Effects of clitorienolactones from Clitoria ternatea root on calcium channel mediating hippocampal long-term potentiation in rats induced chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion (CCH) is a common mechanism of acute brain injury due to impairment of blood flow to the brain. Moreover, a prolonged lack of oxygen supply may result in cerebral infarction or global ischemia, which subsequently causes long-term memory impairment. Research on using Clitoria ternatea root extract for treating long-term memory has been studied extensively. However, the bioactive compound contributing to its neuroprotective effects remains uncertain. In the present study, we investigate the effects of clitorienolactone A (CLA) and B (CLB) from the roots of Clitoria ternatea extract on hippocampal neuroplasticity in rats induced by CCH. CLA and CLB were obtained using column chromatography. The rat model of CCH was induced using two-vessel occlusion surgery (2VO). The 2VO rats were given 10 mg/kg of CLA and CLB orally, followed by hippocampal neuroplasticity recording using in vivo electrophysiological. Rats received CLA and CLB (10 mg/kg) significantly reversed the impairment of long-term potentiation following 2VO surgery. Furthermore, we investigate the effect of CLA and CLB on the calcium channel using the calcium imaging technique. During hypoxia, CLA and CLB sustain the increase in intracellular calcium levels. We next predict the binding interactions of CLA and CLB against NMDA receptors containing GluN2A and GluN2B subunits using in silico molecular docking. Our result found that both CLA and CLB exhibited lower binding affinity against GluN2A and GluN2B subunits. Our findings demonstrated that bioactive compounds from Clitoria ternatea improved long-term memory deficits in the chronic cerebral hypoperfusion rat model via calcium uptake. Hence, CLA and CLB could be potential therapeutic tools for treating cognitive dysfunction.

Binding epitope for recognition of human TRPM4 channel by monoclonal antibody M4M.

Mouse monoclonal antibody M4M was recently designed to block human TRPM4 channel. The polypeptide for generating M4M is composed of peptide A1 between the transmembrane segment 5 (S5) and the pore, and a second peptide A2 between the pore and the transmembrane segment 6 (S6). Using peptide microarray, a 4-amino acid sequence EPGF within the A2 was identified to be the binding epitope for M4M. Substitution of EPGF with other amino acids greatly reduced binding affinity. Structural analysis of human TRPM4 structure indicates that EPGF is located externally to the channel pore. A1 is close to the EPGF binding epitope in space, albeit separated by a 37-amino acid peptide. Electrophysiological study reveals that M4M could block human TRPM4, but with no effect on rodent TRPM4 which shares a different amino acid sequence ERGS for the binding motif. Our results demonstrate that M4M is a specific inhibitor for human TRPM4.

Development and characterization of a monoclonal antibody blocking human TRPM4 channel.

TRPM4 is a calcium-activated non-selective monovalent cation channel implicated in diseases such as stroke. Lack of potent and selective inhibitors remains a major challenge for studying TRPM4. Using a polypeptide from rat TRPM4, we have generated a polyclonal antibody M4P which could alleviate reperfusion injury in a rat model of stroke. Here, we aim to develop a monoclonal antibody that could block human TRPM4 channel. Two mouse monoclonal antibodies M4M and M4M1 were developed to target an extracellular epitope of human TRPM4. Immunohistochemistry and western blot were used to characterize the binding of these antibodies to human TRPM4. Potency of inhibition was compared using electrophysiological methods. We further evaluated the therapeutic potential on a rat model of middle cerebral artery occlusion. Both M4M and M4M1 could bind to human TRPM4 channel on the surface of live cells. Prolonged incubation with TRPM4 blocking antibody internalized surface TRPM4. Comparing to M4M1, M4M is more effective in blocking human TRPM4 channel. In human brain microvascular endothelial cells, M4M successfully inhibited TRPM4 current and ameliorated hypoxia-induced cell swelling. Using wild type rats, neither antibody demonstrated therapeutic potential on stroke. Human TRPM4 channel can be blocked by a monoclonal antibody M4M targeting a key antigenic sequence. For future clinical translation, the antibody needs to be humanized and a transgenic animal carrying human TRPM4 sequence is required for in vivo characterizing its therapeutic potential.

Immature Midbrain Dopaminergic Neurons Derived from Floor-Plate Method Improve Cell Transplantation Therapy Efficacy for Parkinson's Disease.

Recent reports have indicated human embryonic stem cells-derived midbrain dopamine (mDA) neurons as proper cell resources for use in Parkinson's disease (PD) therapy. Nevertheless, no detailed and systematic study has been conducted to identify which differentiation stages of mDA cells are most suitable for transplantation in PD therapy. Here, we transplanted three types of mDA cells, DA progenitors (differentiated in vitro for 16 days [D16]), immature DA neurons (D25), and DA neurons (D35), into PD mice and found that all three types of cells showed high viability and strong neuronal differentiation in vivo. Both D25 and D35 cells showed neuronal maturation and differentiation toward TH+ cells and, accordingly, satisfactory behavioral functional recovery. However, transplanted D16 cells were less capable of producing functional recovery. These findings provide a valuable guideline for standardizing the differentiation stage of the transplantable cells used in clinical cell therapy for PD. Stem Cells Translational Medicine 2017;6:1803-1814.

Synaptotagmin-7 phosphorylation mediates GLP-1-dependent potentiation of insulin secretion from ß-cells.

Glucose stimulates insulin secretion from ß-cells by increasing intracellular Ca(2+). Ca(2+) then binds to synaptotagmin-7 as a major Ca(2+) sensor for exocytosis, triggering secretory granule fusion and insulin secretion. In type-2 diabetes, insulin secretion is impaired; this impairment is ameliorated by glucagon-like peptide-1 (GLP-1) or by GLP-1 receptor agonists, which improve glucose homeostasis. However, the mechanism by which GLP-1 receptor agonists boost insulin secretion remains unclear. Here, we report that GLP-1 stimulates protein kinase A (PKA)-dependent phosphorylation of synaptotagmin-7 at serine-103, which enhances glucose- and Ca(2+)-stimulated insulin secretion and accounts for the improvement of glucose homeostasis by GLP-1. A phospho-mimetic synaptotagmin-7 mutant enhances Ca(2+)-triggered exocytosis, whereas a phospho-inactive synaptotagmin-7 mutant disrupts GLP-1 potentiation of insulin secretion. Our findings thus suggest that synaptotagmin-7 is directly activated by GLP-1 signaling and may serve as a drug target for boosting insulin secretion. Moreover, our data reveal, to our knowledge, the first physiological modulation of Ca(2+)-triggered exocytosis by direct phosphorylation of a synaptotagmin.

Seipin regulates excitatory synaptic transmission in cortical neurons.

Heterozygosity for missense mutations in Seipin, namely N88S and S90L, leads to a broad spectrum of motor neuropathy, while a number of loss-of-function mutations in Seipin are associated with the Berardinelli-Seip congenital generalized lipodystrophy type 2 (CGL2, BSCL2), a condition that is characterized by severe lipoatrophy, insulin resistance, and intellectual impairment. The mechanisms by which Seipin mutations lead to motor neuropathy, lipodystrophy, and insulin resistance, and the role Seipin plays in central nervous system (CNS) remain unknown. The goal of this study is to understand the functions of Seipin in the CNS using a loss-of-function approach, i.e. by knockdown (KD) of Seipin gene expression. Excitatory post-synaptic currents (EPSCs) were impaired in Seipin-KD neurons, while the inhibitory post-synaptic currents (IPSCs) remained unaffected. Expression of a shRNA-resistant human Seipin rescued the impairment of EPSC produced by Seipin KD. Furthermore, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced whole-cell currents were significantly reduced in Seipin KD neurons, which could be rescued by expression of a shRNA-resistant human Seipin. Fluorescent imaging and biochemical studies revealed reduced level of surface AMPA receptors, while no obvious ultrastructural changes in the pre-synapse were found. These data suggest that Seipin regulates excitatory synaptic function through a post-synaptic mechanism.

Enhanced production of neuroprogenitors, dopaminergic neurons, and identification of target genes by overexpression of sonic hedgehog in human embryonic stem cells.

Molecular and cellular signaling pathways are involved in the process of neural differentiation from human embryonic stem cells (hESC) to terminally differentiated neurons. The Sonic hedgehog (SHH) morphogen is required to direct the differentiation of hESC to several neural subtypes, for example, dopaminergic (DA) or motor neurons. However, the roles of SHH signaling and the pathway target genes that regulate the diversity of cellular responses arising from SHH activation during neurogenesis of hESC have yet to be elucidated. In this study, we report that overexpression of SHH in hESC promotes the derivation of neuroprogenitors (NP), increases proliferation of NP, and subsequently increases the yield of DA neurons. Next, gene expression changes resulting from the overexpression of SHH in hESC-derived NP were examined by genome-wide transcriptional profiling. Categorizing the differentially expressed genes according to the Gene Ontology biological processes showed that they are involved in numerous cellular processes, including neural development, NP proliferation, and neural specification. In silico GLI-binding sites analysis of the differentially expressed genes also identified a set of putative novel direct target genes of SHH in hESC-derived NP, which are involved in nervous system development. Electrophoretic mobility shift assays and promoter-luciferase assays confirmed that GLI1 binds to the promoter region and activates transcription of HEY2, a NOTCH signaling target gene. Taken together, our data provide evidence for the first time that there is cross-talk between the NOTCH and SHH signaling pathways in hESC-derived NP and also provide significant new insights into transcriptional targets in SHH-mediated neural differentiation of hESC.

EHD1 is a synaptic protein that modulates exocytosis through binding to snapin.

EHD1 is an EH (Eps15 homology) domain-containing protein involved in endosomal recycling. Our yeast two hybrid screening experiments showed that EHD1 interacts with a synaptic protein, snapin, and the present study was carried out to further elucidate the functional significance of this interaction. Immunoreactivity to EHD1 is observed in the cerebral cortex, hippocampus and striatum, in the rat brain. The protein is colocalized with the axon terminal marker synaptophysin in cultured neurons. EHD1 binds to the C terminus of snapin via its C terminus EH domain. It negatively affects the binding of a SNARE complex protein, SNAP-25, to snapin, probably due to the competition for overlapping binding sites on the C terminus of snapin. EHD1 affects the coupling of synaptotagmin-1 to the SNARE complex, and could be a negative regulator of exocytosis. This is supported by electrophysiological findings that PC-12 cells which overexpress EHD1 show reduced depolarization-induced exocytosis compared to controls, but the reduced exocytosis is not observed in cells which overexpress the N terminus of EHD1 that is unable to bind snapin. Together, the above results indicate that EHD1 is a synaptic protein that negatively affects exocytosis through binding to snapin.

Differential effects of polyunsaturated fatty acids on membrane capacitance and exocytosis in rat pheochromocytoma-12 cells.

The fusion of synaptic vesicles with the plasma membrane during exocytosis can be recorded by membrane capacitance measurements under voltage-clamp conditions. These measurements enable high time-resolution quantitation of exocytosis. The present study was carried out using the above technique to elucidate the effects of various polyunsaturated fatty acids on exocytosis in a neuroendocrine cell, the rat pheochromocytoma-12 (PC12) cell. External application of eicosapentaenoic acid and arachidonic acid resulted in an increase in capacitance of PC12 cells, indicating fusion of secretory vesicles with cell membranes and exocytosis. In contrast, docosahexaenoic acid, linoleic acid, oleic acid, and vehicle control had no significant effect on capacitance. The above findings show differential effects of polyunsaturated fatty acids on exocytosis in PC12 cells. It is postulated that besides arachidonic acid, eicosapentaenoic acid could also play an important role in exocytosis and neurotransmitter release, in neurons and hormone-secreting cells.