Multisite studies for optimization of a highly efficient culture assay used for in vitro detection of residual undifferentiated human pluripotent stem cells intermingled in cell therapy products.

Introduction: MEASURE2 (Multisite Evaluation Study on Analytical Methods for Non-clinical Safety Assessment of HUman-derived REgenerative Medical Products 2) is a Japanese experimental public-private partnership initiative that aims to standardize testing methods for tumorigenicity evaluation of human pluripotent stem cell (hPSC)-derived cell therapy products (CTPs). MEASURE2 organized multisite studies to optimize the methodology of the highly efficient culture (HEC) assay, a sensitive culture-based in vitro assay for detecting residual undifferentiated hPSCs in CTPs. Methods: In these multisite studies, 1) the efficiency of colony formation by human induced pluripotent stem cells (hiPSCs) under two different culture conditions and 2) the sorting efficiency of microbeads conjugated to various anti-hPSC markers during hiPSC enrichment were evaluated using samples in which hiPSCs were spiked into hiPSC-derived mesenchymal stem cells. Results: The efficiency of colony formation was significantly higher under culture conditions with the combination of Chroman 1, Emricasan, Polyamines, and Trans-ISRIB (CEPT) than with Y-27632, which is widely used for the survival of hPSCs. Between-laboratory variance was also smaller under the condition with CEPT than with Y-27632. The sorting efficiency of microbeads conjugated with the anti-Tra-1-60 antibody was sufficiently higher (>80%) than those of the other various microbeads investigated. Conclusions: Results of these multisite studies are expected to contribute to improvements in the sensitivity and robustness of the HEC assay, as well as to the future standardization of the tumorigenicity risk assessment of hPSC-derived CTPs.

Protocol improvement and multisite validation of a digital soft agar colony formation assay for tumorigenic transformed cells intermingled in cell therapy products.

BACKGROUND AIMS: The administration of human cell-processed therapeutic products (hCTPs) is associated with a risk of tumorigenesis due to the transformed cellular contaminants. To mitigate this risk, these impurities should be detected using sensitive and validated assays. The digital soft agar colony formation (D-SAC) assay is an ultrasensitive in vitro test for detecting tumorigenic transformed cells in hCTPs. METHODS: In this study, we first evaluated the colony formation efficiency (CFE) precision of tumorigenic reference cells in positive control samples according to a previously reported D-SAC assay protocol (Protocol I) from multiple laboratories. However, the CFE varied widely among laboratories. Thus, we improved and optimized the test protocol as Protocol II to reduce variability in the CFE of tumorigenic reference cells. Subsequently, the improved protocol was validated at multiple sites. Human mesenchymal stromal cells (hMSCs) were used as model cells, and positive control samples were prepared by spiking them with HeLa cells. RESULTS: Based on the previously reported protocol, the CFE was estimated using an ultra-low concentration (0.0001%) of positive control samples in multiple plates. Next, we improved the protocol to reduce the CFE variability. Based on the CFE results, we estimated the sample size as the number of wells (Protocol II) and assessed the detectability of 0.0001% HeLa cells in hMSCs to validate the protocol at multiple sites. Using Protocol I yielded low CFEs (mean: 30%) and high variability between laboratories (reproducibility coefficient of variance [CV]: 72%). In contrast, Protocol II, which incorporated a relatively high concentration (0.002%) of HeLa cells in the positive control samples, resulted in higher CFE values (mean: 63%) and lower variability (reproducibility CV: 18%). Moreover, the sample sizes for testing were estimated as the number of wells per laboratory (314-570 wells) based on the laboratory-specific CFE (42-76%). Under these conditions, all laboratories achieved a detection limit of 0.0001% HeLa cells in hMSCs in a predetermined number of wells. Moreover, colony formation was not observed in the wells seeded with hMSCs alone. CONCLUSIONS: The D-SAC assay is a highly sensitive and robust test for detecting malignant cells as impurities in hCTPs. In addition, optimal assay conditions were established to test tumorigenic impurities in hCTPs with high sensitivity and an arbitrary false negative rate.

Single-Cell RNA-Seq Reveals LRRC75A-Expressing Cell Population Involved in VEGF Secretion of Multipotent Mesenchymal Stromal/Stem Cells Under Ischemia.

Human multipotent mesenchymal stromal/stem cells (MSCs) have been utilized in cell therapy for various diseases and their clinical applications are expected to increase in the future. However, the variation in MSC-based product quality due to the MSC heterogeneity has resulted in significant constraints in the clinical utility of MSCs. Therefore, we hypothesized that it might be important to identify and ensure/enrich suitable cell subpopulations for therapies using MSC-based products. In this study, we aimed to identify functional cell subpopulations to predict the efficacy of angiogenic therapy using bone marrow-derived MSCs (BM-MSCs). To assess its angiogenic potency, we observed various levels of vascular endothelial growth factor (VEGF) secretion among 11 donor-derived BM-MSC lines under in vitro ischemic culture conditions. Next, by clarifying the heterogeneity of BM-MSCs using single-cell RNA-sequencing analysis, we identified a functional cell subpopulation that contributed to the overall VEGF production in BM-MSC lines under ischemic conditions. We also found that leucine-rich repeat-containing 75A (LRRC75A) was more highly expressed in this cell subpopulation than in the others. Importantly, knockdown of LRRC75A using small interfering RNA resulted in significant inhibition of VEGF secretion in ischemic BM-MSCs, indicating that LRRC75A regulates VEGF secretion under ischemic conditions. Therefore, LRRC75A may be a useful biomarker to identify cell subpopulations that contribute to the angiogenic effects of BM-MSCs. Our work provides evidence that a strategy based on single-cell transcriptome profiles is effective for identifying functional cell subpopulations in heterogeneous MSC-based products.

International evaluation study of a highly efficient culture assay for detection of residual human pluripotent stem cells in cell therapies.

Aim & methods: The Health and Environmental Sciences Institute Cell Therapy-TRAcking, Circulation & Safety Technical Committee launched an international, multisite study to evaluate the sensitivity and reproducibility of the highly efficient culture (HEC) assay, an in vitro assay to detect residual undifferentiated human pluripotent stem cells (hPSCs) in cell therapy products. Results: All facilities detected colonies of human induced pluripotent stem cells (hiPSCs) when five hiPSCs were spiked into 1 million hiPSC-derived cardiomyocytes. Spiking with a trace amount of hiPSCs revealed that repeatability accounts for the majority of reproducibility while the true positive rate was high. Conclusion: The results indicate that the HEC assay is highly sensitive and robust and can be generally applicable for tumorigenicity evaluation of hPSC-derived cell therapy products.

Evaluation of the reproducibility and positive controls of cellular immortality test for the detection of immortalized cellular impurities in human cell-processed therapeutic products.

Introduction: Contamination of human cell-processed therapeutic products (hCTPs) with tumorigenic/immortalized cellular impurities is a major concern in the manufacturing and quality control of hCTPs. The cellular immortality test based on cell growth analysis is a method for detecting tumorigenic/immortalized cellular impurities in hCTPs. However, the performance of the cellular immortality test has not yet been well characterized. In this study, we examined the reproducibility of the cellular immortality test in detecting HeLa cells as a model of tumorigenic cellular impurities, as well as the applicability of other models of cellular impurities with different tumorigenicity to the cellular immortality test. Methods: Using HeLa cells as a model for cellular impurities, we measured the growth rate of human mesenchymal stem cells (hMSCs) supplemented with HeLa cells at concentrations ranging from 0.01 to 0.0001% at each passage in three laboratories and evaluated the reproducibility of the detection of immortalized cellular impurities. In addition, HEK293 cells (another immortalized cell line) and MRC-5 cells (a non-immortalized cell line) were employed as cellular impurity models that exhibit different growth characteristics from HeLa cells, and the ability of the cellular immortality test to detect these different impurities when mixed with hMSCs was examined. Results: In the multisite study, the growth rate of hMSCs supplemented with 1 and 10 HeLa cells (0.0001% and 0.001%) significantly increased and reached a plateau in all three laboratories, whereas those of hMSCs alone eventually decreased. Moreover, when hMSCs were supplemented with 10 and 100 HEK293 and MRC-5 cells (0.001% and 0.01%), the growth rate significantly increased. The growth rate of hMSCs supplemented with HEK293 cells increased with passage and remained high, whereas that of hMSCs supplemented with MRC-5 cells eventually decreased, as in the case of hMSCs alone. Conclusions: These results indicate that the cellular immortality test is reproducible and can detect immortalized (i.e., potentially tumorigenic) cells such as HEK293 cells with a lower growth rate than HeLa cells by discriminating against normal cells, which could contribute to ensuring the safety and quality of hCTPs.

Multisite studies for validation and improvement of a highly efficient culture assay for detection of undifferentiated human pluripotent stem cells intermingled in cell therapy products.

BACKGROUND AIMS: The Multisite Evaluation Study on Analytical Methods for Non-Clinical Safety Assessment of Human-Derived Regenerative Medical Products (MEASURE) is a Japanese experimental public-private partnership initiative, which aims to standardize methodology for tumorigenicity evaluation of human pluripotent stem cell (hPSC)-derived cell therapy products (CTPs). Undifferentiated hPSCs possess tumorigenic potential, and thus residual undifferentiated hPSCs are one of the major hazards for the risk of tumor formation from hPSC-derived CTPs. Among currently available assays, a highly efficient culture (HEC) assay is reported to be one of the most sensitive for the detection of residual undifferentiated hPSCs. METHODS: MEASURE first validated the detection sensitivity of HEC assay and then investigated the feasibility of magnetic-activated cell sorting (MACS) to improve sensitivity. RESULTS: The multisite experiments confirmed that the lower limit of detection under various conditions to which the human induced pluripotent stem cell lines and culture medium/substrate were subjected was 0.001%. In addition, MACS concentrated cells expressing undifferentiated cell markers and consequently achieved a detection sensitivity of 0.00002%. CONCLUSIONS: These results indicate that HEC assay is highly sensitive and robust and that the application of MACS on this assay is a promising tool for further mitigation of the potential tumorigenicity risk of hPSC-derived CTPs.

Tumorigenicity-associated characteristics of human iPS cell lines.

Human induced pluripotent stem cells (hiPSCs) represent promising raw materials of human cell-based therapeutic products (hCTPs). As undifferentiated hiPSCs exhibit intrinsic tumorigenicity properties that enable them to form teratomas, hCTPs containing residual undifferentiated hiPSCs may cause tumor formation following transplantation. We first established quantitative and sensitive tumorigenicity testing of hiPSCs dissociated into single cells using NOD/Shi-scid IL2Rγnull (NOG) mice by inhibiting apoptosis of hiPSCs with a Rho kinase inhibitor. To examine different features in tumorigenicity of various hiPSCs, 10 commonly available hiPSC lines were subjected to in vivo tumorigenicity testing. Transplanted hiPSC lines showed remarkable variation in tumor incidence, formation latency, and volumes. Most of the tumors formed were classified as immature teratomas. However, no signs of malignancies, such as carcinoma and sarcoma, were recognized in the tumors. Characteristics associated tumorigenicity of hiPSCs were investigated with microarray analysis, karyotype analysis, and whole exome sequencing. Gene expression profiling and pathway analysis supported different features of hiPSC lines in tumorigenicity. hiPSC lines showed chromosomal abnormalities in some lines and 61-77 variants of cancer-related genes carrying effective nonsynonymous mutations, which were confirmed in the COSMIC databases. In this study, the chromosomal abnormalities and cancer-related gene mutations observed in hiPSC lines did not lead to the malignancy of tumors derived from hiPSCs. Our results suggest that the potential tumorigenicity risk of hCTPs containing residual undifferentiated hiPSCs is dependent on not only amounts of undifferentiated hiPSCs but also features of the cell lines used as raw materials, a finding that should be considered from the perspective of quality of hCTPs used.

Identification of a Gene Encoding Slow Skeletal Muscle Troponin T as a Novel Marker for Immortalization of Retinal Pigment Epithelial Cells.

Human pluripotent stem cells (hPSCs) are leading candidate raw materials for cell-based therapeutic products (CTPs). In the development of hPSC-derived CTPs, it is imperative to ensure that they do not form tumors after transplantation for safety reasons. Because cellular immortalization is a landmark of malignant transformation and a common feature of cancer cells, we aimed to develop an in vitro assay for detecting immortalized cells in CTPs. We employed retinal pigment epithelial (RPE) cells as a model of hPSC-derived products and identified a gene encoding slow skeletal muscle troponin T (TNNT1) as a novel marker of immortalized RPE cells by comprehensive microarray analysis. TNNT1 mRNA was commonly upregulated in immortalized RPE cells and human induced pluripotent stem cells (hiPSCs), which have self-renewal ability. Additionally, we demonstrated that TNNT1 mRNA expression is higher in several cancer tissues than in normal tissues. Furthermore, stable expression of TNNT1 in ARPE-19 cells affected actin filament organization and enhanced their migration ability. Finally, we established a simple and rapid qRT-PCR assay targeting TNNT1 transcripts that detected as low as 3% of ARPE-19 cells contained in normal primary RPE cells. Purified hiPSC-derived RPE cells showed TNNT1 expression levels below the detection limit determined with primary RPE cells. Our qRT-PCR method is expected to greatly contribute to process validation and quality control of CTPs.

Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation.

Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process.

Characterization of in vivo tumorigenicity tests using severe immunodeficient NOD/Shi-scid IL2Rγnull mice for detection of tumorigenic cellular impurities in human cell-processed therapeutic products.

The contamination of human cell-processed therapeutic products (hCTPs) with tumorigenic cells is one of the major concerns in the manufacturing and quality control of hCTPs. However, no quantitative method for detecting the tumorigenic cellular impurities is currently standardized. NOD/Shi-scid IL2Rγnull (NOG) mice have shown high xeno-engraftment potential compared with other well-known immunodeficient strains, e.g. nude mice. Hypothesizing that tumorigenicity test using NOG mice could be a sensitive and quantitative method to detect a small amount of tumorigenic cells in hCTPs, we examined tumor formation after subcutaneous transplantation of HeLa cells, as a model of tumorigenic cells, in NOG mice and nude mice. Sixteen weeks after inoculation, the 50% tumor-producing dose (TPD50) values of HeLa cells were stable at 1.3 × 104 and 4.0 × 105 cells in NOG and nude mice, respectively, indicating a 30-fold higher sensitivity of NOG mice compared to that of nude mice. Transplanting HeLa cells embedded with Matrigel in NOG mice further decreased the TPD50 value to 7.9 × 10 cells, leading to a 5000-fold higher sensitivity, compared with that of nude mice. Additionally, when HeLa cells were mixed with 106 or 107 human mesenchymal stem cells as well as Matrigel, the TPD50 values in NOG mice were comparable to those of HeLa cells alone with Matrigel. These results suggest that the in vivo tumorigenicity test using NOG mice with Matrigel is a highly sensitive and quantitative method to detect a trace amount of tumorigenic cellular impurities in human somatic cells, which can be useful in the quality assessment of hCTPs.

Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are leading candidate cells as raw materials for cell therapy products, because of their capacity for pluripotent differentiation and unlimited self-renewal. hPSC-derived products have already entered the scope of clinical application. However, the assessment and control of their tumorigenicity remains to be a critical challenge. Sensitive detection of the pluripotent cellular impurities is necessary for the safety and quality control of the hPSC-derived products. In the present study, we established a sensitive assay for detection of the residual undifferentiated hiPSCs in cardiomyocytes, using droplet digital PCR (ddPCR). The ddPCR method with a probe and primers for LIN28 significantly detected as low as 0.001% undifferentiated hiPSCs in primary cardiomyocytes, which is equivalent to the ratio of a single hiPSC to 1 × 105 cardiomyocytes. The ddPCR also showed that LIN28 expression is extremely low in human tissues including liver, heart, pancreas, kidney, spinal cord, corneal epithelium and lung. These results suggest that the ddPCR method targeting LIN28 transcripts is highly sensitive and useful for the quality assessment of various cell therapy products derived from hPSCs.

Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells.

Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs). These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay): soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×104 RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research.

The atypical Guanine-nucleotide exchange factor, dock7, negatively regulates schwann cell differentiation and myelination.

In development of the peripheral nervous system, Schwann cells proliferate, migrate, and ultimately differentiate to form myelin sheath. In all of the myelination stages, Schwann cells continuously undergo morphological changes; however, little is known about their underlying molecular mechanisms. We previously cloned the dock7 gene encoding the atypical Rho family guanine-nucleotide exchange factor (GEF) and reported the positive role of Dock7, the target Rho GTPases Rac/Cdc42, and the downstream c-Jun N-terminal kinase in Schwann cell migration (Yamauchi et al., 2008). We investigated the role of Dock7 in Schwann cell differentiation and myelination. Knockdown of Dock7 by the specific small interfering (si)RNA in primary Schwann cells promotes dibutyryl cAMP-induced morphological differentiation, indicating the negative role of Dock7 in Schwann cell differentiation. It also results in a shorter duration of activation of Rac/Cdc42 and JNK, which is the negative regulator of myelination, and the earlier activation of Rho and Rho-kinase, which is the positive regulator of myelination. To obtain the in vivo evidence, we generated Dock7 short hairpin (sh)RNA transgenic mice. They exhibited a decreased expression of Dock7 in the sciatic nerves and enhanced myelin thickness, consistent with in vitro observation. The effects of the in vivo knockdown on the signals to Rho GTPases are similar to those of the in vitro knockdown. Collectively, the signaling through Dock7 negatively regulates Schwann cell differentiation and the onset of myelination, demonstrating the unexpected role of Dock7 in the interplay between Schwann cell migration and myelination.

Developmental expression of sorting nexin 3 in the mouse central nervous system.

We previously reported that sorting nexin 3 (SNX3), a protein belonging to the sorting nexin family, regulates neurite outgrowth in mouse N1E-115 neuroblastoma cells. The snx3 gene is disrupted in patients with microcephaly, microphthalmia, ectrodactyly, and prognathism (MMEP) and mental retardation, demonstrating that SNX3 plays an important role in the genesis of these organs during development. The present study was designed to determine the expression pattern of snx3 mRNA, particularly in the mouse central nervous system (CNS), from the embryonic stage to adulthood. Whole mount in situ hybridization of embryonic day (E) 9.5 and 10.5 mouse embryos revealed strong positive signals for snx3 mRNA in the forebrain, pharyngeal arches, eyes, and limb buds. In situ hybridization analyses of embryonic and neonatal brain sections revealed that snx3 mRNA is mainly expressed in the cerebral cortex, hippocampus, piriform cortex, cerebellum, and spinal cord. In adulthood, the expression of snx3 mRNA is observed in the cerebral cortex, hippocampus, piriform cortex, and cerebellar neurons. Thus, snx3 mRNA is expressed during neural development and in adult neural tissues, suggesting that SNX3 may play an important role in the development and function of the CNS.

Evaluation of drug toxicity with hepatocytes cultured in a micro-space cell culture system.

A micro-space cell culture system was recently developed in which cells such as hepatocytes can be cultured and formed into a multicellular three-dimensional (3D) architecture. In this study, we assessed the performance of HepG2 cells cultured in this micro-space cell culture system in a drug toxicity test, and evaluated the effects of micro-space culture on their hepatocyte-specific functions. The micro-space cell culture facilitated the formation of 3D HepG2 cell architecture. HepG2 cells cultured in a micro-space culture plate exhibited increased albumin secretion and enhanced mRNA expression levels of cytochrome P450 (CYP) enzyme compared to those cultured in a monolayer culture. When the cells were exposed to acetaminophen, a hepatotoxic drug, the damage to the HepG2 cells grown in micro-space culture was greater than the damage to the HepG2 cells grown in monolayer culture. In addition, human primary hepatocytes grown in micro-space culture also exhibited increased albumin secretion, enhanced CYP mRNA expression levels and increased sensitivity to acetaminophen compared to those grown in monolayer culture. These results suggest that this micro-space culture method enhances the hepatocyte-specific functions of hepatocytes, including drug-metabolizing enzyme activities, making hepatocytes grown in the micro-space culture system a useful tool for evaluating drug toxicity in vitro.

The mood stabilizer valproic acid improves defective neurite formation caused by Charcot-Marie-Tooth disease-associated mutant Rab7 through the JNK signaling pathway.

Charcot-Marie-Tooth (CMT) disease is the most frequent peripheral neuropathy affecting the Schwann cells and neurons. CMT disease type 2 (CMT2) neuropathies are characterized by peripheral nerve aberrance. Four missense mutations of Rab7, a small GTPase of the Rab family involved in intracellular vesicular trafficking, are associated with the CMT2B phenotype. Despite a growing body of evidence concerning the gene structures responsible for genetically heterogenous CMT2B and other CMT2 neuropathies, little is known about the in vitro neuropathy model and how CMT2B-associated mutation-caused aberrant neuritogenesis is properly reversed. Here, we show that valproic acid (VPA), a classical mood-stabilizing drug, improves defective neurite formation in N1E-115 neuroblastoma cells regardless of which CMT2B-associated Rab7 mutant protein is expressed. The effect is mediated by c-Jun N-terminal kinase (JNK) signaling, but not by deacetylase inhibition activity of VPA itself. Furthermore, VPA has similar effects in dorsal root ganglion (DRG) neurons expressing any of the four mutant Rab7 proteins. Thus, VPA has a previously unknown potential to improve defective neuritogenesis associated with CMT2B in vitro, indicating that JNK should be a potential therapeutic target for treatments aimed at improving neuritogenesis.

Sorting nexin 3, a protein upregulated by lithium, contains a novel phosphatidylinositol-binding sequence and mediates neurite outgrowth in N1E-115 cells.

Lithium, a drug in the treatment of bipolar disorder, modulates many aspects of neuronal developmental processes such as neurogenesis, survival, and neuritogenesis. However, the underlying mechanism still remains to be understood. Here, we show that lithium upregulates the expression of sorting nexin 3 (SNX3), one of the Phox (PX) domain-containing proteins involved in endosomal sorting, and regulates neurite outgrowth in mouse N1E-115 neuroblastoma cells. The inhibition of SNX3 function by its knockdown decreases lithium-induced outgrowth of neurites. Transfection of the full-length SNX3 construct into cells facilitates the outgrowth. We also find that the C-terminus, as well as the PX domain, of SNX3 has a functional binding sequence with phosphatidylinositol monophosphates. Transfection of the C-terminal deletion mutant or only the C-terminus does not have an effect on the outgrowth. These results suggest that SNX3, a protein upregulated by lithium, is an as yet unknown regulator of neurite formation and that it contains another functional phosphatidylinositol phosphate-binding region at the C-terminus.

Both V(1A) and V(1B) vasopressin receptors deficiency result in impaired glucose tolerance.

[Arg(8)]-vasopressin (AVP) is involved in the regulation of glucose homeostasis via vasopressin V(1A) and vasopressin V(1B) receptor. Our previous studies have demonstrated that vasopressin V(1A) receptor deficient (V(1A)R(-/-)) mice exhibited hyperglycemia, vasopressin V(1B) receptor deficient (V(1B)R(-/-)) mice, in contrast, exhibited hypoglycemia with hypoinsulinemia. These findings indicate that vasopressin V(1A) receptor deficiency results in decreased insulin sensitivity, whereas vasopressin V(1B) receptor deficiency results in increased insulin sensitivity. In our previous and present studies, we used the glucose tolerance test to investigate glucose tolerance in mutant mice, lacking either the vasopressin V(1A) receptor, the vasopressin V(1B) receptor, or both receptors, that were kept on a high-fat diet. Glucose and insulin levels were lower in V(1B)R(-/-) mice than in wild type (WT) mice when both groups were fed the high-fat diet, which indicates that the insulin sensitivity of the V(1B)R(-/-) mice was enhanced. V(1A)R(-/-) mice on the high-fat diet, on the other hand, exhibited overt obesity, along with an impaired glucose tolerance, while WT mice on the high-fat diet did not. Next, in order to assess the effect of vasopressin V(1B) receptor deficiency on the development of glucose intolerance caused by vasopressin V(1A) receptor deficiency, we generated mice that were deficient for both vasopressin V(1A) receptor and vasopressin V(1B) receptor (V(1AB)R(-/-)), fed them a high-fat diet, and examined their glucose tolerances using the glucose tolerance test. Glucose tolerance was impaired in V(1AB)R(-/-) mice, suggesting that the effects of vasopressin V(1B) receptor deficiency could not influence the development of hyperglycemia promoted by vasopressin V(1A) receptor deficiency, and that blockade of both receptors could lead to impaired glucose tolerance.

Valproic acid-inducible Arl4D and cytohesin-2/ARNO, acting through the downstream Arf6, regulate neurite outgrowth in N1E-115 cells.

The mood-stabilizing agent valproic acid (VPA) potently promotes neuronal differentiation. As yet, however, little is known about the underlying molecular mechanism. Here, we show that VPA upregulates cytohesin-2 and mediates neurite outgrowth in N1E-115 neuroblastoma cells. Cytohesin-2 is the guanine-nucleotide exchange factor (GEF) for small GTPases of the Arf family; it regulates many aspects of cellular functions including morphological changes. Treatment with the specific cytohesin family inhibitor SecinH3 or knockdown of cytohesin-2 with its siRNA results in blunted induction of neurite outgrowth in N1E-115 cells. The outgrowth is specifically inhibited by siRNA knockdown of Arf6, but not by that of Arf1. Furthermore, VPA upregulates Arl4D, an Arf-like small GTPase that has recently been identified as the regulator that binds to cytohesin-2. Arl4D knockdown displays an inhibitory effect on neurite outgrowth resulting from VPA, while expression of constitutively active Arl4D induces outgrowth. We also demonstrate that the addition of cell-permeable peptide, coupling the cytohesin-2-binding region of Arl4D into cells, reduces the effect of VPA. Thus, Arl4D is a previously unknown regulator of neurite formation through cytohesin-2 and Arf6, providing another example that the functional interaction of two different small GTPases controls an important cellular function.

Neurofibromatosis 2 tumor suppressor, the gene induced by valproic acid, mediates neurite outgrowth through interaction with paxillin.

Valproic acid (VPA), the drug for bipolar disorder and epilepsy, has a potent ability to induce neuronal differentiation, yet comparatively little is presently known about the underlying mechanism. We previously demonstrated that c-Jun N-terminal kinase (JNK) phosphorylation of the focal adhesion protein paxillin mediates differentiation in N1E-115 neuroblastoma cells. Here, we show that VPA up-regulates the neurofibromatosis type 2 (NF2) tumor suppressor, merlin, to regulate neurite outgrowth through the interaction with paxillin. The inhibition of merlin function by its knockdown or expression of merlin harboring the Gln-538-to-Pro mutation, a naturally occurring NF2 missense mutation deficient in linking merlin to the actin cytoskeleton, decreases VPA-induced neurite outgrowth. Importantly, the expression of merlin by itself is not sufficient to induce neurite outgrowth, which requires co-expression with paxillin, the binding partner of merlin. In fact, the missense mutation Trp-60-to-Cys or Phe-62-to-Ser, that is deficient in binding to paxillin, reduces neurite outgrowth induced by VPA. In addition, co-expression of a paxillin construct harboring the mutation at the JNK phosphorylation site with merlin results in blunted induction of the outgrowth. We also find that the first LIM domain of paxillin is a major binding region with merlin and that expression of the isolated first LIM domain blocks the effects of VPA. Furthermore, similar findings that merlin regulates neurite outgrowth through the interaction with paxillin have been observed in several kinds of neuronal cells. These results suggest that merlin is an as yet unknown regulator of neurite outgrowth through the interaction with paxillin, providing a possibly common mechanism regulating neurite formation.