Melatonin is protective in necrotic but not in caspasedependent, free radical‐independent apoptotic neuronal cell death in primary neuronal cultures

Substantial evidence indicates that mitochondria are a major checkpoint in several pathways leading to neuronal cell death, but discerning critical propagation stages from downstream consequences has been difficult. The mitochondrial permeability transition (mPT) may be critical in stroke-related injury. To address this hypothesis, identify potential therapeutics, and screen for new uses for established drugs with known toxicity, 1,040 FDA-approved drugs and other bioactive compounds were tested as potential mPT inhibitors. We report the identification of 28 structurally related drugs, including tricyclic antidepressants and antipsychotics, capable of delaying the mPT. Clinically achievable doses of one drug in this general structural class that inhibits mPT, promethazine, were protective in both in vitro and mouse models of stroke. Specifically, promethazine protected primary neuronal cultures subjected to oxygen-glucose deprivation and reduced infarct size and neurological impairment in mice subjected to middle cerebral artery occlusion/reperfusion. These results, in conjunction with new insights provided to older studies, (a) suggest a class of safe, tolerable drugs for stroke and neurodegeneration; (b) provide new tools for understanding mitochondrial roles in neuronal cell death; (c) demonstrate the clinical/experimental value of screening collections of bioactive compounds enriched in clinically available agents; and (d) provide discovery-based evidence that mPT is an essential, causative event in stroke-related injury.

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Astrocytes Protect against Isoflurane Neurotoxicity by Buffering pro-brain–derived Neurotrophic Factor

Anesthesiology ◽

10.1097/aln.0000000000000824 ◽

2015 ◽

Vol 123 (4) ◽

pp. 810-819 ◽

Cited By ~ 15

Author(s):

Creed M. Stary ◽

Xiaoyun Sun ◽

Rona G. Giffard

Keyword(s):

Cell Death ◽

Neurotrophic Factor ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Brain Derived Neurotrophic Factor ◽

Neurotrophin Receptor ◽

Enzyme Linked Immunosorbent Assay ◽

Neuronal Cultures ◽

P75 Neurotrophin Receptor ◽

Primary Neuronal Cultures

Abstract Background: Isoflurane induces cell death in neurons undergoing synaptogenesis via increased production of pro-brain–derived neurotrophic factor (proBDNF) and activation of postsynaptic p75 neurotrophin receptor (p75NTR). Astrocytes express p75NTR, but their role in neuronal p75NTR-mediated cell death remains unclear. The authors investigated whether astrocytes have the capacity to buffer increases in proBDNF and protect against isoflurane/p75NTR neurotoxicity. Methods: Cell death was assessed in day in vitro (DIV) 7 mouse primary neuronal cultures alone or in co-culture with age-matched or DIV 21 astrocytes with propidium iodide 24 h after 1 h exposure to 2% isoflurane or recombinant proBDNF. Astrocyte-targeted knockdown of p75NTR in co-culture was achieved with small-interfering RNA and astrocyte-specific transfection reagent and verified with immunofluorescence microscopy. proBDNF levels were assessed by enzyme-linked immunosorbent assay. Each experiment used six to eight replicate cultures/condition and was repeated at least three times. Results: Exposure to isoflurane significantly (P < 0.05) increased neuronal cell death in primary neuronal cultures (1.5 ± 0.7 fold, mean ± SD) but not in co-culture with DIV 7 (1.0 ± 0.5 fold) or DIV 21 astrocytes (1.2 ± 1.2 fold). Exogenous proBDNF dose dependently induced neuronal cell death in both primary neuronal and co-cultures, an effect enhanced by astrocyte p75NTR inhibition. Astrocyte-targeted p75NTR knockdown in co-cultures increased media proBDNF (1.2 ± 0.1 fold) and augmented isoflurane-induced neuronal cell death (3.8 ± 3.1 fold). Conclusions: The presence of astrocytes provides protection to growing neurons by buffering increased levels of proBDNF induced by isoflurane. These findings may hold clinical significance for the neonatal and injured brain where increased levels of proBDNF impair neurogenesis.

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Augmentation of poly(ADP-ribose) polymerase-dependent neuronal cell death by acidosis

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x16658491 ◽

2016 ◽

Vol 37 (6) ◽

pp. 1982-1993 ◽

Cited By ~ 7

Author(s):

Jian Zhang ◽

Xiaoling Li ◽

Herman Kwansa ◽

Yun Tai Kim ◽

Liye Yi ◽

...

Keyword(s):

Cell Death ◽

Ion Channel ◽

Focal Cerebral Ischemia ◽

Infarct Volume ◽

Neuronal Cell ◽

Neuronal Cultures ◽

Primary Neuronal Cultures ◽

Tissue Acidosis ◽

Nuclear Apoptosis ◽

Apoptosis Inducing Factor

Tissue acidosis is a key component of cerebral ischemic injury, but its influence on cell death signaling pathways is not well defined. One such pathway is parthanatos, in which oxidative damage to DNA results in activation of poly(ADP-ribose) polymerase and generation of poly(ADP-ribose) polymers that trigger release of mitochondrial apoptosis-inducing factor. In primary neuronal cultures, we first investigated whether acidosis per sé is capable of augmenting parthanatos signaling initiated pharmacologically with the DNA alkylating agent, N-methyl- N′-nitro- N-nitrosoguanidine. Exposure of neurons to medium at pH 6.2 for 4 h after N-methyl- N′-nitro- N-nitrosoguanidine washout increased intracellular calcium and augmented the N-methyl- N′-nitro- N-nitrosoguanidine-evoked increase in poly(ADP-ribose) polymers, nuclear apoptosis-inducing factor , and cell death. The augmented nuclear apoptosis-inducing factor and cell death were blocked by the acid-sensitive ion channel-1a inhibitor, psalmotoxin. In vivo, acute hyperglycemia during transient focal cerebral ischemia augmented tissue acidosis, poly(ADP-ribose) polymers formation, and nuclear apoptosis-inducing factor , which was attenuated by a poly(ADP-ribose) polymerase inhibitor. Infarct volume from hyperglycemic ischemia was decreased in poly(ADP-ribose) polymerase 1-null mice. Collectively, these results demonstrate that acidosis can directly amplify neuronal parthanatos in the absence of ischemia through acid-sensitive ion channel-1a . The results further support parthanatos as one of the mechanisms by which ischemia-associated tissue acidosis augments cell death.

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Prion peptide induces neuronal cell death through a pathway involving glycogen synthase kinase 3

Biochemical Journal ◽

10.1042/bj20021596 ◽

2003 ◽

Vol 372 (1) ◽

pp. 129-136 ◽

Cited By ~ 81

Author(s):

Mar PÉREZ ◽

Ana I. ROJO ◽

Francisco WANDOSELL ◽

Javier DÍAZ-NIDO ◽

Jesús AVILA

Keyword(s):

Cell Death ◽

Glycogen Synthase ◽

Neuronal Cell Death ◽

Neuroblastoma Cells ◽

Neuronal Cell ◽

Dominant Negative ◽

Glycogen Synthase Kinase ◽

Glycogen Synthase Kinase 3 ◽

Dominant Negative Mutant ◽

Neuronal Cultures

Prion diseases are characterized by neuronal cell death, glial proliferation and deposition of prion peptide aggregates. An abnormal misfolded isoform of the prion protein (PrP) is considered to be responsible for this neurodegeneration. The PrP 106–126, a synthetic peptide obtained from the amyloidogenic region of the PrP, constitutes a model system to study prion-induced neurodegeneration as it retains the ability to trigger cell death in neuronal cultures. In the present study, we show that the addition of this prion peptide to cultured neurons increases the activity of glycogen synthase kinase 3 (GSK-3), which is accompanied by the enhanced phosphorylation of some microtubule-associated proteins including tau and microtubule-associated protein 2. Prion peptide-treated neurons become progressively atrophic, and die ultimately. Both lithium and insulin, which inhibit GSK-3 activity, significantly decrease prion peptide-induced cell death both in primary neuronal cultures and in neuroblastoma cells. Finally, the overexpression of a dominant-negative mutant of GSK-3 in transfected neuroblastoma cells efficiently prevents prion peptide-induced cell death. These results are consistent with the view that the activation of GSK-3 is a crucial mediator of prion peptide-induced neurodegeneration.

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Fas-Mediated Apoptotic Signaling in the Mouse Brain following Reovirus Infection

Journal of Virology ◽

10.1128/jvi.02488-08 ◽

2009 ◽

Vol 83 (12) ◽

pp. 6161-6170 ◽

Cited By ~ 31

Author(s):

Penny Clarke ◽

J. David Beckham ◽

J. Smith Leser ◽

Cristen C. Hoyt ◽

Kenneth L. Tyler

Keyword(s):

Neuronal Cell Death ◽

Death Receptor ◽

Neuronal Cell ◽

Mitogen Activated Protein Kinase ◽

Neuronal Cultures ◽

Caspase 8 ◽

Primary Neuronal Cultures ◽

Jnk Signaling ◽

Apoptotic Signaling ◽

Induced Apoptosis

ABSTRACT Type 3 (T3) reovirus strains induce apoptotic neuronal cell death and lethal encephalitis in infected mice. T3 strain Dearing (T3D)-induced apoptosis in primary neuronal cultures occurs by a Fas-mediated mechanism and requires the activation of caspase 8. We now show that Fas mRNA is upregulated in the brains of mice infected with encephalitic reovirus T3D and T3 strain Abney (T3A) but not following infection with nonencephalitic reovirus type 1 strain Lang. Fas is upregulated in regions of the brain that are injured during infection with T3 reovirus strains and colocalizes with virus antigen in individual neurons. In contrast, levels of FasL mRNA induced by encephalitic and nonencephalitic reovirus strains do not differ significantly. Caspase 8, the initiator caspase associated with Fas-mediated apoptosis, is activated in the cortex and hippocampal regions of both T3D- and T3A-infected mice. Furthermore, Bid cleavage and the activation of caspase 9 in the brains of T3D-infected mice suggest that the caspase 8-dependent activation of mitochondrial apoptotic signaling contributes to virus-induced apoptosis. We have previously shown that the inhibition of c-Jun N-terminal kinase (JNK) signaling blocks T3D-induced apoptosis and improves the outcome of virus-induced encephalitis. We now show that the reovirus-induced upregulation of Fas requires JNK signaling, thereby providing a link between reovirus-induced death receptor signaling and mitogen-activated protein kinase pathways and a potential mechanism for the therapeutic action of JNK inhibition.

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Pomalidomide Ameliorates H2O2-Induced Oxidative Stress Injury and Cell Death in Rat Primary Cortical Neuronal Cultures by Inducing Anti-Oxidative and Anti-Apoptosis Effects

International Journal of Molecular Sciences ◽

10.3390/ijms19103252 ◽

2018 ◽

Vol 19 (10) ◽

pp. 3252 ◽

Cited By ~ 9

Author(s):

Yan-Rou Tsai ◽

Cheng-Fu Chang ◽

Jing-Huei Lai ◽

John Wu ◽

Yen-Hua Chen ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Mitochondrial Activity ◽

Neuronal Cultures ◽

Nuclear Factor ◽

Neuroprotective Effects ◽

Stress Injury ◽

Anti Inflammatory

Due to its high oxygen demand and abundance of peroxidation-susceptible lipid cells, the brain is particularly vulnerable to oxidative stress. Induced by a redox state imbalance involving either excessive generation of reactive oxygen species (ROS) or dysfunction of the antioxidant system, oxidative stress plays a central role in a common pathophysiology that underpins neuronal cell death in acute neurological disorders epitomized by stroke and chronic ones such as Alzheimer’s disease. After cerebral ischemia, for example, inflammation bears a key responsibility in the development of permanent neurological damage. ROS are involved in the mechanism of post-ischemic inflammation. The activation of several inflammatory enzymes produces ROS, which subsequently suppress mitochondrial activity, leading to further tissue damage. Pomalidomide (POM) is a clinically available immunomodulatory and anti-inflammatory agent. Using H2O2-treated rat primary cortical neuronal cultures, we found POM displayed neuroprotective effects against oxidative stress and cell death that associated with changes in the nuclear factor erythroid derived 2/superoxide dismutase 2/catalase signaling pathway. POM also suppressed nuclear factor kappa-light-chain-enhancer (NF-κB) levels and significantly mitigated cortical neuronal apoptosis by regulating Bax, Cytochrome c and Poly (ADP-ribose) polymerase. In summary, POM exerted neuroprotective effects via its anti-oxidative and anti-inflammatory actions against H2O2-induced injury. POM consequently represents a potential therapeutic agent against brain damage and related disorders and warrants further evaluation.

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The “Dark Side” of Endocannabinoids: A Neurotoxic Role for Anandamide

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-200405000-00011 ◽

2004 ◽

Vol 24 (5) ◽

pp. 564-578 ◽

Cited By ~ 33

Author(s):

Ibolja Cernak ◽

Robert Vink ◽

JoAnne Natale ◽

Bogdan Stoica ◽

M. Lea Paul ◽

...

Keyword(s):

Cell Death ◽

Neuronal Cell ◽

Cell Loss ◽

Dark Side ◽

Neuronal Cultures ◽

Cb1 Receptors ◽

Central Administration ◽

Primary Neuronal Cultures ◽

Neuroprotective Effects

Endocannabinoids, including 2-arachidonoylglycerol and anandamide ( N-arachidonoylethanolamine; AEA), have neuroprotective effects in the brain through actions at CB1 receptors. However, AEA also binds to vanilloid (VR1) receptors and induces cell death in several cell lines. Here we show that anandamide causes neuronal cell death in vitro and exacerbates cell loss caused by stretch-induced axonal injury or trophic withdrawal in rat primary neuronal cultures. Administered intracerebroventricularly, AEA causes sustained cerebral edema, as reflected by diffusion-weighted magnetic resonance imaging, regional cell loss, and impairment in long-term cognitive function. These effects are mediated, in part, through VR1 as well as through calpain-dependent mechanisms, but not through CB1 receptors or caspases. Central administration of AEA also significantly upregulates genes involved in proinflammatory/microglial-related responses. Thus, anandamide produces neurotoxic effects both in vitro and in vivo through multiple mechanisms independent of the CB1 receptor.

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