Human cortical interneurons optimized for grafting specifically integrate, abort seizures, and display prolonged efficacy without over-inhibition.

Previously, we demonstrated the efficacy of human pluripotent stem cell (hPSC)-derived GABAergic cortical interneuron (cIN) grafts in ameliorating seizures. However, a safe and reliable clinical translation requires a mechanistic understanding of graft function, as well as the assurance of long-term efficacy and safety. By employing hPSC-derived chemically matured migratory cINs in two models of epilepsy, we demonstrate lasting efficacy in treating seizures and comorbid deficits, as well as safety without uncontrolled growth. Host inhibition does not increase with increasing grafted cIN densities, assuring their safety without the risk of over-inhibition. Furthermore, their closed-loop optogenetic activation aborted seizure activity, revealing mechanisms of graft-mediated seizure control and allowing graft modulation for optimal translation. Monosynaptic tracing shows their extensive and specific synaptic connections with host neurons, resembling developmental connection specificity. These results offer confidence in stem cell-based therapy for epilepsy as a safe and reliable treatment for patients suffering from intractable epilepsy.

Neurofibromin Deficiency Causes Epidermal Growth Factor Receptor Upregulation through the Activation of Ras/ERK/SP1 Signaling Pathway in Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheet Tumor.

Neurofibromatosis type 1 (NF1) is an autosomal dominant human genetic disorder. The progression of benign plexiform neurofibromas to malignant peripheral nerve sheet tumors (MPNSTs) is a major cause of mortality in patients with NF1. Although elevated epidermal growth factor receptor (EGFR) expression plays a crucial role in the pathogenesis of MPNST, the cause of EGFR overexpression remains unclear. Here, we assessed EGFR expression levels in MPNST tissues of NF1 patients and NF1 patient-derived MPNST cells. We found that the expression of EGFR was upregulated in MPNST tissues and MPNST cells, while the expression of neurofibromin was significantly decreased. Manipulation of NF1 expression by NF1 siRNA treatment or NF1-GAP-related domain overexpression demonstrated that EGFR expression levels were closely and inversely correlated with neurofibromin levels. Notably, knockdown of the NF1 gene by siRNA treatment augmented the nuclear localization of phosphorylated SP1 (pSP1) and enhanced pSP1 binding to the EGFR gene promoter region. Our results suggest that neurofibromin deficiency in NF1-associated MPNSTs enhances the Ras/ERK/SP1 signaling pathway, which in turn may lead to the upregulation of EGFR expression. This study provides insight into the progression of benign tumors and novel therapeutic approaches for treatment of NF1-associated MPNSTs.

Migratory cortical interneuron-specific transcriptome abnormalities in schizophrenia.

Cortical interneurons (cINs) are substantially affected in Schizophrenia (SCZ) and enriched for SCZ heritability during development. To understand SCZ-specific changes in these cells during development, we isolated migratory cINs from cIN spheres derived from 5 healthy control (HC) and 5 SCZ induced pluripotent stem cell lines (iPSCs). Transcriptome analyses show dysregulation in extracellular matrix pathways as the major disturbances in SCZ migratory cINs, whereas sphere cINs show dysregulation in immune pathways. This result suggests the importance of using homogeneous cell populations to identify stage-specific abnormalities and provides a platform to further study the biology of schizophrenia pathogenesis during early development.

A Newly Identified Glutaminase-Free ʟ-Asparaginase (ʟ-ASPG86) from the Marine Bacterium Mesoflavibacter zeaxanthinifaciens.

ʟ-Asparaginase (E.C. 3.5.1.1) is an enzyme involved in asparagine hydrolysis and has the potential to effect leukemic cells and various other cancer cells. We identified the Lasparaginase gene (ʟ-ASPG86) in the genus Mesoflavibacter, which consists of a 1,035 bp open reading frame encoding 344 amino acids. Following phylogenetic analysis, the deduced amino acid sequence of ʟ-ASPG86 (ʟ-ASPG86) was grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The ʟ-ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant ʟ-asparaginase (r-ʟ-ASPG86) showed optimum conditions at 37-40oC, pH 9. Moreover, r-ʟ-ASPG86 did not exhibit glutaminase activity. In the metal ions test, its enzymatic activity was highly improved upon addition of 5 mM manganese (3.97-fold) and magnesium (3.35-fold) compared with the untreated control. The specific activity of r-LASPG86 was 687.1 units/mg under optimum conditions (37°C, pH 9, and 5 mM MnSO4).

TAGLN expression is upregulated in NF1-associated malignant peripheral nerve sheath tumors by hypomethylation in its promoter and subpromoter regions.

Neurofibromatosis type 1 (NF1) caused by NF1 gene mutation is a commonly inherited autosomal dominant disorder. Malignant peripheral nerve sheath tumors (MPNSTs), a type of aggressive sarcoma, are a major cause of mortality in NF1 patients. The malignant transformation of benign plexiform neurofibromas (PNs) to MPNSTs is a marked peculiarity in NF1 patients, yet the pathogenesis remains poorly understood. We found that an actin-associated protein transgelin (SM22) was highly expressed in NF1-deficient MPNST tissues compared to NF1-deficient PN tissues using immunohistological staining and primary cultured MPNST cells in western blot analysis. We further found that this transgelin upregulation was caused by increased transcriptional expression of the TAGLN gene encoding transgelin. Comparison of DNA methylation values in the promoter and subpromoter regions of the TAGLN gene in three types of NF1-deficient primary-cultured cells, derived from an NF1 patient's normal phenotype, a benign PN and MPNST tissues, revealed that the TAGLN gene was hypomethylated in the MPNST cells. Next, to determine the functional role of transgelin in MPNST pathogenesis, we manipulated the TAGLN gene expression and investigated the alteration of the RAS-mitogen-activated protein kinase (MAPK) signaling pathway in the normal-phenotypic and malignant tumor cells. The downregulation of TAGLN expression in NF1-deficient MPNST tumor cells through the treatment of the small interfering RNA resulted in a decrease in the RAS activation (GTP-RAS) and the downstream ERK1/2 activation (phosphorylated ERK1/2), while the overexpression of TAGLN in normal-phenotypic NF1-deficient cells caused an increase in RAS and ERK1/2 activation. These results indicate that upregulation of transgelin caused by hypomethylation of the TAGLN gene is closely involved in tumor progression in NF1.