Erythropoietin prevents early and late neuronal demise through modulation of Akt1 and induction of caspase 1, 3, and 8

Erythropoietin (EPO) modulates primarily the proliferation of immature erythroid precursors, but little is known of the potential protective mechanisms of EPO in the central nervous system. We therefore examined the ability of EPO to modulate a series of death-related cellular pathways during anoxia and free radical induced neuronal degeneration. Neuronal injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidylserine exposure, protein kinase B phosphorylation, cysteine protease activity, mitochondrial membrane potential, and mitogen-activated protein (MAP) kinase phosphorylation. We demonstrate that constitutive neuronal EPO is insufficient to prevent cellular injury, but that signaling through the EPO receptor remains biologically responsive to exogenous EPO administration. Exogenous EPO is both necessary and sufficient to prevent acute genomic DNA destruction and subsequent phagocytosis through membrane PS exposure, because neuronal protection by EPO is completely abolished by co-treatment with an anti-EPO neutralizing antibody. Through pathways that involve the initial activation of protein kinase B, EPO maintains mitochondrial membrane potential. Subsequently, EPO inhibits caspase 8-, caspase 1-, and caspase 3-like activities linked to cytochrome c release through mechanisms that are independent from the MAP kinase systems of p38 and JNK. Elucidating some of the novel neuroprotective pathways employed by EPO may further the development of new therapeutic strategies for neurodegenerative disorders.     

Insulin-like growth factor 1 prevents neuronal cell death induced by corticosterone through activation of the PI3k/Akt pathway

Corticosterone (CORT) is well known to induce neuronal damage in various brain regions including the hippocampus, but the precise mechanism(s) of action underlying these effects has yet to be fully established. Insulin-like growth factor-1 (IGF-1) is a trophic factor promoting cell survival by the activation of the phosphatidylinositide 3-kinase (PI3K)/Akt kinase pathway. We report that IGF-1 prevents neuronal cell death induced by CORT, likely via the stimulation of the PI3K/Akt pathway in primary hippocampal cultured neurons. CORT induced neuronal cell death at a minimal concentration of 50 nM. IGF-1 (10 nM) prevented cell death induced by CORT under serum-free conditions. The neuroprotective effect of IGF-1 was accompanied by reversal of the Akt pathway inhibition induced by CORT. The PI3 kinase inhibitor, LY29004, inhibited the neuroprotective effect of IGF-1 whereas the MEK (MAPK kinase) inhibitor PD98059, an upstream blocker of mitogen-activated protein (MAP) kinase, had no effect. These results suggest that IGF-1 can prevent neuronal cell death induced by CORT in hippocampal neurons by modulating the activity of the PI3K/Akt pathway.     

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.     

Lipopolysaccharide-induced microglial activation induces learning and memory deficits without neuronal cell death in rats

We used lipopolysaccharide (LPS) to activate microglia that play an important role in the brain immune system. LPS injected into the rat hippocampus CA1 region activated microglial cells resulting in an increased production of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha in the hippocampus during the initial stage of treatment. Immunostaining for IL-1beta was increased at 6 hr after LPS injection. IL-1beta-immunopositive cells were co-localized with immunostaining for CD11b. Subacute treatment with LPS by the same route for 5 days caused long-term activation of microglia and induced learning and memory deficits in animals when examined with a step-through passive avoidance test, but histochemical analysis showed that neuronal cell death was not observed under these experimental conditions. The increased expression of the heme oxygenase-1 (HO-1) gene, an oxidative stress maker, was observed. However, the genetic expression of brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, decreased during the course of LPS treatment. We found decreases in [3H]MK801 binding in the hippocampus CA1 region by LPS-treatment for 5 days. The data shows that glutamatergic transmission was attenuated in the LPS-treated rats. These results suggest that long-term activation of microglia induced by LPS results in a decrease of glutamatergic transmission that leads to learning and memory deficits without neuronal cell death. The physiologic significance of these findings is discussed.     

Akt1 protects against inflammatory microglial activation through maintenance of membrane asymmetry and modulation of cysteine protease activity

In several cell systems, protein kinase B (Akt1) can promote cell growth and development, but the "antiapoptotic" pathways of this kinase that may offer protection against cellular inflammatory demise have not been defined. Given that early cellular membrane phosphatidylserine exposure is a critical component of apoptosis, we investigated the role of Akt1 during neuronal apoptotic injury. By employing differentiated SH-SY5Y neuronal cells that overexpress a constitutively active form of Akt1 (myristoylated Akt1), free radical-induced cell injury was assessed through trypan blue dye exclusion, DNA fragmentation, membrane phosphatidylserine exposure, protein kinase B phosphorylation, cysteine protease activity, and mitochondrial membrane potential. Membrane phosphatidylserine exposure was both necessary and sufficient for microglial activation, insofar as cotreatment with an antiphosphatidylserine receptor-neutralizing antibody could prevent microglial activity following neuronal loss of membrane asymmetry. Furthermore, expression of myristoylated Akt1 not only prevented cell injury through the prevention of membrane phosphatidylserine exposure and genomic DNA fragmentation but also inhibited microglial activation and proliferation that required the inhibition of caspase 9-, caspase 3-, and caspase 1-like activities linked to cytochrome c release. Interestingly, Akt1 modulation of membrane phosphatidylserine exposure was primarily through caspase 1 activity. Removal of Akt1 activity abolished neuronal protection, suggesting that Akt1 functions as a critical pathway for the maintenance of cellular integrity and the prevention of phagocytic cellular removal during neurodegenerative insults.     

Automatic counting of microglial cell activation and its applications

Glaucoma is a multifactorial optic neuropathy characterized by the damage and death of the retinal gan-glion cells. This disease results in vision loss and blindness. Any vision loss resulting from the disease cannot be restored and nowadays there is no available cure for glaucoma;however an early detection and treatment, could offer neuronal protection and avoid later serious damages to the visual function. A full understanding of the etiology of the disease will still require the contribution of many scientiifc efforts. Glial activation has been observed in glaucoma, being microglial proliferation a hallmark in this neuro-degenerative disease. A typical project studying these cellular changes involved in glaucoma often needs thousands of images-from several animals-covering different layers and regions of the retina. The gold standard to evaluate them is the manual count. This method requires a large amount of time from special-ized personnel. It is a tedious process and prone to human error. We present here a new method to count microglial cells by using a computer algorithm. It counts in one hour the same number of images that a researcher counts in four weeks, with no loss of reliability.     

Styrene 7,8-oxide induces caspase activation and regular DNA fragmentation in neuronal cells

Neurobehavioral changes have been described in workers occupationally exposed to styrene vapors. Alterations of neurotransmitters and loss of neurons have been observed in brains of styrene-exposed rats. However, the mechanisms of neuronal damage are not yet clearly understood. We have characterized the cellular alterations induced by the main reactive intermediate of styrene metabolism, styrene 7,8-oxide (SO) in the human neuroblastoma SK-N-MC cell line and primary culture of rat cerebellar granule cells (CGC). SK-N-MC cells exposed to SO (0.3-1 mM) displayed apoptotic morphology, together with chromatin condensation and DNA cleavage into high molecular weight fragments of regular size. These features were accompanied by the activation of class II caspases, as detected with the DEVD assay, by following the cleavage of the caspase-substrate poly (ADP-ribose) polymerase (PARP) and by detection of the active fragment of caspase-3. Pre-incubation of the cells with the caspase inhibitor z-VAD-fmk reduced the cellular damage induced by SO, suggesting that caspases play an important role in SO toxicity. Increased proteolysis by class II caspases was detected also in primary culture of CGC exposed to SO. In addition, the presence of the 150-kDa cleavage product of alpha-fodrin suggests a possible activation of calpains in SK-N-MC cells. Moreover, SO did not affect the level of expression of the p53 protein, even though it is known to cause DNA damage. The identified intracellular pathways affected by SO exposure provides end-points that can be used in future studies for the evaluation of the neurotoxic effect of styrene in vivo.     

Differential regulation of trophic and proinflammatory microglial effectors is dependent on severity of neuronal injury

Microglial activation has been reported to promote neurotoxicity and also neuroprotective effects. A possible contributor to this dichotomy of responses may be the degree to which proximal neurons are injured. The aim of this study was to determine whether varying the severity of neuronal injury influenced whether microglia were neuroprotective or neurotoxic. We exposed cortical neuronal cultures to varying degrees of hypoxia thereby generating mild (<20% death, 30 min hypoxia), moderate (40-60% death, 2 h hypoxia), or severe (>70% death, 6 h hypoxia) injuries. Twenty-four hours after hypoxia, the media from the neuronal cultures was collected and incubated with primary microglial cultures for 24 h. Results showed that the classic microglial proinflammatory mediators including inducible nitric oxide synthase, tumor necrosis factor alpha, and interleukin-1-beta were upregulated only in response to mild neuronal injuries, while the trophic microglial effectors brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor were upregulated in response to all degrees of neuronal injury. Microglia stimulated with media from damaged neurons were co-cultured with hypoxic neurons. Microglia stimulated by moderate, but not mild or severe damage were neuroprotective in these co-cultures. We also showed that the severity-dependent phenomenon was not related to autocrine microglial signaling and was dependent on the neurotransmitters released by neurons after injury, namely glutamate and adenosine 5'-triphosphate. Together our results show that severity of neuronal injury is an important factor in determining microglial release of "toxic" versus "protective" effectors and the resulting neurotoxicity versus neuroprotection.     

MCI-186 prevents brain tissue from neuronal damage in cerebral infarction through the activation of intracellular signaling

The mechanism by which MCI-186 (3-methyl-1-phenyl-2-prazolin-5-one) exerts protective effects during cerebral infarction, other than its function as a radical scavenger, has not been fully elucidated. Here, we found that MCI-186 stimulates intracellular survival signaling in vivo and in vitro. In a rat infarction model, the infarct area was significantly smaller and the degree of edema was reduced in MCI-186-treated animals. In the MCI-186-treated rats, the number of single stranded (ss) DNA-positive damaged cells in the peri-infarct area was decreased compared with the control, suggesting that MCI-186 protects cerebral tissues from cell damage. To clarify the mechanisms underlying the effect of MCI-186, we also examined the survival-promoting effect of this agent on cultured cortical neurons. In this in vitro system, MCI-186 blocked serum-free induced neuronal cell death. Interestingly, an increase in the activation of both Akt (a component of the PI3 kinase pathway) and ERK (a component of the MAP kinase pathway) was observed in the cortical cultures after MCI-186 exposure. Furthermore, the MCI-186-dependent survival effect in vitro was blocked by U0126, an MEK (an upstream of ERK) inhibitor, and also by LY294002, a PI3 kinase inhibitor. We also observed similar increases in the activation of Akt and ERK in the in vivo model, further suggesting that the antiapoptotic role of MCI-186 is mediated via the PI3 kinase and MAP kinase signaling pathways. We therefore conclude that, in addition to its role as a free radical scavenger, MCI-186 functions as an antiapoptotic factor by enhancing intracellular survival signaling.     

The metabotropic glutamate system promotes neuronal survival through distinct pathways of programmed cell death

Activation of the metabotropic glutamate receptor (mGluR) system can prevent free radical, nitric oxide (NO)-induced programmed cell death (PCD). To investigate the mechanisms utilized by the mGluR system to regulate the induction of PCD, we examined the course of PCD in real time in individual, living, primary hippocampal neurons. We assessed both phosphatidylserine (PS) externalization, an early event in PCD, and DNA fragmentation during NO toxicity and mGluR modulation to determine the individual contributions of PS externalization and genomic DNA fragmentation during neuronal PCD. Exposure to the NO donors (300 microM SNP or 300 microM NOC-9) induced PCD in approximately 75% of neurons over a 24-h period. The externalization of PS in neurons increased to 21 +/- 2% as early as 3 h following NO exposure and then increased to 80 +/- 2% over a 24-h period. The externalization of PS was independent of the loss of membrane integrity. Agonists for individual mGluR subgroups were equally able to prevent NO-induced neuronal death and DNA degradation, yet they possessed differential abilities to regulate PS externalization. The group I agonist DHPG (750 microM) and the group III agonist L-AP4 (750 microM) both prevented and reversed NO-induced PS externalization. In contrast, activation of group II subtypes using L-CCG-I (750 microM) did not prevent PS externalization. Employing an experimental model that independently led to the externalization of PS residues, we demonstrated that PS externalization does not immediately impact on neuronal survival. Yet, subsequent neuronal survival may ultimately depend upon preventing PS externalization to avoid neuronal tagging for phagocytosis. Since group I and III mGluR subtypes possess the unique ability to maintain genomic integrity and membrane PS asymmetry, these agents may provide superior overall protection against NO-induced neuronal injury.     

Reverse glial glutamate uptake triggers neuronal cell death through extrasynaptic NMDA receptor activation

Evidence have accumulated that reverse glutamate uptake plays a key role in the pathophysiology of cerebral ischemia. Here, we investigated the effects of glial glutamate transporter dysfunction on neuronal survival using the substrate inhibitor of glutamate transporters, l-trans-pyrrolidine,2-4,dicarboxylate (PDC), that partly mimics reverse glutamate uptake. On mice primary cortical co-cultures of neurons and astrocytes, PDC treatment triggered an elevation of extracellular glutamate concentration, induced neuronal calcium influx and a massive NMDA receptor (NMDAR) mediated-neuronal death without having any direct agonist activity on NMDARs. We investigated the NMDAR subpopulation activated by PDC-induced glutamate release. PDC application led to the activation of both subtypes of NMDARs but the presence of astrocytes was required to activate NMDARs located extra-synaptically. Extrasynaptic NMDAR activation was also confirmed by the loss of neuronal mitochondrial membrane potential and the inhibition of pro-survival p-ERK signalling pathway. These data suggest that reverse glial glutamate uptake may trigger neuronal death through preferential activation of extrasynaptic NMDAR-related pathways.     

Carboxyl-terminal fragment of amyloid precursor protein and hydrogen peroxide induce neuronal cell death through different pathways

Summary. Carboxyl-terminal fragments (CTs) of the amyloid precursor protein have been shown to be highly neurotoxic and are though to contribute to the neuropathology of Alzheimer’s disease. We compared the effects of expressing CT99 in the human neuroblastoma MC65 with the effects of hydrogen peroxide on the parental SK-N-MC cells. CT99 and hydrogen peroxide generated a different pattern of free radicals and their toxic effects were differentially protected by a battery of antioxidants. Hydrogen peroxide caused a cell cycle arrest at phase S and apoptosis mediated through caspase-3 activation in a pattern similar to that described for amyloid-β neurotoxicity. However, CT99 apoptosis appeared to be mediated through an unidentified mitochondrial pathway. Both oxidative injury types induced heme oxygenase-1 expression as a neuroprotective response. Overall we found a coincidence in the nonespecific stress oxidative effects of CT99 and hydrogen peroxide, but clear differences on their respective potencies and pathways of neurotoxicity.     

Mild mitochondrial inhibition in vivo enhances glutamate-induced neuronal damage through calpain but not caspase activation: role of ionotropic glutamate receptors

Glutamate neurotoxicity is exacerbated when energy metabolism is impaired. In vitro studies show that neuronal death in these conditions is related to mitochondrial dysfunction, ATP depletion, and the loss of calcium homeostasis. We have recently observed that, in vivo, enhancement of glutamate toxicity elicited by previous mitochondrial inhibition does not involve severe ATP depletion, suggesting the involvement of other processes. Factors such as the activation of different proteases may determine the extent and type of cell death. Protease activation might be triggered by internal or external factors, such as mitochondrial damage or the activation of a particular glutamate receptor subtype. In the present study we aimed to investigate whether moderate inhibition of mitochondrial metabolism facilitates glutamate toxicity through caspase-3 or calpain activation, as well as the contribution of NMDA and non-NMDA glutamate ionotropic receptors to this activation. Rats were pre-treated with a subtoxic dose of 3-NP and 4 h later intrastriatally injected with glutamate. Results show that neither of these treatments alone (3-NP or Glu) or in combination (3-NP+Glu) activated caspase-3. Conversely, calpain activity is induced after glutamate injection both in intact and 3-NP pre-treated rats. Inhibition of calpain activity by MDL-28170 significantly prevented striatal damage. NMDA and non-NMDA receptors contributed equally to calpain activation and to the induction of neuronal death. Results suggest that enhancement of glutamate toxicity due to inhibition of mitochondrial metabolism in vivo, does not recruit caspase-dependent apoptosis but favors calpain activation through the stimulation of both subtypes of glutamate ionotropic receptors.     

Nitric oxide inhibits caspase activation and apoptotic morphology but does not rescue neuronal death.

Nitric oxide (NO) has been shown to inhibit apoptotic cell death by S-nitrosylation of the catalytic-site cysteine residue of caspases. However, it is not clear whether in neurons NO-mediated caspase inactivation leads to improved cell survival. To address this issue, we studied the effect of NO donors on caspase activity and cell survival in cortical neuronal culture treated with the apoptosis inducer staurosporine (STS) and camptothecin. In parallel, cell viability was assessed by the MTS assay and MAP2 staining. We found that NO donors ((+/-)-S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione, and NONOates) dose-dependently inhibited caspase-3 and -9 activity induced by STS and camptothecin. The reduction in caspase-3 activity was, in large part, because of the blockage of the proteolytic conversion of pro-caspase-3 to active caspase-3. NO donors also inhibited the appearance of the classical apoptotic nuclear morphology. However, inhibition of both caspase activity and apoptotic morphology was not associated with enhancement of cell viability. Thus, inhibition of caspase and apoptotic morphology by NO donors does not improve neuronal survival. The data suggest that inhibition of caspase by NO unmasks a caspase-independent form of cell death. A better understanding of this form of cell death may provide new strategies for neuroprotection in neuropathologies, such as ischemic brain injury, associated with apoptosis.     

Tissue plasminogen activator induces microglial inflammation via a noncatalytic molecular mechanism involving activation of mitogen-activated protein kinases and Akt signaling pathways and AnnexinA2 and Galectin-1 receptors

Inflammatory responses mediated by glial cells play a critical role in many pathological situations related to neurodegeneration such as Alzheimer's disease. Tissue plasminogen activator (tPA) is a serine protease which best-known function is fibrinolysis, but it is also involved in many other physiological and pathological events as microglial activation. Here, we found that tPA is required for Aβ-mediated microglial inflammatory response and tumor necrosis factor-α release. We further investigated the molecular mechanism responsible for tPA-mediated microglial activation. We found that tPA induces a catalytic-independent rapid and sustained activation of extracellular signal-regulated kinase (ERK)1/2, Jun N-terminal kinase (JNK), Akt, and p38 signaling pathways. Inhibition of ERK1/2 and JNK resulted in a strong inhibition of microglial activation, whereas Akt inhibition led to increased inflammatory response, suggesting specific functions for each signaling pathway in the regulation of microglial activation. Furthermore, we demonstrated that AnnexinA2 and Galectin-1 receptors are involved in tPA signaling and inflammatory response in glial cells. This study provides new evidences supporting that tPA plays a cytokine-like role in glial activation by triggering receptor-mediated intracellular signaling circuits and opens new therapeutic strategies for the treatment of neurological disorders in which neuroinflammation plays a pathogenic role.     

Subtoxic N-methyl-D-aspartate delayed neuronal death in ischemic brain injury through TrkB receptor- and calmodulin-mediated PI-3K/Akt pathway activation

Previous studies have shown that subtoxic NMDA moderated the neuronal survival in vitro and vivo. We performed this experiment to clarify the precise mechanism underlie subtoxic NMDA delayed neuronal death in ischemic brain injury. We found that pretreatment of NMDA (100 mg/kg) increased the number of the surviving CA1 pyramidal cells of hippocampus at 5 days of reperfusion. This dose of NMDA could also enhance Akt activation after ischemia/reperfusion (I/R). Here, we examined the possible mechanism that NMDA induced Akt activation. On the one hand, we found NMDA receptor-mediated Akt activation was associated with increased expression of BDNF (brain-derived neurotrophic factor) and activation of its high-affinity receptor TrkB after I/R in the hippocampus CA1 region, which could be held down by TrkB receptor antagonist K252a. On the other hand, we found that NMDA enhanced the binding of Ca2+-dependent calmodulin (CaM) to p85 (the regulation subunit of PI-3K), which led to the activation of Akt. W-13, an active CaM inhibitor, prevented the combination of CaM and p85 and subsequent Akt activation. Furthermore, NMDA receptor-mediated Akt activation was reversed by combined treatment with LY294002, the specific blockade of PI-3K. Taken together, our results suggested that subtoxic NMDA exerts the neuroprotective effect via activation of prosurvival PI-3K/Akt pathway against ischemic brain injury, and BDNF-TrkB signaling and Ca2+-dependent CaM cascade might contribute to NMDA induced activation of PI-3K/Akt pathway.     

Characterization of cell death pathways in murine retinal neurodegeneration implicates cytochrome c release, caspase activation, and bid cleavage.

Apoptosis is considered to be the final common pathway of photoreceptor cell death in different inherited retinal diseases. However, apoptosis encompasses diverse pathways of molecular interactions culminating in cellular demise. To begin dissecting these interactions, we have investigated key participants in the rd (retinal degeneration) model of retinal neurodegeneration. By Western blot analysis and immunocytochemistry, we found that cytochrome c release occurs in rd retinas concurrently with the activation of the proapoptotic protein Bid. Active forms of caspase-8 and the mitogen-activated protein kinase p38, both of which are capable of cleaving Bid, were detected in rd retinas at the peak time of photoreceptor death. In addition, the activated form of the cell death effector caspase-3 was detectable particularly at the photoreceptors in parallel with this peak degenerative phase. These data suggest that activation of both major apoptotic pathways occurs during photoreceptor degeneration in the rd mouse model of inherited blindness.     

(-)-Epigallocatechin gallate inhibits lipopolysaccharide-induced microglial activation and protects against inflammation-mediated dopaminergic neuronal injury

Microglial activation is believed to play a pivotal role in the selective neuronal injury associated with several neurodegenerative disorders, including Parkinson's disease (PD) and Alzheimer's disease. We provide evidence that (-)-epigallocatechin gallate (EGCG), a major monomer of green tea polyphenols, potently inhibits lipopolysaccharide (LPS)-activated microglial secretion of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) through the down-regulation of inducible NO synthase and TNF-alpha expression. In addition, EGCG exerted significant protection against microglial activation-induced neuronal injury both in the human dopaminergic cell line SH-SY5Y and in primary rat mesencephalic cultures. Our study demonstrates that EGCG is a potent inhibitor of microglial activation and thus is a useful candidate for a therapeutic approach to alleviating microglia-mediated dopaminergic neuronal injury in PD.