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1.  Mechanism of Okadaic Acid Induced Neuronal Death and the Effect of Estrogens 
Journal of neurochemistry  2008;108(3):732-740.
Serine/threonine protein phosphatases (PPs) are important mediators of general cellular function as well as neurodegenerative processes. We have previously shown inhibition of PPs to be as neurotoxic as glutamate-induced neuronal death but resistant to neuroprotection by estrogens. In this study, the mechanism by which phosphatase inhibition via okadaic acid (OA) induce neurotoxicity is explored. Neurons were exposed to OA or glutamate in the presence or absence of various protein kinases inhibitors, and/or one of four estrogens. Both OA and glutamate induced cell death via increased reactive oxygen species (ROS), protein carbonylation, lipid peroxidation, caspase-3 activity, and mitochondrial dysfunction. All estrogens attenuated glutamate-mediated responses, but not OA-induced responses. In addition, inhibition of PKC and MAPK pathway was neuroprotective against glutamate but not OA toxicity. Interestingly, inhibition of MAPK pathway with PD98096 or U0126 caused a decrease in ROS production suggesting that activation of ERK1/2 could further exacerbate the oxidative stress caused by glutamate-induced toxicity; however, these inhibitors had no effect on OA-induced toxicity. Collectively, these results indicate that both glutamate and OA neurotoxicities are mediated by persistent activation of ERK1/2 and/or PKC and a resulting oxidative stress, and that protein phosphatase activity is an important and necessary aspect of estrogen-mediated neuroprotection.
PMCID: PMC2727740  PMID: 19054278
estradiol; estrogen analogues; okadaic acid; phosphatases; protein kinases; oxidative stress
2.  Pinocembrin protects against β-amyloid-induced toxicity in neurons through inhibiting receptor for advanced glycation end products (RAGE)-independent signaling pathways and regulating mitochondrion-mediated apoptosis 
BMC Medicine  2012;10:105.
It is known that amyloid-β peptide (Aβ) plays a pivotal role in the pathogenesis of Alzheimer's disease (AD). Interaction between Aβ and the receptor for advanced glycation end products (RAGE) has been implicated in neuronal degeneration associated with this disease. Pinocembrin, a flavonoid abundant in propolis, has been reported to possess numerous biological activities beneficial to health. Our previous studies have demonstrated that pinocembrin has neuroprotective effects on ischemic and vascular dementia in animal models. It has been approved by the State Food and Drug Administration of China for clinical use in stroke patients. Against this background, we investigated the effects of pinocembrin on cognitive function and neuronal protection against Aβ-induced toxicity and explored its potential mechanism.
Mice received an intracerebroventricular fusion of Aβ25-35. Pinocembrin was administrated orally at 20 mg/kg/day and 40 mg/kg/day for 8 days. Behavioral performance, cerebral cortex neuropil ultrastructure, neuronal degeneration and RAGE expression were assessed. Further, a RAGE-overexpressing cell model and an AD cell model were used for investigating the mechanisms of pinocembrin. The mechanisms underlying the efficacy of pinocembrin were conducted on target action, mitochondrial function and potential signal transduction using fluorescence-based multiparametric technologies on a high-content analysis platform.
Our results showed that oral administration of pinocembrin improved cognitive function, preserved the ultrastructural neuropil and decreased neurodegeneration of the cerebral cortex in Aβ25-35-treated mice. Pinocembrin did not have a significant effect on inhibiting Aβ1-42 production and scavenging intracellular reactive oxygen species (ROS). However, pinocembrin significantly inhibited the upregulation of RAGE transcripts and protein expression both in vivo and in vitro, and also markedly depressed the activation of p38 mitogen-activated protein kinase (MAPK)-MAPKAP kinase-2 (MK2)-heat shock protein 27 (HSP27) and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK)-c-Jun pathways and the downstream nuclear factor κB (NFκB) inflammatory response subsequent to Aβ-RAGE interaction. In addition, pinocembrin significantly alleviated mitochondrial dysfunction through improving mitochondrial membrane potential and inhibiting mitochondrial oxidative stress, and regulated mitochondrion-mediated apoptosis by restoration of B cell lymphoma 2 (Bcl-2) and cytochrome c and inactivation of caspase 3 and caspase 9.
Pinocembrin was shown to infer cognitive improvement and neuronal protection in AD models. The mechanisms of action of the compound were illustrated on RAGE-dependent transduction inhibition and mitochondrion protection. It appears to be a promising candidate for the prevention and therapy of AD.
PMCID: PMC3542580  PMID: 22989295
Alzheimer's disease; amyloid-β peptide; apoptosis; pinocembrin; receptor for advanced glycation end products
3.  Neuroprotective Activity of pDING in Response to HIV-1 Tat 
Journal of cellular physiology  2014;229(2):153-161.
Although neurons are not productively infected with HIV-1, neuronal injury and death are frequently seen in the brains of AIDS patients with neurological and neurocognitive disorders. Evidently, viral proteins including Tat and cellular inflammatory factors released by activated and/or infected microglia, macrophages, and astrocytes contribute to neuronal cell death. Several studies have demonstrated that HIV-1 associated neuronal cell injury is mediated by dysregulation of signaling pathways that are controlled, in part, by a class of serine/threonine kinases. In this study we demonstrate that pDING, a novel plant-derived phosphate binding protein has the capacity to reduce the severity of injury and death caused by HIV-1 and its neurotoxic Tat protein. We demonstrate that pDING, also called p27SJ/p38SJ, protects cells from the loss of neuronal processes induced by Tat and promotes neuronal outgrowth after Tat-mediated injury. Further, expression of pDING prevents Tat-induced oxidative stress and mitochondrial permeability. With its profound phosphatase activity, pDING controls the activity of several kinases including MAPK, Cdk5 and their downstream target protein, MEF2, which is implicated in neuronal cell protection. Our results show that expression of pDING in neuronal cells diminishes the level of hyperphosphorylated forms of Cdk5 and MEF2 caused by Tat and the other neurotoxic agents that are secreted by the HIV-1 infected cells. These observations suggest that pDING, through its phosphatase activity, has the ability to manipulate the state of phosphorylation and activity of several factors involved in neuronal cell health in response to HIV-1.
PMCID: PMC4204488  PMID: 23955241
HIV-1; DING; Neural degeneration
4.  Selective Inhibition of MAPK Phosphatases by Zinc Accounts for ERK1/2-dependent Oxidative Neuronal Cell Death 
Molecular pharmacology  2008;74(4):1141-1151.
Oxidative stress induced by glutathione depletion in the mouse HT22 neuroblastoma cell line and embryonic rat immature cortical neurons causes a delayed, sustained activation of extracellular signal-regulated kinases-1/2 (ERK1/2), which is required for cell death. This sustained activation of ERK1/2 is mediated primarily by a selective inhibition of distinct ERK1/2-directed phosphatases either by enhanced degradation (i.e. for Mitogen activated protein kinase [MAPK] Phosphatase-1) or as shown here by reductions in enzymatic activity (i.e. for Protein Phosphatase type 2A [PP-2A]). The inhibition of ERK1/2 phosphatases in HT22 cells and immature neurons subjected to glutathione depletion results from oxidative stress as phosphatase activity is restored in cells treated with the antioxidant butylated hydroxyanisole (BHA). This leads to reduced ERK1/2 activation and neuroprotection. Furthermore, an increase in free intracellular zinc that accompanies glutathione-induced oxidative stress in HT22 cells and immature neurons contributes to selective inhibition of ERK1/2 phosphatase activity and cell death. Finally, ERK1/2 also functions to maintain elevated levels of zinc. Thus the elevation of intracellular zinc within neurons subjected to oxidative stress can trigger a robust positive feedback loop operating through activated ERK1/2 that rapidly sets into motion a zinc-dependent pathway of cell death.
PMCID: PMC2575064  PMID: 18635668
5.  Neuroprotective Effects of a Novel Single Compound 1-Methoxyoctadecan-1-ol Isolated from Uncaria sinensis in Primary Cortical Neurons and a Photothrombotic Ischemia Model 
PLoS ONE  2014;9(1):e85322.
We identified a novel neuroprotective compound, 1-methoxyoctadecan-1-ol, from Uncaria sinensis (Oliv.) Havil and investigated its effects and mechanisms in primary cortical neurons and in a photothrombotic ischemic model. In primary rat cortical neurons against glutamate-induced neurotoxicity, pretreatment with 1-methoxyoctadecan-1-ol resulted in significantly reduced neuronal death in a dose-dependent manner. In addition, treatment with 1-methoxyoctadecan-1-ol resulted in decreased neuronal apoptotic death, as assessed by nuclear morphological approaches. To clarify the neuroprotective mechanism of 1-methoxyoctadecan-1-ol, we explored the downstream signaling pathways of N-methyl-D-aspartate receptor (NMDAR) with calpain activation. Treatment with glutamate leads to early activation of NMDAR, which in turn leads to calpain-mediated cleavage of striatal-enriched protein tyrosine phosphatase (STEP) and subsequent activation of p38 mitogen activated protein kinase (MAPK). However, pretreatment with 1-methoxyoctadecan-1-ol resulted in significantly attenuated activation of GluN2B-NMDAR and a decrease in calpain-mediated STEP cleavage, leading to subsequent attenuation of p38 MAPK activation. We confirmed the critical role of p38 MAPK in neuroprotective effects of 1-methoxyoctadecan-1-ol using specific inhibitor SB203580. In the photothrombotic ischemic injury in mice, treatment with 1-methoxyoctadecan-1-ol resulted in significantly reduced infarct volume, edema size, and improved neurological function. 1-methoxyoctadecan-1-ol effectively prevents cerebral ischemic damage through down-regulation of calpain-mediated STEP cleavage and activation of p38 MAPK. These results suggest that 1-methoxyoctadecan-1-ol showed neuroprotective effects through down-regulation of calpain-mediated STEP cleavage with activation of GluN2B-NMDAR, and subsequent alleviation of p38 MAPK activation. In addition, 1-methoxyoctadecan-1-ol might be a useful therapeutic agent for brain disorder such as ischemic stroke.
PMCID: PMC3885700  PMID: 24416390
6.  Nuclear Calcium Signaling Controls Expression of a Large Gene Pool: Identification of a Gene Program for Acquired Neuroprotection Induced by Synaptic Activity 
PLoS Genetics  2009;5(8):e1000604.
Synaptic activity can boost neuroprotection through a mechanism that requires synapse-to-nucleus communication and calcium signals in the cell nucleus. Here we show that in hippocampal neurons nuclear calcium is one of the most potent signals in neuronal gene expression. The induction or repression of 185 neuronal activity-regulated genes is dependent upon nuclear calcium signaling. The nuclear calcium-regulated gene pool contains a genomic program that mediates synaptic activity-induced, acquired neuroprotection. The core set of neuroprotective genes consists of 9 principal components, termed Activity-regulated Inhibitor of Death (AID) genes, and includes Atf3, Btg2, GADD45β, GADD45γ, Inhibin β-A, Interferon activated gene 202B, Npas4, Nr4a1, and Serpinb2, which strongly promote survival of cultured hippocampal neurons. Several AID genes provide neuroprotection through a common process that renders mitochondria more resistant to cellular stress and toxic insults. Stereotaxic delivery of AID gene-expressing recombinant adeno-associated viruses to the hippocampus confers protection in vivo against seizure-induced brain damage. Thus, treatments that enhance nuclear calcium signaling or supplement AID genes represent novel therapies to combat neurodegenerative conditions and neuronal cell loss caused by synaptic dysfunction, which may be accompanied by a deregulation of calcium signal initiation and/or propagation to the cell nucleus.
Author Summary
The dialogue between the synapse and the nucleus plays an important role in the physiology of neurons because it links brief changes in the membrane potential to the transcriptional regulation of genes critical for neuronal survival and long-term memory. The propagation of activity-induced calcium signals to the cell nucleus represents a major route for synapse-to-nucleus communication. Here we identified nuclear calcium-regulated genes that are responsible for a neuroprotective shield that neurons build up upon synaptic activity. We found that among the 185 genes controlled by nuclear calcium signaling, a set of 9 genes had strong survival promoting activity both in cell culture and in an animal model of neurodegeneration. The mechanism through which several genes prevent cell death involves the strengthening of mitochondria against cellular stress and toxic insults. The discovery of an activity-induced neuroprotective gene program suggest that impairments of synaptic activity and synapse-to-nucleus signaling, for example due to expression of Alzheimer's disease protein or in aging, may comprise the cells' own neuroprotective system eventually leading to cell death. Thus, malfunctioning of nuclear calcium signaling could be a key etiological factor common to many neuropathological conditions, providing a simple and unifying concept to explain disease- and aging-related cell loss.
PMCID: PMC2718706  PMID: 19680447
7.  p38 MAPK as a negative regulator of VEGF/VEGFR2 signaling pathway in serum deprived human SK-N-SH neuroblastoma cells 
Neuroscience letters  2007;431(2):95-100.
Evidence suggests that vascular endothelial growth factor (VEGF) mediates neuroprotection to prevent an apoptotic cell death. The p38 mitogen activated protein kinase (MAPK) pathway is implicated as an important mediator of neuronal apoptosis but its role in VEGF-mediated neuroprotection is unclear. Herein, we show that treatments with the p38 MAPK inhibitor, SB202190, enhanced VEGF-mediated survival in serum deprived SK-N-SH neuroblastoma cells by decreasing caspase-3/7 activation while increasing the phosphorylation of the extracellular signal-regulated kinase (ERK1/2) and Akt signaled through the VEGF receptor, VEGFR2. A blockade of VEGFR2 signaling with a selective inhibitor, SU1498 or gene silencing with VEGFR2 siRNA in SB202190 treated cells abrogated this prosurvival response and induced high activation levels of caspase-3/7. These findings suggested that the protection elicited by p38 MAPK inhibition in serum starved cells was dependent on a functional VEGF/VEGFR2 pathway. However, p38 MAPK inhibition attenuated caspase-3 cleavage in SU1498/SB202190 treated cells, indicating that p38 MAPK and caspase-3 only contributed in part to the total levels of caspase-3/7 induced by VEGFR2 inhibition. Pretreatments with the pan caspase inhibitor, z-VAD-fmk, prevented the apoptosis induced by VEGFR2 inhibition and promoted survival in serum starved cells irrespective of p38 MAPK inhibition. Collectively, our findings suggest that p38 MAPK exerts a negative effect on VEGF-mediated signaling through VEGFR2 in serum starved neuroblastoma cells. Furthermore, VEGF signals protection against a caspase-mediated cell death that is regulated by p38 MAPK-dependent and -independent mechanisms.
PMCID: PMC2254182  PMID: 18178312
8.  Adiponectin is Protective against Oxidative Stress Induced Cytotoxicity in Amyloid-Beta Neurotoxicity 
PLoS ONE  2012;7(12):e52354.
Beta-amyloid (Aβ ) neurotoxicity is important in Alzheimer’s disease (AD) pathogenesis. Aβ neurotoxicity causes oxidative stress, inflammation and mitochondrial damage resulting in neuronal degeneration and death. Oxidative stress, inflammation and mitochondrial failure are also pathophysiological mechanisms of type 2 diabetes (T2DM) which is characterized by insulin resistance. Interestingly, T2DM increases risk to develop AD which is associated with reduced neuronal insulin sensitivity (central insulin resistance). We studied the potential protective effect of adiponectin (an adipokine with insulin-sensitizing, anti-inflammatory and anti-oxidant properties) against Aβ neurotoxicity in human neuroblastoma cells (SH-SY5Y) transfected with the Swedish amyloid precursor protein (Sw-APP) mutant, which overproduced Aβ with abnormal intracellular Aβ accumulation. Cytotoxicity was measured by assay for lactate dehydrogenase (LDH) released upon cell death and lysis. Our results revealed that Sw-APP transfected SH-SY5Y cells expressed both adiponectin receptor 1 and 2, and had increased AMP-activated protein kinase (AMPK) activation and enhanced nuclear factor-kappa B (NF-κB) activation compared to control empty-vector transfected SH-SY5Y cells. Importantly, adiponectin at physiological concentration of 10 µg/ml protected Sw-APP transfected SH-SY5Y cells against cytotoxicity under oxidative stress induced by hydrogen peroxide. This neuroprotective action of adiponectin against Aβ neurotoxicity-induced cytotoxicity under oxidative stress involved 1) AMPK activation mediated via the endosomal adaptor protein APPL1 (adaptor protein with phosphotyrosine binding, pleckstrin homology domains and leucine zipper motif) and possibly 2) suppression of NF-κB activation. This raises the possibility of novel therapies for AD such as adiponectin receptor agonists.
PMCID: PMC3531475  PMID: 23300647
9.  The Cystine/Glutamate Antiporter System xc− in Health and Disease: From Molecular Mechanisms to Novel Therapeutic Opportunities 
Antioxidants & Redox Signaling  2013;18(5):522-555.
The antiporter system xc− imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system xc− is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system xc−, including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system xc−. Moreover, the roles of system xc− in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system xc− inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System xc− is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system xc− in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS. Antioxid. Redox Signal. 18, 522–555.
I. Introduction
A. Oxidative stress and antioxidant defense
B. GSH metabolism
C. Glutamate: neurotransmission and neurotoxicity
II. The Cystine/Glutamate Antiporter System xc−
A. Functional and pharmacological characteristics of system xc−
B. The molecular biology of system xc−
C. The phylogeny of xCT, the specific subunit of system xc−
D. Regulation of system xc− by transcriptional regulation of its specific subunit xCT
E. Regulation of system xc− activity by protein trafficking and protein modification
F. Regulation of system xc− activity by substrate availability
III. Expression of System xc− In Vitro and In Vivo and Its Functional Consequences
A. In the absence of disease, system xc− shows a rather restricted expression pattern in vivo
B. System xc− is induced in most cultured cells
C. The role of system xc− in the regulation of GSH synthesis, the extracellular redox milieu, and extracellular glutamate levels
D. Oxidative glutamate toxicity—an in vitro paradigm for neuronal death induced by system xc− inhibition
1. The cell death pathway in oxidative glutamate toxicity
2. Using oxidative glutamate toxicity to identify neuroprotective pathways
3. Using oxidative glutamate toxicity to screen for neuroprotective drugs
4. Oxidative glutamate toxicity in vivo
IV. The Role of System xc− in Health and Disease
A. System xc− in vivo—lessons from xCT-deficient mice
B. The role of system xc− in the immune system and inflammation
C. The role of system xc− in cancer and resistance against anti-cancer drugs
1. System xc− is regulated by potentially oncogenic pathways
2. System xc− mediates the infection of cells by oncogenic Kaposi's sarcoma herpesvirus
3. System xc− plays an important role in the multidrug resistance of cancers
4. Inhibition of system xc− reduces cancer cell replication, tissue invasion, and metastasis
5. System xc− expressed in tumor cells may be used as a target for anticancer drug delivery
6. Up-regulation of system xc− in normal cells provides protection against carcinogenesis—a possible role in cancer prevention
7. Synopsis of the role of system xc− in cancer and resistance against anti-cancer drugs
D. System xc− and diseases of the eye
1. Studies of system xc− in the retina
2. Studies of system xc− in the lens and cornea
3. Synopsis and future directions for system xc− and diseases of the eye
E. The role of system xc− in diseases of the CNS
F. The role of system xc− activity in memory and behavior
V. Conclusion
PMCID: PMC3545354  PMID: 22667998
10.  Differential Expression of Redox Factor-1 Associated with Beta-Amyloid-Mediated Neurotoxicity 
Redox factor-1 (Ref-1), also known as HAP1, APE or APEX, is a multifunctional protein that regulates gene transcription as well as the response to oxidative stress. By interacting with transcription factors such as AP-1, NF-kappaB and p53, and directly participating in the cleavage of apurininic/apyrimidinic DNA lesions, Ref-1 plays crucial roles in both cell death signaling pathways and DNA repair, respectively. Oxidative stress induced by aggregated beta-amyloid (Aβ) peptide, altered DNA repair and transcriptional activation of cell death pathways have been implicated in the pathophysiology of Alzheimer’s disease (AD). Here we show that varying concentrations of Aβ1–42 differentially regulate Ref-1 expression, Ref-1 function and neuronal survival in vitro. Aβ (5.0 μM) caused a relatively rapid decrease in Ref-1 expression and activity associated with extensive DNA damage and neuronal degeneration. In contrast, Ref-1 induction occurred in cells exposed to Aβ (1.0 μM) without significant neuronal cell death. Aβ-induced attenuation of Ref-1 expression and endonuclease activity, and neuronal cell death were prevented by the anti-oxidant, catalase. Similar differential effects on Ref-1 expression and cell viability were observed in N2A neuroblastoma cells treated with either high or low dose hydrogen peroxide. These findings demonstrate the differential regulation of Ref-1 expression by varying degrees of oxidative stress. Parallels between the Ref-1 response to Aβ and H2O2 suggest similarities between DNA repair pathways activated by different inducers of oxidative stress. In AD brain, colocalization of Ref-1 and Aβ the absence of significant DNA damage are consistent with the cell culture results and suggests that Ref-1 may play a more neuroprotective role under these conditions. Modulation of Ref-1 expression and activity by local variations in Aβ concentration may be an important determinant of neuronal vulnerability to oxidative stress in AD.
PMCID: PMC2773510  PMID: 19898678
11.  A neuronal model of Alzheimer’s disease: An insight into the mechanisms of oxidative stress-mediated mitochondrial injury 
Neuroscience  2008;153(1):120-130.
Alzheimer’s disease (AD) is associated with β-amyloid accumulation, oxidative stress and mitochondrial dysfunction. However, the effects of genetic mutation of AD on oxidative status and mitochondrial manganese superoxide dismutase (MnSOD) production during neuronal development are unclear. To investigate the consequences of genetic mutation of AD on oxidative damages and production of MnSOD during neuronal development, we used primary neurons from new born wild-type (WT/WT) and APP (NLh/NLh) and PS1 (P264L) knock-in mice (APP/PS1) which incorporated humanized mutations in the genome. Increasing levels of oxidative damages, including protein carbonyl, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT), were accompanied by a reduction in mitochondrial membrane potential in both developing and mature APP/PS1 neurons compared to WT/WT neurons suggesting mitochondrial dysfunction under oxidative stress. Interestingly, developing APP/PS1 neurons were significantly more resistant to β-amyloid 1-42 treatment, whereas mature APP/PS1 neurons were more vulnerable than WT/WT neurons of the same age. Consistent with the protective function of MnSOD, developing APP/PS1 neurons have increased MnSOD protein and activity, indicating an adaptive response to oxidative stress in developing neurons. In contrast, mature APP/PS1 neurons exhibited lower MnSOD levels compared to mature WT/WT neurons indicating that mature APP/PS1 neurons lost the adaptive response. Moreover, mature APP/PS1 neurons had more co-localization of MnSOD with nitrotyrosine indicating a greater inhibition of MnSOD by nitrotyrosine. Overexpression of MnSOD or addition of MnTE-2-PyP5+ (SOD mimetic) protected against β-amyloid-induced neuronal death and improved mitochondrial respiratory function. Together, the results demonstrate that compensatory induction of MnSOD in response to an early increase in oxidative stress protects developing neurons against β-amyloid toxicity. However, continuing development of neurons under oxidative damage conditions may suppress the expression of MnSOD and enhance cell death in mature neurons.
PMCID: PMC2430183  PMID: 18353561
Alzheimer’s disease; APP/PS1; MnSOD; Oxidative stress; β-amyloid; MnTE-2-PyP5+ (SOD mimetic)
12.  A novel phenoxy thiophene sulphonamide molecule protects against glutamate evoked oxidative injury in a neuronal cell model 
BMC Neuroscience  2013;14:93.
Glutamate is one of the major neurotransmitters in the central nervous system. It is a potent neurotoxin capable of neuronal destruction through numerous signal pathways when present in high concentration. Glutamate-evoked excitotoxicity has been implicated in the etiology of many neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and ischemic stroke. Increasing evidence has shown that reactive oxygen species (ROS) provoked by glutamate-linked oxidative stress plays a crucial role in the pathogenesis of these disorders. We previously reported the discovery of an aryl thiophene compound, 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide (B355252) from a proprietary library of small molecules. We showed that this compound was capable of potentiating nerve growth factor (NGF)-primed neurite outgrowth in neuronal cell models in a low NGF environment. In the present study we investigated the neuroprotective effects and signaling pathways of B355252 on glutamate-evoked excitotoxicity in HT-22, a murine hippocampal neuronal cell line.
Glutamate significantly decreased HT-22 neuronal cell viability in a concentration-dependent manner as measured by the MTT assay. Co-treatment with 2, 4, and 8 μM B355252 protected against cell death caused by glutamate-induced toxicity by 9.1% (p<0.01), 26.0% (p<0.001), and 61.9% (p<0.001) respectively, compared to glutamate-treated control group. B355252 at a concentration of 8 μM fully rescued HT-22 from the neurototoxic effects of glutamate, and by itself increased cell viability by 16% (p<0.001) above untreated control. Glutamate enhanced reduction in glutathione (GSH) synthesis was reversed by 15% (p<0.01) in the presence of B355252. B355252 reduced the expression of apoptosis inducing factor (AIF) by 27%, while the proapoptotic Bcl-2 associated X protein (Bax) was strongly attenuated 3-fold. Glutamate-evoked increase in intracellular calcium (Ca2+) load and subsequent ROS production was inhibited by 71% (p<0.001) and 40% (p<0.001) respectively, to comparable level as untreated control in the presence of B355252. Glutamate significantly upregulated the phosphorylation of extracellular signal regulated kinase Erk1/2 (pERK1/2), while decreasing Erk3. In contrast, B355252 potently attenuated the glutamate-dependent activation of Erk1/2 and robustly increased the level of ERK3 in HT-22.
A novel phenoxy thiophene small molecule, B355252, suppresses glutamate-evoked oxidative stress in HT-22 neurons by blocking Ca2+ and ROS production, and altering the expression or phosphorylation states of Erk kinases. This molecule previously reported to enhance neurite outgrowth in the presence of sub-physiological concentrations of NGF appears to be a promising drug candidate for development as a potential therapeutic and neuroprotective agent for various neurodegenerative disorders.
PMCID: PMC3846642  PMID: 24004478
Glutamate; Neuroprotection; Excitotoxicity; Small molecule; Alzheimer’s disease; Oxidative stress; ERK3; Neurodegenerative disease; Phenoxy thiophene; HT-22
13.  Distinct Roles for μ-Calpain and m-Calpain in Synaptic NMDAR-Mediated Neuroprotection and Extrasynaptic NMDAR-Mediated Neurodegeneration 
The Journal of Neuroscience  2013;33(48):18880-18892.
Prolonged calpain activation is widely recognized as a key component of neurodegeneration in a variety of pathological conditions. Numerous reports have also indicated that synaptic activation of NMDA receptors (NMDARs) provides neuroprotection against a variety of insults. Here, we report the paradoxical finding that such neuroprotection involves calpain activation. NMDAR activation in cultured rat cortical neurons was neuroprotective against starvation and oxidative stress-induced damage. It also resulted in the degradation of two splice variants of PH domain and Leucine-rich repeat Protein Phosphatase 1 (PHLPP1), PHLPP1α and PHLPP1β, which inhibit the Akt and ERK1/2 pathways. Synaptic NMDAR-induced neuroprotection and PHLPP1 degradation were blocked by calpain inhibition. Lentiviral knockdown of PHLPP1 mimicked the neuroprotective effects of synaptic NMDAR activation and occluded the effects of calpain inhibition on neuroprotection. In contrast to synaptic NMDAR activation, extrasynaptic NMDAR activation had no effect on PHLPP1 and the Akt and ERK1/2 pathways, but resulted in calpain-mediated degradation of striatal-enriched protein tyrosine phosphatase (STEP) and neuronal death. Using μ-calpain- and m-calpain-selective inhibitors and μ-calpain and m-calpain siRNAs, we found that μ-calpain-dependent PHLPP1 cleavage was involved in synaptic NMDAR-mediated neuroprotection, while m-calpain-mediated STEP degradation was associated with extrasynaptic NMDAR-induced neurotoxicity. Furthermore, m-calpain inhibition reduced while μ-calpain knockout exacerbated NMDA-induced neurotoxicity in acute mouse hippocampal slices. Thus, synaptic NMDAR-coupled μ-calpain activation is neuroprotective, while extrasynaptic NMDAR-coupled m-calpain activation is neurodegenerative. These results help to reconcile a number of contradictory results in the literature and have critical implications for the understanding and potential treatment of neurodegenerative diseases.
PMCID: PMC3841454  PMID: 24285894
14.  The Pseudophosphatase MK-STYX Induces Neurite-Like Outgrowths in PC12 Cells 
PLoS ONE  2014;9(12):e114535.
The rat pheochromocytoma PC12 cell line is a widely used system to study neuronal differentiation for which sustained activation of the extracellular signaling related kinase (ERK) pathway is required. Here, we investigate the function of MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] in neuronal differentiation. MK-STYX is a member of the MAPK phosphatase (MKP) family, which is generally responsible for dephosphorylating the ERKs. However, MK-STYX lacks catalytic activity due to the absence of the nucleophilic cysteine in the active site signature motif HC(X5)R that is essential for phosphatase activity. Despite being catalytically inactive, MK-STYX has been shown to play a role in important cellular pathways, including stress responses. Here we show that PC12 cells endogenously express MK-STYX. In addition, MK-STYX, but not its catalytically active mutant, induced neurite-like outgrowths in PC12 cells. Furthermore, MK-STYX dramatically increased the number of cells with neurite extensions in response to nerve growth factor (NGF), whereas the catalytically active mutant did not. MK-STYX continued to induce neurites in the presence of a MEK (MAP kinase kinase) inhibitor suggesting that MK-STYX does not act through the Ras-ERK/MAPK pathway but is involved in another pathway whose inactivation leads to neuronal differentiation. RhoA activity assays indicated that MK-STYX induced extensions through the Rho signaling pathway. MK-STYX decreased RhoA activation, whereas RhoA activation increased when MK-STYX was down-regulated. Furthermore, MK-STYX affected downstream players of RhoA such as the actin binding protein cofilin. The presence of MK-STYX decreased the phosphorylation of cofilin in non NGF stimulated cells, but increased its phosphorylation in NGF stimulated cells, whereas knocking down MK-STYX caused an opposite effect. Taken together our data suggest that MK-STYX may be a regulator of RhoA signaling, and implicate this pseudophosphatase as a regulator of neuronal differentiation.
PMCID: PMC4257672  PMID: 25479605
15.  Vitamin E Suppression of Microglial Activation Is Neuroprotective 
Journal of neuroscience research  2001;66(2):163-170.
Neurotoxic microglial-neuronal interactions have been implicated in the pathogenesis of various neurodegenerative diseases such as Alzheimer’s disease, and vitamin E has been shown to have direct neuroprotective effects. To determine whether vitamin E also has indirect neuroprotective effects through suppression of microglial activation, we used a microglial-neuronal coculture. Lipopolysaccharide (LPS) treatment of a microglial cell line (N9) induced a time-dependent activation of both p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-κB (NFκB), with consequent increases in interleukin-1α (IL-1α), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) production. Differentiated neuronal cells (PC12 cells treated with nerve growth factor) exhibited marked loss of processes and decreased survival when cocultured with LPS-activated microglia. Preincubation of microglia with vitamin E diminished this neurotoxic effect, independently of direct effects of the antioxidant on the neuronal cells. Microglial NO production and the induction of IL-1α and TNFα expression also were attenuated by vitamin E. Such antiinflammatory effects of vitamin E were correlated with suppression of p38 MAPK and NFκB activation and were mimicked by an inhibition of either p38 MAPK (by SB203580) or NFκB (by decoy oligonucleotides). These results suggest that, in addition to the beneficial effects of providing direct antioxidant protection to neurons reported by others, vitamin E may provide neuroprotection in vivo through suppression of signaling events necessary for microglial activation.
PMCID: PMC3903400  PMID: 11592111
Alzheimer’s disease; interleukin-1; NFκB; nitric oxide; p38 mitogen-activated protein kinase; tumor necrosis factor; vitamin E
16.  Extracellular progranulin protects cortical neurons from toxic insults by activating survival signaling 
Neurobiology of Aging  2011;32(12):2326.e5-2326.16.
To reduce damage from toxic insults such as glutamate excitotoxicity and oxidative stresses, neurons may deploy an array of neuroprotective mechanisms. Recent reports show that progranulin (PGRN) gene null or missense mutations leading to inactive protein, are linked to frontotemporal lobar degeneration (FTLD), suggesting that survival of certain neuronal populations need full expression of functional PGRN. Here we show that extracellular PRGN stimulates phosphorylation/activation of the neuronal MEK/ERK/p90RSK and PI3K/Akt cell survival pathways and rescues cortical neurons from cell death induced by glutamate or oxidative stresses. Pharmacological inhibition of MEK/ERK/p90RSK signaling blocks the PGRN-induced phosphorylation and neuroprotection against glutamate toxicity whilst inhibition of either MEK/ERK/p90RSK or PI3K/Akt blocks PGRN protection against neurotoxin MPP+. Inhibition of both pathways had synergistic effects on PGRN-dependent neuroprotection against MPP+ toxicity suggesting both pathways contribute to the neuroprotective activities of PGRN. Extracellular PGRN is remarkably stable in neuronal cultures indicating neuroprotective activities are associated with full-length protein. Together, our data show that extracellular PRGN acts as a neuroprotective factor and support the hypothesis that in FTLD, reduction of functional brain PGRN results in reduced survival signaling and decreased neuronal protection against excitotoxicity and oxidative stress leading to accelerated neuronal cell death. That extracellular PGRN has neuroprotective functions against toxic insults suggests that in vitro preparations of this protein may be used therapeutically.
PMCID: PMC3375317  PMID: 21820214
progranulin; neuroprotection; neurodegeneration; frontotemporal lobar degeneration; excitotoxicity; oxidative stress; ERK; Akt
17.  Melatonin Potentiates the Neuroprotective Properties of Resveratrol Against Beta-Amyloid-Induced Neurodegeneration by Modulating AMP-Activated Protein Kinase Pathways 
Background and Purpose
Recent studies have demonstrated that resveratrol (RSV) reduces the incidence of age-related macular degeneration, Alzheimer's disease (AD), and stroke, while melatonin (MEL) supplementation reduces the progression of the cognitive impairment in AD patients. The purpose of this investigation was to assess whether the co-administration of MEL and RSV exerts synergistic effects on their neuroprotective properties against β-amyloid (Aβ)-induced neuronal death.
The neuroprotective effects of co-treatment with MEL and RSV on Aβ1-42-induced cell death, was measured by MTT reduction assay. Aβ1-42 caused an increase in intracellular levels of reactive oxygen species (ROS), as assessed by H2-DCF-DA dye, and a reduction of total glutathione (GSH) levels and mitochondrial membrane potential, as assessed using monochlorobimane and rhodamine 123 fluorescence, respectively. Western blotting was used to investigate the intracellular signaling mechanism involved in these synergic effects.
We treated a murine HT22 hippocampal cell line with MEL or RSV alone or with both simultaneously. MEL and RSV alone significantly attenuated ROS production, mitochondrial membrane-potential disruption and the neurotoxicity induced by Aβ1-42. They also restored the Aβ1-42-induced depletion of GSH, back to within its normal range and prevented the Aβ1-42-induced activation of glycogen synthase kinase 3β (GSK3β). However, co-treatment with MEL and RSV did not exert any significant synergistic effects on either the recovery of the Aβ1-42-induced depletion of GSH or on the inhibition of Aβ1-42-induced GSK3β activation. Aβ1-42 treatment increased AMP-activated protein kinase (AMPK) activity, which is associated with subsequent neuronal death. We demonstrated that MEL and RSV treatment inhibited the phosphorylation of AMPK.
Together, our results suggest that co-administration of MEL and RSV acts as an effective treatment for AD by attenuating Aβ1-42-induced oxidative stress and the AMPK-dependent pathway.
PMCID: PMC2950917  PMID: 20944813
melatonin; resveratrol; neuroprotection; reactive oxygen species; glycogen synthase kinase 3β; AMP-activated protein kinase
18.  The ER stress factor XBP1s prevents amyloid-β neurotoxicity 
Human Molecular Genetics  2011;20(11):2144-2160.
Alzheimer's disease (AD) is an incurable neurodegenerative disorder clinically characterized by progressive cognitive impairment. A prominent pathologic hallmark in the AD brain is the abnormal accumulation of the amyloid-β 1–42 peptide (Aβ), but the exact pathways mediating Aβ neurotoxicity remain enigmatic. Endoplasmic reticulum (ER) stress is induced during AD, and has been indirectly implicated as a mediator of Aβ neurotoxicity. We report here that Aβ activates the ER stress response factor X-box binding protein 1 (XBP1) in transgenic flies and in mammalian cultured neurons, yielding its active form, the transcription factor XBP1s. XBP1s shows neuroprotective activity in two different AD models, flies expressing Aβ and mammalian cultured neurons treated with Aβ oligomers. Trying to identify the mechanisms mediating XBP1s neuroprotection, we found that in PC12 cells treated with Aβ oligomers, XBP1s prevents the accumulation of free calcium (Ca2+) in the cytosol. This protective activity can be mediated by the downregulation of a specific isoform of the ryanodine Ca2+ channel, RyR3. In support of this observation, a mutation in the only ryanodine receptor (RyR) in flies also suppresses Aβ neurotoxicity, indicating the conserved mechanisms between the two AD models. These results underscore the functional relevance of XBP1s in Aβ toxicity, and uncover the potential of XBP1 and RyR as targets for AD therapeutics.
PMCID: PMC3090193  PMID: 21389082
19.  Neuroprotective effects of resveratrol and epigallocatechin gallate polyphenols are mediated by the activation of protein kinase C gamma 
Polyphenols such as epigallocatechin gallate (EGCG) and resveratrol have received a great deal of attention because they may contribute to the purported neuroprotective action of the regular consumption of green tea and red wine. Many studies, including those published by our group, suggest that this protective action includes their abilities to prevent the neurotoxic effects of beta-amyloid, a protein whose accumulation likely plays a pivotal role in Alzheimer's disease. Moreover, the scavenging activities of polyphenols on reactive oxygen species and their inhibitory action of cyclooxygenase likely explain, at least in part, their antioxidant and anti-inflammatory activities. Besides these well-documented properties, the modulatory action of these polyphenols on intracellular signaling pathways related to cell death/survival (e.g., protein kinase C, PKC) has yet to be investigated in detail. Using rat hippocampal neuronal cells, we aimed to investigate here the effects of EGCG and resveratrol on cell death induced by GF 109203X, a selective inhibitor of PKC. The MTT/resazurin and spectrin assays indicated that EGCG and resveratrol protected against GF 109203X-induced cell death and cytoskeleton degeneration, with a maximal effect at 1 and 3 μM, respectively. Moreover, immunofluorescence data revealed that cells treated with these polyphenols increased PKC gamma (γ) activation and promoted neuronal interconnections. Finally, we found that the protective effects of both polyphenols on the cytoskeleton and synaptic plasticity were mediated by the PKCγ subunit. Taken together, the results suggest that PKC, and more specifically its γ subunit, plays a critical role in the protective action of EGCG and resveratrol on neuronal integrity.
PMCID: PMC3872731  PMID: 24421757
PKC; polyphenols; neuroprotection; resveratrol; epigallocatechin gallate; hippocampal cultured cells
20.  Neuroprotective Action of Cycloheximide Involves Induction of Bcl-2 and Antioxidant Pathways 
The Journal of Cell Biology  1997;136(5):1137-1149.
The ability of the protein synthesis inhibitor cycloheximide (CHX) to prevent neuronal death in different paradigms has been interpreted to indicate that the cell death process requires synthesis of “killer” proteins. On the other hand, data indicate that neurotrophic factors protect neurons in the same death paradigms by inducing expression of neuroprotective gene products. We now provide evidence that in embryonic rat hippocampal cell cultures, CHX protects neurons against oxidative insults by a mechanism involving induction of neuroprotective gene products including the antiapoptotic gene bcl-2 and antioxidant enzymes. Neuronal survival after exposure to glutamate, FeSO4, and amyloid β-peptide was increased in cultures pretreated with CHX at concentrations of 50–500 nM; higher and lower concentrations were ineffective. Neuroprotective concentrations of CHX caused only a moderate (20–40%) reduction in overall protein synthesis, and induced an increase in c-fos, c-jun, and bcl-2 mRNAs and protein levels as determined by reverse transcription–PCR analysis and immunocytochemistry, respectively. At neuroprotective CHX concentrations, levels of c-fos heteronuclear RNA increased in parallel with c-fos mRNA, indicating that CHX acts by inducing transcription. Neuroprotective concentrations of CHX suppressed accumulation of H2O2 induced by FeSO4, suggesting activation of antioxidant pathways. Treatment of cultures with an antisense oligodeoxynucleotide directed against bcl-2 mRNA decreased Bcl-2 protein levels and significantly reduced the neuroprotective action of CHX, suggesting that induction of Bcl-2 expression was mechanistically involved in the neuroprotective actions of CHX. In addition, activity levels of the antioxidant enzymes Cu/ Zn-superoxide dismutase, Mn-superoxide dismutase, and catalase were significantly increased in cultures exposed to neuroprotective levels of CHX. Our data suggest that low concentrations of CHX can promote neuron survival by inducing increased levels of gene products that function in antioxidant pathways, a neuroprotective mechanism similar to that used by neurotrophic factors.
PMCID: PMC2132476  PMID: 9060477
21.  Salubrinal attenuates β-amyloid-induced neuronal death and microglial activation by inhibition of the NF-κB pathway 
Neurobiology of Aging  2011;33(5):1007.e9-1007.e17.
Alzheimer’s disease (AD) is characterized by the deposition of β-amyloid (Aβ) peptides in the brain, inducing neuronal cell death and microglial activation. Endoplasmic reticulum (ER) stress has been proposed to be a mediator of Aβ neurotoxicity. In this study, we test whether salubrinal, an ER stress inhibitor, can protect against Aβ-mediated neurotoxicity. We show in rat primary cortical neurons and mouse microglial BV-2 cells that short-term treatment with salubrinal attenuates Aβ-induced neuronal death and microglial activation. Remarkably, our results show that salubrinal’s neuroprotective effects are not due to inhibition of ER stress. Rather, we demonstrate that salubrinal exerts its effects through the inhibition of IκB kinase (IKK) activation, IκB degradation and the subsequent nuclear factor-kappa B (NF-κB) activation. These results elucidate inhibition of the NF-κB pathway as a new mechanism responsible for the protective effects of salubrinal against Aβ neurotoxicity. This study also suggests that modulation of Aβ-induced NF-κB activation could be a potential therapeutic strategy for AD.
PMCID: PMC3294262  PMID: 22056200
Alzheimer’s disease; Salubrinal; β-amyloid; NF-κB
22.  Mitogen-Activated Protein Kinases and Reactive Oxygen Species: How Can ROS Activate MAPK Pathways? 
Journal of Signal Transduction  2011;2011:792639.
Mitogen-activated protein kinases (MAPKs) are serine-threonine protein kinases that play the major role in signal transduction from the cell surface to the nucleus. MAPKs, which consist of growth factor-regulated extracellular signal-related kinases (ERKs), and the stress-activated MAPKs, c-jun NH2-terminal kinases (JNKs) and p38 MAPKs, are part of a three-kinase signaling module composed of the MAPK, an MAPK kinase (MAP2K) and an MAPK kinase (MAP3K). MAP3Ks phosphorylate MAP2Ks, which in turn activate MAPKs. MAPK phosphatases (MKPs), which recognize the TXY amino acid motif present in MAPKs, dephosphorylate and deactivate MAPKs. MAPK pathways are known to be influenced not only by receptor ligand interactions, but also by different stressors placed on the cell. One type of stress that induces potential activation of MAPK pathways is the oxidative stress caused by reactive oxygen species (ROS). Generally, increased ROS production in a cell leads to the activation of ERKs, JNKs, or p38 MAPKs, but the mechanisms by which ROS can activate these kinases are unclear. Oxidative modifications of MAPK signaling proteins and inactivation and/or degradation of MKPs may provide the plausible mechanisms for activation of MAPK pathways by ROS, which will be reviewed in this paper.
PMCID: PMC3100083  PMID: 21637379
23.  Noradrenaline activation of neurotrophic pathways protects against neuronal amyloid toxicity 
Journal of neurochemistry  2010;113(3):649-660.
Degeneration of locus coeruleus (LC) noradrenergic forebrain projection neurons is an early feature of Alzheimer’s disease (AD). The physiological consequences of this phenomenon are unclear, but observations correlating LC neuron loss with increased AD pathology in LC projection sites suggest that noradrenaline (NA) is neuroprotective. To investigate this hypothesis, we determined that NA protected both hNT human neuronal cultures and rat primary hippocampal neurons from amyloid-β (Aβ1–42 and Aβ25–35) toxicity. The noradrenergic co-transmitter galanin was also effective at preventing Aβ-induced cell death. NA inhibited Aβ25–35-mediated increases in intracellular reactive oxygen species, mitochondrial membrane depolarization, and caspase activation in hNT neurons. NA exerted its neuroprotective effects in these cells by stimulating canonical β1 and β2 adrenergic receptor signaling pathways involving the activation of cAMP response element binding protein (CREB) and the induction of endogenous nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Treatment with functional blocking antibodies for either NGF or BDNF blocked NA’s protective actions against Aβ1–42 and Aβ25–35 toxicity in primary hippocampal and hNT neurons, respectively. Taken together, these data suggest that the neuroprotective effects of noradrenergic LC afferents result from stimulating neurotrophic NGF and BDNF autocrine or paracrine loops via β adrenoceptor activation of the CREB pathway.
PMCID: PMC2913691  PMID: 20132474
24.  The Wnt Receptor Ryk Reduces Neuronal and Cell Survival Capacity by Repressing FOXO Activity During the Early Phases of Mutant Huntingtin Pathogenicity 
PLoS Biology  2014;12(6):e1001895.
A study of Huntington's disease reveals that neurons might fail to cope with maintaining their function during the pre-symptomatic, pathogenic phases of HD, possibly due to the early repression of key longevity-promoting transcription factors by abnormal developmental signaling.
The Wnt receptor Ryk is an evolutionary-conserved protein important during neuronal differentiation through several mechanisms, including γ-secretase cleavage and nuclear translocation of its intracellular domain (Ryk-ICD). Although the Wnt pathway may be neuroprotective, the role of Ryk in neurodegenerative disease remains unknown. We found that Ryk is up-regulated in neurons expressing mutant huntingtin (HTT) in several models of Huntington's disease (HD). Further investigation in Caenorhabditis elegans and mouse striatal cell models of HD provided a model in which the early-stage increase of Ryk promotes neuronal dysfunction by repressing the neuroprotective activity of the longevity-promoting factor FOXO through a noncanonical mechanism that implicates the Ryk-ICD fragment and its binding to the FOXO co-factor β-catenin. The Ryk-ICD fragment suppressed neuroprotection by lin-18/Ryk loss-of-function in expanded-polyQ nematodes, repressed FOXO transcriptional activity, and abolished β-catenin protection of mutant htt striatal cells against cell death vulnerability. Additionally, Ryk-ICD was increased in the nucleus of mutant htt cells, and reducing γ-secretase PS1 levels compensated for the cytotoxicity of full-length Ryk in these cells. These findings reveal that the Ryk-ICD pathway may impair FOXO protective activity in mutant polyglutamine neurons, suggesting that neurons are unable to efficiently maintain function and resist disease from the earliest phases of the pathogenic process in HD.
Author Summary
Neuronal cell decline in neurodegenerative disease can be caused by inherited mutations and involves neuronal dysfunction followed by neuronal death. The ability of neurons to cope with the chronic stress induced by mutant protein expression may determine the course of their decline and eventual demise. Although the pathophysiological importance of these stress responses has been previously shown, very little is known about the signaling networks that regulate neuronal homeostasis during the early presymptomatic—but pathogenic—phases of a neurodegenerative disorder such as Huntington's disease (HD). In particular, it remains unclear whether neuronal differentiation factors regulate stress response pathways during neurodegenerative disease and how this might impact the overall capacity of neurons to cope with stress and maintain their function. Here, we show that the Wnt receptor Ryk, a protein known to be important for neurogenesis, is increased in different animal models of HD, before or during the early phases of the disease process. Interestingly, increased levels of Ryk repress activity of the FOXO proteins—a family of transcription factors that play a role in cell survival/longevity and in neuronal homeostasis and protection. Ryk represses FOXO protective activity, possibly directly, through its intracellular domain, a product of γ-secretase–mediated cleavage previously implicated in the birth of new cortical neurons. This highlights the regulation of HD neuron survival by a Ryk-dependent pathway that is distinct from canonical Wnt/Ryk signaling. From our findings, we postulate that neurons are unable to develop an efficient FOXO-mediated survival response during the very early, pathogenic phases of HD.
PMCID: PMC4068980  PMID: 24960609
25.  Ferulic Acid Attenuates the Injury-Induced Decrease of Protein Phosphatase 2A Subunit B in Ischemic Brain Injury 
PLoS ONE  2013;8(1):e54217.
Ferulic acid provides a neuroprotective effect during cerebral ischemia through its anti-oxidant function. Protein phosphatase 2A (PP2A) is a serine and threonine phosphatase that contributes broadly to normal brain function. This study investigated whether ferulic acid regulates PP2A subunit B in a middle cerebral artery occlusion (MCAO) animal model and glutamate toxicity-induced neuronal cell death.
Methodology/Principal Findings
MCAO was surgically induced to yield permanent cerebral ischemic injury in rats. The rats were treated with either vehicle or ferulic acid (100 mg/kg, i.v.) immediately after MCAO, and cerebral cortex tissues were collected 24 h after MCAO. A proteomics approach, RT-PCR, and Western blot analyses performed to identification of PP2A subunit B expression levels. Ferulic acid significantly reduced the MCAO-induced infarct volume of the cerebral cortex. A proteomics approach elucidated the reduction of PP2A subunit B in MCAO-induced animals, and ferulic acid treatment prevented the injury-induced reduction in PP2A subunit B levels. RT-PCR and Western blot analyses also showed that ferulic acid treatment attenuates the injury-induced decrease in PP2A subunit B levels. Moreover, the number of PP2A subunit B-positive cells was reduced in MCAO-induced animals, and ferulic acid prevented these decreases. In cultured neuronal cells, ferulic acid treatment protected cells against glutamate toxicity and prevented the glutamate-induced decrease in PP2A subunit B.
These results suggest that the maintenance of PP2A subunit B by ferulic acid in ischemic brain injury plays an important role for the neuroprotective function of ferulic acid.
PMCID: PMC3547913  PMID: 23349830

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