Mechanism, and treatment of anti-CV2/CRMP5 autoimmune pain.

Paraneoplastic neurological syndromes arise from autoimmune reactions against nervous system antigens due to a maladaptive immune response to a peripheral cancer. Patients with small cell lung carcinoma or malignant thymoma can develop an autoimmune response against the CV2/collapsin response mediator protein 5 (CRMP5) antigen. For reasons that are not understood, approximately 80% of patients experience painful neuropathies. Here, we investigated the mechanisms underlying anti-CV2/CRMP5 autoantibodies (CV2/CRMP5-Abs)-related pain. We found that patient-derived CV2/CRMP5-Abs can bind to their target in rodent dorsal root ganglia (DRG) and superficial laminae of the spinal cord. CV2/CRMP5-Abs induced DRG neuron hyperexcitability and mechanical hypersensitivity in rats that were abolished by preventing binding to their cognate autoantigen CRMP5. The effect of CV2/CRMP5-Abs on sensory neuron hyperexcitability and mechanical hypersensitivity observed in patients was recapitulated in rats using genetic immunization providing an approach to rapidly identify possible therapeutic choices for treating autoantibody-induced pain including the repurposing of a monoclonal anti-CD20 antibody that selectively deplete B-lymphocytes. These data reveal a previously unknown neuronal mechanism of neuropathic pain in patients with paraneoplastic neurological syndromes resulting directly from CV2/CRMP5-Abs-induced nociceptor excitability. CV2/CRMP5-Abs directly sensitize pain responses by increasing sensory neuron excitability and strategies aiming at either blocking or reducing CV2/CRMP5-Abs can treat pain as a comorbidity in patients with paraneoplastic neurological syndromes.

A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

Identification and targeting of a unique NaV1.7 domain driving chronic pain.

Small molecules directly targeting the voltage-gated sodium channel (VGSC) NaV1.7 have not been clinically successful. We reported that preventing the addition of a small ubiquitin-like modifier onto the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 function and was antinociceptive in rodent models of neuropathic pain. Here, we discovered a CRMP2 regulatory sequence (CRS) unique to NaV1.7 that is essential for this regulatory coupling. CRMP2 preferentially bound to the NaV1.7 CRS over other NaV isoforms. Substitution of the NaV1.7 CRS with the homologous domains from the other eight VGSC isoforms decreased NaV1.7 currents. A cell-penetrant decoy peptide corresponding to the NaV1.7-CRS reduced NaV1.7 currents and trafficking, decreased presynaptic NaV1.7 expression, reduced spinal CGRP release, and reversed nerve injury-induced mechanical allodynia. Importantly, the NaV1.7-CRS peptide did not produce motor impairment, nor did it alter physiological pain sensation, which is essential for survival. As a proof-of-concept for a NaV1.7 -targeted gene therapy, we packaged a plasmid encoding the NaV1.7-CRS in an AAV virus. Treatment with this virus reduced NaV1.7 function in both rodent and rhesus macaque sensory neurons. This gene therapy reversed and prevented mechanical allodynia in a model of nerve injury and reversed mechanical and cold allodynia in a model of chemotherapy-induced peripheral neuropathy. These findings support the conclusion that the CRS domain is a targetable region for the treatment of chronic neuropathic pain.

Neuropilin-1 is essential for vascular endothelial growth factor A-mediated increase of sensory neuron activity and development of pain-like behaviors.

ABSTRACT: Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that binds numerous ligands including vascular endothelial growth factor A (VEGFA). Binding of this ligand to NRP-1 and the co-receptor, the tyrosine kinase receptor VEGFR2, elicits nociceptor sensitization resulting in pain through the enhancement of the activity of voltage-gated sodium and calcium channels. We previously reported that blocking the interaction between VEGFA and NRP-1 with the Spike protein of SARS-CoV-2 attenuates VEGFA-induced dorsal root ganglion (DRG) neuronal excitability and alleviates neuropathic pain, pointing to the VEGFA/NRP-1 signaling as a novel therapeutic target of pain. Here, we investigated whether peripheral sensory neurons and spinal cord hyperexcitability and pain behaviors were affected by the loss of NRP-1. Nrp-1 is expressed in both peptidergic and nonpeptidergic sensory neurons. A CRIPSR/Cas9 strategy targeting the second exon of nrp-1 gene was used to knockdown NRP-1. Neuropilin-1 editing in DRG neurons reduced VEGFA-mediated increases in CaV2.2 currents and sodium currents through NaV1.7. Neuropilin-1 editing had no impact on voltage-gated potassium channels. Following in vivo editing of NRP-1, lumbar dorsal horn slices showed a decrease in the frequency of VEGFA-mediated increases in spontaneous excitatory postsynaptic currents. Finally, intrathecal injection of a lentivirus packaged with an NRP-1 guide RNA and Cas9 enzyme prevented spinal nerve injury-induced mechanical allodynia and thermal hyperalgesia in both male and female rats. Collectively, our findings highlight a key role of NRP-1 in modulating pain pathways in the sensory nervous system.

Accurate tumor segmentation and treatment outcome prediction with DeepTOP.

BACKGROUND: Accurate outcome prediction prior to treatment can facilitate trial design and clinical decision making to achieve better treatment outcome. METHOD: We developed the DeepTOP tool with deep learning approach for region-of-interest segmentation and clinical outcome prediction using magnetic resonance imaging (MRI). DeepTOP was constructed with an automatic pipeline from tumor segmentation to outcome prediction. In DeepTOP, the segmentation model used U-Net with a codec structure, and the prediction model was built with a three-layer convolutional neural network. In addition, the weight distribution algorithm was developed and applied in the prediction model to optimize the performance of DeepTOP. RESULTS: A total of 1889 MRI slices from 99 patients in the phase III multicenter randomized clinical trial (NCT01211210) on neoadjuvant treatment for rectal cancer was used to train and validate DeepTOP. We systematically optimized and validated DeepTOP with multiple devised pipelines in the clinical trial, demonstrating a better performance than other competitive algorithms in accurate tumor segmentation (Dice coefficient: 0.79; IoU: 0.75; slice-specific sensitivity: 0.98) and predicting pathological complete response to chemo/radiotherapy (accuracy: 0.789; specificity: 0.725; and sensitivity: 0.812). DeepTOP is a deep learning tool that could avoid manual labeling and feature extraction and realize automatic tumor segmentation and treatment outcome prediction by using the original MRI images. CONCLUSION: DeepTOP is open to provide a tractable framework for the development of other segmentation and predicting tools in clinical settings. DeepTOP-based tumor assessment can provide a reference for clinical decision making and facilitate imaging marker-driven trial design.

Targeting the vascular endothelial growth factor A/neuropilin 1 axis for relief of neuropathic pain.

ABSTRACT: Vascular endothelial growth factor A (VEGF-A) is a pronociceptive factor that causes neuronal sensitization and pain. We reported that blocking the interaction between the membrane receptor neuropilin 1 (NRP1) and VEGF-A-blocked VEGF-A-mediated sensory neuron hyperexcitability and reduced mechanical hypersensitivity in a rodent chronic neuropathic pain model. These findings identified the NRP1-VEGF-A signaling axis for therapeutic targeting of chronic pain. In an in-silico screening of approximately 480 K small molecules binding to the extracellular b1b2 pocket of NRP1, we identified 9 chemical series, with 6 compounds disrupting VEGF-A binding to NRP1. The small molecule with greatest efficacy, 4'-methyl-2'-morpholino-2-(phenylamino)-[4,5'-bipyrimidin]-6(1H)-one, designated NRP1-4, was selected for further evaluation. In cultured primary sensory neurons, VEGF-A enhanced excitability and decreased firing threshold, which was blocked by NRP1-4. In addition, NaV1.7 and CaV2.2 currents and membrane expression were potentiated by treatment with VEGF-A, and this potentiation was blocked by NRP1-4 cotreatment. Neuropilin 1-4 reduced VEGF-A-mediated increases in the frequency and amplitude of spontaneous excitatory postsynaptic currents in dorsal horn of the spinal cord. Neuropilin 1-4 did not bind to more than 300 G-protein-coupled receptors and receptors including human opioids receptors, indicating a favorable safety profile. In rats with spared nerve injury-induced neuropathic pain, intrathecal administration of NRP1-4 significantly attenuated mechanical allodynia. Intravenous treatment with NRP1-4 reversed both mechanical allodynia and thermal hyperalgesia in rats with L5/L6 spinal nerve ligation-induced neuropathic pain. Collectively, our findings show that NRP1-4 is a first-in-class compound targeting the NRP1-VEGF-A signaling axis to control voltage-gated ion channel function, neuronal excitability, and synaptic activity that curb chronic pain.

RAGE signaling pathway is involved in CUS-induced depression-like behaviors by regulating the expression of NR2A and NR2B in rat hippocampus DG.

NMDA receptors play pivotal roles in the neurobiology of chronic stress-induced mood disorders. But the mechanism for chronic stress to disturb the expression of NMDA receptor subunits is still unclear. Recent researches indicated the involvement RAGE signaling pathway in regulation of glutamate system functions. In this study, we hypothesized RAGE signaling pathway mediated chronic stress-induced alteration in the expression of NMDA receptor subunits, leading to depressive-like behaviors. CUS decreased the expression of RAGE, NR2A, and NR2B, inhibited the phosphorylation of transcript factor ERK and CREB in rat hippocampus DG. RAGE knockdown in hippocampus DG by RAGE shRNA lentiviral particles induced depressive-like behaviors, reduced the mRNA and protein expression of NR2A and NR2B, and inhibited the phosphorylation of ERK and CREB. RAGE over-expression in hippocampus DG by RAGE adenovirus particles reversed the effects of CUS on depressive-like behaviors, ERK and CREB phosphorylation, and NR2A and NR2B expression. Our findings suggests that RAGE signaling pathway at least partially participates in the regulation of NR2A and NR2B expression, which mediates the effects of chronic stress on the depressive-like behaviors. These data provide evidence for RAGE signaling as a possible new pathway through which chronic stress results in the maladaptation of NMDA receptors.

The natural product argentatin C attenuates postoperative pain via inhibition of voltage-gated sodium and T-type voltage-gated calcium channels.

BACKGROUND AND PURPOSE: Postoperative pain occurs in as many as 70% of surgeries performed worldwide. Postoperative pain management still relies on opioids despite their negative consequences, resulting in a public health crisis. Therefore, it is important to develop alternative therapies to treat chronic pain. Natural products derived from medicinal plants are potential sources of novel biologically active compounds for development of safe analgesics. In this study, we screened a library of natural products to identify small molecules that target the activity of voltage-gated sodium and calcium channels that have important roles in nociceptive sensory processing. EXPERIMENTAL APPROACH: Fractions derived from the Native American medicinal plant, Parthenium incanum, were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion (DRG) neurons. Further separation of these fractions yielded a cycloartane-type triterpene identified as argentatin C, which was additionally evaluated using whole-cell voltage and current-clamp electrophysiology, and behavioural analysis in a mouse model of postsurgical pain. KEY RESULTS: Argentatin C blocked the activity of both voltage-gated sodium and low-voltage-activated (LVA) calcium channels in calcium imaging assays. Docking analysis predicted that argentatin C may bind to NaV 1.7-1.9 and CaV 3.1-3.3 channels. Furthermore, argentatin C decreased Na+ and T-type Ca2+ currents as well as excitability in rat and macaque DRG neurons, and reversed mechanical allodynia in a mouse model of postsurgical pain. CONCLUSION AND IMPLICATIONS: These results suggest that the dual effect of argentatin C on voltage-gated sodium and calcium channels supports its potential as a novel treatment for painful conditions.

Cell specific regulation of NaV1.7 activity and trafficking in rat nodose ganglia neurons.

The voltage-gated sodium NaV1.7 channel sets the threshold for electrogenesis. Mutations in the gene encoding human NaV1.7 (SCN9A) cause painful neuropathies or pain insensitivity. In dorsal root ganglion (DRG) neurons, activity and trafficking of NaV1.7 are regulated by the auxiliary collapsin response mediator protein 2 (CRMP2). Specifically, preventing addition of a small ubiquitin-like modifier (SUMO), by the E2 SUMO-conjugating enzyme Ubc9, at lysine-374 (K374) of CRMP2 reduces NaV1.7 channel trafficking and activity. We previously identified a small molecule, designated 194, that prevented CRMP2 SUMOylation by Ubc9 to reduce NaV1.7 surface expression and currents, leading to a reduction in spinal nociceptive transmission, and culminating in normalization of mechanical allodynia in models of neuropathic pain. In this study, we investigated whether NaV1.7 control via CRMP2-SUMOylation is conserved in nodose ganglion (NG) neurons. This study was motivated by our desire to develop 194 as a safe, non-opioid substitute for persistent pain, which led us to wonder how 194 would impact NaV1.7 in NG neurons, which are responsible for driving the cough reflex. We found functioning NaV1.7 channels in NG neurons; however, they were resistant to downregulation via either CRMP2 knockdown or pharmacological inhibition of CRMP2 SUMOylation by 194. CRMP2 SUMOylation and interaction with NaV1.7 was consered in NG neurons but the endocytic machinery was deficient in the endocytic adaptor protein Numb. Overexpression of Numb rescued CRMP2-dependent regulation on NaV1.7, rendering NG neurons sensitive to 194. Altogether, these data point at the existence of cell-specific mechanisms regulating NaV1.7 trafficking.

Design, synthesis and biological evaluation of 2,4-pyrimidinediamine derivatives as ALK and HDACs dual inhibitors for the treatment of ALK addicted cancer.

Simultaneous inhibition of histone deacetylases (HDACs) and anaplastic lymphoma kinase (ALK) could enhance therapeutic activity against ALK addicted cancer cells. Herein, a new series of 2,4-pyrimidinediamine derivatives as ALK and HDACs dual inhibitors were designed, synthesised and evaluated. Compound 12a which possessed good inhibitory potency against ALKwt and HDAC1, exhibited stronger antiproliferative activity than Ceritinib on ALK positive cancer cell lines though inducing cell apoptosis and cell cycle arrest in vitro and in vivo. In addition, the mechanism is further verified by the down-regulation of p-ALK protein, and up-regulation of Acetylated histone 3 (Ac-H3) protein in cancer cells. These results suggested that 12a would be a potential candidate for the ALK addicted cancer treatment.

Anaplastic lymphoma kinase tyrosine kinase inhibitors associated gastrointestinal obstruction, perforation, and ulceration: an analysis of the FDA adverse event reporting system database (FAERS).

BACKGROUND : There have been cases reporting anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) and associated serious gastrointestinal (GI) adverse drug reactions (gastrointestinal obstruction, perforation, and ulceration). These adverse drug reactions are not in the drug package inserts, and the drug relationships are not proven in the literature.  AIM: We aimed to examine the potential association between GI obstruction, perforation, and ulceration, and ALK-TKIs by data mining of the US FDA Adverse Event Reporting System (FAERS). METHOD  : We conducted a disproportionality analysis of GI obstruction, perforation, and ulceration by estimating the reporting odds ratios (ROR) and the information component (IC) with 95% confidence intervals. RESULTS : A total of 279 cases of ALK-TKI-associated GI obstruction, perforation, and ulceration from January 1, 2011, to December 31, 2020, were identified. GI obstruction, perforation, and ulceration cause 16% of cases of death. A significantly increased reporting rate for GI obstruction [ROR 1.77 (1.45-2.15); IC 0.82 (0.53-2.03)] and perforation [ROR 1.61 (1.28-2.02); IC 0.68 (0.35-1.92)] was observed for ALK-TKIs as a drug class. The signal of GI ulceration was detected only in crizotinib [ROR 1.23 (1.01-1.50); IC 0.29 (0.01-1.51)]. A statistically significant ROR and IC emerged for the site of the esophagus.  CONCLUSION : Overall, the pharmacovigilance study of the FAERS indicates slightly increased reporting of GI obstruction and perforation, which may cause severe or even fatal outcomes among ALK-TKIs users.

Stereospecific Effects of Benzimidazolonepiperidine Compounds on T-Type Ca2+ Channels and Pain.

T-type calcium channels activate in response to subthreshold membrane depolarizations and represent an important source of Ca2+ influx near the resting membrane potential. These channels regulate neuronal excitability and have been linked to pain. For this reason, T-type calcium channels are suitable molecular targets for the development of new non-opioid analgesics. Our previous work identified an analogue of benzimidazolonepiperidine, 5bk, that preferentially inhibited CaV3.2 channels and reversed mechanical allodynia. In this study, we synthesized and screened a small library of 47 compounds derived from 5bk. We found several compounds that inhibited the Ca2+ influx in DRG neurons of all sizes. After separating the enantiomers of each active compound, we found two compounds, 3-25-R and 3-14-3-S, that potently inhibited the Ca2+ influx. Whole-cell patch clamp recordings from small- to medium-sized DRG neurons revealed that both compounds decreased total Ca2+. Application of 3-14-3-S (but not 3-25-R) blocked transiently expressed CaV3.1-3.3 channels with a similar IC50 value. 3-14-3-S decreased T-type, but not N-type, Ca2+ currents in DRG neurons. Furthermore, intrathecal delivery of 3-14-3-S relieved tonic, neuropathic, and inflammatory pain in preclinical models. 3-14-3-S did not exhibit any activity against G protein-coupled opioid receptors. Preliminary docking studies also suggest that 3-14-3-S can bind to the central pore domain of T-type channels. Together, our chemical characterization and functional and behavioral data identify a novel T-type calcium channel blocker with in vivo efficacy in experimental models of tonic, neuropathic, and inflammatory pain.

Distinct Role of Mono-2-ethylhexyl Phthalate in Neuronal Transmission in Rat CA3 Hippocampal Neurons: Involvement of Ion Channels.

Mono-(2-ethylhexyl) phthalate (MEHP) is one of the main active metabolites of di-(2-ethylhexyl) phthalate (DEHP). In our previous works, by using rat and Drosophila models, we showed a disruption of neural function due to DEHP. However, the exact neural effects of MEHP are still unclear. To explore the effects of MEHP on the central nervous system, the electrophysiological properties of spontaneous action potential (sAP), mini-excitatory postsynaptic currents (mEPSCs), ion channels, including Na+, Ca2+, and K+ channels from rat CA3 hippocampal neurons area were assessed. Our data showed that MEHP (at the concentrations of 100 or 300 µM) decreased the amplitude of sAP and the frequency of mEPSCs. Additionally, MEHP (100 or 300 µM) significantly reduced the peak current density of Ca2+ channels, whereas only the concentration of 300 µM decreased the peak current density of Na+ and K+ channels. Therefore, our results indicate that exposure to MEHP could affect the neuronal excitability and synaptic plasticity of rat CA3 hippocampal neurons by inhibiting ion channels' activity, implying the distinct role of MEHP in neural transmission.

Accelerated biological aging in COVID-19 patients.

Chronological age is a risk factor for SARS-CoV-2 infection and severe COVID-19. Previous findings indicate that epigenetic age could be altered in viral infection. However, the epigenetic aging in COVID-19 has not been well studied. In this study, DNA methylation of the blood samples from 232 healthy individuals and 413 COVID-19 patients is profiled using EPIC methylation array. Epigenetic ages of each individual are determined by applying epigenetic clocks and telomere length estimator to the methylation profile of the individual. Epigenetic age acceleration is calculated and compared between groups. We observe strong correlations between the epigenetic clocks and individual's chronological age (r > 0.8, p < 0.0001). We also find the increasing acceleration of epigenetic aging and telomere attrition in the sequential blood samples from healthy individuals and infected patients developing non-severe and severe COVID-19. In addition, the longitudinal DNA methylation profiling analysis find that the accumulation of epigenetic aging from COVID-19 syndrome could be partly reversed at late clinic phases in some patients. In conclusion, accelerated epigenetic aging is associated with the risk of SARS-CoV-2 infection and developing severe COVID-19. In addition, the accumulation of epigenetic aging from COVID-19 may contribute to the post-COVID-19 syndrome among survivors.

Neuronal allodynic mechanisms of Slc7a5 (LAT1) in the spared nerve injury rodent model of neuropathic pain.

High-impact chronic pain is suffered by 1 in 5 patients in the USA and globally. Effective, non-addictive, non-opioid therapeutics are urgently needed for the treatment of chronic pain. Slc7a5 (Lat1), also known as system L-neutral amino acid transporter, is involved in a number of physiological processes related to inflammation. Transcriptomics studies have shown that Slc7a5 and its binding partner Slc3a2 are expressed in neurons of the dorsal root ganglia (DRG) and spinal dorsal horn, which are critical to the initiation and maintenance of nociception and pathophysiology of chronic pain. In addition, Slc7a5 is a transporter for the first-line anti-allodynic gabapentinoid drugs and binds to ion channels implicated in nociception and chronic pain including the voltage-gated sodium channel Nav1.7 and the voltage-gated potassium channels Kv1.1 and Kv1.2. We found that blocking Slc7a5 with intrathecal administration of the drug JPH203 alleviated allodynia in the spared nerve injury (SNI) rodent model of neuropathic pain. Western blot and immunohistochemistry studies revealed an increase in Slc7a5 protein levels in the spinal cord and DRGs of SNI mice compared to control mice. Using whole-cell current-clamp electrophysiology, we observed that JPH203 treatment reduced excitability of small-diameter (< 30 µm) DRG neurons from SNI mice, in agreement with its behavioral effects. Voltage-clamp recordings from JPH203-treated naïve rat DRGs identified an effect on tetrodotoxin-resistant (TTX-R) sodium currents. Altogether, these results demonstrate that Slc7a5 is dysregulated in chronic neuropathic pain and can be targeted to provide relief of hypersensitivity.

Discovery of 2,4-pyrimidinediamine derivatives as potent dual inhibitors of ALK and HDAC.

Combination of anaplastic lymphoma kinase (ALK) inhibitor with histone deacetylases (HDAC) inhibitor could exert synergistically anti-proliferative effects on ALK positive non-small cell lung cancer (NSCLC) naïve or resistant cells. In this work, we designed and synthesized a series of 2,4-pyrimidinediamine derivatives as dual ALK and HDAC inhibitors based on pharmacophore merged strategy. Among which, compound 10f displayed the most potent and balanced inhibitory activity against ALK (IC50 = 2.1 nM) and HDAC1 (IC50 = 7.9 nM), respectively. In particular, 10f was also potent against the frequently observed Crizotinib-resistant ALKL1196M (IC50 = 1.7 nM) as well as the Ceritinib-resistant ALKG1202R (IC50 = 0.4 nM) mutants. In antiproliferative activity assay, 10f exhibited impressive activity on ALK-addicted cancer cell lines at low micromole concentrations, which was comparable to that of Crizotinib and Ceritinib. Further flow cytometric analysis indicated that 10f could effectively induce cell death via cell apoptosis and cell cycle arrest. Taken together, these results suggested 10f would be a promising lead compound for the ALK-positive NSCLC treatment, especially the Ceritinib- or Crizotinib-resistant NSCLC.

Effects of Mono-2-ethylhexyl Phthalate on the Neural Transmission of PNs in Drosophila Antennal Lobe.

Long-term exposure to different types of chemicals is hazardous to human health. Di(2-ethylhexyl) phthalate (DEHP) could exert pleiotropic deleterious effects on nervous systems. Mono(2-ethylhexyl) phthalate (MEHP), as one of the most toxic metabolites of DEHP, may have similar effects on nervous systems. However, no effects of MEHP on neural circuits have been reported. To uncover the regulation of MEHP on neural transmission, the functional changes of neural excitability and synaptic plasticity of projection neurons (PNs) have been assessed. In the current study, we recorded the action potentials (APs), stimulate action potentials (sti-APs), mini excitement postsynaptic current (mEPSC), calcium currents, and sodium currents from PNs of isolated whole brain of Drosophila model utilizing patch clamp recordings. We found that MEHP-300 (at the concentration of 300 µM), but not MHEP-100 (at the concentration of 100 µM), significantly decreased the frequency and amplitude of APs. Besides, the amplitude and anti-amplitude of sti-APs were reduced with the application of MEHP-300. Meanwhile, MEHP-300 reduced the frequency of mEPSC, but not the amplitude. Furthermore, MEHP-300 reduced the peak current densities of sodium and calcium channels. Therefore, our results indicated that MEHP could alter the neural excitability and synaptic plasticity of PNs by inhibiting the ion channels activities, revealing the potential modulation of MEHP on neural transmission of PNs.

ZNRF2 attenuates focal cerebral ischemia/reperfusion injury in rats by inhibiting mTORC1-mediated autophagy.

Zinc and ring finger 2 (ZNRF2), an E3 ubiquitin ligase, plays a crucial role in many diseases. However, its role in cerebral ischemia/reperfusion injury (CIRI) still remains unknown. In this study, the function and molecular mechanism of ZNRF2 in CIRI in vivo and vitro was studied. ZNRF2 was found to be dramatically downregulated in CIRI. Overexpression of ZNRF2 could significantly reduce the neurological deficit, brain infarct volume and histopathological damage of cortex in middle cerebral artery occlusion/reperfusion. Concomitantly, overexpression of ZNRF2 increased the primary neuronal viability and decreased the neuronal apoptosis induced by oxygen-glucose deprivation and reoxygenation (OGD/R). Mechanistically, overexpression of ZNRF2 inhibited the over-induction of autophagy induced by OGD/R which was abolished by mTORC1 inhibitor rapamycin. It can be concluded that ZNRF2 plays a protective effect in CIRI and the underlying mechanism may be related to the inhibition of mTORC1-mediated autophagy.

Effect of TO901317 on GF to promote the differentiation of human bone marrow mesenchymal stem cells into dopamine neurons on Parkinson's disease.

BACKGROUND: Human bone marrow mesenchymal stem cells (hBMSCs) could differentiate into dopamine-producing cells and ameliorate behavioral deficits in Parkinson's disease (PD) models. Liver X receptors (LXRs) are involved in the maintenance of the normal function of central nervous system myelin. Therefore, the previous work of our team has found the induction of cocktail-induced to dopaminergic (DA) phenotypes from adult rat BMSCs by using sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), basic fibroblast growth factor (bFGF), and TO901317 (an agonist of LXRs) with 87.42% of efficiency in a 6-day induction period. But we did not verify whether the induced cells had the corresponding neural function. METHODS: Expressions of LXRα, LXRß, and tyrosine hydroxylase (TH) were detected by immunofluorescence and western blot. Adenosine triphosphate-binding cassette transporter A1 (ABCA1) was detected by quantitative real-time PCR. The induced cells were transplanted into PD rats to study whether the induced cells are working. RESULTS: The induced cells can release the dopamine transmitter; the maximum induction efficiency of differentiation of hBMSCs into DA neurons was 91.67% under conditions of combined use with TO901317 and growth factors (GF). When the induced-cells were transplanted into PD rats, the expression of TH in the striatum increased significantly, and the behavior of PD rats induced by apomorphine was significantly improved. CONCLUSION: The induced cells have the function of DA neurons and have the potential to treat PD. TO901317 promoted differentiation of hBMSCs into DA neurons, which may be related to activation of the LXR-ABCA1 signaling pathway.

Non-SUMOylated CRMP2 decreases NaV1.7 currents via the endocytic proteins Numb, Nedd4-2 and Eps15.

Voltage-gated sodium channels are key players in neuronal excitability and pain signaling. Functional expression of the voltage-gated sodium channel NaV1.7 is under the control of SUMOylated collapsin response mediator protein 2 (CRMP2). When not SUMOylated, CRMP2 forms a complex with the endocytic proteins Numb, the epidermal growth factor receptor pathway substrate 15 (Eps15), and the E3 ubiquitin ligase Nedd4-2 to promote clathrin-mediated endocytosis of NaV1.7. We recently reported that CRMP2 SUMO-null knock-in (CRMP2K374A/K374A) female mice have reduced NaV1.7 membrane localization and currents in their sensory neurons. Preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in CRMP2K374A/K374A female mice with neuropathic pain. Here we report that inhibiting clathrin assembly in nerve-injured male CRMP2K374A/K374A mice precipitated mechanical allodynia in mice otherwise resistant to developing persistent pain. Furthermore, Numb, Nedd4-2 and Eps15 expression was not modified in basal conditions in the dorsal root ganglia (DRG) of male and female CRMP2K374A/K374A mice. Finally, silencing these proteins in DRG neurons from female CRMP2K374A/K374A mice, restored the loss of sodium currents. Our study shows that the endocytic complex composed of Numb, Nedd4-2 and Eps15, is necessary for non-SUMOylated CRMP2-mediated internalization of sodium channels in vivo.