Fumio Nakamura

Protein tyrosine phosphatase δ (PTPδ) is involved in Sema3A-induced dendritic elaboration of cortical pyramidal neurons through the activation of Fyn tyrosine kinase. However, the in vivo substrates of PTPδ remain largely unknown. Phosphotyrosine proteome analysis of Ptpδ −/− mice brains from both male and female revealed that signal regulatory protein α (SIRPα) was hyperphosphorylated at the carboxy-terminal Tyr501 residue in the knock-outs. Immunohistochemistry with anti-phospho-Tyr501 SIRPα antibody showed the hyperphosphorylation of SIRPα in various regions including the olfactory nerve layer, cortex, striatum, thalamic reticular, and hypothalamic nuclei in Ptpδ −/− brain sections. These regions partially correlated with PTPδ expression. In the primary cultured wild-type (wt) mouse dorsal root ganglion neurons, Sirpα knockdown or overexpression of a cytoplasmic deleted SIRPα mutant partially blocked the Sema3A-induced growth cone collapse response. However, overexpression of nonphosphorylated mutants of SIRPα did not alter the response. This suggests that SIRPα is involved in the Sema3A-induced collapse response in an independent manner of phosphorylation and/or dephosphorylation. In the primary cultured cortical neurons, Sirpα knockdown or the overexpression of SIRPα-Tyr501Phe mutant attenuated Sema3A-induced dendritic growth. In cultured Sirpα −/− cortical neurons, re-expression of wt SIRPα, but not of SIRPα-Tyr501Phe, restored Sema3A-induced dendritic formation. In vivo analyses revealed ectopic expression of SIRPα-Tyr501Phe in cortical layer II/III pyramidal neurons with misoriented apical dendrites and attenuated basal dendrite arborization. Similar irregular cortical dendrites were observed in Sirpα −/− and Ptpδ −/− brains. Collectively, our results demonstrate that PTPδ regulates the dendritic elaboration of cortical pyramidal neurons through the dephosphorylation of SIRPα.

Collapsin response mediator proteins (CRMPs) are key for brain development and function. Here, we link CRMP1 to a neurodevelopmental disorder. We report heterozygous de novo variants in the CRMP1 gene in three unrelated individuals with muscular hypotonia, intellectual disability, and/or autism spectrum disorder. Based on in silico analysis these variants are predicted to affect the CRMP1 structure. We further analyzed the effect of the variants on the protein structure/levels and cellular processes. We showed that the human CRMP1 variants impact the oligomerization of CRMP1 proteins. Moreover, overexpression of the CRMP1 variants affect neurite outgrowth of murine cortical neurons. While altered CRMP1 levels have been reported in psychiatric diseases, genetic variants in CRMP1 gene have never been linked to human disease. We report for the first-time variants in the CRMP1 gene and emphasize its key role in brain development and function by linking directly to a human neurodevelopmental disease.

Emerging evidence suggests that neural activity contributes to tumor initiation and its acquisition of metastatic properties. More specifically, it has been reported that the sympathetic nervous system regulates tumor angiogenesis, tumor growth, and metastasis. The function of the sympathetic nervous system in primary tumors has been gradually elucidated. However, its functions in pre-metastatic environments and/or the preparation of metastatic environments far from the primary sites are still unknown. To investigate the role of the sympathetic nervous system in pre-metastatic environments, we performed chemical sympathectomy using 6-OHDA in mice and observed a decrease in lung metastasis by attenuating the recruitment of myeloid-derived suppressor cells. Furthermore, we note that neuro-immune cell interactions could be observed in tumor-bearing mouse lungs in conjunction with the decreased expression of Sema3A. These data indicate that the sympathetic nervous system contributes to the preparation of pre-metastatic microenvironments in the lungs, which are mediated by neuro-immune cell interactions.

Collapsing response mediator proteins (CRMPs) are key for brain development and function. Here, we link CRMP1 to a neurodevelopmental disorder. We report heterozygous de novo variants in the CRMP1 gene in three unrelated individuals with muscular hypotonia, intellectual disability and/or autism spectrum disorder. Based on in silico analysis these variants are predicted to affect the CRMP1 structure. We further analyzed the effect of the variants on the protein structure/levels and cellular processes. We showed that the human CRMP1 variants are dominant-negative and impact the oligomerization of CRMP1 proteins. Moreover, overexpression of mutant- CRMP1 variants affect neurite outgrowth of murine cortical neurons. While altered CRMP1 levels have been reported in psychiatric diseases, genetic mutation in CRMP1 gene has never been linked to human disease. We report for the first-time mutations in the CRMP1 gene and emphasize its key role in brain development and function by linking directly to a human neurodevelopmental disease.

3D cultured cell aggregates, including spheroids, reflect the gene expression patterns of living tissues/organs. Mass preparation of spheroids enables high-throughput drug screening (HTS). However, conventional optical imaging of spheroids makes it difficult to obtain sufficient resolution of individual living cells in the thick cellular stack. Rapid and accurate assessment of cellular responses in spheroids is required for effective drug screening. Here, we show that negative contrast imaging (NCI) of spheroids overcomes this issue. Hydrophilic fluorescent dye added into the culture medium rapidly diffused into the intercellular space of living spheroids within a few minutes. Confocal microscopy showed the NCI of individual cells as dark and detailed contours clearly separated with fluorescence signals in the intercellular space. NCI enables the visualization of the alteration of cell morphology after anti-tumor drug application to living spheroids and the measurement of the fluorescent dye diffusion rate without any complicated pretreatments. Using this system, we found that the antitumor drug doxorubicin reduced the intercellular space of spheroids consisting of the human hepatocyte carcinoma cell line HepG2, through the activation of TGF-β signaling and upregulation of ECM protein expression, implicating a drug resistance mechanism. Collectively, the combination of NCI of spheroids and HTS may enhance the efficiency of drug discovery.

Collapsin response mediator proteins (CRMPs) have been identified as mediating proteins of repulsive axon guidance cue Semaphorin‐3A (Sema3A). Phosphorylation of CRMPs plays a crucial role in the Sema3A signaling cascade. It has been shown that Fyn phosphorylates CRMP1 at Tyrosine 504 residue (Tyr504); however, the physiological role of this phosphorylation has not been examined. We found that CRMP1 was the most strongly phosphorylated by Fyn among the five members of CRMPs. We confirmed Tyr504 phosphorylation of CRMP1 by Fyn. Immunocytochemistry of mouse dorsal root ganglion (DRG) neurons showed that phosphotyrosine signal in the growth cones was transiently increased in the growth cones upon Sema3A stimulation. Tyr504‐phosphorylated CRMP1 also tended to increase after Sema3A simulation. Ectopic expression of a single amino acid mutant of CRMP1 replacing Tyr504 with phenylalanine (CRMP1‐Tyr504Phe) suppressed Sema3A‐induced growth cone collapse response in chick DRG neurons. CRMP1‐Tyr504Phe expression in mouse hippocampal neurons also suppressed Sema3A but not Sema3F‐induced growth cone collapse response. Immunohistochemistry showed that Tyr504‐phosphorylated CRMP1 was present in the cell bodies and in the dendritic processes of mouse cortical neurons. CRMP1‐Tyr504Phe suppressed Sema3A‐induced dendritic growth of primary cultured mouse cortical neurons as well as the dendritic development of cortical pyramidal neurons in vivo. Fyn^±; Crmp1^± double heterozygous mutant mice exhibited poor development of cortical layer V basal dendrites, which was the similar phenotype observed in Sema3a^‐/‐, Fyn^‐/‐, and Crmp1^‐/‐ mice. These findings demonstrate that Tyr504 phosphorylation of CRMP1 by Fyn is an essential step of Sema3A‐regulated dendritic development of cortical pyramidal neurons. (247 words). image

Mature human erythrocytes circulate in blood for approximately 120 days, and senescent erythrocytes are removed by splenic macrophages. During this process, the cell membranes of senescent erythrocytes express phosphatidylserine, which is recognized as a signal for phagocytosis by macrophages. However, the mechanisms underlying phosphatidylserine exposure in senescent erythrocytes remain unclear. To clarify these mechanisms, we isolated senescent erythrocytes using density gradient centrifugation and applied fluorescence‐labelled lipids to investigate the flippase and scramblase activities. Senescent erythrocytes showed a decrease in flippase activity but not scramblase activity. Intracellular ATP and K⁺, the known influential factors on flippase activity, were altered in senescent erythrocytes. Furthermore, quantification by immunoblotting showed that the main flippase molecule in erythrocytes, ATP11C, was partially lost in the senescent cells. Collectively, these results suggest that multiple factors, including altered intracellular substances and reduced ATP11C levels, contribute to decreased flippase activity in senescent erythrocytes in turn to, present phosphatidylserine on their cell membrane. The present study may enable the identification of novel therapeutic approaches for anaemic states, such as those in inflammatory diseases, rheumatoid arthritis, or renal anaemia, resulting from the abnormally shortened lifespan of erythrocytes.

During development, neurons require axon guidance to the appropriate target cells. This mechanism is regulated by synaptic secretory and adhesion molecules. We have recently reported that protein tyrosine phosphatase δ (PTPδ) mediates semaphorin 3A ( Sema3A) signaling. As PTPδ possesses a sulfated glycan binding region at the N-terminus, we examined the role of this regions in Sema3A-signaling. We constructed a PTPδ mutant harbouring sulfated glycan binding region mutation (AAGAA). Alkaline-phosphatase fused PTPδ ectodomain (AP-PTPδ) bound to Sema3A receptor neuropilin-1 (NRP1) expressed in COS-7 cells. This binding was significantly attenuated in AP-PTPδ (AAGAA) mutant compared with the wild type (Wt). We next investigated Sema3A-induced growth cone collapse activity for cultured dorsal root ganglion (DRG) neurons expressed Wt or AAGAA. As we previously reported, Sema3A response was attenuated in Ptpδ-/- DRG neurons and rescued with the overexpression of Wt. In contrast, AAGAA did not rescue the phenotype. The mutant showed no alteration of the protein localization in the growth cones. These results suggest that glycan binding region of PTPδ may participate in the Sema3A-signaling through the interaction with NRP1.