Adenomatous polyposis coli 2 (APC2) is a family member of APC and mainly expressed in the nervous system. We previously reported that APC2 plays a critical role in axonal projection through the ...regulation of microtubule stability. Here, we show that a lack of Apc2 induces severe laminary defects in some regions of the mouse brain, including the cerebral cortex and cerebellum. In vivo BrdU labeling and immunohistochemical analyses with specific markers revealed that the laminary abnormalities are a result of dysregulated neuronal migration by a cell-autonomous mechanism. Using total internal reflection fluorescent microscopy, we found that APC2 is distributed along actin fibers as well as microtubules. Cerebellar granule cells in dissociated cultures and in vivo showed that BDNF-stimulated directional migration is impaired in Apc2-deficient neurons. We revealed that this impairment stems from the dysregulations of Rho family GTPase activation and TrkB localization, which disrupts the formation of BDNF-stimulated F-actin at the leading edge. Thus, APC2 is an essential mediator of the cytoskeletal regulation at leading edges in response to extracellular signals.
The direction of image motion is coded by direction-selective (DS) ganglion cells in the retina. Particularly, the ON DS ganglion cells project their axons specifically to terminal nuclei of the ...accessory optic system (AOS) responsible for optokinetic reflex (OKR). We recently generated a knock-in mouse in which SPIG1 (SPARC-related protein containing immunoglobulin domains 1)-expressing cells are visualized with GFP, and found that retinal ganglion cells projecting to the medial terminal nucleus (MTN), the principal nucleus of the AOS, are comprised of SPIG1+ and SPIG1(-) ganglion cells distributed in distinct mosaic patterns in the retina. Here we examined light responses of these two subtypes of MTN-projecting cells by targeted electrophysiological recordings. SPIG1+ and SPIG1(-) ganglion cells respond preferentially to upward motion and downward motion, respectively, in the visual field. The direction selectivity of SPIG1+ ganglion cells develops normally in dark-reared mice. The MTN neurons are activated by optokinetic stimuli only of the vertical motion as shown by Fos expression analysis. Combination of genetic labeling and conventional retrograde labeling revealed that axons of SPIG1+ and SPIG1(-) ganglion cells project to the MTN via different pathways. The axon terminals of the two subtypes are organized into discrete clusters in the MTN. These results suggest that information about upward and downward image motion transmitted by distinct ON DS cells is separately processed in the MTN, if not independently. Our findings provide insights into the neural mechanisms of OKR, how information about the direction of image motion is deciphered by the AOS.
The autophosphorylation of specific tyrosine residues occurs in the cytoplasmic region of the insulin receptor (IR) upon insulin binding, and this in turn initiates signal transduction. The R3 ...subfamily (Ptprb, Ptprh, Ptprj and Ptpro) of receptor-like protein tyrosine phosphatases (RPTPs) is characterized by an extracellular region with 6-17 fibronectin type III-like repeats and a cytoplasmic region with a single phosphatase domain. We herein identified the IR as a substrate for R3 RPTPs by using the substrate-trapping mutants of R3 RPTPs. The co-expression of R3 RPTPs with the IR in HEK293T cells suppressed insulin-induced tyrosine phosphorylation of the IR. In vitro assays using synthetic phosphopeptides revealed that R3 RPTPs preferentially dephosphorylated a particular phosphorylation site of the IR: Y960 in the juxtamembrane region and Y1146 in the activation loop. Among four R3 members, only Ptprj was co-expressed with the IR in major insulin target tissues, such as the skeletal muscle, liver and adipose tissue. Importantly, the activation of IR and Akt by insulin was enhanced, and glucose and insulin tolerance was improved in Ptprj-deficient mice. These results demonstrated Ptprj as a physiological enzyme that attenuates insulin signalling in vivo, and indicate that an inhibitor of Ptprj may be an insulin-sensitizing agent.
Protein tyrosine phosphatase receptor type Z (Ptprz/Ptpζ / RPTPβ) is a receptor-like protein tyrosine phosphatase (RPTP) which is predominantly expressed in the central nervous system. ...Tropomyosin-related kinases (Trks) are single-pass transmembrane molecules that are highly expressed in the developing nervous system. Upon the ligand binding of neurotrophins, Trk receptors are activated through autophosphorylation of tyrosine residues; however, the PTPs responsible for the negative regulation of Trk receptors have not been fully elucidated. Here, we identified Ptprz as a specific PTP that efficiently dephosphorylates TrkA as a substrate. Co-expression of Ptprz with Trk receptors in 293T cells showed that Ptprz suppresses the ligand-independent tyrosine phosphorylation of TrkA, but not of TrkB or TrkC, and that Ptprz attenuates TrkA activation induced by nerve growth factor (NGF). Co-expression analyses with TrkA mutants revealed that Ptprz dephosphorylates phosphotyrosine residues in the activation loop of the kinase domain, which are requisite for activation of the TrkA receptor. Consistent with these findings, forced expression of Ptprz in PC12D cells markedly inhibited neurite extension induced by a low dose of NGF. In addition, an increment in the tyrosine phosphorylation of TrkA was observed in the brain of Ptprz-deficient mice. Ptprz thus appears to be one of the PTPs which regulate the activation and signalling of TrkA receptors.
Sotos syndrome, characterized by intellectual disability and characteristic facial features, is caused by haploinsufficiency in the NSD1 gene. We conducted an etiological study on two siblings with ...Sotos features without mutations in NSD1 and detected a homozygous frameshift mutation in the APC2 gene by whole-exome sequencing, which resulted in the loss of function of cytoskeletal regulation in neurons. Apc2-deficient (Apc2−/−) mice exhibited impaired learning and memory abilities along with an abnormal head shape. Endogenous Apc2 expression was downregulated by the knockdown of Nsd1, indicating that APC2 is a downstream effector of NSD1 in neurons. Nsd1 knockdown in embryonic mouse brains impaired the migration and laminar positioning of cortical neurons, as observed in Apc2−/− mice, and this defect was rescued by the forced expression of Apc2. Thus, APC2 is a crucial target of NSD1, which provides an explanation for the intellectual disability associated with Sotos syndrome.
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•The APC2 gene is homozygously mutated in two siblings with Sotos syndrome features•APC2 is downstream of NSD1, the main causative gene of Sotos syndrome•The Apc2-deficient mouse is a model for Sotos syndrome
Sotos syndrome is a genetic disorder characterized by intellectual disability and distinct facial features. Almuriekhi et al. use whole-exome sequencing to identify APC2 mutations in two sibling patients with Sotos features. Apc2-deficient mice exhibit characteristic Sotos-like features. APC2 is a key target of NSD1, the primary gene responsible for Sotos syndrome.
Visual information is transmitted to the brain by roughly a dozen distinct types of retinal ganglion cells (RGCs) defined by a characteristic morphology, physiology, and central projections. However, ...our understanding about how these parallel pathways develop is still in its infancy, because few molecular markers corresponding to individual RGC types are available. Previously, we reported a secretory protein, SPIG1 (clone name; D/Bsp120I #1), preferentially expressed in the dorsal region in the developing chick retina. Here, we generated knock-in mice to visualize SPIG1-expressing cells with green fluorescent protein. We found that the mouse retina is subdivided into two distinct domains for SPIG1 expression and SPIG1 effectively marks a unique subtype of the retinal ganglion cells during the neonatal period. SPIG1-positive RGCs in the dorsotemporal domain project to the dorsal lateral geniculate nucleus (dLGN), superior colliculus, and accessory optic system (AOS). In contrast, in the remaining region, here named the pan-ventronasal domain, SPIG1-positive cells form a regular mosaic and project exclusively to the medial terminal nucleus (MTN) of the AOS that mediates the optokinetic nystagmus as early as P1. Their dendrites costratify with ON cholinergic amacrine strata in the inner plexiform layer as early as P3. These findings suggest that these SPIG1-positive cells are the ON direction selective ganglion cells (DSGCs). Moreover, the MTN-projecting cells in the pan-ventronasal domain are apparently composed of two distinct but interdependent regular mosaics depending on the presence or absence of SPIG1, indicating that they comprise two functionally distinct subtypes of the ON DSGCs. The formation of the regular mosaic appears to be commenced at the end of the prenatal stage and completed through the peak period of the cell death at P6. SPIG1 will thus serve as a useful molecular marker for future studies on the development and function of ON DSGCs.
Two winged-helix transcription factors, FoxG1 (previously called chick brain factor1, CBF1) and FoxD1 (chick brain factor2, CBF2), are expressed specifically in the nasal and temporal regions of the ...developing chick retina, respectively. We previously demonstrated that FoxG1 controls the expression of topographic molecules including FoxD1, and determines the regional specificity of the nasal retina. FoxD1 is known to prescribe temporal specificity, however, molecular mechanisms and downstream targets have not been elucidated. Here we addressed the genetic mechanisms for establishing temporal specificity in the developing retina using an in ovo electroporation technique. Fibroblast growth factor (Fgf) and Wnt first play pivotal roles in inducing the region-specific expression of FoxG1 and FoxD1 in the optic vesicle. Misexpression of FoxD1 represses the expression of FoxG1, GH6, SOHo1, and ephrin-A5, and induces that of EphA3 in the retina. GH6 and SOHo1 repress the expression of FoxD1. In contrast to the inhibitory effect of FoxG1 on bone morphogenic protein (BMP) signaling, FoxD1 does not alter the expression of BMP4 or BMP2. Studies with chimeric mutants of FoxD1 showed that FoxD1 acts as a transcription repressor in controlling its downstream targets in the retina. Taken together with previous findings, our data suggest that FoxG1 and FoxD1 are located at the top of the gene cascade for regional specification along the nasotemporal (anteroposterior) axis in the retina, and FoxD1 determines temporal specificity.
We previously identified SPARC-related protein-containing immunoglobulin domains 1 (SPIG1, also known as Follistatin-like protein 4) as one of the dorsal-retina-specific molecules expressed in the ...developing chick retina. We here demonstrated that the knockdown of SPIG1 in the retinal ganglion cells (RGCs) of developing chick embryos induced the robust ectopic branching of dorsal RGC axons and failed to form a tight terminal zone at the proper position on the tectum. The knockdown of SPIG1 in RGCs also led to enhanced axon branching in vitro. However, this was canceled by the addition of a neutralizing antibody against brain-derived neurotrophic factor (BDNF) to the culture medium. SPIG1 and BDNF were colocalized in vesicle-like structures in cells. SPIG1 bound with the proform of BDNF (proBDNF) but very weakly with mature BDNF in vitro. The expression and secretion of mature BDNF were significantly decreased when SPIG1 was exogenously expressed with BDNF in HEK293T or PC12 cells. The amount of mature BDNF proteins as well as the tyrosine phosphorylation level of the BDNF receptor, tropomyosin-related kinase B (TrkB), in the hippocampus were significantly higher in SPIG1-knockout mice than in wild-type mice. Here the spine density of CA1 pyramidal neurons was consistently increased. Together, these results suggest that SPIG1 negatively regulated BDNF maturation by binding to proBDNF, thereby suppressing axonal branching and spine formation.
Eph receptors are activated by the autophosphorylation of tyrosine residues upon the binding of their ligands, the ephrins; however, the protein tyrosine phosphatases (PTPs) responsible for the ...negative regulation of Eph receptors have not been elucidated. Here, we identified protein tyrosine phosphatase receptor type O (Ptpro) as a specific PTP that efficiently dephosphorylates both EphA and EphB receptors as substrates. Biochemical analyses revealed that Ptpro dephosphorylates a phosphotyrosine residue conserved in the juxtamembrane region, which is required for the activation and signal transmission of Eph receptors. Ptpro thus seems to moderate the amount of maximal activation of Eph receptors. Using the chick retinotectal projection system, we show that Ptpro controls the sensitivity of retinal axons to ephrins and thereby has a crucial role in the establishment of topographic projections. Our findings explain the molecular mechanism that determines the threshold of the response of Eph receptors to ephrins in vivo.
The vacuolating cytotoxin VacA produced by Helicobacter pylori causes massive cellular vacuolation in vitro and gastric tissue damage in vivo, leading to gastric ulcers, when administered ...intragastrically. Here we report that mice deficient in protein tyrosine phosphatase receptor type Z (Ptprz, also called PTP-ζ or RPTP-β, encoded by Ptprz) do not show mucosal damage by VacA, although VacA is incorporated into the gastric epithelial cells to the same extent as in wild-type mice. Primary cultures of gastric epithelial cells from Ptprz+/+ and Ptprz−/− mice also showed similar incorporation of VacA, cellular vacuolation and reduction in cellular proliferation, but only Ptprz+/+ cells showed marked detachment from a reconstituted basement membrane 24 h after treatment with VacA. VacA bound to Ptprz, and the levels of tyrosine phosphorylation of the G protein-coupled receptor kinase-interactor 1 (Git1), a Ptprz substrate, were higher after treatment with VacA, indicating that VacA behaves as a ligand for Ptprz. Furthermore, pleiotrophin (PTN), an endogenous ligand of Ptprz, also induced gastritis specifically in Ptprz+/+ mice when administered orally. Taken together, these data indicate that erroneous Ptprz signaling induces gastric ulcers.
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