Caspase-3 Activation In Neurons Research Articles

Natural Sources, Pharmacological Properties, and Health Benefits of Daucosterol: Versatility of Actions

Daucosterol is a saponin present in various natural sources, including medicinal plant families. This secondary metabolite is produced at different contents depending on species, extraction techniques, and plant parts used. Currently, daucosterol has been tested and explored for its various biological activities. The results reveal potential pharmacological properties such as antioxidant, antidiabetic, hypolipidemic, anti-inflammatory, immunomodulatory, neuroprotective, and anticancer. Indeed, daucosterol possesses important anticancer effects in many signaling pathways, such as an increase in pro-apoptotic proteins Bax and Bcl2, a decrease in the Bcl-2/Bax ratio, upregulation of the phosphatase and tensin homolog (PTEN) gene, inhibition of the PI3K/Akt pathway, and distortion of cell-cycle progression and tumor cell evolution. Its neuroprotective effect is via decreased caspase-3 activation in neurons and during simulated reperfusion (OGD/R), increased IGF1 protein expression (decreasing the downregulation of p-AKT3 and p-GSK-3b4), and activation of the AKT5 signaling pathway. At the same time, daucosterol inhibits key glucose metabolism enzymes to keep blood sugar levels within normal ranges. Therefore, this review describes the principal research on the pharmacological activities of daucosterol and the mechanisms of action underlying some of these effects. Moreover, further investigation of pharmacodynamics, pharmacokinetics, and toxicology are suggested.

MERS-CoV infection causes brain damage in human DPP4-transgenic mice through complement-mediated inflammation.

The highly pathogenic Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a severe respiratory virus. Recent reports indicate additional central nervous system (CNS) involvement. In this study, human DPP4 transgenic mice were infected with MERS-CoV, and viral antigens were first detected in the midbrain-hindbrain 4 days post-infection, suggesting the virus may enter the brainstem via peripheral nerves. Neurons and astrocytes throughout the brain were infected, followed by damage of the blood brain barrier (BBB), as well as microglial activation and inflammatory cell infiltration, which may be caused by complement activation based on the observation of deposition of complement activation product C3 and high expression of C3a receptor (C3aR) and C5a receptor (C5aR1) in neurons and glial cells. It may be concluded that these effects were mediated by complement activation in the brain, because of their reduction resulted from the treatment with mouse C5aR1-specific mAb. Such mAb significantly reduced nucleoprotein expression, suppressed microglial activation and decreased activation of caspase-3 in neurons and p38 phosphorylation in the brain. Collectively, these results suggest that MERS-CoV infection of CNS triggers complement activation, leading to inflammation-mediated damage of brain tissue, and regulating of complement activation could be a promising intervention and adjunctive treatment for CNS injury by MERS-CoV and other coronaviruses.

Rescue from Stx2-Producing E.coli-Associated Encephalopathy by Intravenous Injection of Muse Cells in NOD-SCID Mice.

Shiga toxin-producing Escherichia coli (STEC) causes hemorrhagic colitis, hemolytic uremic syndrome, and acute encephalopathies that may lead to sudden death or severe neurologic sequelae. Current treatments, including immunoglobulin G (IgG) immunoadsorption, plasma exchange, steroid pulse therapy, and the monoclonal antibody eculizumab, have limited effects against the severe neurologic sequelae. Multilineage-differentiating stress-enduring (Muse) cells are endogenous reparative non-tumorigenic stem cells that naturally reside in the body and are currently under clinical trials for regenerative medicine. When administered intravenously, Musecells accumulate to the damaged tissue, where they exert anti-inflammatory, anti-apoptotic, anti-fibrotic, and immunomodulatory effects, and replace damaged cells by differentiating into tissue-constituent cells. Here, severely immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice orally inoculated with 9 × 109 colony-forming units of STEC O111 and treated 48 h later with intravenous injection of 5 × 104 Muse cells exhibited 100% survival and no severe after-effects of infection. Suppression of granulocyte-colony-stimulating factor (G-CSF) by RNAi abolished the beneficial effects of Muse cells, leading to a 40% death and significant body weight loss, suggesting the involvement of G-CSF in the beneficial effects of Muse cells in STEC-infected mice. Thus, intravenous administration of Muse cells could be a candidate therapeutic approach for preventing fatal encephalopathy after STEC infection.

Inhibition of Serine Palmitoyltransferase by a Small Organic Molecule Promotes Neuronal Survival after Astrocyte Amyloid Beta 1-42 Injury.

Alzheimer's disease (AD) is a slow-progressing disease of the brain characterized by symptoms such as impairment of memory and other cognitive functions. AD is associated with an inflammatory process that involves astrocytes and microglial cells, among other components. Astrocytes are the most abundant type of glial cells in the central nervous system (CNS). They are involved in inducing neuroinflammation. The present study uses astrocyte-neuron cocultures to investigate howARN14494, a serine palmitoyltransferase (SPT) inhibitor, affects the CNS in terms of anti-inflammation and neuroprotection. SPT is the first rate-limiting enzyme in the de novo ceramide synthesis pathway. Consistent evidence suggests that ceramide is increased in AD brain patients. After β-amyloid 1-42 injury in an in vitro model of AD, ARN14494 inhibits SPT activity and the synthesis of long-chain ceramides and dihydroceramides that are involved in AD progression. In mouse primary cortical astrocytes, ARN14494 prevents the synthesis of proinflammatory cytokines TNFα and IL1β, growth factor TGFβ1, and oxidative stress-related enzymes iNOS and COX2. ARN14494 also exerts neuroprotective properties in primary cortical neurons. ARN14494 decreases neuronal death and caspase-3 activation in neurons, when the neuroinflammation is attenuated in astrocytes. These findings suggest that ARN14494 protects neurons from β-amyloid 1-42 induced neurotoxicity through a variety of mechanisms, including antioxidation, antiapoptosis, and anti-inflammation. SPT inhibition could therefore be a safe therapeutic strategy for ameliorating the pathology of Alzheimer's disease.

Caspase-dependent degradation of MDMx/MDM4 cell cycle regulatory protein in amyloid β-induced neuronal damage

MDMx/MDM4 is a negative regulator of the p53 tumor suppressor protein and is necessary for survival in dividing cells. MDMx is also expressed in postmitotic neurons, with prosurvival roles that are independent of its extensively described roles in carcinogenesis. We and others have shown a role for MDMx loss in neuronal death in vitro and in vivo in several neurodegenerative diseases. Further, we have recently shown that MDMx is targeted for proteolytic degradation by calcium-dependent proteases, calpains, in neurons in vitro, and that MDMx overexpression provided partial neuroprotection in a model of HIV-associated neurodegeneration. Here, we assessed whether amyloid β (Aβ)-induced MDMx degradation occurred in Alzheimer’s Disease (AD) models. Our data shows an age-dependent reduction in MDMx levels in cholinergic neurons within the cortex of adult mice expressing the swedish mutant of the amyloid precursor protein, APP in the Tg2576 murine model of AD. In vitro, Aβ treatment of primary cortical neurons led to the caspase-dependent MDMx degradation. Our findings suggest that MDMx degradation associated with neuronal death occurs via caspase activation in neurons, and that the progressive loss of MDMx protein represents a potential mechanism of Aβ-induced neuronal death during disease progression in AD.

Different effects of anesthetic isoflurane on caspase-3 activation and cytosol cytochrome c levels between mice neural progenitor cells and neurons

Commonly used anesthetic isoflurane has been reported to promote Alzheimer’s disease (AD) neuropathogenesis by inducing caspase-3 activation. However, the up-stream mechanisms of isoflurane’s effects remain largely to be determined. Specifically, there is a lack of a good model/system to elucidate the underlying mechanism of the isoflurane-induced caspase-3 activation. We therefore set out to assess and compare the effects of isoflurane on caspase-3 activation in neural progenitor cells (NPCs) and in primary neurons from wild-type (WT) and AD transgenic (Tg) mice. The NPCs and neurons were obtained, cultured and then treated with either 2% isoflurane or under control condition for 6 h. The NPCs or neurons were harvested at the end of the treatment and were subjected to Western blot analysis. Here we showed for the first time that the isoflurane treatment induced caspase-3 activation in neurons, but not in NPCs, from either WT or AD Tg mice. Consistently, the isoflurane treatment increased cytosol levels of cytochrome c, a potential up-stream mechanism of isoflurane-induced caspase-3 activation in the mice neurons, but not NPCs. Finally, the isoflurane treatment induced a greater casapse-3 activation in the neurons, but not the NPCs, from AD Tg mice as compared to the WT mice. These data demonstrated that investigation and comparison of isoflurane’s effects between mice NPCs and neurons would serve as a model/system to determine the underlying mechanism by which isoflurane induces caspase-3 activation. These findings would promote more research to investigate the effects of anesthetics on AD neuropathogenesis and the underlying mechanisms.

Mitochondrial Permeability Transition Pore Inhibitors Prevent Ethanol-Induced Neuronal Death in Mice

Ethanol induces brain injury by a mechanism that remains partly unknown. Mitochondria play a key role in cell death processes, notably through the opening of the permeability transition pore (PTP). Here, we tested the effect of ethanol and PTP inhibitors on mitochondrial physiology and cell viability both in vitro and in vivo. Direct addition of ethanol up to 100 mM on isolated mouse brain mitochondria slightly decreased oxygen consumption but did not affect PTP regulation. In comparison, when isolated from ethanol-treated (two doses of 2 g/kg, 2 h apart) 7-day-old mouse pups, brain mitochondria displayed a transient decrease in oxygen consumption but no change in PTP regulation or H2O2 production. Conversely, exposure of primary cultured astrocytes and neurons to 20 mM ethanol for 3 days led to a transient PTP opening in astrocytes without affecting cell viability and to a permanent PTP opening in 10 to 20% neurons with the same percentage of cell death. Ethanol-treated mouse pups displayed a widespread caspase-3 activation in neurons but not in astrocytes and dramatic behavioral alterations. Interestingly, two different PTP inhibitors (namely, cyclosporin A and nortriptyline) prevented both ethanol-induced neuronal death in vivo and ethanol-induced behavioral modifications. We conclude that PTP opening is involved in ethanol-induced neurotoxicity in the mouse.

Caspase-3 activation as a bifurcation point between plasticity and cell death

Death-mediating proteases such as caspases and caspase-3 in particular, have been implicated in neurodegenerative processes, aging and Alzheimer's disease. However, emerging evidence suggests that in addition to their classical role in cell death, caspases play a key role in modulating synaptic function. It is remarkable that active caspases-3, which can trigger widespread damage and degeneration, aggregates in structures as delicate as synapses and persists in neurons without causing acute cell death. Here, we evaluate this dichotomy, and discuss the hypothesis that caspase-3 may be a bifurcation point in cellular signaling, able to orient the neuronal response to stress down either pathological/apoptotic pathways or towards physiological cellular remodeling. We propose that temporal, spatial and other regulators of caspase activity are key determinants of the ultimate effect of caspase-3 activation in neurons. This concept has implications for differential roles of caspase-3 activation across the lifespan. Specifically, we propose that limited caspase-3 activation is critical for synaptic function in the healthy adult brain while chronic activation is involved in degenerative processes in the aging brain.

Activation of microglial cells via protease-activated receptor 2 mediates neuronal cell death in cultured rat primary neuron

The role of protease-activated receptor (PARs) in the regulation of microglial activation process is increasingly evident. In the present study, we have investigated the role of PAR-2, which can be activated by trypsin-like proteases, in microglial activation and neuronal cell death. In cultured rat primary microglia, activation of PAR-2 induced nitrite production by PKC- and MAPKs-dependent mechanism. Among the three members of MAPK pathway, ERK and JNK but not p38 mediated PAR-2-induced microglial activation. The down-stream regulator of PAR-2–PKC–MAPK pathway-induced microglial activation was NF-κB pathway. Besides nitrite, PAR-2 activation increased production of a variety of inflammatory mediators such as ROS and pro-inflammatory cytokines including TNF-α and IL-1β. The addition of culture spent media from PAR-2 activated microglia induced neuronal cell death in primary rat cortical neuron cultures with apoptotic features such as increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive neurons, dissipation of mitochondrial membrane potential, increased expression of pro-apoptotic Bax, decreased expression of anti-apoptotic Bcl-2, Bcl-X L, and activation of caspase-3 in neurons. Interestingly, the increased production of cytoactive molecules as well as the neuronal cell death was normalized by PAR-2 or trypsin inhibitor or an NO synthase inhibitor, N G -nitro- l-arginine-methyl ester. Taken together, these results suggest that overt PAR-2 activation may induce microglial activation, which contributes to neuronal cell death.

Neurobehavioral impairments, generation of oxidative stress and release of pro-apoptotic factors after chronic exposure to sulphur mustard in mouse brain

Recent global events have focused attention on the potential threat of international and domestic chemical terrorism, as well as the possibility of chemical warfare proliferation. Sulphur mustard (SM) is one of the potent chemical warfare agents (CWA), which initiates a cascade of events that converge on the redox mechanisms common to brain injury. The present study was designed to examine the effects of chronic SM exposure on neurobehavioral impairments, mitochondrial oxidative stress in male Swiss Albino mice and its role in inducing apoptotic neuronal cell death. The animals were divided into four groups (control, low, medium and high dose) of 5 animals each. Exposure to SM was given percutaneously daily for 12 weeks. The results demonstrated impairment in neurobehavioral indices viz. rota rod, passive avoidance and water maze tests in a dose dependent manner. There was a significant increase in lipid peroxidation and protein carbonyl content whereas, decrease in the activity of manganese superoxide dismutase (MnSOD), glutathione reductase and glutathione peroxidase suggesting impaired antioxidant defense system. Immunoblotting of cytochrome c, Bcl-2, Bax and activation of caspase-3 suggest induction of apoptosis in a dose dependent manner. Finally, increased p53 expression suggests that it may target the mitochondrial pathway for inducing apoptosis in response to DNA damage signals. In conclusion, chronic SM exposure may have the potential to generate oxidative stress which may trigger the release of cytochrome c as well as caspase-3 activation in neurons leading to cell death by apoptosis in a dose dependent manner which may in the end be responsible for the disruption of cognitive functions in mice.

STI571 prevents apoptosis, tau phosphorylation and behavioural impairments induced by Alzheimer's β-amyloid deposits

There is evidence that amyloid beta-protein (Abeta) deposits or Abeta intermediates trigger pathogenic factors in Alzheimer's disease patients. We have previously reported that c-Abl kinase activation involved in cell signalling regulates the neuronal death response to Abeta fibrils (Abeta(f)). In the present study we investigated the therapeutic potential of the selective c-Abl inhibitor STI571 on both the intrahippocampal injection of Abeta(f) and APPsw/PSEN1DeltaE9 transgenic mice Alzheimer's disease models. Injection of Abeta(f) induced an increase in the numbers of p73 and c-Abl immunoreactive cells in the hippocampal area near to the lesion. Chronic intraperitoneal administration of STI571 reduced the rat behavioural deficit induced by Abeta(f), as well as apoptosis and tau phosphorylation. Our in vitro studies suggest that inhibition of the c-Abl/p73 signalling pathway is the mechanism underlying of the effects of STI571 on Abeta-induced apoptosis for the following reasons: (i) Abeta(f) induces p73 phosphorylation, the TAp73 isoform levels increase so as to enhance its proapoptotic function, and all these effects where reduced by STI571; (ii) c-Abl kinase activity is required for neuronal apoptosis and (iii) STI571 prevents the Abeta-induced increase in the expression of apoptotic genes. Furthermore, in the Abeta-injected area there was a huge increase in phosphorylated p73 and a larger number of TAp73-positive cells, with these changes being prevented by STI571 coinjection. Moreover, the intraperitoneal administration of STI571 rescued the cognitive decline in APPsw/PSEN1DeltaE9 mice, p73 phosphorylation, tau phosphorylation and caspase-3 activation in neurons around Abeta deposits. Besides, we observed a decrease in the number and size of Abeta deposits in the APPsw/PSEN1DeltaE9-STI571-treated mice. These results are consistent with the role of the c-Abl/p73 signalling pathway in Abeta neurodegeneration, and suggest that STI571-like compounds would be effective in therapeutic treatments of Alzheimer disease.

A role for synGAP in regulating neuronal apoptosis

The brain-specific Ras/Rap GTPase-activating protein synGAP is a major component of the postsynaptic density at glutamatergic synapses. It is a target for phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II, which up-regulates its GTPase-activating activity. Thus, SynGAP may play an important role in coupling N-methyl-D-aspartate-type glutamate receptor activation to signaling pathways downstream of Ras or Rap. Homozygous deletion of synGAP is lethal within the first few days after birth. Therefore, to study the functions of synGAP, we used the cre/loxP recombination system to produce conditional mice mutants in which gradual loss of synGAP begins at approximately 1 week, and usually becomes maximal by 3 weeks, after birth. The resulting phenotypes fall into two groups. In a small group, the level of synGAP protein is reduced to 20-25% of wild type, and they die at 2-3 weeks of age. In a larger group, the levels remain higher than approximately 40% of wild type, and they survive and remain healthy. In all mutants, however, an abnormally high number of neurons in the hippocampus and cortex undergo apoptosis, as detected by caspase-3 activation. The effect is cell autonomous, occurring only in neuronal types in which the synGAP gene is eliminated. The level of caspase-3 activation in neurons correlates inversely with the level of synGAP protein measured at 2 and 8 weeks after birth, indicating that neuronal apoptosis is enhanced by reduction of synGAP. These data show that synGAP plays a role in regulation of the onset of apoptotic neuronal death.

5-s-Cysteinyl-conjugates of catecholamines induce cell damage, extensive DNA base modification and increases in caspase-3 activity in neurons.

A decrease in reduced glutathione levels in dopamine containing nigral cells in Parkinson's disease may result from the formation of cysteinyl-adducts of catecholamines, which in turn exert toxicity on nigral cells. We show that exposure of neurons (CSM 14.1) to 5-S-cysteinyl conjugates of dopamine, L-DOPA, DOPAC or DHMA causes neuronal damage, increases in oxidative DNA base modification and an elevation of caspase-3 activity in cells. Damage to neurons was apparent 12-48 h of post-exposure and there were increases in caspase-3 activity in neurons after 6 h. These changes were paralleled by large increases in pyrimidine and purine base oxidation products, such as 8-OH-guanine suggesting that 5-S-cysteinyl conjugates of catecholamines are capable of diffusing into cells and stimulating the formation of reactive oxygen species (ROS), which may then lead to a mechanism of cell damage involving caspase-3. Indeed, intracellular ROS were observed to rise sharply on exposure to the conjugates. These results suggest one mechanism by which oxidative stress may occur in the substantia nigra in Parkinson's disease.

Clusterin contributes to caspase-3-independent brain injury following neonatal hypoxia-ischemia.

Clusterin, also known as apolipoprotein J, is a ubiquitously expressed molecule thought to influence a variety of processes including cell death. In the brain, it accumulates in dying neurons following seizures and hypoxic-ischemic (H-I) injury. Despite this, in vivo evidence that clusterin directly influences cell death is lacking. Following neonatal H-I brain injury in mice (a model of cerebral palsy), there was evidence of apoptotic changes (neuronal caspase-3 activation), as well as accumulation of clusterin in dying neurons. Clusterin-deficient mice had 50% less brain injury following neonatal H-I. Surprisingly, the absence of clusterin had no effect on caspase-3 activation, and clusterin accumulation and caspase-3 activation did not colocalize to the same cells. Studies with cultured cortical neurons demonstrated that exogenous purified astrocyte-secreted clusterin exacerbated oxygen/glucose-deprivation-induced necrotic death. These results indicate that clusterin may be a new therapeutic target to modulate non-caspase-dependent neuronal death following acute brain injury.

Activation of Neuronal Caspase-3 by Intracellular Accumulation of Wild-Type Alzheimer Amyloid Precursor Protein

Forced overexpression of wild-type Alzheimer amyloid precursor protein (APP) causes postmitotic neurons to degenerate. Caspase-3 (CPP32) is a principal cell death protease involved in neuronal apoptosis during physiological development and under pathological conditions. Here, we investigated whether APP overexpression activates caspase-3 in human postmitotic neurons using adenovirus-mediated gene transfer. When a recombinant adenovirus vector expressing human wild-type APP695 was infected in vitro into neurally differentiated embryonal carcinoma NT2 cells, only postmitotic neurons underwent severe degeneration. Before neurodegeneration, full-length APP- and Abeta-immunoreactive peptides were accumulated in infected neurons, and caspase-3-like protease activity was markedly elevated. Western blot analysis revealed that activated caspase-3 subunits were generated in APP-accumulating neurons. Such neuronal caspase-3 activation was undetectable in NT2 neurons infected with beta-galactosidase-expressing adenovirus. Addition of the caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde to the culture medium significantly reduced the severity of degeneration exhibited by APP-overexpressing neurons. Immunocytochemical analyses revealed that some APP-accumulating neurons contained activated caspase-3 subunits and exhibited the characteristics of apoptosis, such as chromatin condensation and DNA fragmentation. Activation of caspase-3 was also observed in vivo in rat hippocampal neurons infected with the APP-expressing adenovirus. These results suggest that wild-type APP is an intrinsic activator of caspase-3-mediated death machinery in postmitotic neurons.

Contribution of p53-Dependent Caspase Activation to Neuronal Cell Death Declines with Neuronal Maturation

Caspases play a pivotal role in neuronal cell death during development and after trophic factor withdrawal. However, the mechanisms regulating caspase activity and the role played by caspase activation in response to neuronal injury is poorly understood. The tumor suppressor gene p53 has been implicated in the loss of neuronal viability caused by excitotoxic and DNA damaging agents. In the present study we determined if p53-mediated neuronal cell death required caspase activation. DNA damage increased caspase activity in both cultured embryonic telencephalic and postnatal cortical neurons in a p53-dependent manner. Caspase inhibitors protected embryonic telencephalic neurons, but not postnatal cortical neurons, from DNA damage-induced cell death as measured by direct cell counting and annexin V staining. In marked contrast to the caspase inhibitors, an inhibitor of the DNA repair enzyme, poly(ADP-ribose) polymerase, conferred significant protection from genotoxic and excitotoxic cell death on postnatal cortical neurons but had no effect on embryonic neurons. Glutamate-mediated excitotoxicity in postnatal neurons was not associated with measurable changes in caspase activity, consistent with the failure of caspase inhibitors to prevent cell death under these conditions. Moreover, adenovirus-mediated overexpression of p53 killed embryonic and postnatal neurons without activating caspases. Thus, p53-mediated neuronal cell death may occur via both caspase-dependent and caspase-independent pathways. These results demonstrate that p53 is required for caspase activation in response to some forms of neuronal injury. However, the relative importance of caspase activation in neurons depends on the developmental status of the cell and the specific nature of the death stimulus.