A Neuroprotective Herbal Mixture Inhibits Caspase-3-independent Apoptosis in Retinal Ganglion Cells

It was previously demonstrated that Menta-FX, a mixture of Panax quinquefolius L. (PQE), Ginkgo biloba (GBE), and Hypericum perforatum extracts (HPE), enhances retinal ganglion cell survival after axotomy. However, the mechanisms of neuroprotection remain unknown. The aim of this study is to elucidate the neuroprotective mechanisms of Menta-FX. Since PQE, GBE and HPE have all been observed to display anti-oxidative property, the involvement of anti-oxidation in Menta-FX’s neuroprotective effect was investigated. Menta-FX lowered nitric oxide (NO) content in axotomized retinas without affecting nitric oxide synthase activity, suggesting that Menta-FX possibly exhibited a NO scavenging property. In addition, the effect of Menta-FX on the frequency of axotomy-induced nuclear fragmentation and caspase-3 activation was investigated. Menta-FX treatment significantly reduced nuclear fragmentation in axotomized retinas. Surprisingly, Menta-FX had no effect on caspase-3 activation, but selectively lowered caspase-3-independent nuclear fragmentation in axotomized retinal ganglion cells. In addition, inhibition of PI3K activity by intravitreal injection of wortmannin, a phosphoinositide-3 kinase (PI3K) inhibitor, completely abolished the neuroprotective effect of Menta-FX, indicating that Menta-FX’s neuroprotective effect was PI3K-dependent. Data here suggest that Menta-FX displayed a PI3K-dependent, selective inhibition on a caspase-3-independent apoptotic pathway in axotomized RGCs, thus, highlighting the potential use of herbal remedies as neuroprotective agents for other neurodegenerative diseases.     

Mutually exclusive expression of sex-specific and non-sex-specific fruitless gene products in the Drosophila central nervous system

The fruitless gene of Drosophila produces multiple protein isoforms, which are classified into two major classes, sex-specific Fru proteins (FruM) and non-sex specific proteins (FruCOM). Whereas FruM proteins are expressed in ∼2000 neurons to masculinize their structure and function, little is known about FruCOM's roles. As an attempt to obtain clues to the roles of FruCOM, we compared expression patterns of FruCOM and FruM in the central nervous system at the late larval stage. We found that nearly all neuroblasts express FruCOM but not FruM, whereas a subset of ganglion mother cells and differentiated neurons express FruM but not FruCOM. It is inferred that FruCOM proteins support fundamental stem cell functions, contrasting to FruM proteins, which play major roles in sex-specific differentiation of neurons.     

Phosphorylation of tyrosine hydroxylase in the superior cervical ganglion

We studied the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat. Ganglia were preincubated with [³²P]P_i and were then incubated in non-radioactive medium containing a variety of agents that are known to activate tyrosine hydroxylase in this tissue. Tyrosine hydroxylase was isolated from homogenates of the ganglia by immunoprecipitation followed by polyacrylamide gel electrophoresis. ³²P-labelled tyrosine hydroxylase was visualized by radioautography, and the incorporation of ³²P into the enzyme was quantitated by densitometry of the autoradiograms. Veratridine produced a concentration-dependent increase in the incorporation of ³²P into tyrosine hydroxylase, with 50 μM veratridine producing a 5-fold increase in ³²P incorporation. The nicotinic agonist, dimethylphenylpiperazinium (100 μM), caused a 7-fold increase in the phosphorylation of tyrosine hydroxylase. The effect of dimethylphenylpiperazinium was maximal within 1 min and decreased upon continued exposure of the ganglia to this agent. The actions of dimethylphenylpiperazinium and of veratridine were dependent on extracellular Ca²⁺. Muscarine, 8-Br-cAMP, forskolin, vasoactive intestinal peptide, isoproterenol, deoxycholate and phospholipase C also stimulated the incorporation of ³²P into tyrosine hydroxylase. These data support the hypothesis that phosphorylation plays a role in activation of tyrosine hydroxylase produced by all of these agents.     

Green synthesis of selenium-N-heterocyclic carbene compounds: Evaluation of antimicrobial and anticancer potential

Three benzimidazolium salts (III-V) and respective selenium adducts (VI-VIII) were designed, synthesized and characterized by various analytical techniques (FT-IR and NMR ¹H, ¹³C). Selected salts and respective selenium N-Heterocyclic carbenes (selenium-NHC) adducts were tested in vitro against Cervical Cancer Cell line (Hela), Breast Adenocarcinoma cell line (MCF-7), Retinal Ganglion Cell line (RGC-5) and Mouse Melanoma Cell line (B16F10) using MTT assay and the results were compared with standard drug 5-Fluorouracil. Se-NHC compounds and azolium salts showed significant anticancer potential. Molecular docking studies of compounds (VI, VII and VIII) showed strong binding energies and ligand affinity toward following angiogenic factors: VEGF-A (vascular endothelial growth factor A), EGF (human epidermal growth factor), HIF (Hypoxia-inducible factor) and COX-1 (Cyclooxygenase-1) suggesting that the anticancer activity of adducts (VI, VII and VIII) may be due to their strong anti-angiogenic effect. In addition, compounds III-VIII were screened for their antibacterial and antifungal potential. Adduct VI was found to be potent anti-fungal agent against A. Niger with zone of inhibition (ZI) value 27.01 ± 0.251 mm which is better than standard drug Clotrimazole tested in parallel.     

Phosphorylation of Rat Melanopsin at Ser-381 and Ser-398 by Light/Dark and Its Importance for Intrinsically Photosensitive Ganglion Cells (ipRGCs) Cellular Ca2+ Signaling

The G protein-coupled light-sensitive receptor melanopsin is involved in non-image-forming light responses including circadian timing. The predicted secondary structure of melanopsin indicates a long cytoplasmic tail with many potential phosphorylation sites. Using bioinformatics, we identified a number of amino acids with a high probability of being phosphorylated. We generated antibodies against melanopsin phosphorylated at Ser-381 and Ser-398, respectively. The antibody specificity was verified by immunoblotting and immunohistochemical staining of HEK-293 cells expressing rat melanopsin mutated in Ser-381 or Ser-398. Using the antibody recognizing phospho-Ser-381 melanopsin, we demonstrated by immunoblotting and immunohistochemical staining in HEK-293 cells expressing rat melanopsin that the receptor is phosphorylated in this position during the dark and dephosphorylated when light is turned on. On the contrary, we found that melanopsin at Ser-398 was unphosphorylated in the dark and became phosphorylated after light stimulation. The light-induced changes in phosphorylation at both Ser-381 and Ser-398 were rapid and lasted throughout the 4-h experimental period. Furthermore, phosphorylation at Ser-381 and Ser-398 was independent of each other. The changes in phosphorylation were confirmed in vivo by immunohistochemical staining of rat retinas during light and dark. We further demonstrated that mutation of Ser-381 and Ser-398 in melanopsin-expressing HEK-293 cells affected the light-induced Ca²⁺ response, which was significantly reduced as compared with wild type. Examining the light-evoked Ca²⁺ response in a melanopsin Ser-381 plus Ser-398 double mutant provided evidence that the phosphorylation events were independent.     

A potential neuroprotective role of apolipoprotein E-containing lipoproteins through low density lipoprotein receptor-related protein 1 in normal tension glaucoma

Glaucoma is an optic neuropathy and the second major cause of blindness worldwide next to cataracts. The protection from retinal ganglion cell (RGC) loss, one of the main characteristics of glaucoma, would be a straightforward treatment for this disorder. However, the clinical application of neuroprotection has not, so far, been successful. Here, we report that apolipoprotein E-containing lipoproteins (E-LPs) protect primary cultured RGCs from Ca(2+)-dependent, and mitochondrion-mediated, apoptosis induced by glutamate. Binding of E-LPs to the low density lipoprotein receptor-related protein 1 recruited the N-methyl-d-aspartate receptor, blocked intracellular Ca(2+) elevation, and inactivated glycogen synthase kinase 3β, thereby inhibiting apoptosis. When compared with contralateral eyes treated with phosphate-buffered saline, intravitreal administration of E-LPs protected against RGC loss in glutamate aspartate transporter-deficient mice, a model of normal tension glaucoma that causes glaucomatous optic neuropathy without elevation of intraocular pressure. Although the presence of α2-macroglobulin, another ligand of the low density lipoprotein receptor-related protein 1, interfered with the neuroprotective effect of E-LPs against glutamate-induced neurotoxicity, the addition of E-LPs overcame the inhibitory effect of α2-macroglobulin. These findings may provide a potential therapeutic strategy for normal tension glaucoma by an LRP1-mediated pathway.     

Na+ channel Scn1b gene regulates dorsal root ganglion nociceptor excitability in vivo

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na(+) current (I(Na)) mediated by voltage-gated Na(+) channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S I(Na) inactivation, reduced persistent TTX-R I(Na), a prolonged rate of recovery of TTX-R I(Na) from inactivation, and reduced cell surface expression of Na(v)1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K(+) current (I(to)) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both I(Na) and I(K).     

Endocytosis-independent mechanisms of Delta ligand proteolysis

Delta proteins function as cell surface ligands for Notch receptors in a highly conserved signal transduction mechanism. Delta activates Notch by "trans-endocytosis", whereby endocytosis of Delta that is in complex with Notch on a neighboring cell induces activating cleavages in Notch. Alternatively, proteolysis of Delta renders the ligand inactive by dissociating the extracellular and cytosolic domains. How proteolysis and trans-endocytosis cooperate in Delta function is not well understood. We now show that Drosophila Delta proteolysis occurs independent of and prior to endocytosis in neuroblasts and ganglion mother cells in vivo and cells in culture. Delta cleavage occurs at two novel sites that we identify in the juxtamembrane (JM) and transmembrane (TM) domains. In addition to the previously identified Kuzbanian ADAM protease, which acts on the JM domain, proteolysis in the TM domain is facilitated by a thiol-sensitive aspartyl protease that is distinct from Presenilin. Furthermore, cleavage in the TM domain is upregulated in the presence of Notch. Overall, Drosophila Delta proteolysis differs from the conventional regulated intramembrane proteolysis (RIP) mechanism by two criteria: (1) TM-domain processing of Delta is not sensitive to Presenilin, and (2) TM and JM domain cleavages occur independently of each other. Altogether, these data support a model whereby proteolysis can modulate Delta ligand activity independently of endocytosis.     

北京鸭视网膜节细胞的大小、密度和分布

采用Nissl染色法、视神经溃变法和神经元逆行追踪标记辣根过氧化物酶(HRP)法,研究了北京鸭视网膜节细胞(RGCs)的大小和密度及其分布的变化。北京鸭RGCs形态多样,有圆形、椭圆形和多角形等,RGCs总数为1.3×106个(P0),RGCs平均密度为5370个/mm2(P0),在视网膜中央有一个偏向鼻侧的高密度区即中央高密度区(8860个/mm2),由中央区至周边部,细胞密度逐渐降低,颞侧周边部最低(3440个/mm2)。不同区域RGCs大小差异显著,中央区以小细胞为主(62.2±23.3μm2,P0),而周边部RGCs逐渐增大,颞侧周边部最大(P0:133.7±75.7μm2;P8:152.9±55.9μm2)。由此可见,伴随RGCs大小由中央区至周边部的递增而细胞密度呈现递减的变化,这种变化趋势在颞侧周边部最明显。与此同时,随日龄增长,RGCs总数和密度均递减而细胞大小递增。dACs是位于视网膜节细胞层的小神经元,细胞大小为23.7±4.0μm2