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Supratentorial extraventricular ependymoma (STEE) and supratentorial intraventricular ependymoma (STIE) are two subsets of supratentorial ependymoma (SE). These two subsets have similar gene features and only differ in original sites: STEE occurs in the brain parenchyma, and STIE is located in ventricles and surrounded by cerebral spinal fluid. The present study aims to depict the diversities of these two subsets and elucidate the potential effects of the anatomic site on the tumor with the same type, grade, and molecular features. Sixty-six consecutive adult SE patients from 2008 to 2021 were enrolled in our study. Clinical data, pathological features, and long-term outcomes were analyzed retrospectively. Results demonstrated that adult STEE presented with a higher proportion of WHO grade 3 (P = .028) and higher Ki-67 index (≥10%) (P = .019) compared to adult STIE. Survival analysis demonstrated that patients of grade 3 STEE exhibited a significantly longer overall survival (OS) than patients of grade 3 STIE (median OS, 24.4 months vs. 13.0 months; P = .004). Grade 2 (hazard ratio (HR) = 0.217; P.001) and gross total resection (GTR) (HR = 0.156; P.001) were identified as favorable prognostic factors for all adult SE. The STEE was also associated with a lesser hazard of death for patients of grade 3 on multivariate analysis (HR = 0.263; P = .047). These findings suggested that the extraventricular site was an indicator for higher grade and better prognosis in adult supratentorial ependymoma.

Oxidative stress in spermatozoa is a major contributor to male subfertility, which makes it an informed choice to generate animal models of male subfertility with targeted modifications of the antioxidant systems. However, the critical male germ cell-specific antioxidant mechanisms have not been well defined yet. Here we identify LanCL1 as a major male germ cell-specific antioxidant gene, reduced expression of which is related to human male infertility. Mice deficient in LanCL1 display spermatozoal oxidative damage and impaired male fertility. Histopathological studies reveal that LanCL1-mediated antioxidant response is required for mouse testicular homeostasis, from the initiation of spermatogenesis to the maintenance of viability and functionality of male germ cells. Conversely, a mouse model expressing LanCL1 transgene is protected against high-fat-diet/obesity-induced oxidative damage and subfertility. We further show that germ cell-expressed LanCL1, in response to spermatogenic reactive oxygen species, is regulated by transcription factor specific protein 1 (SP1) during spermatogenesis. This study demonstrates a critical role for the SP1-LanCL1 axis in regulating testicular homeostasis and male fertility mediated by redox balance, and provides evidence that LanCL1 genetically modified mice have attractive applications as animal models of male subfertility.

Purpose: Glioma is a common type of malignant tumor in the brain, of which glioblastoma (GBM) is the most aggressive and lethal form. Despite current progress in the clinical treatment, the prognosis of GBM is still not satisfied, mainly due to the genomic alternations and complicated gene regulatory network. MEOX2 was originally identified as a homeobox transcriptional factor, that was reported to play a role in several types of cancers. However, the expression pattern and biological functions of MEOX2 in glioma are not well elucidated. This study aims to clarify the role of MEOX2 in glioma.Methods: Bioinformatic analysis from TCGA and CGGA databases was used to investigate the correlation of MEOX2 with glioma malignancy and clinical prognosis. The expression of MEOX2 was confirmed from clinical samples by qPCR and western blot. Lentivirus-mediated MEOX2 overexpression and knockdown were employed to investigate the biological functions of MEOX2 in glioma malignant behaviors. RNA-seq and signaling investigations were performed to reveal the molecular mechanisms.Results: Bioinformatic data from public databases and our cohort showed that MEOX2 expression was highly associated with glioma malignancy and could predict the poor prognosis. MEOX2 positively regulates glioma cell proliferation, migration/invasion and chemoresistance to temozolomide. Mechanistically, MEOX2 regulates gene profiles related to PI3K/AKT/mTORC1 pathways and thus promotes glioma malignancy.Conclusion: Our work identifies the novel function of MEOX2 in gliomas, and provides a potential target for glioma prognosis and clinical interventions.

Glioma is a common type of malignant and aggressive tumor in the brain. Despite progress on mechanistic studies, current understanding of the initiation and progression of glioma remains incomplete. GIGYF2 is a critical regulator in neural development and degeneration, however, its contribution in glioma is not yet elucidated. In this study, using an integrative approach spanning bioinformatic analysis and functional approaches, we explored the potential contribution of GIGYF2 in glioma. Bioinformatic data from public database and our cohort showed that GIGYF2 expression was closely associated with low glioma malignancy and better patient survival. Elevation of GIGYF2 expression impaired cell migration and enhanced temozolomide sensitivity of human glioma cells. We further establish its molecular mechanism by demonstrating that GIGYF2 inhibits MMP-9 mediated cell migration pathway and pro-survival AKT/Bax/Caspase-3 signaling. Our work identifies the suppressive role of GIGYF2 in gliomas, and clarifies the relationship between GIGYF2 expression and glioma malignancy, which may provide a potential target for future interventions.

Background Glioma is one of the most malignant brain tumors and accounts for the majority of brain cancer related death. Despite progress on mechanistic studies, current understandings of the initiation and progression of glioma are still incomplete. Previous studies demonstrate that Engrailed-2 (EN2), a homeobox-containing transcription factor, is associated with tumorigenesis in a range of cancers heterogeneously, however, the profiles of EN2 expression and its potential functions in gliomas remain unclear. Methods Real-time PCR was used to identify the expression of EN2 in glioma tissues. To study the biological function of EN2 in glioma, we compared the cell viability and proliferation profiles between EN2 overexpressed and control cells using cell counting kit-8 (CCK8) assay, EdU incorporation assay and colony formation assay. Flow cytometry and Hoechst staining assays were performed to investigate the role of EN2 on glioma cell death. Finally, wound healing and transwell assays were carried out to investigate the role of EN2 on glioma cell invasion. Results We identified that EN2 was downregulated in human gliomas compared with paired adjacent normal tissues and negatively associated with glioma malignancy. Elevated EN2 expression inhibits cell proliferation, enhances glioma sensitivity to temozolomide and inhibits migration/invasion of glioma cells. Conclusions Our data identify a novel function of EN2 in glioma suppression and provide potential therapeutic targets for glioma therapy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. Improving neuronal survival in ALS remains a significant challenge. Previously, we identified Lanthionine synthetase C-like protein 1 (LanCL1) as a neuronal antioxidant defense gene, the genetic deletion of which causes apoptotic neurodegeneration in the brain. Here, we report in vivo data using the transgenic SOD1G93A mouse model of ALS indicating that CNS-specific expression of LanCL1 transgene extends lifespan, delays disease onset, decelerates symptomatic progression, and improves motor performance of SOD1G93A mice. Conversely, CNS-specific deletion of LanCL1 leads to neurodegenerative phenotypes, including motor neuron loss, neuroinflammation, and oxidative damage. Analysis reveals that LanCL1 is a positive regulator of AKT activity, and LanCL1 overexpression restores the impaired AKT activity in ALS model mice. These findings indicate that LanCL1 regulates neuronal survival through an alternative mechanism, and suggest a new therapeutic target in ALS.

The metabolic coupling of Schwann cells (SCs) and peripheral axons is poorly understood. Few molecules in SCs are known to regulate axon stability. Using SC-specific Rheb knockout mice, we demonstrate that Rheb-regulated mitochondrial pyruvate metabolism is critical for SC-mediated non-cell-autonomous regulation of peripheral axon stability. Rheb knockout suppresses pyruvate dehydrogenase (PDH) activity (independently of mTORC1) and shifts pyruvate metabolism toward lactate production in SCs. The increased lactate causes age-dependent peripheral axon degeneration, affecting peripheral nerve function. Lactate, as an energy substrate and a potential signaling molecule, enhanced neuronal mitochondrial metabolism and energy production of peripheral nerves. Albeit beneficial to injured peripheral axons in the short term, we show that persistently increased lactate metabolism of neurons enhances ROS production, eventually damaging mitochondria, neuroenergetics, and axon stability. This study highlights the complex roles of lactate metabolism to peripheral axons and the importance of lactate homeostasis in preserving peripheral nerves.

Background Malignant glioma exerts a metabolic shift from oxidative phosphorylation (OXPHOs) to aerobic glycolysis, with suppressed mitochondrial functions. This phenomenon offers a proliferation advantage to tumor cells and decrease mitochondria-dependent cell death. However, the underlying mechanism for mitochondrial dysfunction in glioma is not well elucidated. MTCH2 is a mitochondrial outer membrane protein that regulates mitochondrial metabolism and related cell death. This study aims to clarify the role of MTCH2 in glioma. Methods Bioinformatic analysis from TCGA and CGGA databases were used to investigate the association of MTCH2 with glioma malignancy and clinical significance. The expression of MTCH2 was verified from clinical specimens using real-time PCR and western blots in our cohorts. siRNA-mediated MTCH2 knockdown were used to assess the biological functions of MTCH2 in glioma progression, including cell invasion and temozolomide-induced cell death. Biochemical investigations of mitochondrial and cellular signaling alternations were performed to detect the mechanism by which MTCH2 regulates glioma malignancy. Results Bioinformatic data from public database and our cohort showed that MTCH2 expression was closely associated with glioma malignancy and poor patient survival. Silencing of MTCH2 expression impaired cell migration/invasion and enhanced temozolomide sensitivity of human glioma cells. Mechanistically, MTCH2 knockdown may increase mitochondrial OXPHOs and thus oxidative damage, decreased migration/invasion pathways, and repressed pro-survival AKT signaling. Conclusion Our work establishes the relationship between MTCH2 expression and glioma malignancy, and provides a potential target for future interventions.

Background: An increasing incidence of pancreatic cancer has been observed worldwide over the last few decades. Previous reports suggested that hay fever, a common allergic disease, may function in pancreatic cancer. Data on hay fever as a risk or protective factor for pancreatic cancer was controversial in several case–control reports. So, we here did a meta-analysis on published studies to evaluate the association of hay fever and the risk of pancreatic cancer. Methods: A comprehensive literature search was performed through public databases. The association between hay fever and pancreatic cancer was evaluated by odds ratios (ORs) and 95% confidence intervals (CIs). The Cochran's Q test and I2 index were used to evaluate heterogeneity. Results: We included 8 population-based case–control studies involving 10,454 participants from 1986 to 2014. A history of hay fever was associated with a decreased risk of pancreatic cancer (OR, 0.57; 95% CI, 0.50–0.64, P < 0.00001) through fixed effect model. Conclusion: The result of our study suggested that hay fever may significantly decrease the risk of pancreatic cancer.

Neuronal activity increases energy consumption and requires balanced production to maintain neuronal function. How activity is coupled to energy production remains incompletely understood. Here, we report that Rheb regulates mitochondrial tricarboxylic acid cycle flux of acetyl-CoA by activating pyruvate dehydrogenase (PDH) to increase ATP production. Rheb is induced by synaptic activity and lactate and dynamically trafficked to the mitochondrial matrix through its interaction with Tom20. Mitochondria-localized Rheb protein is required for activity-induced PDH activation and ATP production. Cell-type-specific gain- and loss-of-function genetic models for Rheb reveal reciprocal changes in PDH phosphorylation/activity, acetyl-CoA, and ATP that are not evident with genetic or pharmacological manipulations of mTORC1. Mechanistically, Rheb physically associates with PDH phosphatase (PDP), enhancing its activity and association with the catalytic E1α-subunit of PDH to reduce PDH phosphorylation and increase its activity. Findings identify Rheb as a nodal point that balances neuronal activity and neuroenergetics via Rheb-PDH axis.

BACKGROUND Malignant glioma display a metabolic shift towards aerobic glycolysis with reprogramming of mitochondrial oxidative phosphorylation (OXPHOs). However, the underlying mechanism for this metabolic switch in glioma is not well elucidated. Mitochondrial translocases of the outer/inner membrane (TOMs/TIMs) import proteins into mitochondria, and could thereby regulate OXPHOs. The objective of this study is to investigate the expression of TOM/TIM members in glioma, as well as their functional and therapeutic implications. METHODS Transcriptome sequencing (RNA-seq), real-time PCR, Western blot, and immunohistochemistry were used to identify Tom20 as a significantly downregulated TOM/TIM protein in 20 paired glioma/Peritumoral tissues. To study the biological function of Tom20 in glioma, we interrogated metabolic alterations in Tom20 overexpressed glioma cells by GC-MS metabolomics, acetyl-CoA assay, and Seahorse assay. We compared the cell proliferation and viability profiles between Tom20 overexpressed and control cells in vitro and in vivo. To investigate the therapeutic implication of Tom20 expression, we tested OXPHOs inhibitor metformin in Tom20 overexpressed cells and xenograft mouse models. RESULTS We find that Tom20, a critical component of the mitochondrial outer membrane translocases, is downregulated in malignant gliomas. Using an integrative approach spanning bioinformatic analysis, metabolomics, and functional approaches, we reveal that Tom20 elevation activates mitochondrial OXPHOs in glioma cells and reduces tumor malignancy. We also find that Tom20 upregulation sensitizes glioma cells to metformin in vitro, and improves the therapeutic efficacy of metformin in glioma in vivo. CONCLUSION Our work defines Tom20 as a glioma suppressor and an indicator of metformin treatment in glioma.

Myelin Deficit of the central nervous system leads to several severe diseases in human, but lack definitive diagnostic test. In this work, we proposed and performed terahertz spectroscopy and sub-wavelength imaging combined with chemometric techniques to discriminate and evaluate the severity of myelin deficit in and rhesus monkey brains. We demonstrated THz imaging with sub-100 μm spatial resolution of brain tissues. And our spectroscopy results well match with pathological analysis, indicating that THz is a quick, powerful, evolving tool for identification and evaluation in preclinical animals and potentially in para-clinical biopsy of humans.

While myelin deficit of the central nervous system leads to several severe diseases, the definitive diagnostic means are lacking. We proposed and performed terahertz time-domain spectroscopy (THz-TDS) combined with chemometric techniques to discriminate and evaluate the severity of myelin deficit in mouse and rhesus monkey brains. The THz refractive index and absorption coefficient of paraffin-embedded brain tissues from both normal and mutant dysmyelinating mice are shown. Principal component analysis of time-domain THz signal (PCA-tdTHz) and absorption-refractive index relation of THz spectrum identified myelin deficit without exogenous labeling or any pretreatment. Further, with the established PCA-tdTHz, we evaluated the severity of myelin deficit lesions in rhesus monkey brain induced by experimental autoimmune encephalomyelitis, which is the most-studied animal model of multiple sclerosis. The results well matched the pathological analysis, indicating that PCA-tdTHz is a quick, powerful, evolving tool for identification and evaluation myelin deficit in preclinical animals and potentially in para-clinical human biopsy.

We investigated a possible role of hMLH1 hypermethylation and microsatellite instability in meningioma progression. Fifty meningomas were examined for methylation of hMLH1 using a methylation-specific PCR; 43 of them were analyzed for microsatellite instability using nine microsatellite markers. Loss of heterozygosity on chromosome 22q was detected using two markers. Two atypical meningiomas showed microsatellite instability at four loci; one was methylated on hMLH1 and the other was unmethylated. Nine meningiomas were found to have methylated hMLH1; the frequencies in the different grades of meningioma were one of 20, two of 16, and six of 14, respectively. We concluded that the methylation status of hMLH1 is associated with the meningioma grade but not with microsatellite instability. Loss of heterozygosity was detected in 22 cases in at least one marker. The frequency of loss of heterozygosity increased with meningioma grade, but the tendency was not significant. The correlation between loss of heterozygosity and methylation of the hMLH1 gene was also not significant. We conclude that hypermethylation of the promoter of hMLH1 is an epigenetic change in meningiomas and is associated with the tumor grade, while microsatellite instability is an uncommon event in meningiomas.

It is recognized that expression of AQP4 protein is much greater in gliomas than in normal tissue. The relationship between AQP4 and glioma-associated brain edema is affected by osmotic pressure and hypoxia. In this study, we detected changes of AQP4 expression in tumor and peritumoral edematous tissues to analyze the relationship between AQP4 protein and the edema index (EI). We also detected expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) to investigate their relationship with AQP4 protein, and thus to uncover the molecular biological mechanisms of AQP4 expression in glioma-associated brain edema. Sixty-five patients with brain glioma were divided into tumor and peritumor groups. Fresh tumor specimens, including six cases of grade I glioma, 18 of grade II, 11 of grade III and 30 of grade IV, and peritumoral edematous tissue specimens (1 cm distant from the tumor) were resected from these patients, and AQP4 protein expression levels were detected by western blot. Different AQP4 expression in the tumor and peritumor groups were compared. The relationship between AQP4 expression levels and the degree of peritumoral edema, and expression differences in different grades, were analyzed. Immunofluorescence cytochemistry was used to detect positive expression of AQP4 protein, VEGF protein, and HIF-1α protein in tumor tissue, and differences between expression were analyzed. Western blot showed that AQP4 expression in the peritumor (0.7697 ± 0.0941) and tumor (0.6934 ± 0.0625) groups was higher than in the control group (0.6215 ± 0.0884), and was highest in the peritumor group (both P0.01). AQP4 expression level in the peritumor group was positively correlated with EI (r = 0.677, P0.001) whereas AQP4 expression level in the tumor group was not correlated with EI (r = 0.096, P0.05). AQP4 expression increased with higher tumor grades in the peritumor group, but differences were not significant in the tumor group. Immunofluorescence cytochemical staining revealed that AQP4 protein in normal brain tissue was mainly expressed in the cell membrane surface, and that cytoplasm and nuclear staining was shallow. In glioma cells, AQP4 was widely distributed in the cytoplasm, particularly in the edematous area around the tumor. AQP4 protein expression in the tumor was significantly positively correlated with both VEGF protein (r = 0.877, P0.001) and HIF-1α protein (r = 0.876, P0.001). AQP4 expression was higher in brain tumor, especially peritumor. The degree of peritumoral edema correlates with AQP4 protein expression only in peritumor, whereas AQP4 expression is in accordance with expression of VEGF and HIF-1α. In glioma-associated brain edema, AQP4 is coregulated by osmotic pressure and hypoxia, with predominance of osmotic regulation, and is redistributed in glioma cells, mainly in the cytoplasm, and its expression level increased with higher glioma grades.

Rheb1is an immediate early gene that functions to activate mammalian target of rapamycin (mTor) selectively in complex 1 (mTORC1). We have demonstrated previously that Rheb1 is essential for myelination in the CNS using aNestin-Credriver line that deletesRheb1in all neural cell lineages, and recent studies using oligodendrocyte-specificCNP-Crehave suggested a preferential role for mTORC1 is myelination in the spinal cord. Here, we examine the role of Rheb1/mTORC1 in mouse oligodendrocyte lineage using separate Cre drivers for oligodendrocyte progenitor cells (OPCs) includingOlig1-CreandOlig2-Creas well as differentiated and mature oligodendrocytes includingCNP-Cre and Tmem10-Cre. Deletion ofRheb1in OPCs impairs their differentiation to mature oligodendrocytes. This is accompanied by reduced OPC cell-cycle exit suggesting a requirement for Rheb1 in OPC differentiation. The effect of Rheb1 on OPC differentiation is mediated by mTor sinceOlig1-Credeletion ofmTorphenocopiesOlig1-Cre Rheb1deletion. Deletion ofRheb1in mature oligodendrocytes, in contrast, does not disrupt developmental myelination or myelin maintenance. Loss ofRheb1in OPCs or neural progenitors does not affect astrocyte formation in gray and white matter, as indicated by the pan-astrocyte marker Aldh1L1. We conclude that OPC-intrinsic mTORC1 activity mediated by Rheb1 is critical for differentiation of OPCs to mature oligodendrocytes, but that mature oligodendrocytes do not require Rheb1 to make myelin or maintain it in the adult brain. These studies reveal mechanisms that may be relevant for both developmental myelination and impaired remyelination in myelin disease.

SummaryProduction of reactive oxygen species (ROS) increases with neuronal activity that accompanies synaptic development and function. Transcription-related factors and metabolic enzymes that are expressed in all tissues have been described to counteract neuronal ROS to prevent oxidative damage. Here, we describe the antioxidant gene LanCL1 that is prominently enriched in brain neurons. Its expression is developmentally regulated and induced by neuronal activity, neurotrophic factors implicated in neuronal plasticity and survival, and oxidative stress. Genetic deletion of LanCL1 causes enhanced accumulation of ROS in brain, as well as development-related lipid, protein, and DNA damage; mitochondrial dysfunction; and apoptotic neurodegeneration. LanCL1 transgene protects neurons from ROS. LanCL1 protein purified from eukaryotic cells catalyzes the formation of thioether products similar to glutathione S-transferase. These studies reveal a neuron-specific glutathione defense mechanism that is essential for neuronal function and survival.

Excessive food/energy intake is linked to obesity and metabolic disorders, such as diabetes. The hypothalamus in the brain plays a critical role in the control of food intake and peripheral metabolism. The signaling pathways in hypothalamic neurons that regulate food intake and peripheral metabolism need to be better understood for developing pharmacological interventions to manage eating behavior and obesity. Mammalian target of rapamycin (mTOR), a serine/threonine kinase, is a master regulator of cellular metabolism in different cell types. Pharmacological manipulations of mTOR complex 1 (mTORC1) activity in hypothalamic neurons alter food intake and body weight. Our previous study identified Rheb1 (Ras homolog enriched in brain 1) as an essential activator of mTORC1 activity in the brain. Here we examine whether central Rheb1 regulates food intake and peripheral metabolism through mTORC1 signaling. We find that genetic deletion of Rheb1 in the brain causes a reduction in mTORC1 activity and impairs normal food intake. As a result, Rheb1 knockout mice exhibit hypoglycemia and increased lipid mobilization in adipose tissue and ketogenesis in the liver. Our work highlights the importance of central Rheb1 signaling in euglycemia and energy homeostasis in animals.

Oligodendrocytes ensheath axons to form compact insulating multilamellar structures known as myelin. Tmem10 is a novel type I transmembrane glycoprotein that is highly expressed in oligodendrocytes and whose biological function remains largely unknown. Furthermore, the expression pattern of Tmem10 remains a matter of some controversy. Given the inconsistency of its expression pattern and the lack of validated specific antibodies, Tmem10 is not widely accepted as a marker for mature oligodendrocytes. As a means to solve these problems and to aid future studies of oligodendrocyte-associated diseases, we have generated a highly specific Tmem10 antibody. Using this Tmem10 antibody, we clarify that Tmem10 protein is firstly expressed at 2 weeks in the postnatal mouse brain with age-related increase, only in the central nervous system (CNS). We also reveal that Tmem10 is expressed specifically in late stage oligodendrocytes and later than MAG, a late-stage myelin marker. Finally, we show that Tmem10 co-expresses with MOG- and MBP-positive myelin fibers and is dramatically reduced in a hypomyelination mouse model. In conclusion, our study demonstrates that Tmem10 can be used as a specific marker for myelinating oligodendrocytes and perhaps for the evaluation of myelination diseases, such as multiple sclerosis.

Peritumoral brain edema is a common complication of meningiomas. It is believed that vascular endothelial growth factor (VEGF), as an angiogenic factor, plays a vital role in edema formation. Aquaporin-4 (AQP4) is a small integral membrane protein that regulates water in the normal brain. However, the expression of AQP4 and its relationship to VEGF in edematous meningiomas are not well known. We studied tumor specimens of 59 human supratentorial meningiomas. Western blot analysis was used to detect the expression of AQP4, and double-labeling immunofluorescence histochemical staining was performed to determine the relationship between AQP4 and VEGF. The AQP4 expression was significantly higher in the edema group, in which the protein level was correlated with the extent of edema. Greater VEGF expression was also observed in the edema group, and a relationship between AQP4 and VEGF was found. We conclude that AQP4 is involved in peritumoral brain edema formation in meningiomas and is also closely related to the expression of VEGF.

Apoptosis is one kind of programmed cell death and contributes to development of a variety of organs such as brain. PNAS4 has been reported to be a novel apoptosis-related gene. Overexpression and knocking down of PNAS4 would cause zebrafish and Xenopus lavis developmental abnormalities. But its function and apoptotic mechanism in mammals are still unknown. Here, we first reported that established PNAS4 CKO (conditional knock out) mice using recombineering technology. We prepared its polyclonal antibodies which recognized both myc-PNAS4 overexpression protein and WT and CKO mice brain tissue and MEFS cells with high titre and specificity. Further we detected that PNAS4 was highly expressed in the embryonic period. However, we observed neither neural structural abnormality nor apoptosis signal in PNAS4 CKO mice brain. Our data suggested that PNAS4 was not involved in mice brain development and apoptosis.

To study the methods for preparing the monoclonal antibody against synthetic antigen of cyclosporine A.These included using photochemical reaction to couple cyclosporine A with poly-L-lysine as synthetic antigen, immunizing BALB/c mice, fusing spleen cells and SP2/0 cells. Then hybridoma cells were repeatedly screened and subcloned. The monoclonal antibodies were identified by indirect ELISA.Four hybridoma cells in secreting anti-cyclosporine A monoclonal antibodies were obtained. Only two showed high titer and some specificity.The methods of antigen synthesis and immunization were practicable. But the methods of antibody preparation and preservation need to be improved.

SummarymTor kinase is involved in cell growth, proliferation, and differentiation. The roles of mTor activators, Rheb1 and Rheb2, have not been established in vivo. Here, we report that Rheb1, but not Rheb2, is critical for embryonic survival and mTORC1 signaling. Embryonic deletion of Rheb1 in neural progenitor cells abolishes mTORC1 signaling in developing brain and increases mTORC2 signaling. Remarkably, embryonic and early postnatal brain development appears grossly normal in these Rheb1f/f,Nes-cre mice with the notable exception of deficits of myelination. Conditional expression of Rheb1 transgene in neural progenitors increases mTORC1 activity and promotes myelination in the brain. In addition the Rheb1 transgene rescues mTORC1 signaling and hypomyelination in the Rheb1f/f,Nes-cre mice. Our study demonstrates that Rheb1 is essential for mTORC1 signaling and myelination in the brain, and suggests that mTORC1 signaling plays a role in selective cellular adaptations, rather than general cellular viability.