Phosphorylation In PC12 Cells Research Articles

MiR-200a-3p Regulates PRKACB and Participates in Aluminium-Induced Tau Phosphorylation in PC12 Cells

Aluminium (Al) is an environmental neurotoxin that humans are widely exposed to, but the molecular mechanism of its toxic effects is not fully understood. Many studies have shown that exposure to Al can cause abnormal phosphorylation of the tau protein that is believed as one of pathological features of Alzheimer’s disease. Increasing evidence indicates that microRNAs (miRNAs) may be involved in the pathological processes of neurodegenerative diseases and are potential regulatory factors for related target genes. Phosphorylation at Ser-133 of cAMP response element-binding protein (CREB) is one of the major pathways of CREB activation, and phosphorylation at this site is controlled by protein kinase A (PKA). The catalytic subunit of PKA, cAMP-dependent protein kinase catalytic subunit beta (PRKACB), phosphorylates CREB. The target gene prediction software TargetScan showed that PRKACB was one of the target mRNAs of miR-200a-3p. The purpose of this study was to investigate whether miR-200a-3p regulates the PKA/CREB pathway by targeting PRKACB and leads to abnormal phosphorylation of the tau protein in nerve cells. The results showed that Al exposure increased the expression level of miR-200a-3p, and miR-200a-3p increased the expression of targeted downregulated PRKACB, and then decreased the PKA/CREB signalling pathway activity, leading to abnormal hyperphosphorylation of tau.Graphical

Amphetamine-induced neurite injury in PC12 cells through inhibiting GAP-43 pathway

Amphetamine (AMPH) causes the degeneration of dopamine terminals in the central nervous system. The mechanisms for this damage are unclear. We found AMPH reduced level of GAP-43 in the striatum of rats that receives rich dopaminergic terminals. Using PC12 cells as dopaminergic neuronal models, we further found that AMPH inhibited GAP-43 and GAP-43 phosphorylation in PC12 cells. The reduced GAP-43 was correlated with neurite injury of PC12 cells. The PKCβ1, an upstream molecule of GAP-43, was also inhibited by AMPH. Phorbol 12-myristate 13-acetate (PMA) as a specific activator of PKC increased levels of PKCβ1 and GAP-43, and efficiently prevented neurite degeneration of PC12 cells induced by AMPH. On the other side, enzastuarin, an inhibitor of PKC, decreased levels of PKCβ1 and GAP-43, and caused neurite injury of PC12 cells. Together, our results suggest that AMPH induces neurite injury in PC12 cells through inhibiting PKCβ1/GAP-43 pathway.

The Neuroprotective Effect of the Extract of Traditional Mongolian Medicine Eerdun Wurile in PC12 Cells

<i>Background</i>: Traditional Mongolian medicine Eerdun Wurile is one of the ideal drugs for cerebral stroke and is effective on treating vascular and nervous system diseases. Numerous studies have proved that Eerdun Wurile has neuroprotective effect. However, the biological mechanism of which is still unclear. <i>Objective</i>: To investigatethe molecular mechanism underlying the neuroprotective effect of Eerdun Wurile. <i>Methods</i>: The extract from Eerdun Wurile was prepared by simulated gastric fluid first and its effect on PC12 cells differentiaton and MAPK phosphorylation was tested by neurite outgrowth assay and western blotting. Also, the target of active compounds was predicted by moleculr modeling system and the content of active compounds was examined by LC-MS/MS. <i>Result</i>: Result showed that the extract stimulated the differentiation of PC12 cells, exhibited nerve growth factor (NGF)-like effect. Meanwhile, the extract induced the MAPK phosphorylation in PC12 cells. In-depth analysis by molecular modeling technology predicted that genipin interact with MAPK as a target. Consistent with the prediction, genipin induced the phosphorylation of MAPK in PC12 cells. Besides, genipin was detected both in the extract and Eerdun Wurile pills. <i>Conclusion</i>: These results proved that the extract of Eerdun Wurile has neuroprotective effect in PC12 cells, and genipin in the extract and Eerdun Wurile might be one of the active compounds that show neuroprotective effect.

Modulation of ERK1/2 and Akt Pathways Involved in the Neurotrophic Action of Caffeic Acid Alkyl Esters.

Neurodegenerative diseases affect millions of human lives all over the world. The number of afflicted patients is rapidly growing, and disease-modifying agents are urgently needed. Caffeic acid, an important member of the hydroxycinnamic acid family of polyphenols, has considerable neurotrophic effects. We have previously shown how caffeate alkyl ester derivatives significantly promote survival and differentiation in neuronal cells. In this study, the mechanisms by which these ester derivatives exert their neurotrophic effects are examined. A series of eight caffeic acid esters with different alkyl chain lengths, ranging from methyl (CAF1) to dodecyl esters (CAF8), were synthesized and studied for their influence on neurotrophic signaling pathways. Caffeate esters did not induce tropomyosin-receptor kinase A (TrkA) phosphorylation, which was assessed by immunoblotting up to a concentration of 25 µM. NIH/3T3 cells overexpressing TrkA were generated to further examine phosphorylation of this receptor tyrosine kinase. None of the esters induced TrkA phosphorylation in these cells either. Assessment of the effect of caffeate derivatives on downstream neurotrophic pathways by immunoblotting showed that the most potent esters, decyl caffeate (CAF7) and dodecyl caffeate (CAF8) caused extracellular signal-regulated kinase (ERK1/2) and Akt serine threonine kinase phosphorylation in PC12 cells at 5 and 25 µM concentrations. In conclusion, this study shows that caffeate esters exert their neurotrophic action by modulation of ERK1/2 and Akt signaling pathways in neuronal cells, and further demonstrates the potential therapeutic implications of these derivatives for neurodegenerative diseases.

Hyperforin attenuates aluminum-induced Aβ production and Tau phosphorylation via regulating Akt/GSK-3β signaling pathway in PC12 cells

Aluminum (Al) is a neurotoxicant and cause β-amyloid (Aβ) peptides aggregation and tau hyperphosphorylation. Hyperforin (HF) is one of the major active constituents of the extracts of St. John’s Wort (Hypericum perforatum), can treat Alzheimer’s disease (AD) and other diseases involving peptide accumulation and cognition impairment. To determine the effects of HF on Al-induced Aβ formation and tau hyperphosphorylation, PC12 cells were cultured and treated with Al-malt (500μM) and/or HF (1μM). The results showed that HF treatment significantly attenuated Al-malt-induced Aβ1-42 production by reducing the expressions of APP, BACE1 and PS1, while increasing the expressions of sAPPα, ADAM9/10/17, and tau phosphorylation in PC12 cells. In addition, HF treatment also increased phosphorylation of AKT (Ser473) and inhibited GSK-3β activity by increasing phosphorylation of GSK-3β (Ser9). These results indicated that HF may exert the protection via regulating the AKT/GSK-3β signaling to reduce Aβ production and tau phosphorylation in PC12 cells. Furthermore, these results could lead a possible therapeutics for the management of Al neurotoxicity.

Selenium and Zinc against Aβ25-35-Induced Cytotoxicity and Tau Phosphorylation in PC12 Cells and Inhibits γ-cleavage of APP.

Amyloid beta (Aβ) is the main component of the amyloid plaques that accumulate in the brains of Alzheimer patients. The present study was conducted to investigate whether the combined treatment with selenium (Se) and zinc (Zn) offers more beneficial effects than that provided by either of them alone in reversing Aβ25-35-induced neurotoxicity in PC12 cells. Cells were pretreated with 0.1μmol/L of Se and Zn for 4h, after treated with 10mmol/L Aβ25-35 for 24h. Cells were divided into control and five treated groups, and received either 10mmol/L Aβ25-35,10mmol/L Aβ25-35+0.1μmol/L Se, 10mmol/L Aβ25-35+0.1μmol/L Zn, 10mmol/LAβ25-35+0.1μmol/L Se+0.1μmol/L Zn, or 0.1μmol/L Se+0.1μmol/L Zn. The result showed that cell viability was decreased in MTT metabolic rate; LDH release and MDA, H2O2, and NO levels were increased and the GSK-3β and phosphorylated tau protein level were increased in Aβ25-35-treated group (P<0.05 or P<0.01), which whole changes were attenuated by Se and Zn and Se combined Zn. In order to evaluate whether the Se and Zn have an effect on processing pathway of amyloid precursor protein (APP), we examined the activity of γ-secretase in primary cultured cortical neuron cells. ELISA analysis showed that Se and Zn could inhibit the activity of γ-secretase. Then we also investigated the effect of Se and Zn on the Aβ1-40 concentration and APP-N-terminal fragment expression from APP695 stably transfected Chinese hamster ovary (CHO) cells. APP695 stably transfected CHO cells were treated with 0.1μmol/L Se and Zn; cells were divided into control and four treated groups, which received either 0.5M DAPT, 0.1μmol/L Se, 0.1μmol/L Zn, or 0.1μmol/L Se+0.1μmol/L Zn. Se and Zn could decrease Aβ1-40 production and increase the APP-N-terminal fragment protein expression. These experiments indicate that Se and Zn have a protective effect on AD pathology that a possible mechanism is inhibiting the activity of γ-secretase to decreasing Aβ1-40 production further influencing the APP processing. Altogether, our findings may provide a novel therapeutic target to treat AD sufferers.

Excessive nNOS/NO/AMPK signaling activation mediated by the blockage of the CBS/H2S system contributes to oxygen‑glucose deprivation‑induced endoplasmic reticulum stress in PC12cells.

Hypoxic‑ischemia stress causes severe brain injury, leading to death and disability worldwide. Although it has been reported that endoplasmic reticulum(ER) stress is an essential step in the progression of hypoxia or ischemia‑induced brain injury, the underlying molecular mechanisms are and have not yet been fully elucidated. Accumulating evidence has indicated that both nitric oxide(NO) and hydrogen sulfide(H2S) play an important role in the development of cerebral ischemic injury. In the present study, we aimed to investigate the effect of the association between NO signaling and the cystathionine β‑synthase(CBS)/H2S system on ER stress in a cell model of cerebral hypoxia‑ischemia injury. We found that oxygen‑glucose deprivation(OGD) markedly increased the NO level and neuronal NO synthase(nNOS) activity. 3‑Bromo‑7‑nitroindazole(3‑Br‑7‑NI), a relatively selective nNOS inhibitor, abolished the OGD‑induced inhibition of cell viability and the increased expression of ER stress‑related proteins, including glucose‑regulated protein78(GRP78), C/EBP homologous protein(CHOP) and cleaved caspase‑12 in PC12 cells, indicating the contribution of excessive nNOS/NO signaling to OGD‑induced ER stress. Furthermore, we found that OGD increased the phosphorylated AMP‑activated protein kinase(p‑AMPK)/AMPK ratio, and the AMPK activator, 5‑aminoimidazole‑4‑carboxamide‑1‑β‑D‑ribofuranoside (AICAR), attenuated the effects on OGD‑induced ER stress, suggesting that OGD‑induced NO overproduction results in AMPK activation in PC12 cells. We also found that OGD induced the downregulation of the CBS/H2S system, as indicated by the decreased H2S level in the culture supernatant and CBS activity in PC12 cells. In addition, we found that treatment with NaHS(a H2S donor) or S‑adenosyl‑L‑methionine(SAM, a CBS agonist) mitigated OGD‑induced ER stress, aswellas the NO level, nNOS activity and AMPK phosphorylation in PC12 cells. On the whole, these results suggest that the inhibition of the CBS/H2S system, which facilitated excessive nNOS/NO/AMPK activation, contributes to OGD‑induced ER stress.

Derivatives of caffeic acid, a natural antioxidant, as the basis for the discovery of novel nonpeptidic neurotrophic agents

Neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease, threaten the lives of millions of people and the number of affected patients is constantly growing with the increase of the aging population. Small molecule neurotrophic agents represent promising therapeutics for the pharmacological management of neurodegenerative diseases. In this study, a series of caffeic acid amide analogues with variable alkyl chain lengths, including ACAF3 (C3), ACAF4 (C4), ACAF6 (C6), ACAF8 (C8) and ACAF12 (C12) were synthesized and their neurotrophic activity was examined by different methods in PC12 neuronal cells. We found that all caffeic acid amide derivatives significantly increased survival in PC12 neuronal cells in serum-deprived conditions at 25μM, as measured by the MTT assay. ACAF4, ACAF6 and ACAF8 at 5µM also significantly enhanced the effect of nerve growth factor (NGF) in inducing neurite outgrowth, a sign of neuronal differentiation. The neurotrophic effects of amide derivatives did not seem to be mediated by direct activation of tropomyosin receptor kinase A (TrkA) receptor, since K252a, a potent TrkA antagonist, did not block the neuronal survival enhancement effect. Similarly, the active compounds did not activate TrkA as measured by immunoblotting with anti-phosphoTrkA antibody. We also examined the effect of amide derivatives on signaling pathways involved in survival and differentiation by immunoblotting. ACAF4 and ACAF12 induced ERK1/2 phosphorylation in PC12 cells at 5 and 25µM, while ACAF12 was also able to significantly increase AKT phosphorylation at 5 and 25µM. Molecular docking studies indicated that compared to the parental compound caffeic acid, ACAF12 exhibited higher binding energy with phosphoinositide 3-kinase (PI3K) as a putative molecular target. Based on Lipinski’s rule of five, all of the compounds obeyed three molecular descriptors (HBD, HBA and MM) in drug-likeness test. Taken together, these findings show for the first time that caffeic amides possess strong neurotrophic effects exerted via modulation of ERK1/2 and AKT signaling pathways presumably by activation of PI3K and thus represent promising agents for the discovery of neurotrophic compounds for management of neurodegenerative diseases.

Functional interaction between Lypd6 and nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain‐containing 6 (Lypd6) with nAChRs in human brain extracts, identifying Lypd6 as a novel regulator of nAChR function. Using protein cross‐linking and affinity purification from human temporal cortical extracts, we demonstrate that Lypd6 is a synaptically enriched membrane‐bound protein that binds to multiple nAChR subtypes in the human brain. Additionally, soluble recombinant Lypd6 protein attenuates nicotine‐induced hippocampal inward currents in rat brain slices and decreases nicotine‐induced extracellular signal‐regulated kinase phosphorylation in PC12 cells, suggesting that binding of Lypd6 is sufficient to inhibit nAChR‐mediated intracellular signaling. We further show that perinatal nicotine exposure in rats (4 mg/kg/day through minipumps to dams from embryonic day 7 to post‐natal day 21) significantly increases Lypd6 protein levels in the hippocampus in adulthood, which did not occur after exposure to nicotine in adulthood only. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain, and that Lypd6 is dysregulated by nicotine exposure during early development. Regulatory proteins of the Lynx family modulate the function of nicotinic receptors (nAChRs). We report for the first time that the Lynx protein Lypd6 binds to nAChRs in human brain extracts, and that recombinant Lypd6 decreases nicotine‐induced ERK phosphorylation and attenuates nicotine‐induced hippocampal inward currents. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain.

Lynx1 and Aβ1–42 bind competitively to multiple nicotinic acetylcholine receptor subtypes

Lynx1 regulates synaptic plasticity in the brain by regulating nicotinic acetylcholine receptors (nAChRs). It is not known to which extent Lynx1 can bind to endogenous nAChR subunits in the brain or how this interaction is affected by Alzheimer's disease pathology. We apply affinity purification to demonstrate that a water-soluble variant of human Lynx1 (Ws-Lynx1) isolates α3, α4, α5, α6, α7, β2, and β4 nAChR subunits from human and rat cortical extracts, and rat midbrain and olfactory bulb extracts, suggesting that Lynx1 forms complexes with multiple nAChR subtypes in the human and rodent brain. Incubation with Ws-Lynx1 decreases nicotine-mediated extracellular signal–regulated kinase phosphorylation in PC12 cells and striatal neurons, indicating that binding of Ws-Lynx1 is sufficient to inhibit signaling downstream of nAChRs. The effect of nicotine in PC12 cells is independent of α7 or α4β2 nAChRs, suggesting that Lynx1 can affect the function of native non-α7, non-α4β2 nAChR subtypes. We further show that Lynx1 and oligomeric β-amyloid1–42 compete for binding to several nAChR subunits, that Ws-Lynx1 prevents β-amyloid1–42–induced cytotoxicity in cortical neurons, and that cortical Lynx1 levels are decreased in a transgenic mouse model with concomitant β-amyloid and tau pathology. Our data suggest that Lynx1 binds to multiple nAChR subtypes in the brain and that this interaction might have functional and pathophysiological implications in relation to Alzheimer's disease.

Leptin-Sensitive JAK2 Activation in the Regulation of Tau Phosphorylation in PC12 Cells

Background/Aims: Alzheimer's disease (AD) is characterized by two major hallmarks: the deposition and accumulation of β-amyloid (Aβ) peptide and hyperphosphorylated tau in intracellular neurofibrillary tangles. Sets of evidence show that leptin reduces Aβ production and tau phosphorylation. Herein, we investigated the signaling pathways activated by leptin, to extensively understand its mechanism. Methods: Western blotting was employed to assess the protein abundance of p-tau and BAX, MTT assay to decipher the cells viability. Results: Leptin decreased tau phosphorylation, an effect was dependent on the activation of JAK2. Conclusion: The data suggest that JAK2 is involved in AD-related pathways.

Parkin represses 6-hydroxydopamine-induced apoptosis via stabilizing scaffold protein p62 in PC12 cells.

Parkin has been shown to exert protective effects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in different models of Parkinson disease. In the present study we investigated the molecular mechanisms underlying the neuroprotective action of parkin in vitro. HEK293, HeLa and PC12 cells were transfected with parkin, parkin mutants, p62 or si-p62. Protein expression and ubiquitination were assessed using immunoblot analysis. Immunoprecipitation assay was performed to identify the interaction between parkin and scaffold protein p62. PC12 and SH-SY5Y cells were treated with 6-OHDA (200 μmol/L), and cell apoptosis was detected using PI and Hoechst staining. In HEK293 cells co-transfected with parkin and p62, parkin was co-immunoprecipitated with p62, and parkin overexpression increased p62 protein levels. In parkin-deficient HeLa cells, transfection with wild-type pakin, but not with ligase activity-deficient pakin mutants, significantly increased p62 levels, suggesting that parkin stabilized p62 through its E3 ligase activity. Transfection with parkin or p62 significantly repressed ERK1/2 phosphorylation in HeLa cells, but transfection with parkin did not repress ERK1/2 phosphorylation in p62-knockdown HeLa cells, suggesting that p62 was involved in parkin-induced inhibition on ERK1/2 phosphorylation. Overexpression of parkin or p62 significantly repressed 6-OHDA-induced ERK1/2 phosphorylation in PC12 cells, and parkin overexpression inhibited 6-OHDA-induced apoptosis in PC12 and SH-SY5Y cells. Parkin protects PC12 cells against 6-OHDA-induced apoptosis via ubiquitinating and stabilizing scaffold protein p62, and repressing ERK1/2 activation.

Pharmacological characterization of conotoxin lt14a as a potent non-addictive analgesic.

Conotoxin lt14a is a small peptide consisting of 13 amino acids. It was originally identified from the cDNA of Conus litteratus in the South China Sea. Previous reports showed lt14a exhibited antinociceptive activity using a hot plate-induced pain mouse model and acted as an antagonist of neuronal nicotinic acetylcholine receptors. We confirmed that conotoxin lt14a administration resulted in antinociception activity using a mouse inflammatory pain model and a rat model of mechanically-induced pain. The mRNA expression of c-fos and NOS in the spinal cord of rats was suppressed by lt14a. Labeling of lt14a with an Alexa Fluor 488 ester showed that lt14a was bound to the surface of PC12 cells and that this binding was inhibited by pre-application of the nicotinic acetylcholine receptor (nAChR) antagonist tubocurarine chloride (TUB) and the nAChR blocker hexamethonium bromide (HB). These data confirm previous reports that showed lt14a binds to the surface of PC12 cells via nAChRs with patch clamp whole-cell recordings. Additional results showed that lt14a suppressed extracellular signal-regulated kinase (ERK1/2) phosphorylation in PC12 cells activated by Ach. Our results showed that lt14a did not induce drug dependence but rather suppressed morphine withdrawal symptoms. Our work suggests that lt14a is a novel antinociceptive agent that targets the nAChR receptor without inducing drug dependence.

Prostate stem cell antigen interacts with nicotinic acetylcholine receptors and is affected in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder involving impaired cholinergic neurotransmission and dysregulation of nicotinic acetylcholine receptors (nAChRs). Ly-6/neurotoxin (Lynx) proteins have been shown to modulate cognition and neural plasticity by binding to nAChR subtypes and modulating their function. Hence, changes in nAChR regulatory proteins such as Lynx proteins could underlie the dysregulation of nAChRs in AD. Using Western blotting, we detected bands corresponding to the Lynx proteins prostate stem cell antigen (PSCA) and Lypd6 in human cortex indicating that both proteins are present in the human brain. We further showed that PSCA forms stable complexes with the α4 nAChR subunit and decreases nicotine-induced extracellular-signal regulated kinase phosphorylation in PC12 cells. In addition, we analyzed protein levels of PSCA and Lypd6 in postmortem tissue of medial frontal gyrus from AD patients and found significantly increased PSCA levels (approximately 70%). In contrast, no changes in Lypd6 levels were detected. In concordance with our findings in AD patients, PSCA levels were increased in the frontal cortex of triple transgenic mice with an AD-like pathology harboring human transgenes that cause both age-dependent β-amyloidosis and tauopathy, whereas Tg2576 mice, which display β-amyloidosis only, had unchanged PSCA levels compared to wild-type animals. These findings identify PSCA as a nAChR-binding protein in the human brain that is affected in AD, suggesting that PSCA-nAChR interactions may be involved in the cognitive dysfunction observed in AD.

Role of the Insulin-Like Growth Factor Type 1 Receptor in the Pathogenesis of Diabetic Encephalopathy.

Defective cognitive function is common in patients with diabetes, suggesting that insulin normally exerts anabolic actions in neuron, namely, diabetic encephalopathy. However, because insulin can cross-activate the insulin-like growth factor type 1 receptor (IGF-1R), which also functions in most of tissues, such as muscle and bone, it has been difficult to establish the direct (IGF-1-independent) actions of insulin in the pathogenesis of diabetic encephalopathy. To overcome this problem, we examined insulin signaling and action in primary PC-12 cells engineered for conditional disruption of the IGF-1 receptor (ΔIGF-1R). The results showed that the lower glucose metabolism and high expression of IGF-1R occurred in the brain of the DE rat model. The results also showed the defect of IGF-1R could significantly improve the ability of glucose consumption and enhance sensitivity to insulin-induced IR and Akt phosphorylation in PC12 cells. And meanwhile, IGF-1R allele gene knockout (IGF-1Rneo) mice treated with HFD/STZ had better cognitive abilities than those of wild mice. Those results indicate that insulin exerts direct anabolic actions in neuron-like cells by activation of its cognate receptor and prove that IGF-1R plays an important role in the pathogenesis of diabetic encephalopathy.

Puerarin induces the upregulation of glutathione levels and nuclear translocation of Nrf2 through PI3K/Akt/GSK-3β signaling events in PC12 cells exposed to lead

Oxidative stress is thought to be involved in lead-induced toxicity, especially affecting the brain. We reported previously that puerarin possesses antioxidative properties in the nervous system. Therefore, the aim of the present study was to test the hypothesis that puerarin inhibits lead acetate-induced oxidative stress in PC12 cells by interrupting phosphatidylinositol-3 kinase (PI3K)/Akt signaling through increasing glutathione (GSH) synthesis. Our results showed that puerarin attenuates oxidative stress in a concentration-dependent manner in PC12 cells exposed to lead acetate demonstrated by scavenging reactive oxygen species (ROS) and reducing lipid peroxidation (LPO). Treatment with puerarin significantly up-regulates glutamate cysteine ligase catalytic subunit (GCLc) expression both at its mRNA and protein levels, but not glutamate cysteine ligase modifier (GCLm) subunit, accompanying the elevation of cellular glutathione level. The increased nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) was not because of increased transcription of Nrf2 as Nrf2 transcript levels did not change after puerarin treatment. The effects of puerarin could be partially blocked by pharmacologic inhibition of PI3K and the glycogen synthase kinase 3β (GSK-3β) pathways with LY294002 and LiCl, respectively. On the other hand, puerarin treatment promoted Akt and GSK-3β phosphorylation in PC12 cells exposed to lead acetate. Moreover, puerarin failed to modulate the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p-c-Jun N-terminal kinases (JNK), and p-p38 mitogen-activated protein kinase (MAPK) demonstrating some specificity for its action on the PI3K/GSK-3β pathway. These findings suggest that puerarin as a phytoestrogen might be an attractive agent for prevention and treatment of chronic diseases related to lead neurotoxicity.

Multiple treatments with L-3,4-dihydroxyphenylalanine modulate dopamine biosynthesis and neurotoxicity through the protein kinase A-transient extracellular signal-regulated kinase and exchange protein activation by cyclic AMP-sustained extracellular signal-regulated kinase signaling pathways.

Multiple treatments with L-3,4-dihydroxyphenylalanine (L-DOPA; 20 µM) induce neurite-like outgrowth and reduce dopamine biosynthesis in rat adrenal pheochromocytoma (PC) 12 cells. We therefore investigated the effects of multiple treatments with L-DOPA (MT-LD) on cell survival and death over a duration of 6 days by using PC12 cells and embryonic rat midbrain primary cell cultures. MT-LD (10 and 20 µM) decreased cell viability, and both types of cells advanced to the differentiation process at 4-6 days. MT-LD induced cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylation and exchange protein activation by cAMP (Epac) expression at 1-3 days, which led to transient extracellular signal-regulated kinase (ERK1/2) phosphorylation in both cells. In these states, MT-LD activated cAMP-response element binding protein (CREB; Ser133) and tyrosine hydroxylase (Ser40) phosphorylation in PC12 cells, which led to an increase in intracellular dopamine levels. In contrast, MT-LD induced prolonged Epac expression at 4-5 days in both cells, which led to sustained ERK1/2 phosphorylation. In these states, the dopamine levels were decreased in PC12 cells. In addition, MT-LD induced c-Jun N-terminal kinase1/2 phosphorylation and cleaved caspase-3 expression at 4-6 days in both cells. These results suggest that MT-LD maintains cell survival via PKA-transient ERK1/2 activation, which stimulates dopamine biosynthesis. In contrast, at the later time period, MT-LD induces differentiation via both prolonged Epac and sustained ERK1/2 activation, which subsequently leads to the cell death process. Our data demonstrate that L-DOPA can cause neurotoxicity by modulating the Epac-ERK pathways in neuronal and PC12 cells.

The PERK-eIF2α signaling pathway is involved in TCDD-induced ER stress in PC12 cells

Studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces apoptotic cell death in neuronal cells. However, whether this is the result of endoplasmic reticulum (ER) stress-mediated apoptosis remains unknown. In this study, we determined whether ER stress plays a role in the TCDD-induced apoptosis of pheochromocytoma (PC12) cells and primary neurons. PC12 cells were exposed to different TCDD concentrations (1, 10, 100, 200, or 500nM) for varying lengths of time (1, 3, 6, 12, or 24h). TCDD concentrations much higher than 10nM (100, 200, or 500nM) markedly increased glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) levels, which are hallmarks of ER stress. We also evaluated the effects of TCDD on ER morphology in PC12 cells and primary neurons that were treated with different TCDD concentrations (1, 10, 50, or 200nM) for 24h. Ultrastructural ER alterations were observed with transmission electron microscopy in PC12 cells and primary neurons treated with high concentrations of TCDD. Furthermore, TCDD-induced ER stress significantly promoted the activation of the PKR-like ER kinase (PERK), a sensor for the unfolded protein response (UPR), and its downstream target eukaryotic translation initiation factor 2 α (eIF2α); in contrast, TCDD did not appear to affect inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6), two other UPR sensors. Importantly, TCDD significantly inhibited eIF2α phosphorylation and triggered apoptosis in PC12 cells after 6–24h of treatment. Salubrinal, which activates the PERK-eIF2α pathway, significantly enhanced eIF2α phosphorylation in PC12 cells and attenuated the TCDD-induced cell death. In contrast, knocking down eIF2α using small interfering RNA markedly enhanced TCDD-induced cell death. Together, these results indicate that the PERK-eIF2α pathway plays an important role in TCDD-induced ER stress and apoptosis in PC12 cells.

Extracellular α-synuclein leads to microtubule destabilization via GSK-3β-dependent Tau phosphorylation in PC12 cells.

α-Synuclein (ASN) plays an important role in pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. Novel and most interesting data showed elevated tauopathy in PD and suggested relationship between ASN and Tau protein. However, the mechanism of ASN-evoked Tau protein modification is not fully elucidated. In this study we investigated the role of extracellular ASN in Tau hyperphosphorylation in rat pheochromocytoma (PC12) cells and the involvement of glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase 5 (CDK5) in ASN-dependent Tau modification. Our results indicated that exogenously added ASN increases Tau phosphorylation at Ser396. Accordingly, the GSK-3β inhibitor (SB-216763) prevented ASN-evoked Tau hyperphosphorylation, but the CDK5 inhibitor had no effect. Moreover, western blot analysis showed that ASN affected GSK-3β via increasing of protein level and activation of this enzyme. GSK-3β activity evaluated by its phosphorylation status assay showed that ASN significantly increased the phosphorylation of this enzyme at Tyr216 with parallel decrease in phosphorylation at Ser9, indicative of stimulation of GSK-3β activity. Moreover, the effect of ASN on microtubule (MT) destabilization and cell death with simultaneous the involvement of GSK-3β in these processes were analyzed. ASN treatment increased the amount of free tubulin and concomitantly reduced the amount of polymerized tubulin and SB-216763 suppressed these ASN-induced changes in tubulin, indicating that GSK-3β is involved in ASN-evoked MT destabilization. ASN-induced apoptotic processes lead to decrease in PC12 cells viability and SB-216763 protected those cells against ASN-evoked cytotoxicity. Concluding, extracellular ASN is involved in GSK-3β-dependent Tau hyperphosphorylation, which leads to microtubule destabilization. GSK-3β inhibition may be an effective strategy for protecting against ASN-induced cytotoxicity.

Cytocompatibility of novel extracellular matrix protein analogs of biodegradable polyester polymers derived from α-hydroxy amino acids

One of the challenges in regenerative medicine is the development of novel biodegradable materials to build scaffolds that will support multiple cell types for tissue engineering. Here we describe the preparation, characterization, and cytocompatibility of homo- and hetero-polyesters of α-hydroxy amino acid derivatives with or without lactic acid conjugation. The polymers were prepared by a direct condensation method and characterized using gel permeation chromatography, 1H-nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, optical activity, and solubility. The surface charge of the polymers was evaluated using zeta potential measurements. The polymers were coated onto glass cover slips followed by characterization using nano-surface profiler, thin film reflectometry, and atomic force microscopy (AFM). Their interaction with endothelial and neuronal cells was assessed using adhesion, proliferation, and differentiation assays. Of the characterized polymers, Poly-HOVal-LA, but not Poly-(D)HOPhe, significantly augmented nerve growth factor (NGF)-induced neuronal differentiation of the PC12 pheochromcytoma cells. In contrast, Poly-HOLeu increased by 20% the adhesion of endothelial cells, but did not affect PC12 cell differentiation. NGF-induced Erk1/2 phosphorylation in PC12 cells grown on the different polymers was similar to the effect observed for cells cultured on collagen type I. While no significant association could be established between charge and the differentiative/proliferative properties of the polymers, AFM analysis indicated augmentation of NGF-induced neuronal differentiation on smooth polymer surfaces. We conclude that overall selective cytocompatibility and bioactivity might render α-hydroxy amino acid polymers useful as extracellular matrix-mimicking materials for tissue engineering.