Polychlorinated biphenyls (PCBs) are a group of organic chlorine chemicals that can induce various adverse health effects in animals and humans. The toxicology of PCBs is a significant public health concern because of their long-term presence in the environment. Among the 209 PCB congeners, PCB95 has been reported to be neurotoxic, however, there has been limited researches on evaluating whether and how PCB95 affects cellular lipids, the most abundant components of the brain. In this study, PCB95 was found to inhibit cell proliferation at concentrations of 0.1 μM, 2 μM and 10 μM for 120 h. Additionally, there may be a shift in apoptosis to necrosis at 2 μM PCB95 exposure for 24 h. However, lipid peroxidation was found not dominant for PCB95 exposure, especially at the concentrations of 0.1 μM and 2 μM. Because of playing vital roles in cell metabolism, 20 glycerophospholipids in PC12 cells were investigated after exposure to PCB95 for 120 h. The distinctions in the orthogonal projection to latent structures-discriminant analysis (OPLS-DA) models indicated that different concentrations of PCB95 leaded to aberrant glycerophospholipid metabolism. Based on the principles of t-test P-value < 0.05, variable importance at projection (VIP) value >1 and fold change >1, PC (14:0/14:0) and PC (16:0/14:0) were screened as potential biomarkers from all the target glycerophospholipids. This study is the first time that identifies the effects of PCB95 on specific glycerophospholipids in PC12 cells, and the observed changes in glycerophospholipids provides the basis for further evaluation of PCB95-induced neurotoxicity mechanisms.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Currently, there is only one FDA-approved treatment for ALS (riluzole), and that drug only extends life, on average, by 2-3 months. Mutations in Cu/Zn superoxide dismutase (SOD1) are found in familial forms of the disease and have played an important role in the study of ALS pathophysiology. On the basis of their activity in a PC12-G93A-YFP high-throughput screening assay, several bioactive compounds have been identified and classified as cyclohexane-1,3-dione (CHD) derivatives. A concise and efficient synthetic route has been developed to provide diverse CHD analogs. The structural modification of the CHD scaffold led to the discovery of a more potent analog (26) with an EC(50) of 700 nM having good pharmacokinetic properties, such as high solubility, low human and mouse metabolic potential, and relatively good plasma stability. It was also found to efficiently penetrate the blood-brain barrier. However, compound 26 did not exhibit any significant life span extension in the ALS mouse model. It was found that, although 26 was active in PC12 cells, it had poor activity in other cell types, including primary cortical neurons, indicating that it can penetrate into the brain, but is not active in neuronal cells, potentially due to poor selective cell penetration. Further structural modification of the CHD scaffold was aimed at improving global cell activity as well as maintaining potency. Two new analogs (71 and 73) were synthesized, which had significantly enhanced cortical neuronal cell permeability, as well as similar potency to that of 26 in the PC12-G93A assay. These CHD analogs are being investigated further as novel therapeutic candidates for ALS.

Oxidative stress is a major cause of cellular injury in a variety of human diseases including neurodegenerative disorders. Thus, removal of excessive reactive oxygen species (ROS) or suppression of ROS generation may be effective in preventing oxidative stress-induced cell death. This study was designed to investigate the effect of icariside II (ICS II), a novel phosphodiesterase 5 inhibitor, on hydrogen peroxide (H2 O2 )-induced death of highly differentiated rat neuronal PC12 cells, and to further examine the underlying mechanisms. We found that ICS II pre-treatment significantly abrogated H2 O2 -induced PC12 cell death as demonstrated by the increase of the number of metabolically active cells and decrease of intracellular lactate dehydrogenase (LDH) release. Furthermore, ICS II inhibited H2 O2 -induced cell death through attenuating intracellular ROS production, mitochondrial impairment, and activating glycogen synthase kinase-3β (GSK-3β) as demonstrated by reduced intracellular and mitochondrial ROS levels, restored mitochondrial membrane potential (MMP), decreased p-tyr216-GSK-3β level and increased p-ser9-GSK-3β level respectively. The GSK-3β inhibitor SB216763 abrogated H2 O2 -induced cell death. Moreover, ICS II significantly inhibited H2 O2 -induced autophagy by the reducing autophagosomes number and the LC3-II/LC3-I ratio, down-regulating Beclin-1 expression, and up-regulating p62/SQSTM1 and HSP60 expression. The autophagy inhibitor 3-methyl adenine (3-MA) blocked H2 O2 -induced cell death. Altogether, this study demonstrated that ICS II may alleviate oxidative stress-induced autophagy in PC12 cells, and the underlying mechanisms are related to its antioxidant activity functioning via ROS/GSK-3β/mitochondrial signalling pathways.

More than 300 missense mutations in PSEN1 gene have been correlated to the early-onset Alzheimer's disease (EOAD), but given the high diversity of PS1 (the PSEN1 gene product) substrates and the involvement of PS1 in multiple biological functions, different mutants may represent different EOAD etiologies, and how each mutant contributes to the EOAD remains to be further investigated. Here we report the identification of a novel PSEN1 p.Tyr159Ser in a family with multiple EOAD cases. The mutant PS1 protein (PS1Y159S) was analyzed for its activity in producing amyloid-β (Aβ) and for the efficiency in maturation in vitro. We also screened other mutations and SNPs that may modify the effect of PSEN1 p.Tyr159Ser on AD pathogenesis. The blood samples of the family were collected for whole-exome gene sequencing and analysis. The identified mutant PS1 and several other PS1 mutants were co-expressed with the APP Swedish mutant to compare the effects on APP processing and PS1 maturation.1. The proband and her siblings over 50 years old showed typical AD or MCI symptoms. Exon sequencing identified the p.Tyr159Ser mutation in the PSEN1 gene. As not until the age of 78 did the proband's mother who carried this mutation displayed the symptoms of uncharacterized neuropsychiatry instead of AD, but all the mutation bearing lower generation developed AD or MCI after the age of 50, we also analyzed mutations/SNPs that are different between the mother and the lower generation. By in vitro assays, we found that the Y159S substitution strongly increased Aβ42/Aβ40 ratio and significantly affected PS1 maturation. The newly discovered PSEN1 p.Tyr159Ser is an AD-causing mutation, yet, the carriers are not obligated AD patients. Mutations/SNPs in other gene may modify the effects of this mutation, and the identification of these mutations/SNPs may facilitate the discovery of AD-preventing mechanisms and methods.

An on-demand anesthetic that would only take effect when needed and where the intensity of anesthesia could be easily adjustable according to patients' needs would be highly desirable. Here, we design and synthesize a macromolecular prodrug (P407-CM-T) in which the local anesthetic tetracaine (T) is attached to the polymer poloxamer 407 (P407) via a photo-cleavable coumarin linkage (CM). P407-CM-T solution is an injectable liquid at room temperature and gels near body temperature. The macromolecular prodrug has no anesthetic effect itself unless irradiated with a low-power blue light emitting diode (LED), resulting in local anesthesia. By adjusting the intensity and duration of irradiation, the anesthetic effect can be modulated. Local anesthesia can be repeatedly triggered.

Oxidative stress plays a critical role in cerebral ischemia-reperfusion injury. We have previously developed a powerful antioxidant, HL-008. This study aimed to investigate the neuroprotective function of HL-008. HL-008 efficacy in vitro and in vivo was evaluated using a PC-12 cell oxidative stress model induced by hydrogen peroxide and a rat model of middle cerebral artery occlusion, respectively. The MTT assay was used to analyze cell viability. 2,3,5-Triphenyltetrazolium chloride and Hematoxylin and Eosin staining, immunofluorescence, western blot, and proteomics were used to evaluate the infarction volume, brain tissue morphology, apoptosis, inflammation, and related pathways. Indicators related to oxidative levels were also detected. HL-008 significantly reduced the cerebral infarction volume induced by ischemia-reperfusion, improved the neurological score, alleviated oxidative stress and inflammation in the brain tissue, reduced glial cell activation, inhibited brain tissue apoptosis by influencing multiple signaling pathways, and had a neuroprotective effect. If HL-008 is successfully developed, it could significantly improve stroke patients' quality of life.

Emerging evidence has implicated poly-(ADP-ribose) polymerase 1 (PARP-1), a transcriptional coregulator, in a variety of inflammatory diseases. In the current study, the role of PARP-1 in neuropathic pain and the underlying mechanisms were investigated. Neuropathic pain was determined by assessing the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) following lumbar 5 spinal nerve ligation (SNL) in male rates. Western blotting, qRT-PCR, immunohistochemistry, chromatin immunoprecipitation (ChIP), and Co-IP assays were performed to elucidate the mechanisms. The results showed that SNL resulted in a significant increase in the expression and activation of PARP-1 in the ipsilateral L4/5 dorsal root ganglia (DRG) and spinal dorsal horn, which occurred on day one, reached peak on day 7, and persisted more than 2 weeks after surgery. Double immunofluorescence staining revealed that PARP-1 was expressed exclusively in DRG A-type and C-type neurons. In the spinal cord, PARP-1 mainly colocalized with the neuronal marker NeuN and the astrocytic marker GFAP specifically in the superficial lamina. Prior intrathecal (i.t.) injection of PJ-34, a PARPs inhibitor, or Tiq-A, a specific PARP-1 inhibitor, dose-dependently prevented the reductions in PWT and PWL following SNL. Established neuropathic pain-like hypersensitivity was also attenuated with i.t. injection of PJ-34 and Tiq-A starting on day 7 following SNL, a timepoint at which neuropathic pain was fully established. SNL-induced mechanical allodynia and thermal hyperalgesia were also alleviated by i.t. injection of PARP-1 siRNA following a reduction in PARP-1 expression in the dorsal horn. Moreover, the SNL-induced increases in TNF-α protein and mRNA in the dorsal horn and DRG were dramatically suppressed by i.t. injection of Tiq-A or PARP-1 siRNA. The i.t. lipopolysaccharide (LPS)-induced increase in the production of TNF-α in the dorsal horn was also inhibited by prior to i.t. injection of PARP-1 siRNA. Results of ChIP assay showed that SNL-induced PARP-1 activation promoted the binding of NF-κB p65 with the TNF-α promoter in the dorsal horn and that PARP-1 inhibition reduced this binding and suppressed TNF-α expression. Co-IP assay revealed that SNL caused a significant increase in the level of histone H1 poly(ADP)-ribosylation. Together, these results indicate that PARP-1-regulated TNF-α expression in the DRG and spinal dorsal horn following SNL contributes to the development and maintenance of neuropathic pain. Targeting PARP-1 might be a promising therapeutic strategy for the treatment of the chronic pain.

Stroke is the major cause of permanent disability and mortality in China. Apigenin-7-O-β-D-(-6''-p-coumaroyl)-glucopyranoside (APG) is a glycoside subtype of apigenin and has the antioxidant activity; however, whether and how it plays a neuroprotective role following cerebral ischemia remains unknown. In present study, we adopted the oxygen glucose/reperfusion model in PC12 cells, bilateral common carotid artery occlusion model in C57B6 mice and middle cerebral artery occlusion model in SD rats to observe the therapeutic effects of APG on ischemic stroke. We also discussed the underlying mechanism. Treatment with 0.4 μg/ml or 0.8 μg/ml APG promoted cell viability and proliferation, reduced LDH release and apoptotic cell death levels in PC12 cells. Treatment with 50 mg/kg or 100 mg/kg APG at 30 minutes after reperfusion improved neurological outcomes in vivo, as demonstrated by elevation of neurological scores in both mice and rats. It also increased the number of survival neurons in mice and reduced infarct volume in rats. APG also increased the contents of Mn-SOD and the phosphorylation level of STAT3, elevated the antioxidant activity and reduced oxidative productions. These findings revealed a neuroprotective effect of APG, which possibly induced by the STAT3 phosphorylation-mediated Mn-SOD up-regulation.

Insulin-like growth factor 1 (IGF-1) has neuroprotective actions, including vasodilatory, anti-inflammatory, and antithrombotic effects, following ischemic stroke. However, the molecular mechanisms underlying the neuroprotective effects of IGF-1 following ischemic stroke remain unknown. Therefore, in the present study, we investigated whether IGF-1 exerted its neuroprotective effects by regulating the Hippo/YAP signaling pathway, potentially via activation of the PI3K/AKT cascade, following ischemic stroke. In the in vitro study, we exposed cultured PC12 and SH-5YSY cells, and cortical primary neurons, to oxygen-glucose deprivation. Cell viability was measured using CCK-8 assay. In the in vivo study, Sprague-Dawley rats were subjected to middle cerebral artery occlusion. Neurological function was assessed using a modified neurologic scoring system and the modified neurological severity score (mNSS) test, brain edema was detected by brain water content measurement, infarct volume was measured using triphenyltetrazolium chloride staining, and neuronal death and apoptosis were evaluated by TUNEL/NeuN double staining, HE and Nissl staining, and immunohistochemistry staining for NeuN. Finally, western blot analysis was used to measure the level of IGF-1 in vivo and levels of YAP/TAZ, PI3K and phosphorylated AKT (p-AKT) both in vitro and in vivo. IGF-1 induced activation of YAP/TAZ, which resulted in improved cell viability in vitro, and reduced neurological deficits, brain water content, neuronal death and apoptosis, and cerebral infarct volume in vivo. Notably, the neuroprotective effects of IGF-1 were blocked by an inhibitor of the PI3K/AKT cascade, LY294002. LY294002 treatment not only downregulated PI3K and p-AKT, but YAP/TAZ as well, leading to aggravation of neurological dysfunction and worsening of brain damage. Our findings indicate that the neuroprotective effects of IGF-1 are, at least in part mediated by upregulation of YAP/TAZ via activation of the PI3K/AKT cascade following cerebral ischemic stroke.

Rotenone (ROT) is an environmental neurotoxin which has been demonstrated to cause characteristic loss of dopamine (DA) neurons in Parkinson's disease (PD). Icariin (ICA) is a flavonoid glucoside isolated from Herba Epimedii that has been shown to display neuroprotective functions. The present study evaluated protective effects of ICA on ROT-induced neurotoxicity and determined the modulation of ICA on the regulation of autophagy in vivo and in vitro. Rats were treated with ROT (1.0 mg/kg/day) with a co-administration of ICA (15 or 30 mg/kg/day) for 5 weeks. Immunohistochemical analysis showed a significant loss in DA neurons in the substantia nigra (SN) of rats treated with ROT, accompanied by an increase in the accumulation of α-synuclein and a compromised mitochondrial respiration. However, co-administration of ICA potently ameliorated the ROT-induced neuronal cell injury and improved mitochondrial function and decreased the accumulation of α-synuclein. ROT treatment resulted in a decrease in the protein expression of LC3-II and Beclin-1, and an increase in the protein level of P62, and upregulated the activation of mammalian target of rapamycin (mTOR), whereas ICA significantly reversed these aberrant changes caused by ROT. Furthermore, the neuroprotective effect of ICA was further verified in PC12 cells. Cells treated with ROT displayed an increased cytotoxicity and a decreased oxygen consumption which were rescued by the presence of ICA. Furthermore, ROT decreased the protein expression level of LC3-II, enhanced Beclin-1 expression, and activated phosphorylation of mTOR, whereas ICA markedly reversed this dysregulation of autophagy caused by ROT in the PC12 cells. Collectively, these results suggest that ICA mediated activation of autophagic flux confers a neuroprotective action on ROT-induced neurotoxicity.

Background Severe postoperative pain remains a clinical problem that impacts patient's rehabilitation. The present work aims to investigate the role of Toll-like receptor-4 (TLR4) activation in wounded plantar tissue and dorsal root ganglion (DRG) in the genesis of postoperative pain and its underlying mechanisms. Results Postoperative pain was induced by plantar incision in rat hind paw. Plantar incision led to increased expression of TLR4 in ipsilateral lumbar 4-5 (L4/L5) DRGs, which occurred at 2 h and was persistent to the third day after surgery. Similar to the change in TLR4 expression, there was also significant increase in phosphorylated nuclear factor-kappa B p65 (p-p65) in DRGs after surgery. Immunofluorescence staining revealed that the increased expressions of TLR4 and p-p65 not only in neuronal cells but also in satellite glial cells in DRG. Furthermore, the enhanced expressions of TLR4 and p-p65 were also detected in plantar tissues around the incision, which was observed starting at 2 h and lasting until the third day after surgery. Prior intrathecal (i.t.) injections of TAK-242 (a TLR4-specific antagonist) or 4',6-diamidino-2-phenylindole-dihydrochloride (PDTC, a nuclear factor-kappa B activation inhibitor) dose dependently alleviated plantar incision-induced mechanical allodynia and thermal hyperalgesia and inhibited the increased expressions of p-p65, tumor necrosis factor-alpha, and interleukin-1 beta in DRG. Prior subcutaneous (s.c.) plantar injection of TAK-242 or PDTC also ameliorated pain-related hypersensitivity following plantar incision. Moreover, the plantar s.c. injection of TAK-242 or PDTC inhibited the increased expressions of p-p65, tumor necrosis factor-alpha, and interleukin-1 beta not only in local wounded plantar tissue but also dramatically in ipsilateral lumbar 4-5 DRGs. Conclusion TLR4/ nuclear factor-kappa B signaling activation in local injured tissue and DRG contribute to the development of postoperative pain via regulating pro-inflammatory cytokines release. Targeting TLR4/ nuclear factor-kappa B signaling in local tissue at early stage of surgery may be an effective strategy for the treatment of postoperative pain.

MicroRNAs are small non-coding RNAs that repress the expression of their target proteins. The roles of microRNAs in the development of Alzheimer's disease (AD) are not clear. In this study we show that miR-200c represses the expression of PTEN protein. PTEN downregulation by miR-200c supports the survival and differentiation of cultured neurons. AD is a progressive neurodegenerative disease signified by beta amyloid (Aβ) peptide aggregation and deposition. In a mouse model of AD that is induced by APPswe and PS1ΔE9 double transgenes, we found Aβ deposition results in neuronal ER stress that induces miR200c. Pharmacological blockade of ER stress inhibited Aβ-induced miR-200c overexpression in AD brains. MiR-200c was detected in the serum of both AD mice and human AD patients. These findings suggest that miR-200c functions as part of the neuronal cell-intrinsic adaptive machinery, and supports neuronal survival and differentiation in response to Aβ induced ER-stress by downregulating PTEN.

Dopamine transporter (DAT) is the target of cocaine and HIV-1 transactivator of transcription (Tat) protein. Identifying allosteric modulatory molecules with potential attenuation of cocaine and Tat binding to DAT are of great scientific and clinical interest. We demonstrated that tyrosine 470 and 88 act as functional recognition residues in human DAT (hDAT) for Tat-induced inhibition of DA transport and transporter conformational transitions. Here we investigated the allosteric modulatory effects of two allosteric ligands, SRI-20041 and SRI-30827 on cocaine binding on wild type (WT) hDAT, Y470 H and Y88 F mutants. Effect of SRI-30827 on Tat-induced inhibition of [3H]WIN35,428 binding was also determined. Compared to a competitive DAT inhibitor indatraline, both SRI-compounds displayed a similar decrease (30%) in IC50 for inhibition of [3H]DA uptake by cocaine in WT hDAT. The addition of SRI-20041 or SRI-30827 following cocaine slowed the dissociation rate of [3H]WIN35,428 binding in WT hDAT relative to cocaine alone. Moreover, Y470H and Y88F hDAT potentiate the inhibitory effect of cocaine on DA uptake and attenuate the effects of SRI-compounds on cocaine-mediated dissociation rate. SRI-30827 attenuated Tat-induced inhibition of [3H]WIN35,428 binding. These observations demonstrate that tyrosine 470 and 88 are critical for allosteric modulatory effects of SRI-compounds on the interaction of cocaine with hDAT.

Delivery of hydrophilic small molecule therapeutics by traditional drug delivery systems is challenging. Herein, we have used the specific interaction between DNA aptamers and drugs to create simple and effective drug depot systems. The specific binding of a phosphorothioate-modified aptamer to drugs formed non-covalent aptamer/drug complexes, which created a sustained release system. We demonstrated the effectiveness of this system with small hydrophilic molecules, the site 1 sodium channel blockers tetrodotoxin and saxitoxin. The aptamer-based delivery system greatly prolonged the duration of local anesthesia and reduced systemic toxicity. The beneficial effects of the aptamers were restricted to the compounds they were specific to. These studies establish aptamers as a class of highly specific, modifiable drug delivery systems, and demonstrate potential usefulness in the management of postoperative pain.