Potential Alzheimer's early biomarkers in a transgenic rat model and benefits of diazoxide/dibenzoylmethane co-treatment on spatial memory and AD-pathology.

Alzheimer's disease (AD) is the major form of dementia prevalent in older adults and with a high incidence in females. Identification of early biomarkers is essential for preventive intervention to delay its progression. Furthermore, due to its multifactorial nature, a multi-target approach could be therapeutically beneficial. Our studies included 4- (pre-pathology) and 11-month (mild-pathology) TgF344-AD rats, a transgenic Alzheimer's model that exhibits age-dependent AD progression. We identified two potential early biomarker genes for AD, early growth response 2 (EGR2) and histone 1H2AA (HIST1H2AA), in the hippocampus of 4-month females. Out of 17,168 genes analyzed by RNA sequencing, expression of these two genes was significantly altered in 4-month TgF344-AD rats compared to wild-type littermates. We also evaluated co-treatment with diazoxide (DZ), a potassium channel activator, and dibenzoylmethane (DIB), which inhibits eIF2α-P activity, on TgF344-AD and wild-type rats. DZ/DIB-treatment mitigated spatial memory deficits and buildup of hippocampal Aß plaques and tau PHF in 11-month TgF344-AD rats but had no effect on wild-type littermates. To our knowledge, this preclinical study is the first to report EGR2 and HIST1H2AA as potential AD biomarkers in females, and the benefits of DZ/DIB-treatment in AD. Evaluations across multiple AD-related models is warranted to corroborate our findings.

Repurposing ibudilast to mitigate Alzheimer's disease by targeting inflammation.

Alzheimer's disease is a multifactorial disease that exhibits cognitive deficits, neuronal loss, amyloid plaques, neurofibrillary tangles and neuroinflammation in the brain. Hence, a multi-target drug would improve treatment efficacy. We applied a new multi-scale predictive modelling framework that integrates machine learning with biophysics and systems pharmacology to screen drugs for Alzheimer's disease using patients' tissue samples. Our predictive modelling framework identified ibudilast as a drug with repurposing potential to treat Alzheimer's disease. Ibudilast is a multi-target drug, as it is a phosphodiesterase inhibitor and toll-like receptor 4 (TLR4) antagonist. In addition, we predict that ibudilast inhibits off-target kinases (e.g. IRAK1 and GSG2). In Japan and other Asian countries, ibudilast is approved for treating asthma and stroke due to its anti-inflammatory potential. Based on these previous studies and on our predictions, we tested for the first time the efficacy of ibudilast in Fisher transgenic 344-AD rats. This transgenic rat model is unique as it exhibits hippocampal-dependent spatial learning and memory deficits and Alzheimer's disease pathology, including hippocampal amyloid plaques, tau paired-helical filaments, neuronal loss and microgliosis, in a progressive age-dependent manner that mimics the pathology observed in Alzheimer's disease patients. Following long-term treatment with ibudilast, transgenic rats were evaluated at 11 months of age for spatial memory performance and Alzheimer's disease pathology. We demonstrate that ibudilast-treatment of transgenic rats mitigated hippocampal-dependent spatial memory deficits, as well as hippocampal (hilar subregion) amyloid plaque and tau paired-helical filament load, and microgliosis compared to untreated transgenic rat. Neuronal density analysed across all hippocampal regions was similar in ibudilast-treated transgenic compared to untreated transgenic rats. Interestingly, RNA sequencing analysis of hippocampal tissue showed that ibudilast-treatment affects gene expression levels of the TLR and ubiquitin-proteasome pathways differentially in male and female transgenic rats. Based on the TLR4 signalling pathway, our RNA sequencing data suggest that ibudilast-treatment inhibits IRAK1 activity by increasing expression of its negative regulator IRAK3, and/or by altering TRAF6 and other TLR-related ubiquitin ligase and conjugase levels. Our results support that ibudilast can serve as a repurposed drug that targets multiple pathways including TLR signalling and the ubiquitin/proteasome pathway to reduce cognitive deficits and pathology relevant to Alzheimer's disease.

Histone deacetylase inhibitor RG2833 has therapeutic potential for Alzheimer's disease in females.

Nearly two-thirds of patients with Alzheimer's are women. Identifying therapeutics specific for women is critical to lowering their elevated risk for developing this major cause of adult dementia. Moreover, targeting epigenetic processes that regulate multiple cellular pathways is advantageous given Alzheimer's multifactorial nature. Histone acetylation is an epigenetic process heavily involved in memory consolidation. Its disruption is linked to Alzheimer's. Through our computational studies, we predicted that the investigational drug RG2833 (N-[6-(2-aminoanilino)-6-oxohexyl]-4-methylbenzamide) has repurposing potential for Alzheimer's. RG2833 is a histone deacetylase HDAC1/3 inhibitor that is orally bioavailable and permeates the blood-brain-barrier. We investigated the RG2833 therapeutic potential in TgF344-AD rats, which are a model of Alzheimer's that exhibits age-dependent progression, thus mimicking this aspect of Alzheimer's patients that is difficult to establish in animal models. We investigated the RG2833 effects on cognitive performance, gene expression, and AD-like pathology in 11-month TgF344-AD female and male rats. A total of 89 rats were used: wild type n = 45 (17 females, 28 males), and TgF344-AD n = 44 (24 females, 20 males)] across multiple cohorts. No obvious toxicity was detected in the TgF344-AD rats up to 6 months of RG2833-treatment starting at 5 months of age administering the drug in rodent chow at ∼30mg/kg of body weight. We started treatment early in the course of pathology when therapeutic intervention is predicted to be more effective than in later stages of the disease. The drug-treatment significantly mitigated hippocampal-dependent spatial memory deficits in 11-month TgF344-AD females but not in males, compared to wild type littermates. This female sex-specific drug effect has not been previously reported. RG2833-treatment failed to ameliorate amyloid beta accumulation and microgliosis in female and male TgF344-AD rats. However, RNAseq analysis of hippocampal tissue from TgF344-AD rats showed that drug-treatment in females upregulated the expression of immediate early genes, such as Arc, Egr1 and c-Fos, and other genes involved in synaptic plasticity and memory consolidation. Remarkably, out of 17,168 genes analyzed for each sex, no significant changes in gene expression were detected in males at P < 0.05, false discovery rate < 0.05, and fold-change ≥ 1.5. Our data suggest that histone modifying therapeutics such as RG2833 improve cognitive behavior by modulating the expression of immediate early, neuroprotective and synaptic plasticity genes. Our preclinical study supports that RG2833 has therapeutic potential specifically for female Alzheimer's patients. RG2833 evaluations using other AD-related models is necessary to confirm our findings.

Females exhibit higher GluA2 levels and outperform males in active place avoidance despite increased amyloid plaques in TgF344-Alzheimer's rats.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is most prevalent in females. While estrogen provides neuroprotection in females, sex mediated differences in the development of AD pathology are not fully elucidated. Therefore, comparing events between sexes in early-stage AD pathology may reveal more effective therapeutic targets of intervention. To address sex differences, we analyzed early-stage 9-month male and female TgF344-AD (Tg-AD) rats, an AD model carrying the APPswe and Presenilin 1 (PS1ΔE9) mutations that develops progressive age-dependent AD pathology similar to humans. Tg-AD females significantly outperformed Tg-AD males in the active place avoidance (aPAT) test that assesses hippocampal-dependent spatial learning and memory. However, comparisons between Tg-AD male or female rats and their WT counterparts showed significant deficits for female but not male rats. Nevertheless, Tg-AD females experienced significantly less hippocampal neuronal loss with higher GluA2 subunit levels than Tg-AD males. Unexpectedly, Tg-AD females displayed higher levels of hippocampal amyloid plaques than Tg-AD males. Thus, we propose that GluA2 may provide a neuroprotective function for Tg-AD females in our rat model by mitigating cognitive impairment independently of amyloid plaques. Elucidating this protective mechanism in AD could lead to new targets for early intervention.

Timapiprant, a prostaglandin D2 receptor antagonist, ameliorates pathology in a rat Alzheimer's model.

We investigated the relevance of the prostaglandin D2 pathway in Alzheimer's disease, because prostaglandin D2 is a major prostaglandin in the brain. Thus, its contribution to Alzheimer's disease merits attention, given the known impact of the prostaglandin E2 pathway in Alzheimer's disease. We used the TgF344-AD transgenic rat model because it exhibits age-dependent and progressive Alzheimer's disease pathology. Prostaglandin D2 levels in hippocampi of TgF344-AD and wild-type littermates were significantly higher than prostaglandin E2. Prostaglandin D2 signals through DP1 and DP2 receptors. Microglial DP1 receptors were more abundant and neuronal DP2 receptors were fewer in TgF344-AD than in wild-type rats. Expression of the major brain prostaglandin D2 synthase (lipocalin-type PGDS) was the highest among 33 genes involved in the prostaglandin D2 and prostaglandin E2 pathways. We treated a subset of rats (wild-type and TgF344-AD males) with timapiprant, a potent highly selective DP2 antagonist in development for allergic inflammation treatment. Timapiprant significantly mitigated Alzheimer's disease pathology and cognitive deficits in TgF344-AD males. Thus, selective DP2 antagonists have potential as therapeutics to treat Alzheimer's disease.

PACAP27 mitigates an age-dependent hippocampal vulnerability to PGJ2-induced spatial learning deficits and neuroinflammation in mice.

BACKGROUND: Inflammation in the brain is mediated by the cyclooxygenase pathway, which leads to the production of prostaglandins. Prostaglandin (PG) D2, the most abundant PG in the brain, increases under pathological conditions and is spontaneously metabolized to PGJ2. PGJ2 is highly neurotoxic, with the potential to transition neuroinflammation into a chronic state and contribute to neurodegeneration as seen in many neurological diseases. Conversely, PACAP27 is a lipophilic peptide that raises intracellular cAMP and is an anti-inflammatory agent. The aim of our study was to investigate the therapeutic potential of PACAP27 to counter the behavioral and neurotoxic effects of PGJ2 observed in aged subjects. METHODS: PGJ2 was injected bilaterally into the hippocampal CA1 region of 53-week-old and 12-week-old C57BL/6N male mice, once per week over 3 weeks (three total infusions) and included co-infusions of PACAP27 within respective treatment groups. Our behavioral assessments looked at spatial learning and memory performance on the 8-arm radial maze, followed by histological analyses of fixed hippocampal tissue using Fluoro-Jade C and fluorescent immunohistochemistry focused on IBA-1 microglia. RESULTS: Aged mice treated with PGJ2 exhibited spatial learning and long-term memory deficits, as well as neurodegeneration in CA3 pyramidal neurons. Aged mice that received co-infusions of PACAP27 exhibited remediated learning and memory performance and decreased neurodegeneration in CA3 pyramidal neurons. Moreover, microglial activation in the CA3 region was also reduced in aged mice cotreated with PACAP27. CONCLUSIONS: Our data show that PGJ2 can produce a retrograde spread of damage not observed in PGJ2-treated young mice, leading to age-dependent neurodegeneration of hippocampal neurons producing learning and memory deficits. PACAP27 can remediate the behavioral and neurodegenerative effects that PGJ2 produces in aged subjects. Targeting specific neurotoxic prostaglandins, such as PGJ2, offers great promise as a new therapeutic strategy downstream of cyclooxygenases, to combat the neuronal deficits induced by chronic inflammation.

Mitochondrial and calcium perturbations in rat CNS neurons induce calpain-cleavage of Parkin: Phosphatase inhibition stabilizes pSer65Parkin reducing its calpain-cleavage.

Mitochondrial impairment and calcium (Ca++) dyshomeostasis are associated with Parkinson's disease (PD). When intracellular ATP levels are lowered, Ca++-ATPase pumps are impaired causing cytoplasmic Ca++ to be elevated and calpain activation. Little is known about the effect of calpain activation on Parkin integrity. To address this gap, we examined the effects of mitochondrial inhibitors [oligomycin (Oligo), antimycin and rotenone] on endogenous Parkin integrity in rat midbrain and cerebral cortical cultures. All drugs induced calpain-cleavage of Parkin to ~36.9/43.6 kDa fragments. In contrast, treatment with the proinflammatory prostaglandin J2 (PGJ2) and the proteasome inhibitor epoxomicin induced caspase-cleavage of Parkin to fragments of a different size, previously shown by others to be triggered by apoptosis. Calpain-cleaved Parkin was enriched in neuronal mitochondrial fractions. Pre-treatment with the phosphatase inhibitor okadaic acid prior to Oligo-treatment, stabilized full-length Parkin phosphorylated at Ser65, and reduced calpain-cleavage of Parkin. Treatment with the Ca++ ionophore A23187, which facilitates Ca++ transport across the plasma membrane, mimicked the effect of Oligo by inducing calpain-cleavage of Parkin. Removing extracellular Ca++ from the media prevented oligomycin- and ionophore-induced calpain-cleavage of Parkin. Computational analysis predicted that calpain-cleavage of Parkin liberates its UbL domain. The phosphagen cyclocreatine moderately mitigated Parkin cleavage by calpain. Moreover, the pituitary adenylate cyclase activating peptide (PACAP27), which stimulates cAMP production, prevented caspase but not calpain-cleavage of Parkin. Overall, our data support a link between Parkin phosphorylation and its cleavage by calpain. This mechanism reflects the impact of mitochondrial impairment and Ca++-dyshomeostasis on Parkin integrity and could influence PD pathogenesis.

Prostaglandin J2 promotes O-GlcNAcylation raising APP processing by α- and ß-secretases: relevance to Alzheimer's disease.

Regulation of the amyloid precursor protein (APP) processing by α- and ß-secretases is of special interest to Alzheimer's disease (AD), as these proteases prevent or mediate amyloid beta formation, respectively. Neuroinflammation is also implicated in AD. Our data demonstrate that the endogenous mediator of inflammation prostaglandin J2 (PGJ2) promotes full-length APP (FL-APP) processing by α- and ß-secretases. The decrease in FL-APP was independent of proteasomal, lysosomal, calpain, caspase, and γ-secretase activities. Moreover, PGJ2-treatment promoted cleavage of secreted APP, specifically sAPPα and sAPPß, generated by α and ß-secretase, respectively. Notably, PGJ2-treatment induced caspase-dependent cleavage of sAPPß. Mechanistically, PGJ2-treatment selectively diminished mature (O- and N-glycosylated) but not immature (N-glycosylated only) FL-APP. PGJ2-treatment also increased the overall levels of protein O-GlcNAcylation, which occurs within the nucleocytoplasmic compartment. It is known that APP undergoes O-GlcNAcylation and that the latter protects proteins from proteasomal degradation. Our results suggest that by increasing protein O-GlcNAcylation levels, PGJ2 renders mature APP less prone to proteasomal degradation, thus shunting APP toward processing by α- and ß-secretases.

CCR 20th anniversary commentary: a chimeric antibody, C225, inhibits EGFR activation and tumor growth.

Murine mAb 225 was effective against the EGFR tyrosine kinase and inhibited tumor growth in preclinical studies. A phase I trial showed safety, tumor localization, and satisfactory pharmacokinetics. Human:murine chimeric C225 retained biologic activity, which was essential for the conduct of subsequent combination therapy trials and eventual regulatory approval.

Neuroinflammation and J2 prostaglandins: linking impairment of the ubiquitin-proteasome pathway and mitochondria to neurodegeneration.

The immune response of the CNS is a defense mechanism activated upon injury to initiate repair mechanisms while chronic over-activation of the CNS immune system (termed neuroinflammation) may exacerbate injury. The latter is implicated in a variety of neurological and neurodegenerative disorders such as Alzheimer and Parkinson diseases, amyotrophic lateral sclerosis, multiple sclerosis, traumatic brain injury, HIV dementia, and prion diseases. Cyclooxygenases (COX-1 and COX-2), which are key enzymes in the conversion of arachidonic acid into bioactive prostanoids, play a central role in the inflammatory cascade. J2 prostaglandins are endogenous toxic products of cyclooxygenases, and because their levels are significantly increased upon brain injury, they are actively involved in neuronal dysfunction induced by pro-inflammatory stimuli. In this review, we highlight the mechanisms by which J2 prostaglandins (1) exert their actions, (2) potentially contribute to the transition from acute to chronic inflammation and to the spreading of neuropathology, (3) disturb the ubiquitin-proteasome pathway and mitochondrial function, and (4) contribute to neurodegenerative disorders such as Alzheimer and Parkinson diseases, and amyotrophic lateral sclerosis, as well as stroke, traumatic brain injury (TBI), and demyelination in Krabbe disease. We conclude by discussing the therapeutic potential of targeting the J2 prostaglandin pathway to prevent/delay neurodegeneration associated with neuroinflammation. In this context, we suggest a shift from the traditional view that cyclooxygenases are the most appropriate targets to treat neuroinflammation, to the notion that J2 prostaglandin pathways and other neurotoxic prostaglandins downstream from cyclooxygenases, would offer significant benefits as more effective therapeutic targets to treat chronic neurodegenerative diseases, while minimizing adverse side effects.

Signaling through the vascular endothelial growth factor receptor VEGFR-2 protects hippocampal neurons from mitochondrial dysfunction and oxidative stress.

Vascular endothelial growth factor VEGF (VEGF-A or VEGF165) is a potent angiogenic factor that also signals neuroprotection through activation of its cognate receptor VEGFR-2. In this capacity, VEGF signaling can rescue neurons from the damage induced by stressful stimuli many of which elicit oxidative stress. However, the regulatory role that VEGFR-2 plays in providing neuroprotection remains elusive. Therefore, we investigated the effects of VEGFR-2 inhibition on primary cultures of mature hippocampal neurons undergoing nutritional stress. We found that neurons cultured under nutritional stress had increased expression of VEGF and its receptors, VEGFR-1, VEGFR-2, and NP-1, as well as enhanced levels of VEGFR-2 phosphorylation. These neurons also showed increased activation of the prosurvival pathways for MEK/ERK1/2 and PI3K/Akt, enhanced phosphorylation (inactivation) of the proapoptotic BAD, and higher levels of the antiapoptotic protein Bcl-xL, all of which were augmented by treatments with exogenous VEGF and blocked by VEGFR-2 inhibition. The blockade of VEGFR-2 function also elicited a cytotoxicity that was accompanied by caspase-3 activation, induction of hemeoxygenase-1 (HO-1), oxidative stress, and a collapse in the mitochondrial membrane potential (ΔΨ(m)). Knockdown of VEGFR-2 by siRNA generated a similar pattern of redox change and mitochondrial impairment. Pretreatments with VEGF, VEGF-B, or the antioxidant N-acetylcysteine (NAC) rescued SU1498 or siRNA-treated neurons from the mitochondrial dysfunction and oxidative stress induced by VEGFR-2 inhibition in a timely fashion. These findings suggested that VEGF or VEGF-B can provide neuroprotection by signaling through an alternate VEGF receptor. Together, our findings suggest that VEGF signaling through VEGFR-2 plays a critical regulatory role in protecting stressed hippocampal neurons from the damaging effects of an oxidative insult. These findings also implicate VEGFR-1 or NP-1 as compensatory receptors that mediate neuroprotection when VEGFR-2 function is blocked.

Okadaic acid induces Akt hyperphosphorylation and an oxidative stress-mediated cell death in serum starved SK-N-SH human neuroblastoma cells that are augmented by rapamycin.

Using a neuronal model of serum starved SK-N-SH neuroblastoma cells, we showed previously that the phosphorylation of Akt and the mTOR substrates S6K and S6 through the vascular endothelial growth factor receptor VEGFR2 was enhanced by treatments with the phosphatase PP2A inhibitor okadaic acid (OA). These findings suggested that PP2A inhibition uncouples the regulation of Akt signaling by mTOR and affects cell survival. We therefore examined the effects of mTOR inhibition on Akt phosphorylation at sites threonine 308 (T308) and serine 473 (S473) and survival in OA treated cells. OA induced a loss in cell viability, the accumulation of hyperactivated Akt as monomeric and ubiquitinated forms and an increase in the total levels of ubiquitinated proteins. These events were exacerbated by treatments with an allosteric (rapamycin) but not an active-site inhibitor (PP242) of mTOR. Notably, rapamycin augmented the OA-induced hyperphosphorylation of Akt by suppressing a negative feedback loop of Akt activation through VEGFR2 and its downstream target phosphatidylinositol 3-kinase (PI3K). Treatments with the antioxidant N-acetlycysteine but not the pan caspase inhibitor Z-VAD-FMK promoted survival. Unlike reports that rapamycin promotes survival through increased Akt activation, these findings show that rapamycin-induced hyperphosphorylation of Akt fails to rescue our neuronal model from an oxidative stress-induced and caspase-independent cell death mediated by PP2A inhibition. Moreover, the exacerbation of OA-induced events by rapamycin suggests that mTOR and PP2A work in concert to regulate cell survival, activated Akt and the levels of ubiquitinated proteins.

Crosstalk between VEGFR2 and muscarinic receptors regulates the mTOR pathway in serum starved SK-N-SH human neuroblastoma cells.

Muscarinic acetylcholine receptors (mAchRs) are guanosine nucleotide-binding protein (G protein) coupled receptors that crosstalk with receptor tyrosine kinases (RTKs) to signal mitogenic pathways. In particular, mAchRs are known to couple with RTKs for several growth factors to activate the mammalian target of rapamycin (mTOR)/Akt pathway, a regulator of protein synthesis. The RTK for the vascular endothelial growth factor (VEGF), VEGFR2, can signal protein synthesis but whether it cooperates with mAchRs to mediate mTOR activation has not been demonstrated. Using serum starved SK-N-SH neuroblastoma cells, we show that the muscarinic receptor agonists carbachol and pilocarpine enhance the activation of the mTOR substrate p70 S6 Kinase (S6K) and its target ribosomal protein S6 (S6) in a VEGFR2 dependent manner. Treatments with carbachol increased VEGFR2 phosphorylation, suggesting that mAchRs stimulate VEGFR2 transactivation to enhance mTOR signaling. Inhibitor studies revealed that phosphatidylinositol 3 kinase resides upstream from S6K, S6 and Akt phosphorylation while protein kinase C (PKC) functions in an opposing fashion by positively regulating S6K and S6 phosphorylation and suppressing Akt activation. Treatments with the phosphatase inhibitors sodium orthovanadate and okadaic acid increase S6, Akt and to a lesser extent S6K phosphorylation, indicating that tyrosine and serine/threonine dephosphorylation also regulates their activity. However, okadaic acid elicited a far greater increase in phosphorylation, implicating phosphatase 2A as a critical determinant of their function. Finally, pilocarpine but not carbachol induced a time and dose dependent cell death that was associated with caspase activation and oxidative stress but independent of S6K and S6 activation through VEGFR2. Accordingly, our findings suggest that mAchRs crosstalk with VEGFR2 to enhance mTOR activity but signal divergent effects on survival through alternate mechanisms.

Persistent mitochondrial dysfunction and oxidative stress hinder neuronal cell recovery from reversible proteasome inhibition.

Oxidative stress, proteasome impairment and mitochondrial dysfunction are implicated as contributors to ageing and neurodegeneration. Using mouse neuronal cells, we showed previously that the reversible proteasome inhibitor, [N-benzyloxycarbonyl-Ile-Glu (O-t-bytul)-Ala-leucinal; (PSI)] induced excessive reactive oxygen species (ROS) that mediated mitochondrial damage and a caspase-independent cell death. Herein, we examined whether this insult persists in neuronal cells recovering from inhibitor removal over time. Recovery from proteasome inhibition showed a time and dose-dependent cell death that was accompanied by ROS overproduction, caspase activation and mitochondrial membrane permeabilization with the subcellular relocalizations of the proapoptotic proteins, Bax, cytochrome c and the apoptosis inducing factor (AIF). Caspase inhibition failed to promote survival indicating that cell death was caspase-independent. Treatments with the antioxidant N-acetyl-cysteine (NAC) were needed to promote survival in cell recovering from mild proteasome inhibition while overexpression of the antiapoptotic protein Bcl-xL together with NAC attenuated cell death during recovery from potent inhibition. Whereas inhibitor removal increased proteasome function, cells recovering from potent proteasome inhibition showed excessive levels of ubiquitinated proteins that required the presence of NAC for their removal. Collectively, these results suggest that the oxidative stress and mitochondrial inhibition induced by proteasome inhibition persists to influence neuronal cell survival when proteasome function is restored.

p38 MAPK as a negative regulator of VEGF/VEGFR2 signaling pathway in serum deprived human SK-N-SH neuroblastoma cells.

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.

The VEGFR2 and PKA pathways converge at MEK/ERK1/2 to promote survival in serum deprived neuronal cells.

Identifying prosurvival mechanisms in stressed neuronal cells would provide protective strategies to hinder neurodegeneration. Recent evidence shows that vascular endothelial growth factor (VEGF), a well-established mitogen in endothelial cells, can mediate neuroprotection against damaging insults through the activation of its cognate receptor VEGFR2. In addition, growth factor receptor signaling pathways have been shown to crosstalk with cAMP-dependent Protein Kinase A (PKA) to protect neuronal cells from harmful stimuli. Whether a relationship exists between VEGFR2 and PKA in mediating neuroprotection under stressful conditions is unknown. Using SK-N-SH neuronal cells as a model system, we show that serum deprivation induces an upregulation in VEGF and VEGFR2 that concomitantly serves as a prosurvival signaling pathway. Inhibitor studies revealed that PKA functioned concurrently with VEGFR2 pathway to signal the activation of the extracellular signal-regulated protein kinases (ERK1/2) as protection against caspase-3/7 activation and a subsequent cell death. The loss in cell viability induced by VEGFR2 and PKA inhibition was prevented by caspase inhibition or overexpression of ERK1. Overexpression of the antiapoptotic protein Bcl-xL also promoted survival when VEGFR2 function was blocked. However, the protection elicited by all three treatments were prevented by the inclusion of a selective inhibitor of mitogen-activated protein kinase kinase (MEK), the upstream kinase that activates ERK1/2. Taken together, these findings suggested that PKA and VEGFR2 converge at the MEK/ERK1/2 pathway to protect serum starved neuronal cells from a caspase-dependent cell death.

Reactive oxygen species induced by proteasome inhibition in neuronal cells mediate mitochondrial dysfunction and a caspase-independent cell death.

While increasing evidence shows that proteasome inhibition triggers oxidative damage, mitochondrial dysfunction and death in neuronal cells, the regulatory relationship among these events is unclear. Using mouse neuronal cells we show that the cytotoxicity induced by mild (0.25 microM) and potent (5.0 microM) doses of the proteasome inhibitor, N-Benzyloxycarbonyl-Ile-Glu (O-t-butyl)-Ala-leucinal, (PSI) involved a dose-dependent increase in caspase activation, overproduction of reactive oxygen species (ROS) and a mitochondrial dysfunction manifested by the translocation of the proapoptotic protein, Bax, from the cytoplasm to the mitochondria, membrane depolarization and the release of cytochrome c and the apoptosis inducing factor (AIF) from mitochondria to the cytoplasm and nucleus, respectively. Whereas caspase or Bax inhibition failed to prevent mitochondrial membrane depolarization and neuronal cell death, pretreatments with the antioxidant N-acetyl-L-cysteine (NAC) or overexpression of the antiapoptotic protein Bcl-xL abrogated these events in cells exposed to mild levels of PSI. These findings implicated ROS as a mediator of PSI-induced cytotoxicity. However, depletions in glutathione and Bcl-xL with potent proteasome inhibition exacerbated this response whereupon survival required the cooperative protection of NAC with Bcl-xL overexpression. Collectively, ROS induced by proteasome inhibition mediates a mitochondrial dysfunction in neuronal cells that culminates in death through caspase- and Bax-independent mechanisms.

Vocal features of conversational sarcasm: a comparison of methods.

This study investigated vocal cues that differentiate sarcastic utterances from non-sarcastic utterances. Utterances were drawn from videotapes of participant interviews and arranged on a master tape for analysis. Utterances that were identified as sarcastic by speakers and recognized as sarcastic by listeners were randomly arranged with utterances identified and recognized as non-sarcastic by the same participants. Both sarcastic and non-sarcastic utterances were analyzed by two methods-acoustic analysis and perceptual coding. The acoustic analysis proved slightly more successful than the perceptual coding in discriminating between sarcastic and non-sarcastic utterances. The acoustic analysis indicated that fundamental frequency, frequency range, length of utterance, and total amount of sound significantly discriminated sarcastic from non-sarcastic utterances. The perceptual coding method revealed that pitch range, length of utterance, and total amount of sound significantly discriminated sarcastic from non-sarcastic utterances. Moderate correlations were found between the acoustic and perceptual variables.

Myc stabilization in response to estrogen and phospholipase D in MCF-7 breast cancer cells.

Estrogen, which has been strongly implicated in breast cancer, suppresses apoptosis in estrogen receptor (ER) positive MCF-7 breast cancer cells. Phospholipase D (PLD), which is commonly elevated in ER negative breast cancer cells, also suppresses apoptosis. Survival signals generated by both estrogen and PLD are dependent upon elevated Myc expression. We report here that estrogen- and PLD-induced increases in Myc expression are due to reduced turnover of Myc protein. Estrogen and PLD suppressed phosphorylation of Myc at Thr58--a site that targets Myc for degradation by the proteasome. The data provide a mechanism for elevated Myc expression in hormone-dependent and hormone-independent breast cancer.

A single amino acid substitution in a proteasome subunit triggers aggregation of ubiquitinated proteins in stressed neuronal cells.

Accumulation of ubiquitinated proteins in inclusions is common to various neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis, although it occurs in selective neurons in each disease. The mechanisms generating such abnormal aggregates and their role in neurodegeneration remain unclear. Inclusions appear in familial and non-familial cases of neurodegenerative disorders, suggesting that factors other than particular mutations contribute to protein accumulation and aggregation. Proteasome impairment triggered by aging or conditions such as oxidative stress may contribute to protein accumulation and aggregation in neurodegeneration. To test this hypothesis in mouse neuronal cells, we overexpressed a 20S proteasome beta5 subunit with an active site mutation. The N-terminal threonine to alanine substitution resulted in impairment of the chymotrypsin-like activity, which is a rate-limiting step in protein degradation by the proteasome. The Thr1Ala mutation was not lethal under homeostatic conditions. However, this single amino acid substitution significantly hypersensitized the cells to oxidative stress, triggering not only the accumulation and aggregation of ubiquitinated proteins, including synuclein, but also cell death. Our results demonstrate that this genetic manipulation of proteasome activity involving a single amino acid substitution causes the formation of protein aggregates in stressed neuronal cells independently of the occurrence of mutations in other cellular proteins. These results support the notion that proteasome disruption may be central to the development of familial as well as sporadic cases of neurodegeneration.