Generation of an induced pluripotent stem cell (iPSC) line from a Parkinson's disease patient with a pathogenic LRP10/c.688C > T(p.Arg230Trp) mutation.

Parkinson's disease (PD) is a highly prevalent and severe neurodegenerative disease that affects more than 10 million individuals worldwide. Pathogenic mutations in LRP10 have been associated with autosomal dominant PD. Here, we report an induced pluripotent stem cell (iPSC) line generated from a PD patient harboring the LRP10 c.688C > T (p.Arg230Trp) variant. Skin fibroblasts from the PD patient were successfully reprogrammed into iPSCs that expressed pluripotency markers, a normal karyotype, and the capacity to differentiate into the three germ layers in vivo. This iPSC line is a potential resource for studying the pathogenic mechanisms of PD.

Epigenetic modification in Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder caused by genetic, epigenetic, and environmental factors. Recent advance in genomics and epigenetics have revealed epigenetic mechanisms in PD. These epigenetic modifications include DNA methylation, post-translational histone modifications, chromatin remodeling, and RNA-based mechanisms, which regulate cellular functions in almost all cells. Epigenetic alterations are involved in multiple aspects of neuronal development and neurodegeneration in PD. In this review, we discuss current understanding of the epigenetic mechanisms that regulate gene expression and neural degeneration and then highlight emerging epigenetic targets and diagnostic and therapeutic biomarkers for treating or preventing PD.

Both prolonged high-fat diet consumption and calorie restriction boost hepatic NAD+ metabolism in mice.

Hepatic NAD+ homeostasis is essential to metabolic flexibility upon energy balance challenges. The molecular mechanism is unclear. This study aimed to determine how the enzymes involved in NAD+ salvage (Nampt, Nmnat1, Nrk1), clearance (Nnmt, Aox1, Cyp2e1), and consumption pathways (Sirt1, Sirt3, Sirt6, Parp1, Cd38) were regulated in the liver upon energy overload or shortage, as well as their relationships with glucose and lipid metabolism. Male C57BL/6N mice were fed ad libitum with the CHOW diet, high-fat diet (HFD), or subjected to 40% calorie restriction (CR) CHOW diet for 16 weeks respectively. HFD feeding increased hepatic lipids content and inflammatory markers, while lipids accumulation was not changed by CR. Both HFD feeding and CR elevated the hepatic NAD+ levels, as well as gene and protein levels of Nampt and Nmnat1. Furthermore, both HFD feeding and CR lowered acetylation of PGC-1α in parallel with the reduced hepatic lipogenesis and enhanced fatty acid oxidation, while CR enhanced hepatic AMPK activity and gluconeogenesis. Hepatic Nampt and Nnmt gene expression negatively correlated with fasting plasma glucose levels concomitant with positive correlations with Pck1 gene expression. Nrk1 and Cyp2e1 gene expression positively correlated with fat mass and plasma cholesterol levels, as well as Srebf1 gene expression. These data highlight that hepatic NAD+ metabolism will be induced for either the down-regulation of lipogenesis upon over nutrition or up-regulation of gluconeogenesis in response to CR, thus contributing to the hepatic metabolic flexibility upon energy balance challenges.

Current Approaches and Molecular Mechanisms for Directly Reprogramming Fibroblasts Into Neurons and Dopamine Neurons.

Parkinson's disease is mainly caused by specific degeneration of dopaminergic neurons (DA neurons) in the substantia nigra of the middle brain. Over the past two decades, transplantation of neural stem cells (NSCs) from fetal brain-derived neural stem cells (fNSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs) has been shown to improve the symptoms of motor dysfunction in Parkinson's disease (PD) animal models and PD patients significantly. However, there are ethical concerns with fNSCs and hESCs and there is an issue of rejection by the immune system, and the iPSCs may involve tumorigenicity caused by the integration of the transgenes. Recent studies have shown that somatic fibroblasts can be directly reprogrammed to NSCs, neurons, and specific dopamine neurons. Directly induced neurons (iN) or induced DA neurons (iDANs) from somatic fibroblasts have several advantages over iPSC cells. The neurons produced by direct transdifferentiation do not pass through a pluripotent state. Therefore, direct reprogramming can generate patient-specific cells, and it can overcome the safety problems of rejection by the immune system and teratoma formation related to hESCs and iPSCs. However, there are some critical issues such as the low efficiency of direct reprogramming, biological functions, and risks from the directly converted neurons, which hinder their clinical applications. Here, the recent progress in methods, mechanisms, and future challenges of directly reprogramming somatic fibroblasts into neurons or dopamine neurons were summarized to speed up the clinical translation of these directly converted neural cells to treat PD and other neurodegenerative diseases.

Generation of integration-free human iPSC line LCPHi001-A from a Parkinson's disease patient carrying the RecNciI mutation in GBA gene.

Parkinson's disease (PD) is a neurodegenerative disease caused by environmental and genetic factors. The identified PD genes include SNCA, LRRK2, Parkin, DJ-1, PINK1, and ATP13A2. Mutations in the glucocerebrosidase (GBA) gene were reported to be associated with PD in different ethnic populations. Here we generated a novel induced pluripotent stem cell (iPSC) line LCPHi001-A from a PD patient carrying RecNciI mutation (c.1448 T > C, c.1483G > C, and c.1497G > C) in GBA by non-integrative episomal plasmids. The LCPHi001-A line expressed pluripotency markers, displayed differentiation capacity to three germ layers in vivo, and had the normal karyotype.

The Interaction of EphA4 With PDGFRß Regulates Proliferation and Neuronal Differentiation of Neural Progenitor Cells in vitro and Promotes Neurogenesis in vivo.

Neural progenitor cells (NPCs) have great potentials in cell replacement therapy for neurodegenerative diseases, such as Alzheimer's disease (AD), by promoting neurogenesis associated with hippocampal memory improvement. Ephrin receptors and angiogenic growth factor receptors have a marked impact on the proliferation and differentiation of NPCs. Although ephrin receptor A4 (EphA4) was shown to directly interact with platelet-derived growth factor receptor ß (PDGFRß), the functional effects of this interaction on neurogenesis in cultured NPCs and adult hippocampus have not yet been studied. Immunoprecipitation demonstrated that EphA4 directly interacted with PDGFRß in NPCs under ligand stimulation. Ephrin-A1 and PDGF-platelet-derived growth factor BB (BB) significantly increased proliferation and neuronal differentiation of NPCs, which was further augmented by combined treatment of Ephrin-A1 and PDGF-BB. We also found that ligand-dependent proliferation and neuronal differentiation were inhibited by the dominant-negative EphA4 mutant or a PDGFR inhibitor. Most importantly, injection of ephrin-A1 and/or PDGF-BB promoted hippocampal NPC proliferation in the APP/PS1 mouse model of AD, indicating that direct interaction of EphA4 with PDGFRß plays a functional role on neurogenesis in vivo. Finally, studies in NPCs showed that the EphA4/PDGFRß/FGFR1/FRS2α complex formed by ligand stimulation is involved in neurogenesis via ERK signaling. The present findings provided a novel insight into the functional role of direct interaction of EphA4 and PDGFRß in neurogenesis, implicating its potential use for treating neurodegenerative diseases.

Possible role of EphA4 and VEGFR2 interactions in neural stem and progenitor cell differentiation.

Neural stem and progenitor cells (NSPCs) are important pluripotent stem cells, which have potential applications for cell replacement therapy. Ephrin receptors (Ephs) and angiogenic growth factor receptors have a major impact on the proliferation and differentiation of NSPCs. Potential interactions between EphA4 and vascular endothelial growth factor (VEGF) receptor (VEGFR) 2, and their roles in NSPC differentiation in vitro remain unknown. In the present study, mouse embryonic NSPCs were treated with ephrin-A1 or VEGF165 alone as well as with combination treatment (ephrin-A1 + VEGF165). Immunoprecipitation and immunoblot assays demonstrated that wild-type EphA4, but not the EphA4 kinase-dead mutant, interacted with VEGFR2 when overexpressed in 293T cells. This interaction was inhibited by dominant-negative EphA4. The percentage of ß-tubulin III (Tuj1)+, but not glial fibrillary acid protein (GFAP)+ cells, was increased in the ephrin-A1 + VEGF165 combination group as compared to the VEGF165 alone group in mouse embryonic NSPCs. VEGF165-induced neuronal differentiation was potentiated by ephrin-A1 in NSPCs in vitro and ephrin-A1- or VEGF165-stimulated EphA4 and VEGFR2 interactions may mediate the signaling pathway.

Activation of GABAA Receptors in Colon Epithelium Exacerbates Acute Colitis.

Emerging evidence indicates that gamma-aminobutyric acid (GABA) has many beneficial effects such as ameliorating immune and inflammatory response. But, here we reported that activation of GABAA receptors (GABAA Rs) aggravated dextran sulfate sodium (DSS)-induced colitis, although the expression of pro-inflammatory cytokines was inhibited. By contrast, blocking of GABAA Rs markedly alleviated DSS-induced colitis. Notably, GABAA Rs and glutamic acid decarboxylase 65/67 were significantly increased in colon mucosa of ulcerative colitis patients and the mouse model of colitis. Further studies showed that GABA treatment resulted in an increment of serum FITC-dextran following its oral administration, a decrement of transepithelial electrical resistance, and an increment of bacterial invasion, effects which were blocked by bicuculline. In addition, GABA inhibited the expression of tight junction proteins and mucin secretion in colitis colon. GABA also decreased the expression of ki-67 and increased cleaved-caspase 3 expression in intestinal epithelia. Our data indicate that the GABAA Rs activation within colon mucosa disrupts the intestinal barrier and increases the intestinal permeability which facilitates inflammatory reaction in colon. Meanwhile, the suppression effect of GABA on pro-inflammatory cytokines leads to insufficient bacteria elimination and further aggravated the bacteria invasion and inflammatory damage.

The antinociception of oxytocin on colonic hypersensitivity in rats was mediated by inhibition of mast cell degranulation via Ca(2+)-NOS pathway.

This study was conducted to investigate the effects of oxytocin (OT) on visceral hypersensitivity/pain and mast cell degranulation and the underlying mechanisms. We found that oxytocin receptor (OTR) was expressed in colonic mast cells in humans and rats, as well as in human mast cell line-1 (HMC-1), rat basophilic leukemia cell line (RBL-2H3) and mouse mastocytoma cell line (P815). OT decreased 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced visceral hypersensitivity, colonic mast cell degranulation and histamine release after mast cell degranulation in rats. Also, OT attenuated the compound 48/80 (C48/80)-evoked histamine release in P815 cells and inward currents, responsible for the mast cell degranulation, in HMC-1, RBL-2H3 and P815 cells. Moreover, these protective effects of OT against visceral hypersensitivity and mast cell degranulation were eliminated by coadministration of OTR antagonist atosiban or a nonselective inhibitor of nitric oxide synthase (NOS), NG-Methyl-L-arginine acetate salt (L-NMMA). Notably, OT evoked a concentration-dependent increase of intracellular Ca(2+) in HMC-1, RBL-2H3 and P815 cells, which was responsible for the activation of neuronal NOS (NOS1) and endothelial NOS (NOS3). Our findings strongly suggest that OT might exert the antinociception on colonic hypersensitivity through inhibition of mast cell degranulation via Ca(2+)-NOS pathway.

Up-Regulation of GABAergic Signal Events in Bone Marrow Lymphocytes in Childhood Acute Lymphoblastic Leukemia.

Gamma-aminobutyric acid (GABA) is involved in the proliferation, differentiation, and migration of several cell types including cancer cells. Whether GABA may be involved with acute lymphoblastic leukemia (ALL) is unclear. Therefore, the goal of this report was to examine if GABAergic signaling expression is altered in bone marrow lymphocytes of ALL children. RT-PCR and western blot analysis were used to examine the expression of the GABA synthetizing enzyme glutamic acid decarboxylase (GAD) isoforms (GAD65 and GAD67), and type-A GABA receptor (GABAAR) subunits [α(1-6), ß(1-3), γ(1-3), δ, ε, θ, π, and ρ(1-3)] in bone marrow lymphocytes of 19 ALL children before chemotherapy. The data obtained were compared with those in 13 age-matched non-ALL children. Immunofluorescent staining was used to examine the cellular localization of GAD. We found that GAD and GABAAR subunits were expressed in bone marrow lymphocytes of ALL children. Moreover, RT-PCR and western blot showed that GAD and several GABAAR subunits were significantly increased in ALL children as compared with the data of non-ALL children. Our present study reveals up-regulation of GABAergic signaling events in bone marrow lymphocytes in ALL children. However, the role of this signaling system in lymphocyte proliferation and invasion as related to the progression of ALL requires further investigation.

L-Cysteine enhances nutrient absorption via a cystathionine-ß-synthase-derived H2 S pathway in rodent jejunum.

Hydrogen sulphide (H2 S) is generated endogenously from L-cysteine (L-Cys) by the enzymes cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE). In addition, L-Cys is commonly used as a precursor in the food and pharmaceutical industries. The aim of the present study is to determine whether L-Cys regulates intestinal nutrient transport. To that end, the presence of CBS and CSE in the jejunum epithelium was assessed by immunohistochemistry, Western blotting and the methylene blue assay. In addition, in vivo L-Cys (100 mg/kg, administered immediately after the glucose load) significantly increased blood glucose levels 30 min after the oral administration of glucose to mice. This effect of L-Cys was completely blocked by amino-oxyacetic acid (AOA; 50 mg/kg; administered at the same time as L-Cys) an inhibitor of CBS. Measurements of the short-circuit current (Isc) in the rat jejunum epithelium revealed that L-Cys (1 mmol/L; 6 min before the administration of L-alanine) enhances Na(+)-coupled L-alanine or glucose transport, and that this effect is inhibited by AOA (1 mmol/L;10 min before the administration of L-Cys), but not D,L-propargylglycine (PAG;1 mmol/L; 10 min before the administration of L-Cys), a CSE inhibitor. Notably, L-Cys-evoked enhancement of nutrient transport was alleviated by glibenclamide (Gli;0.1 mmol/L; 10 min before the administration of L-Cys), a K(+) channel blocker. Together, the data indicate that L-Cys enhances jejunal nutrient transport, suggesting a new approach to future treatment of nutrition-related maladies, including a range of serious health consequences linked to undernutrition.

Identification of a novel protein complex containing ASIC1a and GABAA receptors and their interregulation.

Acid-sensing ion channels (ASICs) belong to the family of the epithelial sodium channel/degenerin (ENaC/DEG) and are activated by extracellular protons. They are widely distributed within both the central and peripheral nervous systems. ASICs were modified by the activation of γ-aminobutyric acid receptors (GABAA), a ligand-gated chloride channels, in hippocampal neurons. In contrast, the activity of GABAA receptors were also modulated by extracellular pH. However so far, the mechanisms underlying this intermodulation remain obscure. We hypothesized that these two receptors-GABAA receptors and ASICs channels might form a novel protein complex and functionally interact with each other. In the study reported here, we found that ASICs were modified by the activation of GABAA receptors either in HEK293 cells following transient co-transfection of GABAA and ASIC1a or in primary cultured dorsal root ganglia (DRG) neurons. Conversely, activation of ASIC1a also modifies the GABAA receptor-channel kinetics. Immunoassays showed that both GABAA and ASIC1a proteins were co-immunoprecipitated mutually either in HEK293 cells co-transfected with GABAA and ASIC1a or in primary cultured DRG neurons. Our results indicate that putative GABAA and ASIC1a channels functionally interact with each other, possibly via an inter-molecular association by forming a novel protein complex.

GABAergic signaling facilitates breast cancer metastasis by promoting ERK1/2-dependent phosphorylation.

The present study aims to determine the role of γ-aminobutyric acid (GABA) signaling molecules in breast cancer metastasis. Our results reveal that GABAergic system exists in breast cancer cells. Both the GABA synthetic enzyme. (GAD65/67) and GABAB receptor are expressed in 4T1 mouse breast cancer cells, MCF-7 human breast cancer cells and human breast cancer tissue. Baclofen, a GABABR agonist, significantly promoted 4T1 cells invasion and migration in vitro and metastasis in vivo, an event that was attenuated by GABABR antagonist CGP55845. Baclofen-induced breast cancer metastasis was mediated by ERK1/2 pathway.