Compartment-specific regulation of NaV1.7 in sensory neurons after acute exposure to TNF-α.

Tumor necrosis factor α (TNF-α) is a major pro-inflammatory cytokine, important in many diseases, that sensitizes nociceptors through its action on a variety of ion channels, including voltage-gated sodium (NaV) channels. We show here that TNF-α acutely upregulates sensory neuron excitability and current density of threshold channel NaV1.7. Using electrophysiological recordings and live imaging, we demonstrate that this effect on NaV1.7 is mediated by p38 MAPK and identify serine 110 in the channel's N terminus as the phospho-acceptor site, which triggers NaV1.7 channel insertion into the somatic membrane. We also show that the N terminus of NaV1.7 is sufficient to mediate this effect. Although acute TNF-α treatment increases NaV1.7-carrying vesicle accumulation at axonal endings, we did not observe increased channel insertion into the axonal membrane. These results identify molecular determinants of TNF-α-mediated regulation of NaV1.7 in sensory neurons and demonstrate compartment-specific effects of TNF-α on channel insertion in the neuronal plasma membrane.

esRAGE-expressing oHSV enhances anti-tumor efficacy by inhibition of endothelial cell activation.

High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) molecule that plays an important role in inflammation and tumorigenesis. Receptor for advanced glycation end products (RAGE) is one of the major receptors to which extracellular HMGB1 binds to mediate its activity. RAGE is highly expressed on the endothelial cells (ECs) and regulates endothelial permeability during inflammation. Here, we introduced the endogenous secretory form of RAGE (esRAGE) as a decoy receptor for RAGE ligands into an oncolytic herpes simplex virus 1 (oHSV) (OVesRAGE), which, upon release, can function to block RAGE signaling. OVesRAGE significantly decreased phosphorylation of MEK1/2 and Erk and increased cleaved PARP in glioblastoma (GBM) cells in vitro and in vivo. oHSV-infected GBM cells co-cultured with ECs were used to test OVesRAGE effect on EC activation, vessel leakiness, virus replication, and tumor cell killing. OVesRAGE could effectively secrete esRAGE and rescue virus-induced EC migration and activation. Reduced EC activation facilitated virus replication in tumor cells when co-cultured with ECs. Finally, OVesRAGE significantly enhanced therapeutic efficacy in GBM-bearing mice. Collectively, our data demonstrate that HMGB1-RAGE signaling could be a promising target and that its inhibition is a feasible approach to improve the efficacy of oHSV therapy.

Phosphorylation states greatly regulate the activity and gating properties of Cav 3.1 T-type Ca2+ channels.

Cav 3.1 T-type Ca2+ channels play pivotal roles in neuronal low-threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Cav 3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Cav 3.1 channel has been poorly investigated. In this work, we analyzed rat Cav 3.1 protein expressed in HEK-293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Cav 3.1 phosphorylation map which includes the reported mouse Cav 3.1 phosphosites. Site-directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho-silent Cav 3.1 mutants expressed in Xenopus oocytes showed that the phospho-silent mutation of the N-terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho-silent mutations of the C-terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage-dependent gating properties. In contrast, the phosphomimetic Asp mutations of Cav 3.1 on the N- and C-terminal Ser residues reversed the effects of the phospho-silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Cav 3.1 at the N- and C-terminal regions play crucial roles in the regulation of the channel activity and voltage-dependent gating properties.

Global analysis of ginsenoside Rg1 protective effects in ß-amyloid-treated neuronal cells.

BACKGROUND: A number of reports have described the protective effects of ginsenoside Rg1 (Rg1) in Alzheimer's disease (AD). However, the protective mechanisms of Rg1 in AD remain elusive. METHODS: To investigate the potential mechanisms of Rg1 in ß-amyloid peptide-treated SH-SY5Y cells, a comparative proteomic analysis was performed using stable isotope labeling with amino acids in cell culture combined with nano-LC-MS/MS. RESULTS: We identified a total of 1,149 proteins in three independent experiments. Forty-nine proteins were significantly altered by Rg1 after exposure of the cells to ß-amyloid peptides. The protein interaction network analysis showed that these altered proteins were clustered in ribosomal proteins, mitochondria, the actin cytoskeleton, and splicing proteins. Among these proteins, mitochondrial proteins containing HSD17B10, AARS2, TOMM40, VDAC1, COX5A, and NDUFA4 were associated with mitochondrial dysfunction in the pathogenesis of AD. CONCLUSION: Our results suggest that mitochondrial proteins may be related to the protective mechanisms of Rg1 in AD.

Proteomic analysis reveals that the protective effects of ginsenoside Rb1 are associated with the actin cytoskeleton in ß-amyloid-treated neuronal cells.

BACKGROUND: The ginsenoside Rb1 (Rb1) is the most abundant compound in the root of Panax ginseng. Recent studies have shown that Rb1 has a neuroprotective effect. However, the mechanisms underlying this effect are still unknown. METHODS: We used stable isotope labeling with amino acids in cell culture, combined with quantitative mass spectrometry, to explore a potential protective mechanism of Rb1 in ß-amyloid-treated neuronal cells. RESULTS: A total of 1,231 proteins were commonly identified from three replicate experiments. Among these, 40 proteins were significantly changed in response to Rb1 pretreatment in ß-amyloid-treated neuronal cells. Analysis of the functional enrichments and protein interactions of altered proteins revealed that actin cytoskeleton proteins might be linked to the regulatory mechanisms of Rb1. The CAP1, CAPZB, TOMM40, and DSTN proteins showed potential as molecular target proteins for the functional contribution of Rb1 in Alzheimer's disease (AD). CONCLUSION: Our proteomic data may provide new insights into the protective mechanisms of Rb1 in AD.

PTPσ functions as a presynaptic receptor for the glypican-4/LRRTM4 complex and is essential for excitatory synaptic transmission.

Leukocyte common antigen-related receptor protein tyrosine phosphatases--comprising LAR, PTPδ, and PTPσ--are synaptic adhesion molecules that organize synapse development. Here, we identify glypican 4 (GPC-4) as a ligand for PTPσ. GPC-4 showed strong (nanomolar) affinity and heparan sulfate (HS)-dependent interaction with the Ig domains of PTPσ. PTPσ bound only to proteolytically cleaved GPC-4 and formed additional complex with leucine-rich repeat transmembrane protein 4 (LRRTM4) in rat brains. Moreover, single knockdown (KD) of PTPσ, but not LAR, in cultured neurons significantly reduced the synaptogenic activity of LRRTM4, a postsynaptic ligand of GPC-4, in heterologous synapse-formation assays. Finally, PTPσ KD dramatically decreased both the frequency and amplitude of excitatory synaptic transmission. This effect was reversed by wild-type PTPσ, but not by a HS-binding-defective PTPσ mutant. Our results collectively suggest that presynaptic PTPσ, together with GPC-4, acts in a HS-dependent manner to maintain excitatory synapse development and function.