2a Cells Research Articles

Neuropharmacological evaluation on four traditional herbs used as nervine tonic and commonly available as Shankhpushpi in India

BackgroundShankhpushpi is an Ayurvedic drug, widely used for its actions on the central nervous system, especially to improve intellect and boost memory. Four botanicals viz. Canscora decussata Schult. (CD), Clitorea ternatea Linn. (CT), Convolvulus pluricaulis Choisy. (CP) and Evolvulus alsinoides Linn. (EA) are considered as sources of Shankhpushpi by Indian practitioners on the basis of their morphological descriptions given in ancient texts. ObjectiveThe present study was undertaken to evaluate the neuropharmacological effect of four herbs commonly identified as source of Shankhpushpi. Materials and methodsMethanol extracts of all four varieties were tested and evaluated in vitro and in vivo for their neuropharmacological effects. Experiments such as protection against β-amyloid induced neurotoxicity on brain cell line (Neuro 2A), antioxidant potential, AchE (acetylcholinesterase enzyme) inhibition, and 5-LOX (lipoxygenase) enzyme inhibition were conducted for in vitro evaluation. For in vivo evaluation, scopolamine (0.3 mg/kg i.p.) induced memory retrieval using pole climbing apparatus and Morris water maze were performed in rat models. ResultsIt was found that protective effects of EA and CD against β-amyloid induced neurotoxicity in Neuro 2A cells were significantly higher than CT and CP. EA proved to be superior than other varieties on the basis of antioxidant activity, AchE inhibitory and LOX inhibitory activities. The preventive activity of EA on scopolamine induced memory retrieval in pole climbing and Morris water maze task in rats was found to be higher than that of CD, CT and CP. ConclusionEA has remarkable neuropharmacological effect as compared to other three varieties of Shankhpushpi. This effect may be attributed due to the presence of steroids (stigmasterol and betulinic acid), coumarins (scopoletin) and flavonoids (β-carotene and chlorogenic acid). Hence it can be used as a promising lead in development and management of neuronal disorders including Alzheimer's disease.

Post-translational incorporation of 3,4-dihydroxyphenylalanine into the C terminus of α-tubulin in living cells.

The C-terminal tyrosine (Tyr) of the α-tubulin chain is subjected to post-translational removal and readdition in a process termed the "detyrosination/tyrosination cycle". We showed in previous studies using soluble rat brain extracts that l-3,4-dihydroxyphenylalanine (l-Dopa) is incorporated into the same site as Tyr. We now demonstrate that l-Dopa incorporation into tubulin also occurs in living cells. We detected such incorporation by determining the "tyrosination state" of tubulin before and after incubation of cells in the presence of l-Dopa. The presence of a tubulin isospecies following l-Dopa incubation that was not recognized by antibodies specific to Tyr- and deTyr-tubulin was presumed to reflect formation of Dopa-tubulin. l-Dopa was identified by HPLC as the C-terminal compound bound to α-tubulin. l-Dopa incorporation into tubulin was observed in Neuro 2A cells and several other cell lines, and was not due to denovo protein biosynthesis. Dopa-tubulin had microtubule-forming capability similar to that of Tyr- and deTyr-tubulin. l-Dopa incorporation into tubulin did not notably alter cell viability, morphology, or proliferation rate. CAD cells (a neuron-like cell line derived from mouse brain) are easily cultured under differentiating and nondifferentiating conditions, and can be treated with l-Dopa. Treatment of CAD cells with l-Dopa and consequent increase in l-Dopa-tubulin resulted in reduction of microtubule dynamics in neurite-like processes.

Adenosine monophosphate-activated protein kinase modulation by berberine attenuates mitochondrial deficits and redox imbalance in experimental diabetic neuropathy

Adenosine monophosphate-activated protein kinase (AMPK) has been studied for its myriad metabolic and mitochondrial benefits in several chronic diseases. Recent studies have uncovered its therapeutic potential against mitochondrial dysfunction in cultured dorsal root ganglion (DRG) neurons isolated from streptozotocin (STZ) induced diabetic rats. The present study is aimed at evaluating the pharmacological efficacy of berberine (BRB), a natural AMPK activator against experimental diabetic neuropathy (DN) phenotype developed in STZ (55 mg/kg, i.p.) induced diabetic rats and neurotoxicity in high-glucose (30 mM) stimulated neuro 2a (N2A) cells. Diabetic-rats have shown reduced expression of p-AMPK (Thr 172) in sciatic nerves with a consequent reduction in mitochondrial biogenesis and autophagy. BRB (50 & 100 mg/kg, po) administration to diabetic rats for 2-weeks rescued mitochondrial functional deficits and autophagy impairment by increasing the p-AMPK expression. BRB administration also augmented the NEF-2 related factor 2 (Nrf2) mediated endogenous antioxidant defence systems to restrain neuronal damage and neuroinflammation. These effects after BRB administration resulted in enhanced conduction velocity, improved nerve blood flow and attenuated hyperalgesia. Similarly, BRB exposure (5 & 10 μM) to N2A cells attenuated high glucose induced ROS generation, mitochondrial membrane depolarization through the promotion of Nrf2 mediated antioxidant defence systems. BRB exposure augmented peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) mediated mitochondrial biogenesis in neuronal cells. Results from this study signify the importance of mitoprotection conferred by BRB in DN and can be used as a preliminary basis for further molecular exploration.

RGS2 modulates the selection of Gαi/o subtype involved in dopamine D2 receptor signaling

IntroductionRegulator of G‐protein signaling (RGS) proteins modulate GPCR activity by terminating G protein signaling through acceleration of GTP hydrolysis. It is believed that RGS proteins are recruited by and bind to activated Gα subunits to exert GTPase‐activating (GAP) activity. However, recent literature has indicated that certain RGS proteins bind to GPCRs and this binding is critical for receptor‐mediated G protein signaling. Previously, we reported that RGS2 is a negative modulator of dopamine D2 receptor‐mediated Gαi/o signaling. The present study aimed to investigate the mechanism underlying the interaction of RGS2 with D2Rs and Gαi/o proteins.MethodsIn the present study, we utilized a neuroblastoma 2a cell line stably expressing the short form of dopamine D2 receptors (N2A‐D2S). RGS2 N‐terminus truncation (partial and full truncation) mutants, RGS2 siRNA and corresponding controls were transiently transfected into N2A‐D2S cells. Immunoprecipitation was performed to evaluate the coupling between D2Rs and RGS2. Immunocytochemistry and confocal microscopy were utilized to study agonist (quinpirole) stimulated recruitment of Gαi/o subtypes to surface D2Rs. [35S] GTPγS binding was performed to measure D2R‐stimulated Gαi/o activation.ResultsOur data indicate that RGS2 interacts with D2Rs via its N‐terminus and modulates D2R‐stimulated recruitment of Gαi/o subtypes. Specifically, we found that: 1) RGS2 couples to D2Rs via its N‐terminus. RGS2 N‐terminus truncation abolishes the co‐localization of RGS2 with surface D2Rs 2) RGS2 N‐terminus truncation attenuated D2R‐stimulated Gαi/o activation; and 3) RGS2 knockdown alters the selection of Gα subtype recruited to activated D2Rs. D2R stimulation by quinpirole (5 min, 1 μM) induced translocation of Gαi2, not Gαo, towards the plasma membrane in the control cells. However, RGS2 knockdown by siRNA resulted in the translocation of Gαo, not Gαi2, towards the plasma membrane after D2R stimulation.These results suggest that the interaction between RGS2 and D2Rs mediates the selection of the Gαi/o subtype engaged in D2R‐mediated signaling. The finding of this study highlights a novel mechanistic action of RGS2 in regulation of D2R‐mediated Gαi/o signaling.Support or Funding InformationThis work is supported by NIH NIAAA training grant T32 AA007565, DA006634 and a pilot grant from Center for Molecular Communication and Signaling at Wake Forest University.

25-Hydroxy vitamin D suppresses hepatitis C virus replication and contributes to rapid virological response of treatment efficacy.

25-Hydroxy vitamin D (Vit D) plays a role in treatment outcomes in chronic hepatitis C virus (HCV) infection. We aimed to clarify whether HCV replication is inhibited by Vit D in HCV replicon cells. Clinical implication was assessed for rapid virological response (RVR) and sustained virological response (SVR) among those patients receiving antiviral therapy. Cell survival and viral loads were observed in Con1 (genotype 1b) and J6/JFH (genotype 2a) cells treated with different doses of Vit D. Three groups of patients with different treatment responses were recruited to assess their Vit D levels: group A, RVR-/SVR-; group B, RVR+/SVR-; and group C, RVR+/SVR+. The viral load of Con1 cells decreased by 69%, 80%, and 86% following treatment with 1μM, 5μM, and 10μM Vit D, respectively (P <0.0001). In J6/JFH cells, it decreased by 12%, 55%, and 80.5% following treatment with 1μM, 5μM, and 10μM Vit D, respectively (P <0.0001). There was a significant increase of Vit D between chronic hepatitis C groups, ranging from 4.4 ± 5.6ng/mL in group A (n = 44), to 17.2 ± 11.6ng/mL in group B (n = 44), and 32.5 ± 37.5ng/mL of group C (n = 44) (P <0.001). Advanced fibrosis (odds ratio = 0.13, 95% confidence interval = 0.04-0.41, P <0.001) and Vit D deficiency (<10ng/mL) (odds ratio = 0.11, 95% confidence interval = 0.03-0.43, P = 0.001) were predictive of SVR in the multivariate regression analysis. Vitamin D decreases HCV replication and also contributes to early treatment viral kinetics.

PGC-1α or FNDC5 Is Involved in Modulating the Effects of Aβ1-42 Oligomers on Suppressing the Expression of BDNF, a Beneficial Factor for Inhibiting Neuronal Apoptosis, Aβ Deposition and Cognitive Decline of APP/PS1 Tg Mice.

Alzheimer's disease (AD) is generally defined as the aberrant production of β-amyloid protein (Aβ) and hyperphosphorylated tau protein, which are deposited in β-amyloid plaques (APs) and neurofibrillary tangles (NFTs), respectively. Decreased levels of brain-derived neurotrophic factor (BDNF) have been detected in patients with AD compared to control subjects. However, the underlying molecular mechanisms driving the downregulation of the BDNF remain unknown. Therefore, we explored the mechanisms underlying the regulation of BDNF in the neurons of APP/PS1 transgenic (Tg) mice, an AD experimental model. Using the APP/PS1 Tg mice, we found that BDNF expression was markedly downregualted at the age of 3- and 9-month-old. After cerebroventricular injection (i.c.v) of Aβ1−42 oligomers into the mice, BDNF was also found to be decreased, which demonstrated the critical roles of the Aβ1−42 oligomers in regulating the expression of BDNF. In neuronal culture, peroxisome proliferators-activated receptor γ coactivator 1α (PGC-1α) and fibronectin type III domain-containing 5 (FNDC5) were found to be downregulated by treatment with the Aβ1−42 oligomers. In addition, overexpression of either PGC-1α or FNDC5 reversed the suppressive effects of the Aβ1−42 oligomers on the expression of BDNF in neuroblastoma 2a (n2a) cells. More importantly, elevating the levels of PGC-1α, FNDC5 or BDNF in the n2a cells counteracted the effects of the Aβ1−42 oligomers on neuronal apoptosis. Additionally, intranasal administration BDNF in the APP/PS1 Tg mice decreased the Aβ deposition and reduced the cognitive decline of the mice.

Inhibition of veratridine-induced delayed inactivation of the voltage-sensitive sodium channel by synthetic analogs of crambescin B

Crambescin B carboxylic acid, a synthetic analog of crambescin B, was recently found to inhibit the voltage-sensitive sodium channels (VSSC) in a cell-based assay using neuroblastoma Neuro 2A cells. In the present study, whole-cell patch-clamp recordings were conducted with three heterologously expressed VSSC subtypes, Nav1.2, Nav1.6 and Nav1.7, in a human embryonic kidney cell line HEK293T to further characterize the inhibition of VSSC by crambescin B carboxylic acid. Contrary to the previous observation, crambescin B carboxylic acid did not inhibit peak current evoked by depolarization from the holding potential of −100mV to the test potential of −10mV in the absence or presence of veratridine (VTD). In the presence of VTD, however, crambescin B carboxylic acid diminished VTD-induced sustained and tail currents through the three VSSC subtypes in a dose-dependent manner, whereas TTX inhibited both the peak current and the VTD-induced sustained and tail currents through all subtypes of VSSC tested. We thus concluded that crambescin B carboxylic acid does not block VSSC in a similar manner to TTX but modulate the action of VTD, thereby causing an apparent block of VSSC in the cell-based assay.

Isolation, characterization using LC-ESI-QTOF, NMR and in vitro cytotoxicity assay of niclosamide forced degradation products

The present study describes the isolation, characterization and in vitro cytotoxic effect of all forced degradation products of niclosamide (NCM) an anthelmintic class of drug used specifically to treat tapeworms. NCM was subjected to forced degradation involving hydrolysis (acidic, alkaline and neutral), oxidative, photolysis and thermal stress, as per ICH (Q1A (R2)) suggested conditions. The drug under hydrolytic (acidic and basic) conditions showed extensive degradation, while it was stable under neutral hydrolytic, oxidative, photolytic and thermal stress conditions. A total of four degradation products (DPs) were observed and chromatographic separation of drug and its degradation products were achieved on a reverse phase Fortis diphenyl column (150×4.6mm, 5μm) using 0.1% formic acid and acetonitrile as mobile phase in gradient mode. All the four degradation products were isolated by semi preparative LC and its structures were characterized and confirmed by high resolution MS and 1H NMR spectroscopic techniques. In view of safety aspects, cytotoxicity assay were carried out for NCM and its four degradation products on human mononuclear cells and cell lines depicting the major organelle: neuronal (Neuro 2a), hepatic (HepG 2) and alveolar (A549). NCM was found to be non toxic on human mononuclear cells and cell lines at tested concentrations. However DP-1, DP-2, DP-3 and DP-4 showed significant increase in LDH release as compared to control at a concentration of 100μM. DP-1 and DP-3 exhibited toxicity on A549 cells with an IC50 of 92.18±4.93μM and 65.42±6.29μM respectively. DP-2, DP-3 and DP-4 were cytotoxic to Neuro 2a cells with an IC50 of 63.62±3.85μM, 86.09±6.19μM and 42.81±8.10μM respectively. The degradation products were found to be nontoxic on HepG 2 cells.

Pharmacological characterization of rat VD-hemopressin(α), an α-hemoglobin-derived peptide exhibiting cannabinoid agonist-like effects in mice

Hemopressin and related peptides have shown to function as the endogenous ligands or the regulator of cannabinoid receptors. Moreover, hemopressin and its truncated peptides were also reported to produce a slight modulatory effect on opioid system. In the present work, based on the amino acid sequence analyses of hemoglobin subunit α, rat VD-hemopressin(α) [(r)VD-Hpα] was predicted as a cannabinoid peptide derived from rat α-hemoglobin. Furthermore, (r)VD-Hpα was synthesized and characterized in a series of in vitro and in vivo assays. Our results demonstrated that (r)VD-Hpα induced neurite outgrowth in Neuro 2A cells via CB1 receptor. In the tail-flick assay, (r)VD-Hpα dose-dependently exerted central antinociception through CB1 receptor, but not CB2 and opioid receptors. In mice, supraspinal administration of (r)VD-Hpα produced dose-dependent hypothermia, which was partially reduced by the CB1 receptor antagonist AM251, but not by the antagonists of CB2 and opioid receptors. In addition, (r)VD-Hpα caused hypoactivity after intracerebroventricular injection, and this effect was insensitive to the antagonists of cannabinoid and opioid receptors. Further assessment of the side-effects demonstrated that (r)VD-Hpα evoked the limited effects on gastrointestinal transit at antinociceptive doses, but repeated i.c.v. injection of (r)VD-Hpα induced development of antinociceptive tolerance. Taken together, these data suggest that the predicted peptide (r)VD-Hpα produces antinociception, hypothermia and hypoactivity via different pharmacological mechanisms, at least partially, which may offer an attractive strategy for separating cannabinoid analgesia from hypoactivity. Moreover, it implies that (r)VD-Hpα has therapeutic potential in pain management with limited side-effects.

Synthesis of silica-PAMAM dendrimer nanoparticles as promising carriers in Neuro blastoma cells

Mesoporous silica carriers are emerging as therapeutic drug delivery systems. The objective of this study was to develop a formulation for synthesizing silica-PAMAM dendrimer hybrid nanoparticles with sol-gel technique. Subsequently, black carrot anthocyanins were encapsulated and investigated for their capability in terms of inhibiting the proliferative effects of neuroblastoma (Neuro 2A). In this context, particle size distributions were ascertained followed by thermal analysis (DSC), scanning electron microscopy and encapsulation efficiency. Subsequently, in vitro release kinetics was determined along with cytotoxicity of empty and anthocyanin doped hybrid nanoparticles. The lowest particle size was 134.8 nm with a zeta potential of +19.78 mV which enhanced electrostatic interaction with the cell membrane in the cytotoxicity analyses. As the anthocyanin content was totally released at the end of 6 days, the cytotoxicity was observed for 134 h, reaching an inhibition of 87.9%. On the other hand, Neuro 2A cells incubated with empty nanoparticles exhibited a high proliferation indicating that hybrid nanoparticles were not toxic to the cells and the inhibitory effect was associated with the anthocyanins.

Overexpression of Tau Rescues Nogo-66-Induced Neurite Outgrowth Inhibition In Vitro.

Nogo-66 plays a central role in the myelin-mediated inhibition of neurite outgrowth. Tau is a microtubule-associated protein involved in microtubule assembly and stabilization. It remains unverified whether tau interacts directly with growth factor receptors, or engages in cross-talk with regeneration inhibitors like Nogo-66. Here, we report that plasmid overexpression of tau significantly elevated the protein levels of total tau, phosphorylated tau, and microtubule-affinity regulating kinase (MARK). Nogo-66 transiently elevated the total tau protein level and persistently reduced the level of p-S262 tau (tau phosphorylated at serine 262), whereas it had little influence on the level of p-T205 tau (tau phosphorylated at threonine 205). Nogo-66 significantly decreased the protein level of MARK. Hymenialdisine, an inhibitor of MARK, significantly reduced the level of p-S262 tau. Overexpression of tau rescued the Nogo-66-induced inhibition of neurite outgrowth in neuroblastoma 2a (N2a) cells and primary cortical neurons. However, concomitant inhibition of MARK abolished the rescue of neurite outgrowth by tau in N2a cells. We conclude that dephosphorylation of tau at S262 is able to regulate Nogo-66 signaling, and that overexpression of tau can rescue the Nogo-66-induced inhibition of neurite outgrowth in vitro.

Activation of NPFF2 receptor stimulates neurite outgrowth in Neuro 2A cells through activation of ERK signaling pathway

Neurite outgrowth is an important process in neural regeneration and plasticity, especially after neural injury, and recent evidence indicates that several Gαi/o protein-coupled receptors play an important role in neurite outgrowth. The neuropeptide (NP)FF system contains two Gαi/o protein-coupled receptors, NPFF1 and NPFF2 receptors, which are mainly distributed in the central nervous system. The aim of the present study was to determine whether the NPFF system is involved in neurite outgrowth in Neuro 2A cells. We showed that Neuro 2A cells endogenously expressed NPFF2 receptor, and the NPFF2 receptor agonist dNPA inhibited cyclic adenosine monophosphate (cAMP) production stimulated by forskolin in Neuro 2A cells. We also demonstrated that NPFF and dNPA dose-dependently induced neurite outgrowth in Neuro 2A cells, which was completely abolished by the NPFF receptor antagonist RF9. Pretreatment with mitogen-activated protein kinase inhibitors PD98059 and U0126 decreased dNPA-induced neurite outgrowth. In addition, dNPA increased phosphorylation of extracellular signal-regulated kinase (ERK) in Neuro 2A cells, which was completely antagonized by pretreatment with U0126. Our results suggest that activation of NPFF2 receptor stimulates neurite outgrowth in Neuro 2A cells through activation of the ERK signaling pathway. Moreover, NPFF2 receptor may be a potential therapeutic target for neural injury and degeneration in the future.

Profiles of miRNAs matched to biology in aromatase inhibitor resistant breast cancer.

Aromatase inhibitor (AI) resistance during breast cancer treatment is mimicked by MCF-7:5C (5C) and MCF-7:2A (2A) cell lines that grow spontaneously. Survival signaling is reconfigured but cells are vulnerable to estradiol (E2)-inducible apoptosis. These model systems have alterations of stress related pathways including the accumulation of endoplasmic reticulum, oxidative, and inflammatory stress that occur prior to E2-induced apoptosis. We investigated miRNA expression profiles of 5C and 2A to characterize their AI resistance phenotypes. Affymetrix GeneChip miRNA2.0 arrays identified 184 miRNAs differentially expressed in 2A and 5C compared to E2-free wild-type MCF-7:WS8. In 5C, 34 miRNAs of the DLK1-DIO3 locus and miR-31 were overexpressed, whereas miR-222 was low. TCGA data revealed poor and favorable overall survival for low miR-31 and miR-222 levels, respectively (HR=3.0, 95% CI:1.9-4.8; HR=0.3, 95% CI:0.1-0.6). Targets of deregulated miRNAs were identified using CLIP-confirmed TargetScan predictions. KEGG enrichment analyses for 5C- and 2A-specific target gene sets revealed pathways associated with cell proliferation including insulin, mTOR, and ErbB signaling as well as immune response and metabolism. Key genes overrepresented in 5C- and 2A-specific pathway interaction networks including EGFR, IGF1R and PIK3R1 had lower protein levels in 5C compared to 2A and were found to be differentially modulated by respective miRNA sets. Distinct up-regulated miRNAs from the DLK1-DIO3 locus may cause these attenuative effects as they are predicted to interact with corresponding 3′ untranslated regions. These new miRNA profiles become an important regulatory database to explore E2-induced apoptotic mechanisms of clinical relevance for the treatment of resistant breast cancer.

Effects of membrane properties on the binding activities of the HN and HC heavy-chain domains of botulinum neurotoxin A

Binding behaviors of the HN and the HC domains of BoNT/A were investigated individually to identify if there exist any differences in their interaction with the cell membrane. Recombinant fragments corresponding to both BoNT/A HN and HC regions were prepared (HN519–845 and HC967–1296) and their binding to synaptic proteins was verified. The binding behaviors of these heavy-chain domains were analyzed by treating the Neuro 2a, a murine neuroblastoma cell line, with compounds known to alter membrane properties. Cholesterol depletion and lipid raft inhibition increased the binding of HN519–845 to Neuro 2a cells without affecting HC967–1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both HC967–1296 and HN519–845 whereas, loading exogenous GD1a, on to the Neuro 2a cells, increased the binding of both the peptides to cells. Microtubule disruption of the Neuro 2a cells by nocodazole decreased the binding of both HC967–1296 and HN519–845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K+) depletion buffer lowered the binding of both HC967–1296 and HN519–845 to the cells, but seemed to exert a more pronounced effect on the binding of HC967–1296 than on the binding of HN519–845. Results indicate that while both the HN and HC domains are involved in the binding of the toxin to neuronal cells there are differences in their behavior which probably stem from their respective amino acid composition and structural location in the toxin three-dimensional structure along with their intended role in translocation and internalization into the cells.

RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells

Dysregulated expression and function of dopamine D2 receptors (D2Rs) are implicated in drug addiction, Parkinson's disease and schizophrenia. In the current study, we examined whether D2Rs are modulated by regulator of G protein signaling 2 (RGS2), a member of the RGS family that regulates G protein signaling via acceleration of GTPase activity. Using neuroblastoma 2a (N2A) cells, we found that RGS2 was immunoprecipitated by aluminum fluoride-activated Gαi2 proteins. RGS2 siRNA knockdown enhanced membrane [35S] GTPγS binding to activated Gαi/o proteins, augmented inhibition of cAMP accumulation and increased ERK phosphorylation in the presence of a D2/D3R agonist quinpirole when compared to scrambled siRNA treatment. These data suggest that RGS2 is a negative modulator of D2R-mediated Gαi/o signaling. Moreover, RGS2 knockdown slightly increased constitutive D2R internalization and markedly abolished quinpirole-induced D2R internalization assessed by immunocytochemistry. RGS2 knockdown did not compromise agonist-induced β-arrestin membrane recruitment; however, it prevents β-arrestin dissociation from the membrane after prolonged quinpirole treatment during which time β-arrestin moved away from the membrane in control cells. Additionally, confocal microscopy analysis of β-arrestin post-endocytic fate revealed that quinpirole treatment caused β-arrestin to translocate to the early and the recycling endosome in a time-dependent manner in control cells whereas translocation of β-arrestin to these endosomes did not occur in RGS2 knockdown cells. The impaired β-arrestin translocation likely contributed to the abolishment of quinpirole-stimulated D2R internalization in RGS2 knockdown cells. Thus, RGS2 is integral for β-arrestin-mediated D2R internalization. The current study revealed a novel regulation of D2R signaling and internalization by RGS2 proteins.

MARK2 Rescues Nogo-66-Induced Inhibition of Neurite Outgrowth via Regulating Microtubule-Associated Proteins in Neurons In Vitro.

The ability of neurons in the adult mammalian central nervous system (CNS) to regenerate after injury is limited by inhibitors in CNS myelin. Nogo-66 is the most important myelin inhibitor but the mechanisms of Nogo-66 inhibition of neurite outgrowth remain poorly understood. Particularly, the relationship between Nogo-66 and microtubule-affinity regulating kinase 2 (MARK2) has not been examined. This study investigated the role of MARK2 in Nogo-66 inhibition and the function of MARK2 in neurite elongation in neurons in vitro. MARK2 and phosphorylated MARK2 at Ser212 (p-Ser212) alterations in Neuro 2a cells were assessed at different Nogo-66 exposure times; the relationships between MARK2 and microtubule-associated proteins (MAPs) were determined via the overexpression or interference of MARK2. Our study reports that Nogo-66 inhibited the expression of total MARK2 but also reduced Ser212 phosphorylation of MARK2, whereas levels of MAP1-b and tau varied depending on MARK2 overexpression or reduced expression. Furthermore, MARK2 increased the proportion of tyrosinated α-tubulin, thereby disrupting the stability of tubulin, most likely affecting axonal growth. In line with these results, overexpression of MARK2 promoted neurite elongation and therefore is able to rescue the inhibitory effect of Nogo-66 on neurite growth. In conclusion, the intracellular PKB/MARK2/MAPs/α-tubulin pathway appears to be essential for neurite elongation in neurons in vitro. These results suggest a critical role for MARK2 in overcoming Nogo-66-induced inhibition of axon outgrowth in neurons. Pharmacological activators of MARK2 may be applicable to promote successful axonal outgrowth following many types of CNS injuries.

Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury.

BackgroundMethyl-CpG-binding protein 2 (MeCP2), a protein with affinity for methylated cytosines, is crucial for neuronal development and function. MeCP2 regulates gene expression through activation, repression and chromatin remodeling. Mutations in MeCP2 cause Rett syndrome, and these patients display impaired nociception. We observed an increase in MeCP2 expression in mouse dorsal root ganglia (DRG) after peripheral nerve injury. The functional implication of increased MeCP2 is largely unknown. To identify regions of the genome bound by MeCP2 in the DRG and the changes induced by nerve injury, a chromatin immunoprecipitation of MeCP2 followed by sequencing (ChIP-seq) was performed 4 weeks after spared nerve injury (SNI).ResultsWhile the number of binding sites across the genome remained similar in the SNI model and sham control, SNI induced the redistribution of MeCP2 to transcriptionally relevant regions. To determine how differential binding of MeCP2 can affect gene expression in the DRG, we investigated mmu-miR-126, a microRNA locus that had enriched MeCP2 binding in the SNI model. Enriched MeCP2 binding to miR-126 locus after nerve injury repressed miR-126 expression, and this was not mediated by alterations in methylation pattern at the miR-126 locus. Downregulation of miR-126 resulted in the upregulation of its two target genes Dnmt1 and Vegfa in Neuro 2A cells and in SNI model compared to control. These target genes were significantly downregulated in Mecp2-null mice compared to wild-type littermates, indicating a regulatory role for MeCP2 in activating Dnmt1 and Vegfa expression. Intrathecal delivery of miR-126 was not sufficient to reverse nerve injury-induced mechanical and thermal hypersensitivity, but decreased Dnmt1 and Vegfa expression in the DRG.ConclusionsOur study shows a regulatory role for MeCP2 in that changes in global redistribution can result in direct and indirect modulation of gene expression in the DRG. Alterations in genome-wide binding of MeCP2 therefore provide a molecular basis for a better understanding of epigenetic regulation-induced molecular changes underlying nerve injury.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0073-5) contains supplementary material, which is available to authorized users.

Urokinase and urokinase receptor participate in regulation of neuronal migration, axon growth and branching

PurposeRecent findings indicate the significant contribution of urokinase and urokinase receptor (uPA and uPAR) in the processes of nerve regeneration, however, their role in axonal growth and branching is unclear. Using a 3D model of mouse Dorsal Root Ganglia (DRG) explants, differentiated into neurons Neuro 2a cells and transgenic mice lacking the urokinase gene, we studied the involvement of the uPA/uPAR system in the neural cell migration, neurite outgrowth, elongation and branching. ResultsuPA and uPAR are expressed in the growth cones of axons. Using an ex vivo model of DRG explants in Matrigel we have found that uPA inhibition attenuates neural cell migration and axonal growth, pointing to an important role of urokinase in these processes. Apparently, uPA mediates its effects through its specific receptor uPAR: anti-uPAR antibody, which blocks the uPA binding to uPAR, stimulates axon branching and attenuates neural cell migration from DRG explants. Simultaneous inhibition of uPA and uPAR almost completely prevents the axonal outgrowth from explants into the Matrigels. Experiments in vitro using Neuro 2a cells differentiated into neurons demonstrate that administration of exogenous uPA increases the neurite growth rate (elongation), most likely via the interaction of uPA with uPAR. Blocking of uPAR stimulates neurite formation and enhances branching of preexisting neurites. The results obtained on DRG explants from transgenic mice lacking uPA gene support the assumption that uPA stimulates neurite growth via uPA/uPAR interaction and uPAR role in axons branching and neural cell migration. ConclusionsThe uPA/uPAR system plays an essential role in neural cell migration, axonal growth and branching.

Aβ-Induced Insulin Resistance and the Effects of Insulin on the Cholesterol Synthesis Pathway and Aβ Secretion in Neural Cells.

Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) toxicity, tau pathology, insulin resistance, neuroinflammation, and dysregulation of cholesterol homeostasis, all of which play roles in neurodegeneration. Insulin has polytrophic effects on neurons and may be at the center of these pathophysiological changes. In this study, we investigated possible relationships among insulin signaling and cholesterol biosynthesis, along with the effects of Aβ42 on these pathways in vitro. We found that neuroblastoma 2a (N2a) cells transfected with the human gene encoding amyloid-β protein precursor (AβPP) (N2a-AβPP) produced Aβ and exhibited insulin resistance by reduced p-Akt and a suppressed cholesterol-synthesis pathway following insulin treatment, and by increased phosphorylation of insulin receptor subunit-1 at serine 612 (p-IRS-S612) as compared to parental N2a cells. Treatment of human neuroblastoma SH-SY5Y cells with Aβ42 also increased p-IRS-S612, suggesting that Aβ42 is responsible for insulin resistance. The insulin resistance was alleviated when N2a-AβPP cells were treated with higher insulin concentrations. Insulin increased Aβ release from N2a-AβPP cells, by which it may promote Aβ clearance. Insulin increased cholesterol-synthesis gene expression in SH-SY5Y and N2a cells, including 24-dehydrocholesterol reductase (DHCR24) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) through sterol-regulatory element-binding protein-2 (SREBP2). While Aβ42-treated SH-SY5Y cells exhibited increased HMGCR expression and c-Jun phosphorylation as pro-inflammatory responses, they also showed down-regulation of neuro-protective/anti-inflammatory DHCR24. These results suggest that Aβ42 may cause insulin resistance, activate JNK for c-Jun phosphorylation, and lead to dysregulation of cholesterol homeostasis, and that enhancing insulin signaling may relieve the insulin-resistant phenotype and the dysregulated cholesterol-synthesis pathway to promote Aβ release for clearance from neural cells.

Th17 cytokine differentiation and loss of plasticity after SOCS1 inactivation in a cutaneous T-cell lymphoma.

We propose that deregulated T-helper-cell (Th) signaling underlies evolving Th17 cytokine expression seen during progression of cutaneous T-cell lymphoma (CTCL). Accordingly, we developed a lymphoma progression model comprising cell lines established at indolent (MAC-1) and aggressive (MAC-2A) CTCL stages. We discovered activating JAK3 (V722I) mutations present at indolent disease, reinforced in aggressive disease by novel compound heterozygous SOCS1 (G78R/D105N) JAK-binding domain inactivating mutations. Though isogenic, indolent and aggressive-stage cell lines had diverged phenotypically, the latter expressing multiple Th17 related cytokines, the former a narrower profile. Importantly, indolent stage cells remained poised for Th17 cytokine expression, readily inducible by treatment with IL-2 - a cytokine which mitigates Th17 differentiation in mice. In indolent stage cells JAK3 expression was boosted by IL-2 treatment. Th17 conversion of MAC-1 cells by IL-2 was blocked by pharmacological inhibition of JAK3 or STAT5, implicating IL2RG - JAK3 – STAT5 signaling in plasticity responses. Like IL-2 treatment, SOCS1 knockdown drove indolent stage cells to mimic key aggressive stage properties, notably IL17F upregulation. Co-immunoprecipitation experiments showed that SOCS1 mutations abolished JAK3 binding, revealing a key role for SOCS1 in regulating JAK3/STAT5 signaling. Collectively, our results show how JAK/STAT pathway mutations contribute to disease progression in CTCL cells by potentiating inflammatory cytokine signaling, widening the potential therapeutic target range for this intractable entity. MAC-1/2A cells also provide a candidate human Th17 laboratory model for identifying potentally actionable CTCL markers or targets and testing their druggability in vitro.