Caspr4 interaction with LNX2 modulates the proliferation and neuronal differentiation of mouse neural progenitor cells.

Contactin-associated protein 4 (Caspr4), also known as contactin-associated protein-like protein (CNTNAP4), is expressed in various regions of the brain. Recent reports suggest that CNTNAP4 is a susceptibility gene of autism spectrum disorders (ASDs). However, the molecular function of Caspr4 in the brain has yet to be identified. In this study, we show an essential role of Caspr4 in neural progenitor cells (NPCs). Caspr4 is expressed in NPCs in the subventricular zone (SVZ), a neurogenic region in the developing cortex. Knocking down of Caspr4 enhances the proliferation of NPCs derived from the SVZ of embryonic day 14 mouse. Neuronal differentiation is increased by overexpression of Caspr4, but decreased by knocking down of Caspr4 in cultured mouse NPCs. Transfection of the intracellular domain of Caspr4 (C4ICD) rescues the abnormal decreased neuronal differentiation of Caspr4-knocking down NPCs. Ligand of Numb protein X2 (LNX2), a binding partner of Numb, interacts with Caspr4 in a PDZ domain-dependent manner and plays a similar role to Caspr4 in NPCs. Moreover, transfection of LNX2 rescues the decreased neuronal differentiation in Caspr4-knocking down NPCs. In contrast, transfection of C4ICD fails to do so in LNX2-knocking down NPCs. These results indicate that Caspr4 inhibits neuronal differentiation in a LNX-dependent manner. Therefore, this study reveals a novel role of Caspr4 through LNX2 in NPCs, which may link to the pathogenesis of ASDs.

Characterization of the testis-specific promoter region in the human pituitary adenylate cyclase-activating polypeptide (PACAP) gene.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide localized in the testis at concentration comparable to that found in the brain, suggesting involvement in spermatogenesis. In this study, we identified the human PACAP testis-specific exon (TSE) 10.9 kb upstream from the translational start site and found that the testis-specific transcript of the human PACAP gene was found to be spliced from the TSE into a region of intron 2 without a frameshift. The resulting PACAP precursor has no signal peptide, suggesting that PACAP functions physiologically in an intracrine manner in the testis. The 5'-flanking region of the TSE contains an 80-bp fragment with potent promoter activity in testicular F9 cell. Electrophoresis mobility shift assays showed that proteins from the F9 nuclear extract interacted specifically with the 80-bp fragment. DNA affinity chromatography allowed isolation of the specific proteins bound to the 80-bp fragment, two of which were identified as Poly (ADP-ribose) polymerase-1 (PARP-1) and TIA-1-related protein (TIAR) by mass spectrometry. By using their siRNAs, the depletion of their proteins in F9 cells affected the potent promoter activity of the 80-bp fragment, suggesting that they might be involved in the testis-specific gene expression of PACAP.

Contactin-associated protein (Caspr) 2 interacts with carboxypeptidase E in the CNS.

To identify proteins interacting with the intracellular domain of the neural cell adhesion molecule contactin-associated protein 2 (Caspr2), yeast two-hybrid screening was performed. We identified carboxypeptidase E (CPE) as a Caspr2-interacting candidate protein. Glutathione S-transferase pull-down and immunoprecipitation analyses indicated that Caspr2 was associated with CPE in vitro and in vivo. Both Caspr2 and CPE were expressed predominantly in the CNS. Immunohistochemical analyses revealed that both Caspr2- and CPE-like immunoreactivities were found to co-localize in the apical dendrites and cell bodies of rat cortical neurons. In subcellular localization analysis, Caspr2- and CPE-like immunoreactivities were co-migrated in the fractions of Golgi/ER. Additionally, in COS-7 cells co-transfected with CPE and Caspr2 cDNAs, Caspr2- and CPE-immunoreactivities were co-localized in both Golgi and membrane, whereas it was only observed in Golgi of either COS-7 cell transfected with CPE or Caspr2 cDNA alone. It is known that the membrane-bound form of CPE functions as a sorting receptor of prohormones in the trans-Golgi network. Taken together, our data suggest that CPE may be a key molecule to regulate Caspr2 trafficking to the cell membrane.

Up-regulation of matrix metalloproteinase-3 in the dorsal root ganglion of rats with paclitaxel-induced neuropathy.

Paclitaxel-induced painful peripheral neuropathy is a major dose-limiting factor. Recently, it has been reported that macrophages accumulated in the dorsal root ganglion of paclitaxel-treated rats, and their activation is suggested to contribute to generation and development of the neuropathy. However, the mechanism for macrophage activation is still unknown. In this study, to explore candidate genes involved in the mechanism for macrophage activation in the dorsal root ganglion of paclitaxel-treated rats, we developed model rats for paclitaxel-induced neuropathic pain and performed a microarray assay to analyze the changes of gene expressions in the dorsal root ganglion. Among the genes with changed expression levels, we focused on matrix metalloproteinase-3 (MMP-3, stromelysin-1) and CD163, a macrophage marker. By reverse transcription-polymerase chain reaction, the expression levels of MMP-3 and CD163 were markedly up-regulated in paclitaxel-treated dorsal root ganglion. As a result of immunohistochemical study, large ganglion neurons, but neither Schwann cells nor macrophages, predominantly expressed MMP-3. This MMP-3 up-regulation occurred prior to macrophage accumulation in the dorsal root ganglion. In addition, recombinant MMP-3 led to the activation of RAW264 macrophages in vitro. Taken together, the up-regulation of MMP-3 and following macrophage activation caused in the dorsal root ganglion might be a significant event to trigger a series of reactions developing paclitaxel-induced peripheral neuropathic pain.

A TAG1-APP signalling pathway through Fe65 negatively modulates neurogenesis.

The release of amyloid precursor protein (APP) intracellular domain (AICD) may be triggered by extracellular cues through gamma-secretase-dependent cleavage. AICD binds to Fe65, which may have a role in AICD-dependent signalling; however, the functional ligand has not been characterized. In this study, we have identified TAG1 as a functional ligand of APP. We found that, through an extracellular interaction with APP, TAG1 increased AICD release and triggered Fe65-dependent activity in a gamma-secretase-dependent manner. TAG1, APP and Fe65 colocalized in the neural stem cell niche of the fetal ventricular zone. Neural precursor cells from TAG1-/-, APP-/- and TAG1-/-;APP-/- mice had aberrantly enhanced neurogenesis, which was significantly reversed in TAG1-/- mice by TAG1 or AICD but not by AICD mutated at the Fe65 binding site. Notably, TAG1 reduced normal neurogenesis in Fe65+/+ mice. Abnormally enhanced neurogenesis also occurred in Fe65-/- mice but could not be reversed by TAG1. These results describe a TAG1-APP signalling pathway that negatively modulates neurogenesis through Fe65.

The small heat shock protein alphaB-crystallin inhibits differentiation-induced caspase 3 activation and myogenic differentiation.

Myoblasts respond to growth factor deprivation either by diffentiation into multinucleated myotubes or by undergoing apoptosis. The induction of apoptosis and differentiation in myogenic lineage may use overlapping cellular mechanisms. Here we demonstrate that the expression of the small heat shock protein alphaB-crystallin as well as MyoD and myogenin is induced during myogenic differentiation in C2C12 cells, and these inductions occur at an early stage in the differentiation in vitro. To investigate the effect of alphaB-crystallin on myogenic differentiation and apoptosis, C2C12 cells were infected with adenovirus vector bearing full-length alphaB-crystallin cDNA. Overexpression of alphaB-crystallin in C2C12 cells suppressed differentiation-induced apoptosis and activation of caspase 3, and also decreased the expression of MyoD and myogenin during myogenic differentiation of C2C12 cells induced by the differentiation medium. Our findings suggest that stress such as growth factor deprivation plays an important role in triggering apoptosis associated with myogenic differentiation and alphaB-crystallin suppressed the differentiation, apoptosis and caspase 3 activity.