Expression Of NeuroD1 Research Articles

Preliminary Assessment of DNA Profile Patterns of Neuronal Differentiation Factor (NeuroD1) Gene in the Hippocampus of Wistar Rats (Rattus norvegicus) Ingested Hyoscyamine Fraction of Datura stramonium Seeds

The study was designed to find out how oral ingestion of hyoscyamine fraction of D. stramonium seeds affect the DNA profiles patterns and expression of NeuroD1 gene in the hippocampus of adult Wistar rats. Fresh D. stramonium seeds were procured, identified, macerated and fractionated. Thirty-two (32) male adult Wistar rats weighed 200-250 grams were procured and divided into four groups comprised of control and three treatment groups for the study. The control received an equivalent bodyweight of normal saline, while the treatment groups received 200, 400 and 800 mg/kgbwt of hyoscyamine fraction for three weeks respectively. The animals were sacrificed, tissues harvested for histology, gel electrophoresis and gene expression studies. The fold changes were calculated and DNA profiles were determined using LabImage bio-imaging software. One-way ANOVA followed by Fisher’s multiple comparisons post-hoc test was used to obtain the difference in the expression of the gene across the groups using Minitab 17 (LLC., U.K.) statistical package software. P < 0.05 was considered statistically significant. There was a statistically significant downregulation (p = 0.001) in the NeuroD1 expression, increased DNA fragment size, decreased DNA migration distance and band volume, and necrosis of the dentate gyrus (DG) region in the hippocampus of the treated animals. In conclusion, oral ingestion of hyoscyamine fraction of D. stramonium seeds is toxic to the DNA profile patterns and expression of the NeuroD1 gene in Wistar rats.

LncRNA SOX2OT Knockdown Alleviates Lipopolysaccharide-Induced Damage of PC12 Cells by Regulating miR-331-3p/Neurod1 Axis

Long noncoding RNAs (lncRNAs) serve as crucial regulators in the pathogenesis of spinal cord injury (SCI). However, the role of lncRNA SOX2 overlapping transcript (SOX2OT) in SCI remains to be well revealed. An SCI rat model was established and assessed by the Basso-Beattie-Bresnahan (BBB) method. An SCI PC12 cell model was established through lipopolysaccharide (LPS) treatment. Quantitative real-time polymerase chain reaction assay was used for SOX2OT, miR-331-3p, and neurogenic differentiation 1 (Neurod1) mRNA levels. Cell counting kit-8 assay and flow cytometry analysis were performed for cell viability and apoptosis, respectively. Enzyme-linked immunosorbent assay was performed for the levels of inflammatory cytokines. The production of superoxide dismutase and malondialdehyde was determined with relevant kits. Dual-luciferase reporter and RNA immunoprecipitation assays were conducted for the relationships among SOX2OT, miR-331-3p, and Neurod1. Western blot assay was employed for protein levels. SOX2OT was elevated in SCI rat and cell models. SOX2OT knockdown relieved the injury of SCI in SCI rat model. Moreover, the suppressive role in PC12 cell viability and the promotional roles in apoptosis, inflammation, and oxidative stress mediated by LPS were all restored by silencing SOX2OT. For mechanism analysis, SOX2OT was identified as a sponge of miR-331-3p to positively regulate Neurod1 expression. Inhibition of miR-331-3p reversed the effect of SOX2OT knockdown on LPS-induced PC12 damage. Overexpression of miR-331-3p protected PC12 cells from LPS-induced damage by binding to Neurod1. In addition, SOX2OT knockdown relieved PC12 cell injury by inactivation of Janus kinase-signal transducer and activator of transcription pathway. SOX2OT promoted PC12 cell injury through modulating miR-331-3p/Neurod1 axis and activating Janus kinase-signal transducer and activator of transcription pathway.

UTX/KDM6A deletion promotes the recovery of spinal cord injury by epigenetically triggering intrinsic neural regeneration

Interrupted axons that fail to regenerate mainly cause poor recovery after spinal cord injury (SCI). How neurons epigenetically respond to injury determines the intrinsic growth ability of axons. However, the mechanism underlying epigenetic regulation of axonal regeneration post-SCI remains largely unknown. In this study, we elucidated the role of the epigenetic regulatory network involving ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX)/microRNA-24 (miR-24)/NeuroD1 in axonal regeneration and functional recovery in mice following SCI. Our results showed that UTX was significantly increased post-SCI and repressed axonal regeneration in vitro. However, downregulation of UTX remarkably promoted axonal regeneration. Furthermore, miR-24 was increased post-SCI and positively regulated by UTX. miR-24 also inhibited axonal regeneration. Chromatin immunoprecipitation (ChIP) indicated that UTX binds to the miR-24 promoter and regulates miR-24 expression. Genome sequencing and bioinformatics analysis suggested that NeuroD1 is a potential downstream target of UTX/miR-24. A dual-luciferase reporter assay indicated that miR-24 binds to NeuroD1; moreover, it represses axonal regeneration by negatively regulating the expression of NeuroD1 via modulation of microtubule stability. UTX deletion in vivo prominently promoted axonal regeneration and improved functional recovery post-SCI, and silencing NeuroD1 restored UTX function. Our findings indicate that UTX could be a potential target in SCI.

MBRS-62. CURAXIN CBL0137 INHIBITS THE VIABILITY OF CANCEROUS CELLS IN PRE-CLINIC MODELS OF MYC-AMPLIFIED MEDULLOBLASTOMA

Medulloblastoma is a malignant brain tumour that mostly occurs in children, and MYC-amplified medulloblastoma is characterized by pronounced invasiveness and dismal prognosis. There is no effective treatment for Medulloblastoma and its precise pathological mechanism remains obscure. Previous studies indicated that the altered epigenetic machinery manifested in the neoplastic transformation of MYC-amplified MB has become increasingly evident. It is hypothesized that epigenetic genes dependencies associated with small molecule inhibitors that have been approved or are in advanced development may help identify the potential therapeutic targets. By integrating mRNA expression profiles and the corresponding clinic-pathologic information of patients suffering from medulloblastoma, and analysing prior CRISPR screening results, we demonstrated that SSRP1 is a negative prognostic factor that functions to stimulate the viability of MB cells. SSRP1 is a subunit of the FACT complex, an important histone chaperone required for transcriptional regulation, DNA replication and damage repair. Its biological effect on tumour proliferation was assessed by using RNA interference and administering CBL0137, a small molecular inhibitor of FACT. Gene expression analysis also demonstrated that CBL0137 selectively downregulated the expression of MYC and NEUROD1. Furthermore, the administration of CBL0137 suppressed tumour growth in mouse xenograft models. This pharmacological method to selectively target MYC transcription was demonstrated in our study, and therefore can be applied as a promising treatment strategy for MYC-amplified medulloblastoma. In Conclusion, we identified an attractive strategy of selectively downregulating MYC transcription by applying inhibitor CBL0137, thereby revealing the potential clinical utility of CBL0137 to improve the prognosis of MYC-amplified medulloblastoma patients.

PCP4/PEP19 downregulates neurite outgrowth via transcriptional regulation of Ascl1 and NeuroD1 expression in human neuroblastoma M17 cells

AbstractPurkinje cell protein 4/peptide 19 (PCP4/PEP19) is 7.6 kDa peptide originally found in Purkinje cells. PCP4/PEP19 is a differentiation maker of Purkinje cells, where it functions as an antiapoptotic factor. Cerebral neuronal cells also express PCP4/PEP19, which may be related to neuronal cell survival. However, evidence suggests that PCP4/PEP19 may also be involved in neuronal differentiation. Here, we investigated the effects of PCP4/PEP19 expression on neuronal differentiation by analyzing neurite outgrowth, and expression of neuronal differentiation markers in cultured human neuroblastoma M17 cells. When PCP4/PEP19 expression was reduced by siRNA-mediated knockdown, neurite outgrowth was significantly increased. Among many differentiation markers tested, expression of NeuroD1 was increased, while that of Ascl1 was decreased upon PCP4/PEP19 knockdown. Furthermore, luciferase reporter assays revealed that PCP4/PEP19 knockdown upregulated NeuroD1 and downregulated Ascl1 expression, at the transcriptional level. These results suggest a new function of PCP4/PEP19, which suppresses neurite outgrowth and neuronal differentiation through the regulation of NeuroD1 and Ascl1 expression in M17 cells. Furthermore, immunohistochemical studies showed that PCP4/PEP19 localizes in the nuclei of human neuroblastoma cells. Therefore, PCP4/PEP19 may also be an intranuclear negative regulator of neuronal differentiation and may thus be a potential therapeutic target to promote cellular differentiation in human neuroblastoma.PCP4/PEP19 knockdown in neuroblastoma cells induce neurite outgrowth, upregulation of NeuroD1 and downregulation of Ascl1 expression, suggesting that PCP4/PEP19 can suppress neurite outgrowth and neuronal differentiation through the regulation of NeuroD1 and Ascl1. Immunohistochemistry shows nuclear localization of PCP4/PEP19 in neuroblastoma cells. PCP4/PEP19 may therefore be an intranuclear negative regulator of neuronal differentiation.

Pathomorphology of the atypical form of congenital hyperinsulinism on the example of three clinical cases

Background . Congenital hyperinsulinism (CH) is characterized by persistent hypoglycemia, leading to irreversible damage to the cerebral cortex. The atypical form of CH (ACH) remains practically unexplored. Objective . Description of histological and immunohistochemical features of the pancreas in ACH. Design and methods . Material for research — fragments of the resected pancreas of 3 children with ACH aged 3, 6 and 7 months. Control group — pancreas of 9 children who died from heart malformations. Pancreatic tissue was stained with hematoxylin and eosin, an immunohistochemical reaction was performed with antibodies to chromogranin A, somatostatin, and transcription factors (TF) NeuroD1, Nkx2.2, and Isl1. Results . In all patients, numerous hyperplastic islets of Langerhans were observed, which in places merged with each other. In two patients, the lesion was limited to the pancreas area, and in one it covered the entire gland. All patients showed a sharp increase (p < 0.01) in the expression of NeuroD1 both in the exocrine and endocrine parts of the pancreas in the affected area: in 79.0 ± 23.4 % of endocrinocytes and in 88.0 ± 8.2 % of exocrinocytes (in the control 10.5 ± 7.9 % and 4.0 ± 4.9 % respectively). The expression of chromogranin A and TF Isl1 and Nkx2.2 increased statistically significantly (p < 0.01) only in affected endocrinocytes (chromogranin A — 62.3 ± 15.4 % of endocrinocytes, Isl1 — 82.0 ± 4.6 %, Nkx2.2 — 81.3 ± 4.6 %; in the control — 39.4 ± 7.9 %, 30.2 ± 16.2 % and 26.4 ± 11.9 % respectively). In 2 patients, there was a tendency to an increase in the number of somatostatin + endocrinocytes. Conclusion . ACH is morphologically very heterogeneous. Like other forms of CH, ACH was characterized by a statistically significant increase in the expression of chromogranin A and TF NeuroD1, Nkx2.2, Isl1, while the expression of somatostatin in general remained unchanged.

YAP1 Expression in SCLC Defines a Distinct Subtype With T-cell-Inflamed Phenotype.

The clinical and biological significance of the newly described SCLC subtypes, SCLC-A, SCLC-N, SCLC-Y, and SCLC-P, defined by the dominant expression of transcription factors ASCL1, NeuroD1, YAP1, and POU2F3, respectively, remain to be established. We generated new RNA sequencing expression data from a discovery set of 59 archival tumor samples of neuroendocrine tumors and new protein expression data by immunohistochemistry in 99 SCLC cases. We validated the findings from this discovery set in two independent validation sets consisting of RNA sequencing data generated from 51 SCLC cell lines and 81 primary human SCLC samples. We successfully classified 71.8% of SCLC and 18.5% of carcinoid cases in our discovery set into one of the four SCLC subtypes. Gene set enrichment analysis for differentially expressed genes between the SCLC survival outliers (top and bottom deciles) matched for clinically relevant prognostic factors revealed substantial up-regulation of interferon-γ response genes in long-term survivors. The SCLC-Y subtype was associated with high expression of interferon-γ response genes, highest weighted score on a validated 18-gene T-cell-inflamed gene expression profile score, and high expression of HLA and T-cell receptor genes. YAP1 protein expression was more prevalent and more intensely expressed in limited-stage versus extensive-stage SCLC (30.6% versus 8.5%; p= 0.0058) indicating good prognosis for the SCLC-Y subtype. We replicated the inflamed phenotype of SCLC-Y in the two independent validation data sets from the SCLC cell lines and tumor samples. SCLC subtyping using transcriptional signaling holds clinical relevance with the inflamed phenotype associated with the SCLC-Y subset.

Development of Neuroregenerative Gene Therapy to Reverse Glial Scar Tissue Back to Neuron-Enriched Tissue.

Injuries in the central nervous system (CNS) often causes neuronal loss and glial scar formation. We have recently demonstrated NeuroD1-mediated direct conversion of reactive glial cells into functional neurons in adult mouse brains. Here, we further investigate whether such direct glia-to-neuron conversion technology can reverse glial scar back to neural tissue in a severe stab injury model of the mouse cortex. Using an adeno-associated virus (AAV)-based gene therapy approach, we ectopically expressed a single neural transcription factor NeuroD1 in reactive astrocytes in the injured areas. We discovered that the reactive astrocytes were efficiently converted into neurons both before and after glial scar formation, and the remaining astrocytes proliferated to repopulate themselves. The astrocyte-converted neurons were highly functional, capable of firing action potentials and establishing synaptic connections with other neurons. Unexpectedly, the expression of NeuroD1 in reactive astrocytes resulted in a significant reduction of toxic A1 astrocytes, together with a significant decrease of reactive microglia and neuroinflammation. Furthermore, accompanying the regeneration of new neurons and repopulation of new astrocytes, new blood vessels emerged and blood-brain-barrier (BBB) was restored. These results demonstrate an innovative neuroregenerative gene therapy that can directly reverse glial scar back to neural tissue, opening a new avenue for brain repair after injury.

In vivo Neuroregeneration to Treat Ischemic Stroke Through NeuroD1 AAV-Based Gene Therapy in Adult Non-human Primates.

Stroke may cause severe death and disability but many clinical trials have failed in the past, partially because the lack of an effective method to regenerate new neurons after stroke. In this study, we report an in vivo neural regeneration approach through AAV NeuroD1-based gene therapy to repair damaged brains after ischemic stroke in adult non-human primates (NHPs). We demonstrate that ectopic expression of a neural transcription factor NeuroD1 in the reactive astrocytes after monkey cortical stroke can convert 90% of the infected astrocytes into neurons. Interestingly, astrocytes are not depleted in the NeuroD1-converted areas, consistent with the proliferative capability of astrocytes. Following ischemic stroke in monkey cortex, the NeuroD1-mediated astrocyte-to-neuron (AtN) conversion significantly increased local neuronal density, reduced microglia and macrophage, and surprisingly protected parvalbumin interneurons in the converted areas. Furthermore, the NeuroD1 gene therapy showed a broad time window in AtN conversion, from 10 to 30 days following ischemic stroke. The cortical astrocyte-converted neurons showed Tbr1+ cortical neuron identity, similar to our earlier findings in rodent animal models. Unexpectedly, NeuroD1 expression in converted neurons showed a significant decrease after 6 months of viral infection, indicating a downregulation of NeuroD1 after neuronal maturation in adult NHPs. These results suggest that in vivo cell conversion through NeuroD1-based gene therapy may be an effective approach to regenerate new neurons for tissue repair in adult primate brains.

RUES2 hESCs exhibit MGE-biased neuronal differentiation and muHTT-dependent defective specification hinting at SP1

RUES2 cell lines represent the first collection of isogenic human embryonic stem cells (hESCs) carrying different pathological CAG lengths in the HTT gene. However, their neuronal differentiation potential has yet to be thoroughly evaluated. Here, we report that RUES2 during ventral telencephalic differentiation is biased towards medial ganglionic eminence (MGE). We also show that HD-RUES2 cells exhibit an altered MGE transcriptional signature in addition to recapitulating known HD phenotypes, with reduced expression of the neurodevelopmental regulators NEUROD1 and BDNF and increased cleavage of synaptically enriched N-cadherin. Finally, we identified the transcription factor SP1 as a common potential detrimental co-partner of muHTT by de novo motif discovery analysis on the LGE, MGE, and cortical genes differentially expressed in HD human pluripotent stem cells in our and additional datasets. Taken together, these observations suggest a broad deleterious effect of muHTT in the early phases of neuronal development that may unfold through its altered interaction with SP1.

Lineage tracing of direct astrocyte-to-neuron conversion in the mouse cortex.

Regenerating functional new neurons in the adult mammalian central nervous system has been proven to be very challenging due to the inability of neurons to divide and repopulate themselves after neuronal loss. Glial cells, on the other hand, can divide and repopulate themselves under injury or diseased conditions. We have previously reported that ectopic expression of NeuroD1 in dividing glial cells can directly convert them into neurons. Here, using astrocytic lineage-tracing reporter mice (Aldh1l1-CreERT2 mice crossing with Ai14 mice), we demonstrate that lineage-traced astrocytes can be successfully converted into NeuN-positive neurons after expressing NeuroD1 through adeno-associated viruses. Retroviral expression of NeuroD1 further confirms that dividing glial cells can be converted into neurons. Importantly, we demonstrate that for in vivo cell conversion study, using a safe level of adeno-associated virus dosage (1010–1012 gc/mL, 1 µL) in the rodent brain is critical to avoid artifacts caused by toxic dosage, such as that used in a recent bioRxiv study (2 × 1013 gc/mL, 1 µL, mouse cortex). For therapeutic purpose under injury or diseased conditions, or for non-human primate studies, adeno-associated virus dosage needs to be optimized through a series of dose-finding experiments. Moreover, for future in vivo glia-to-neuron conversion studies, we recommend that the adeno-associated virus results are further verified with retroviruses that mainly express transgenes in dividing glial cells in order to draw solid conclusions. The study was approved by the Laboratory Animal Ethics Committee of Jinan University, China (approval No. IACUC-20180330-06) on March 30, 2018.

SCLC Subtypes Defined by ASCL1, NEUROD1, POU2F3, and YAP1: A Comprehensive Immunohistochemical and Histopathologic Characterization.

Recent studies have identified subtypes of small cell lung carcinoma (SCLC) defined by the RNA expression of ASCL1, NEUROD1, POU2F3, and YAP1 transcriptional regulators. There are only limited data on the distribution of these markers at the protein level and associated pathologic characteristics in clinical SCLC samples. The expression of ASCL1, NEUROD1, POU2F3, and YAP1 was analyzed by immunohistochemistry in 174 patient samples with SCLC. Subtypes defined by these markers were correlated with histologic characteristics, expression of classic neuroendocrine markers (synaptophysin, chromogranin A, CD56, INSM1), and other SCLC markers, including the neuroendocrine phenotype-associated markers TTF-1 and DLL3. ASCL1 and NEUROD1 expression had the following distribution: (1) 41% ASCL1+/NEUROD1-; (2) 37% ASCL1+/NEUROD1+; (3) 8% ASCL1-/NEUROD1+; and (4) 14% ASCL1-/NEUROD1-. On the basis of their relative expression, 69% of cases were ASCL1-dominant and 17%were NEUROD1-dominant. POU2F3 was expressed in 7% of SCLC and was mutually exclusive of ASCL1 and NEUROD1. YAP1 was expressed at low levels, primarily in combined SCLC, and was not exclusive of other subtypes. Both ASCL1-dominant and NEUROD1-dominant subtypes were associated with neuroendocrine markerhigh/TTF-1high/DLL3high profile, whereas POU2F3 and other ASCL1/NEUROD1 double-negative tumors were neuroendocrine markerlow/TTF-1low/DLL3low. This is the first comprehensive immunohistochemical and histopathologic analysis of novel SCLC subtypes in patient samples. We confirm that ASCL1/NEUROD1 double-negative tumors represent a distinct neuroendocrine-low subtype of SCLC, which is either uniquely associated with POU2F3 or lacks a known dominant regulator. The expression profiles of these markers appear more heterogeneous in native samples than in experimental models, particularly with regard to the high prevalence of ASCL1/NEUROD1 coexpression. These findings may have prognostic and therapeutic implications and warrant further clinical investigation.

Neuronal transcription program induced in hippocampal cells cocultured with bone marrow derived mesenchymal cells

Several approaches have been applied to harvest bone marrow stromal cells (BMSCs) and to differentiate into neurons or neuronal-like cells through chemical stimulation or exposing to growth factors. To date, the data regarding induction or regulation of neuronal transcription program in neuronal-like cells derived from BMSCs is yet unknown. The objective of this study is to co-culture BMSCs with neonatal hippocampal cells and generate neuronal-like cells by direct cell-to-cell contact system without using neuronal growth factors or neurobasal medium. Here, we proposed a role for NeuroD1 and Neurogenin -2 bHLH family of transcription factors implicated in onset of neurogenesis and differentiation of cells into neurons in promoting the interaction of hippocampal cells with BMSCs and their differentiation in to neurons. The proliferation of the cells was assessed with MTT assay and the role of neuronal induction and differentiation transcription regulators NeuroD1 and Neurogenin-2 in cocultured cells was determined through immunocytochemical analysis. We observed activation and expression of the neurogenic transcription factors NeuroD1 and NGN-2 associated with neuronal activation program to initiate the onset of neurogenesis in cocultured cells. Further, our results have shown a significant expression of neuronal progenitor and immature neuronal marker i.e., nestin and tubulin respectively in cocultured cells endorsing the initiation of neuronal activation.

SCLC-CellMiner: A Resource for Small Cell Lung Cancer Cell Line Genomics and Pharmacology Based on Genomic Signatures

SUMMARYCellMiner-SCLC (https://discover.nci.nih.gov/SclcCellMinerCDB/) integrates drug sensitivity and genomic data, including high-resolution methylome and transcriptome from 118 patient-derived small cell lung cancer (SCLC) cell lines, providing a resource for research into this “recalcitrant cancer.” We demonstrate the reproducibility and stability of data from multiple sources and validate the SCLC consensus nomenclature on the basis of expression of master transcription factors NEUROD1, ASCL1, POU2F3, and YAP1. Our analyses reveal transcription networks linking SCLC subtypes with MYC and its paralogs and the NOTCH and HIPPO pathways. SCLC subsets express specific surface markers, providing potential opportunities for antibody-based targeted therapies. YAP1-driven SCLCs are notable for differential expression of the NOTCH pathway, epithelial-mesenchymal transition (EMT), and antigen-presenting machinery (APM) genes and sensitivity to mTOR and AKT inhibitors. These analyses provide insights into SCLC biology and a framework for future investigations into subtype-specific SCLC vulnerabilities.

Medulloblastoma cells resemble neuronal progenitors in their differentiation

ABSTRACT Medulloblastoma (MB) often originate from cerebellar granule neuron precursors (GNPs). We recently found that medulloblastoma cells undergo differentiation as GNPs. Differentiated MB cells have permanently lost their proliferative capacity and tumorigenicity. The differentiation of MB cells is driven by the transcription factor NeuroD1 (Neurogenic differentiation 1), and NeuroD1 expression in MB cells is repressed by EZH2-mediated H3K27me3.

Conversion of Neural Stem Cells into Functional Neuron-Like Cells by MicroRNA-218: Differential Expression of Functionality Genes.

Conversion of mesenchymal stem cells (MSC) into neuron-like cells (NLC) is a feasible cell therapy strategy for replacing lost neurons in neuronal disorders. In this study, adipose-derived MSC (ADMSC) were converted into neural stem cells (NSC) via neurosphere. The resulting NSC were then differentiated into NLC by transduction with microRNA-218, using a lentiviral vector. ADMSC, NSC, and NLC were first characterized by flow cytometry, RT-PCR, and immunocytochemistry. The functionality of the NLC was evaluated by qRT-PCR and patch clamp recording. Immunophenotyping of ADMSC showed their immunoreactivity to MSC markers CD90, CD73, CD105, and CD49d, but not to CD31 and CD45. RT-PCR results demonstrated the expression of nestin, neurogenin, neurod1, neurofilament light, and GAP43 genes in NSC while NLC expressed synaptophysin, neurofilament heavy, and GAP43. In addition, NSC morphology changed into multipolar with long processes after transduction with miR-218. Moreover, using qRT-PCR, the expression levels of miR-218 and functionality genes CACNA1C, SNAP25, KCNH1, KCNMA1, and SCN9A were significantly increased in NLC, compared with NSC, and ADMSC at 3weeks and 5months post-transduction. Furthermore, the generated NLC expressed significantly higher protein levels of neurofilament heavy polypeptide (NFh) and enolase 2 (Eno2) neuronal markers, compared with ADMSC and NSC. Finally, action potentials were successfully recorded by the generated NLC, using patch clamp. In summary, ADMSC-derived NSC differentiated into functional NLC by transduction with miR-218. The generated NLC expressed functional SNAP25, CACNA1C, KCNH1, KCNMA1, and SCN9A and produced an action potential, which provides useful insights into the generation of functional neuronal cells.

Pathophysiological and morphological aspects of congenital hyperinsulinism. Review

Congenital hyperinsulinism is one of the most common causes of hypoglycemia in infants. In the absence of adequate pharmacological correction of hypoglycemic attacks, the development of irreversible changes in the central nervous system with the subsequent disability of such patients is possible. Due to the ineffectiveness of conservative therapy, in many cases surgical intervention is required. However, due to the imperfection of preoperative diagnosis, pathologists intraoperatively perform the differentiation between focal and diffuse forms of congenital hyperinsulinism. But the pancreatic pathology with congenital hyperinsulinism remains poorly understood, therefore histological and further immunohistochemical diagnosis presents significant difficulties.This review presents data on the histological, histochemical, and immunohistochemical characteristics of the endocrine pancreas, which may be useful in the further studies of congenital hyperinsulinism. In particular, we describe in detail the current approaches to morphological classification and intraoperative histological diagnosis of various forms of congenital hyperinsulinism. In addition, we give a detailed description of the expression of transcription factors NeuroD1, Nkx2.2 and Isl1 in pancreatic tissue during immunohistochemical study. Also, we describe the profile of immunohistochemical staining of dopamine and somatostatin receptors, as well as somatostatin and chromogranin A.The data of numerous studies combined in this review can help researchers in the further search for solutions to the diagnostic and therapeutic problems associated with congenital hyperinsulinism.

1731-P: The Mechanism of the Protective Effect of GLP-1 on the Amelioration of Alzheimer’s Disease by Enhancing Brain Insulin Production

Background: Growing evidence supported that type 2 diabetes mellitus (T2DM) patients had a greater risk of developing Alzheimer’s disease (AD). Insulin secreted by “β cells” in brain played a critical role in T2DM patients and in AD patients. GLP-1 could improve cognition in AD patients via enhancing brain insulin signaling. We hypothesize the therapeutic effect of GLP-1 in T2DM might be due to promoting the insulin secretion of “β cells” in the nervous system. However, the molecular mechanism is unclear. Methods: The hippocampus cell- HT-22 cell line and T2DM rats were used in this study. The genes of insulin syntheses, animals’ cognitive function and the phosphorylation of tau protein in hippocampus of T2DM brain were examined. Results: Firstly, HT22 cell line was intervened by GLP-1 or high glucose or GLP-1+ high glucose. When stimulated by GLP-1 combined with high glucose, IRS1/PI3K/AKT located in insulin signal pathway could be activated and the expression of neurod-1 and ngn3 (insulin synthesis genes) were notably increased. These results indicated that neuronal cell could secrete insulin under a certain condition. Secondly, RAAV-sh- neurod-1 and RAAV-sh-ngn3 were icv to the T2DM rats to prevent the production of brain insulin. The Barnes Maze test indicated the T2DM rats with peripheral GLP-1 and icv RAAV-sh-nc injection had a more intelligence than those with peripheral GLP-1 and icv RAAV-sh- neurod-1 and RAAV-sh-ngn3. Thirdly, the T2DM rats with peripheral GLP-1 and icv RAAV-sh- neurod-1 and RAAV-sh-ngn3 had a significantly hyperphsphorylation of tau protein than those rats with peripheral GLP-1 and icv RAAV-sh-nc injection. This result showed that brain insulin could improve cognition by GLP-1 in T2DM rats. Conclusions: These results suggested that neuronal cells might synthesis and secrete insulin under the condition of high glucose and GLP-1. And GLP-1 could not improve cognitive ability in T2DM when brain insulin synthesized was prevented. Disclosure J. Huang: None. Y. Yang: None. Funding National Natural Science Foundation of China (81670754)

NeuroD1 Dictates Tumor Cell Differentiation in Medulloblastoma

SUMMARYTumor cells are characterized by unlimited proliferation and perturbed differentiation. Using single-cell RNA sequencing, we demonstrate that tumor cells in medulloblastoma (MB) retain their capacity to differentiate in a similar way as their normal originating cells, cerebellar granule neuron precursors. Once they differentiate, MB cells permanently lose their proliferative capacity and tumorigenic potential. Differentiated MB cells highly express NeuroD1, a helix-loop-helix transcription factor, and forced expression of NeuroD1 promotes the differentiation of MB cells. The expression of NeuroD1 in bulk MB cells is repressed by trimethylation of histone 3 lysine-27 (H3K27me3). Inhibition of the histone lysine methyltransferase EZH2 prevents H3K27 trimethylation, resulting in increased NeuroD1 expression and enhanced differentiation in MB cells, which consequently reduces tumor growth. These studies reveal the mechanisms underlying MB cell differentiation and provide rationales to treat MB (potentially other malignancies) by stimulating tumor cell differentiation.

MYC Drives Temporal Evolution of Small Cell Lung Cancer Subtypes by Reprogramming Neuroendocrine Fate

Small cell lung cancer (SCLC) is a neuroendocrine tumor treated clinically as a single disease with poor outcomes. Distinct SCLC molecular subtypes have been defined based on expression of ASCL1, NEUROD1, POU2F3, or YAP1. Here, we use mouse and human models with a time-series single-cell transcriptome analysis to reveal that MYC drives dynamic evolution of SCLC subtypes. In neuroendocrine cells, MYC activates Notch to dedifferentiate tumor cells, promoting a temporal shift in SCLC from ASCL1+ to NEUROD1+ to YAP1+ states. MYC alternatively promotes POU2F3+ tumors from a distinct cell type. Human SCLC exhibits intratumoral subtype heterogeneity, suggesting that this dynamic evolution occurs in patient tumors. These findings suggest that genetics, cell of origin, and tumor cell plasticity determine SCLC subtype.