3.7-fold). These findings demonstrate that memantine promotes the proliferation of neural progenitor cells and the production of mature granule neurons in the adult hippocampus.

NMDA receptor regulates migration of newly generated neurons in the adult hippocampus via Disrupted-In-Schizophrenia 1 (DISC1)

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07282.x

Inhibition of NMDA receptors induces delayed neuronal maturation and sustained proliferation of progenitor cells during neocortical development.

To elucidate the role of N‐methyl‐D‐aspartate (NMDA) receptors during the early stage of cerebral neocortical development, we investigated the effect of an NMDA receptor antagonist, D(−)‐2‐amino‐5‐phosphonopentanoic acid (D‐APV), on cell migration and proliferation in slice cultures and dissociated primary cultures prepared from rat cerebral neocortex at embryonic Day 17. Pulse‐labeling experiments with 5‐bromo‐2′‐deoxyuridine (BrdU) showed that chronic exposure to D‐APV in slices delayed neuronal migration. Calcium imaging experiments revealed that functional NMDA receptors were expressed in neurons and the treatment with D‐APV delayed neuronal maturation judging from the subunit composition of NMDA receptor subtypes. The results using pulse‐labeling with BrdU indicated that exposure to D‐APV for 3 days induced a clear increase in the number of proliferating progenitor cells in the ventricular zone in neocortical slices. Exposure to D‐APV in primary cultures also increased the proliferation of progenitor cells. The effect of D‐APV on progenitor cell proliferation was possibly mediated through neuronal cells. To elucidate the mechanism of enhanced progenitor cell proliferation induced by D‐APV, we investigated expression of Hes1 and Hes5 mRNA in the ventricular zone of neocortical slices by reverse transcription‐polymerase chain reaction. Tissue exposed to D‐APV for 3 days showed higher expression of Hes1 and Hes5 mRNA than did unexposed control tissue. These results suggest that NMDA receptors expressed in neurons function in neuronal migration and maturation and in the proliferation of progenitor cells.

Appearance of Phagocytic Microglia Adjacent to Motoneurons in Spinal Cord Tissue From a Presymptomatic Transgenic Rat Model of Amyotrophic Lateral Sclerosis

Microglial activation occurs early during the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent evidence indicates that the expression of mutant Cu2+/Zn2+ superoxide dismutase 1 (SOD1) in microglia contributes to the late disease progression of ALS. However, the mechanism by which microglia influence the neurodegenerative process and disease progression in ALS remains unclear. In this study, we revealed that activated microglia aggregated in the lumbar spinal cord of presymptomatic mutant SOD1H46R transgenic rats, an animal model of familial ALS. The aggregated microglia expressed a marker of proliferating cell, Ki67, and phagocytic marker proteins ED1 and major histocompatibility complex (MHC) class II. The motoneurons near the microglial aggregates showed weak choline acetyltransferase (ChAT) immunoreactivity and contained reduced granular endoplasmic reticulum and altered nucleus electron microscopically. Furthermore, immunopositive signals for tumor necrosis factor‐α (TNFα) and monocyte chemoattractant protein‐1 (MCP‐1) were localized in the aggregated microglia. These results suggest that the activated and aggregated microglia represent phagocytic features in response to early changes in motoneurons and possibly play an important role in ALS disease progression during the presymptomatic stage.

Fyn Is Required for Haloperidol-induced Catalepsy in Mice

Fyn-mediated tyrosine phosphorylation of N-methyl-d-aspartate (NMDA) receptor subunits has been implicated in various brain functions, including ethanol tolerance, learning, and seizure susceptibility. In this study, we explored the role of Fyn in haloperidol-induced catalepsy, an animal model of the extrapyramidal side effects of antipsychotics. Haloperidol induced catalepsy and muscle rigidity in the control mice, but these responses were significantly reduced in Fyn-deficient mice. Expression of the striatal dopamine D2 receptor, the main site of haloperidol action, did not differ between the two genotypes. Fyn activation and enhanced tyrosine phosphorylation of the NMDA receptor NR2B subunit, as measured by Western blotting, were induced after haloperidol injection of the control mice, but both responses were significantly reduced in Fyn-deficient mice. Dopamine D2 receptor blockade was shown to increase both NR2B phosphorylation and the NMDA-induced calcium responses in control cultured striatal neurons but not in Fyn-deficient neurons. Based on these findings, we proposed a new molecular mechanism underlying haloperidol-induced catalepsy, in which the dopamine D2 receptor antagonist induces striatal Fyn activation and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing the behavioral changes that are manifested as a cataleptic response.

Novel variants of the SHANK3 gene in Japanese autistic patients with severe delayed speech development.

The 22q13.3 deletion syndrome is characterized by a significant delay in language development, mental retardation, hypotonia, and autistic features. Cumulative evidence has shown that haploinsufficiency of the SHANK3 gene is a major cause of the neurological symptoms of the 22q13.3 deletion syndrome. Shank3, a multidomain protein containing the SH3 and PDZ domains, is thought to play an important role in the formation and function of synapses in the developing brain. In this study, we analyzed the SHANK3 gene in 128 autistic patients with manifestations similar to those seen in the 22q13.3 deletion syndrome. The results showed a 6-amino acid deletion upstream of the SH3 domain, a missense variant (arginine to histidine at amino acid position 656) in the PDZ domain, and the insertion or deletion of a repeated 10-bp GC sequence located 9-bp downstream from the 3′ end of exon 11. None of these variants was found in 228 controls.