Ceramide-induced cell death in primary neuronal cultures: upregulation of ceramide levels during neuronal apoptosis

Ceramide is a sphingolipid that has been implicated both in apoptosis and protection from cell death. We show that in both rat cerebellar granule cells and cortical neuronal cultures application of C(2)-ceramide causes cell death in a dose- and time-dependent manner. Similar effects were observed with the exogenous application of bacterial sphingomyelinase, which hydrolyzes sphingomyelin located on the outer leaflet of the plasma membrane and leads to endogenous ceramide accumulation. Furthermore, endogenous ceramide levels were increased during apoptosis induced by nutrient deprivation or etoposide treatment. These findings suggest that upregulation of ceramide levels, which may be generated through activation of sphingomyelinase, contributes to neuronal apoptosis.     

Induction of neuronal cell death by paraneoplastic Ma1 antigen

Paraneoplastic Ma1 (PNMA1) is a member of a family of proteins involved in an autoimmune disorder called paraneoplastic neurological syndrome. Although it is widely expressed in brain, nothing is known about the function of PNMA1 in neurons. We find that PNMA1 expression is highest in the perinatal brain, a period during which developmentally regulated neuronal death occurs. PNMA1 expression increases in cerebellar granule neurons (CGNs) induced to die by low potassium (LK) and in cortical neurons following homocysteic acid (HCA) treament. Elevated PNMA1 expression is also observed in the degenerating striatum in two separate mouse models of Huntington's disease, the R6/2 transgenic model and the 3-nitropropionic acid-induced chemical model. Suppression of endogenous PNMA1 expression inhibits LK-induced neuronal apoptosis. Ectopic expression of PNMA1 promotes apoptosis even in medium containing high potassium, a condition that normally ensures survival of CGNs. Deletion of the N-terminal half of the PNMA1 protein abrogates its apoptotic activity, whereas deletion of the C-terminal half renders the protein more toxic. Within the N-terminal half, the ability to induce neuronal death depends on the presence of a BH3-like domain. In addition to being necessary for apoptosis, the BH3-like domain is necessary for self-association of PNMA1. Apoptosis by PNMA1 expression is inhibited by overexpression of Bcl2, suggesting that PNMA1-induced neuronal death may depend on the binding of a proapoptotic member of the Bcl2 family to the BH3 domain. Taken together, our results suggest that PNMA1 is a proapoptotic protein in neurons, elevated expression of which may contribute to neurodegenerative disorders.     

p21-Activated kinase-1 is necessary for depolarization-mediated neuronal survival

Cerebellar granule neurons undergo apoptosis when switched from culture medium containing high potassium (HK) to medium that contains low potassium (LK). HK treatment leads to an activation of p21-activated kinase-1 (PAK-1). Overexpression of a constitutively active form of PAK-1 protects against apoptosis in LK medium. Overexpression of a dominant-negative form of PAK-1 blocks survival in HK. Although PAK-1 is usually considered to be a downstream effector of Rac and Cdc42, we were unable to detect association between PAK-1 and either Rac1 or Cdc42 in cerebellar granule neurons. Interaction between PAK-1 and PDK1 is detected in granule neurons, although there is no change in the extent of interaction in neurons primed to die. Neuronal survival by PAK-1 overexpression is not inhibited by PD98059 or LY294002, which inhibit the activity of MEK and PI-3 kinase, respectively. The ability of PAK-1 to maintain neuronal survival is, however, blocked by ML-9, a compound known to inhibit Akt. Our results show that that PAK-1 is necessary for neuronal survival in HK and suggest that its neuroprotective action may be mediated by a GTPase-independent, but Akt-dependent, mechanism.     

Role of oxidative stress in the apoptotic cell death of cultured cerebellar granule neurons

When cultured cerebellar granule neurons (CGN) are transferred from 25 mM KCl (K25) to 5 mM KCl (K5) caspase-3 and caspase-8, but not caspase-1 or caspase-9,activities are induced and cells die apoptotically. CGN death was triggered by a [Ca(2+)](i) modification when [Ca(2+)](i) was reduced from 300 nM to 50 nM in a K5 medium. The [Ca(2+)](i) changes were followed by an increase in ROS levels. The generation of both cytosolic and mitochondrial reactive oxygen species (ROS) occurred at three different times, 10 min, 30 min and 3--4 hr but only those ROS produced after 3--4 hr are involved in the process of cell death. When CGN cultured in a K5 medium are treated with different antioxidants like scavengers of ROS (mannitol, DMSO) or antioxidant enzymes (superoxide dismutase and catalase) phosphatidylserine translocation, caspase activity, chromatin condensation and cell death is markedly diminished. The protective effect of antioxidants is not mediated through a modification in [Ca(2+)](i). Caspase activation, PS translocation and chromatin condensation were downstream of ROS production. In contrast to H(2)O(2), ROS produced by a xanthine/xanthine oxidase system in CGN cultured in K25 were able to directly induce caspase-3 activation and death that resulted sensitive to z-VAD, a caspase inhibitor. These findings indicate that a reduction in [Ca(2+)](i) triggers CGN death by inducing a generation of ROS after 3--4 hr, which could play a critical role in the initial phases of the apoptotic process including PS translocation, chromatin condensation and the activation of initiator and executor caspases.     

Up-regulation of lysosomal cathepsin L and autophagy during neuronal death induced by reduced serum and potassium

Serum and potassium deprivation-induced neuronal death on the primary culture of rat cerebellar granule neurons is being widely used as an in vitro model of neurodegeneration and neuronal apoptosis. In our experiments, serum and potassium deprivation for 12 h induced neuronal death in approximately 20% of cerebellar granule neurons as demonstrated by Trypan Blue assay. Neuronal death was accompanied by a transient increase in the intralysosomal cathepsin L activity, which preceded neuronal death. During this time, the lysosomal membrane integrity remained preserved and no leakage of cathepsin L into the cytosol was seen. Ultrastructural analysis revealed the appearance of multiple vacuoles and autophagosomes in the cytoplasmatic compartment of serum- and potassium-deprived granule neurons. Addition of selective cathepsin L inhibitors or of the autophagy inhibitor 3-methyladenine provided partial protection against serum and potassium deprivation-induced death. Our data also show that combining cathepsin L inhibitors and caspase-3 inhibitors leads to a synergistic neuroprotective effect against serum and potassium deprivation. The results of the current study suggest that activation of the autophagosomal--lysosomal compartment plays an important role in neuronal death induced by serum and potassium deprivation in cultured cerebellar granule cells.     

Conditional deletion of histone deacetylase‐4 in the central nervous system has no major effect on brain architecture or neuronal viability

Evidence from different laboratories using cell culture and in vivo model systems indicates that histone deacetylase‐4 (HDAC4) plays an essential role in maintaining neuronal survival. Indeed, HDAC4 null knockout mice, which die within 2 weeks of birth, display cerebellar degeneration, whereas RNAi‐mediated knockdown of HDAC4 expression in the retina of normal mice leads to apoptosis of retinal neurons. As a step toward analyzing the role of HDAC4 in the regulation of neuronal survival in more detail, we generated two separate lines of conditional knockout mice by breeding HDAC4‐flox mice with mice expressing Cre recombinase through a Thy1 or nestin promoter. Surprisingly, both Thy1‐Cre/HDAC4^?/? mice, in which HDAC4 is ablated in neurons of the cortex and hippocampus, as well as Nes‐Cre/HDAC4^?/? mice, in which HDAC4 is ablated in neural progenitor cells of the CNS, appear normal at birth, have normal body weight, are fertile, and perform normally in locomotor activity assays. Histological analysis of the brains of Nes‐Cre/HDAC4^?/? mice revealed no obvious abnormalities in cytoarchitecture. Immunohistological analysis of tyrosine hydroxylase and calbindin also showed no discernible defects. Terminal deoxynucleotidyl transferase dUTP nick end‐labeling staining showed no difference in the level of neuronal death in the cortex and cerebellum of Nes‐Cre/HDAC4^?/? mice compared with controls. These results indicate that neurons are less dependent on HDAC4 expression for their survival than previously believed and suggest that neuronal death observed in HDAC4 null knockout mice and after RNAi injection may result from HDAC4 deficiency in nonneural cells. © 2012 Wiley Periodicals, Inc.     

Balancing neuronal death

Although it is apparent that neuronal death must be tightly regulated to ensure the proper development and mature functions of the nervous system, the molecular details of this regulation are not fully understood. In multiple neurodegenerative diseases, there is inappropriate death of cells in the nervous system. A better understanding of how death is regulated in the normal nervous system can provide a framework for determining how this regulation can go awry during neurodegenerative disease. The key executioners of neuronal apoptosis, the caspases, are regulated at several levels. The endogenous inhibitor of apoptosis family of proteins, the IAPs, can suppress caspase activity. In this Mini-Review, we examine what is known about the function of IAPs in normal neuronal function and in disease.     

Taurine deficiency in dissociated mouse cerebellar cultures affects neuronal migration

The role of taurine in the process of neuronal migration was studied in a microwell cell culture system. Immunocytochemical analysis of the cellular composition of this culture system revealed the presence of the astrocytic marker GFAP in some structures such as the aggregates of neuronal bodies and in those cables used for migration, resembling what is described in vivo. The neuronal marker γ-enolase stained practically all structures, including the aggregates and all cables. The intracellular taurine concentration was reduced by 60% in mouse cerebellar granule cells treated with a blocker of taurine transport, guanidinoethane sulfonate (GES). Under these conditions cell migration was markedly reduced to approximately 50% of that in untrated cultures. Both, taurine depletion and impairment of cell migration induced by GES were prevented by adding taurine to the culture medium. Taurine deficiency similarly affected different morphological parameters such as the number of cables suitable for neuronal migration as well as the number of migrating neurons. The number of aggregates of neuronal bodies was significantly increased, by about 30%, as a consequence of the reduced migration. Taurine alone did not exert any effect on the parameters evaluated. GES treatment of granule cells did not affect mitochondrial metabolism or K⁺-stimulated Ca²⁺-dependent [³H]-d-aspartate release. This suggests that the described effects of taurine deficiency were not due to an alteration of neuronal viability and that the action of GES was not simply due to unspecific and deleterious effects. These results are in agreement with those obtained in in vivo studies. This approach represents a useful model to investigate the role played by taurine in the process of neuronal migration.     

Induction of neuronal death by alpha-synuclein

The molecular and cellular mechanisms underlying neuronal loss in neurodegenerative diseases are unclear. It is generally thought that aggregation of mutated, abnormally modified or abnormally folded proteins leads to the accumulation of extracellular, intracellular or intranuclear deposits that severely compromise cell physiology, leading to the death of the affected neurons. However, there is growing evidence that neuronal apoptosis in the absence of obvious pathological deposits could have a serious impact on the pathogenesis of neurodegenerative diseases. alpha-Synuclein has been implicated in aetiology and pathogenesis of certain neurodegenerative diseases, although the precise role of this protein in neurodegeneration is uncertain. The normal functions of alpha-synuclein and other members of the synuclein family in the development and function of the nervous system also remain elusive. Here we show that overexpression of wild-type and mutant forms of alpha-synuclein in cultured neurons, but not the closely related persyn (gamma-synuclein), causes apoptosis. These findings suggest that abnormalities of alpha-synuclein metabolism could lead to the neuronal loss occurring in certain forms of neurodegeneration before the formation of characteristic pathological lesions.     

Metabotropic glutamate receptors prevent nitric oxide-induced programmed cell death

Activation of metabotropic glutamate receptor (mGluR) subtypes can prevent neuronal injury through the signal transduction pathways of nitric oxide (NO). It is this link to NO free radical injury and subsequent DNA damage that is the most intriguing. We therefore examined whether neuronal protection through mGluR activation was dependent on the molecular mechanisms of programmed cell death (PCD). The NO generators sodium nitroprusside and 3-morpholino-sydnonimine were administered to induce NO toxicity in primary hippocampal neurons. PCD was documented by hematoxylin and eosin nuclear staining, DNA gel electrophoresis, transmission electron microscopy, and protein synthesis assays. Following NO exposure, PCD induction was rapid and robust in approximately 70% of the neuronal population. Activation of specific mGluR subtypes with 1S,3R-ACPD and L-AP4, agents that are neuroprotective against NO, significantly limited the progression of PCD. In contrast, antagonism of mGluRs with L-AP3 did not prevent the development of PCD. Induction of new protein synthesis, a common requisite for PCD, was evident following NO exposure, but did not appear to represent a principal pathway of modulation by the mGluR agonists. Our studies suggest that mGluR modulation of NO-induced PCD represents a primary molecular pathway responsible for neuronal survival. Further elucidation of the molecular mGluR signaling pathways may yield new insight into specific genetic regulatory mechanisms responsible for neuronal injury. J. Neurosci. Res. 50: 549–564, 1997. © 1997 Wiley-Liss, Inc.     

磷酸化C—JUN不参与谷氨酸诱导的小脑颗粒神经元凋亡

观察磷酸化 C- JUN与谷氨酸诱导的小脑颗粒神经元凋亡的关系。在培养的小脑颗粒神经元建立谷氨酸凋亡模型 ;采用 MTT法分析细胞存活率 ,相差显微镜观察形态学 ,DNA凝胶电泳法分析细胞凋亡和原位细胞荧光免疫组织化学法检测磷酸化 C- JUN。结果显示 ,谷氨酸诱导大鼠小脑颗粒神经元细胞体积缩小 ,突触断裂、消失 ,DNA电泳呈典型的“梯状”条带 ;谷氨酸处理 2 4 h后细胞存活率为 2 8.6%± 5.2 %。神经元在谷氨酸处理 5,30 min及 1 ,2 ,4,8,1 6和 2 4 h后均未检测到有磷酸化 C- JUN阳性细胞 ,与去极化组 ( 2 5mmol/L KCl)相同。而复极化组 ( 5mmol/L KCl)则在 30 min检测到大量的磷酸化 C- JUN阳性细胞 ,4h荧光最强并持续。处理 4h后 ,40 0倍荧光显微镜下 ,复极化组、去极化组和谷氨酸组的磷酸化 C- JUN阳性细胞数分别为 1 2 4± 1 7,8± 3,5± 3。上述结果提示 ,谷氨酸诱导小脑颗粒神经元凋亡 ,磷酸化 C- JUN不参与谷氨酸诱导的大鼠小脑颗粒神经元凋亡。     

Caspase activation pathways induced by staurosporine and low potassium: role of caspase-2

Apoptotic death is a physiological process with regulatory mechanisms that are under the control of different molecules such as caspases. These are classified as initiators, such as caspases-8 and -9, and effectors, such as caspases-3 and -7. The participation of caspase-2 in the effector phase of apoptosis has been commonly observed in many cell types; however, it is able to act as an initiator caspase, depending on the apoptotic stimulus. Cerebellar granule cells (CGCs) undergo apoptosis when they are transferred from high potassium (K25) to low potassium (K5); this process seems to be mediated by caspase-3 activation. Staurosporine (STS), a full strength inhibitor of kinase proteins, also induces apoptosis in these cells. To characterize the caspase cascade induced by two stimuli in the same cell type we studied the activation of different caspases in CGCs treated with STS or K5. We found that both K5 and STS induce the activation of caspase-3. This result was confirmed by the proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), an endogenous caspase-3 substrate. Caspase-2 was activated preferentially by STS, which showed a temporal course suggesting that this caspase was induced before caspase-3. The initiator caspase-9 was also activated by both K5 and STS, as well as cytochrome-c release. The results obtained in this study suggest that STS and K5 induced different activation caspase pathways for apoptotic cell death of CGCs.     

Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death

We have shown previously that pigment epithelium-derived factor (PEDF) acts as a survival factor for cerebellar granule cell neurons in culture, as well as protecting them against glutamate toxicity. In this study we have examined effects of PEDF on apoptotic cell death. We find that the granule cells die of apoptosis throughout the culture period, what we have termed “natural” apoptosis. PEDF prevents this natural apoptosis if added to immature cells, within the first 2 days in vitro (DIV), and the effect is maintained for up to DIV12. However, PEDF has no effect if added to mature cells at DIV5. Similar results are obtained when apoptosis is induced by shifting the cells from a serum- and 25 mM KCl-containing medium to serum-free medium with 5 mM KCl. PEDF most effectively blocks induced apoptosis in immature cells (DIV2) when added 24 hr prior to the change of medium, but still provides some protection when added simultaneously. However, 24 hr pretreatment with PEDF has a minimal effect when apoptosis is induced in mature DIV6 cells; addition at the same time is completely ineffective. Two polypeptide fragments of PEDF, only one of which contains the serine-protease inhibitory site, are equally active, supporting previous results which suggest that the neurotrophic effects of PEDF are not mediated by protease inhibition. We conclude that PEDF protects immature but not mature granule cells against both natural and induced apoptosis. J. Neurosci. Res. 53:7–15, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Akt mediates the anti-apoptotic effect of NMDA but not that induced by potassium depolarization in cultured cerebellar granule cells

Apoptosis of cultured cerebellar granule neurons (CGNs) deprived of serum is prevented by K+ depolarization or moderate concentrations of N-methyl-d-aspartate (NMDA). Here, we have examined the role of the serine/threonine kinase Akt in these protective effects. The exposure of mouse CGNs to NMDA or K+ depolarization increased the phosphorylation of Akt, compared with that measured in cells incubated in a physiological K+ concentration. Only the NMDA-evoked response was reduced by inhibitors of phosphatidylinositol 3-kinase (wortmannin and LY294002) and mitogen-activated protein kinase (PD98059 and U0126). Similarly, the capacity of NMDA to inhibit apoptosis of CGNs deprived of serum was greatly reduced by these inhibitors as well as by the transfection of neurons with a catalytically inactive mutant of Akt, whereas the protective effect of K+ depolarization remained unaffected. These findings indicate that K+ depolarization and NMDA activate Akt through different signalling pathways in CGNs. Moreover, Akt mediates the anti-apoptotic effect of NMDA, but not that evoked by K+ depolarization.     

Ataxia Jackson (<Emphasis Type="Italic">ax</Emphasis><Superscript><Emphasis Type="Italic">J</Emphasis></Superscript>): a genetic model for apoptotic neuronal cell death

Programmed cell death or apoptosis is an important process to form normal adult cytoarchitecture. But in vivo analysis of neuronal apoptosis has not been well advanced. Therefore, apoptotic cell death of a particular neuronal system or anatomical part in a mutant is an invaluable target to learn about a link between a gene and neuronal apoptosis. Ataxia (ax) is an autosomal recessive neurological mutant mouse. We recently investigated brains of homozygotes for ataxia Jackson (ax(J)), an allele of ax, using TUNEL method. A few TUNEL-positive cells were observed in the granular cell layer of the cerebellum, the dentate gyrus, and the olfactory bulb of phenotypically normal littermates (ax(J)/+ or +/+) aged at 23-38 days. In affected ax(J)/ax(J) mice, however, the number of TUNEL-positive cells was significantly increased in the cerebellum, particularly in the granular cell layer (p < 0.05). The ax(J) mouse will be an in vivo unique model for studies on the genetic basis of apoptotic neuronal cell death, and identification of the ax gene is desired to elucidate molecular basis of the apoptosis.     

Neuronal NOS activation during oxygen and glucose deprivation triggers cerebellar granule cell death in the later reoxygenation phase

The present study investigated the temporal relationship between neuronal nitric oxide synthase (nNOS) activity and expression and the development of neuronal damage occurring during anoxia and anoxia followed by reoxygenation. For this purpose, cerebellar granule cells were exposed to 2 hr of oxygen and glucose deprivation (OGD) and 24 hr of reoxygenation. To clarify the consequences of nNOS activity inhibition on neuronal survival, cerebellar granule cells were exposed to OGD, both in the absence of extracellular Na(+) ([Na(+)](e)), a condition that by reducing intracellular Ca(2+) ([Ca(2+)](I)) prevents Ca(2+)-dependent nNOS activation, and in the presence of selective and nonselective nNOS inhibitors, such as N(omega)-L-allyl-L-arginine (L-ALA), N(omega)-propyl-L-arginine (NPLA), and L-nitro-arginine-methyl-ester (L-NAME), respectively. The results demonstrated that the removal of [Na(+)](e) hampered the [Ca(2+)](i) increase and decreased expression and activity of nNOS. Similarly, the increase of free radical production present in cerebellar neurons, exposed previously to OGD and OGD/reoxygenation, was abolished completely in the absence of [Na(+)](e). Furthermore, the absence of [Na(+)](e) in cerebellar neurons exposed to 2 hr of OGD led to the improvement of mitochondrial activity and neuronal survival, both after the OGD phase and after 24 hr of reoxygenation. Finally, the exposure of cerebellar neurons to L-ALA (200 nM), and L-NAME (500 microM) was able to effectively reduce NO(*) production and caused an increase in mitochondrial oxidative activity and an improvement of neuronal survival not only during OGD, but also during reoxygenation. Similar results during OGD were obtained also with NPLA (5 nM), another selective nNOS inhibitor. These data suggest that the activation of nNOS is highly accountable for the neuronal damage occurring during the OGD and reoxygenation phases.     

Characterization of neuronal death induced by focally evoked limbic seizures in the C57BL/6 mouse

Research into the molecular mechanisms of epileptic brain injury is hampered by the resistance of key mouse strains to seizure-induced neuronal death evoked by systemically administered excitotoxins such as kainic acid. Because C57BL/6 mice are extensively employed as the genetic background for transgenic/knockout modeling in cell death research but are seizure resistant, we sought to develop a seizure model in this strain characterized by injury to the hippocampal CA subfields. Adult male C57BL/6 mice underwent focally evoked seizures induced by intraamygdala microinjection of kainic acid. Kainic acid (KA) effectively elicited ipsilateral CA3 pyramidal neuronal death within a narrow dose range of 0.1-0.3 microg, with mortality < 10%. With employment of the most consistent (0.3 microg) dose, seizures were terminated 15, 30, 60, or 90 min after KA by diazepam. Damage was largely restricted to the ipsilateral CA3 subfield of the hippocampus, but injury was also consistent within CA1, suggesting that this mouse model better reflects the hippocampal neuropathology of human temporal lobe epilepsy than does the rat, in which CA1 is typically spared. Confirming this CA1 injury as seizure specific and not a consequence of ischemia, we used laser-Doppler flowmetry to determine that cerebral perfusion did not significantly change (97% to 118%) over control. Degenerating cells were > 95% neuronal as determined by neuron-specific nuclear protein (NeuN) counterstaining of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeled (TUNEL) brain sections. Furthermore, TUNEL-positive cells often exhibited the morphological features of apoptosis, and small numbers were positive for cleaved caspase-3. These data establish a mouse model of focally evoked seizures in the C57BL/6 strain associated with a restricted pattern of apoptotic neurodegeneration within the hippocampal subfields that may be applied to research into the molecular basis of neuronal death after seizures.     

Vitamin K has the potential to protect neurons from methylmercury-induced cell death in vitro

Vitamin K (VK) has a protective effect on neural cells. Methylmercury is a neurotoxicant that directly induces neuronal death in vivo and in vitro. Therefore, in the present study, we hypothesized that VK inhibits the neurotoxicity of methylmercury. To prove our hypothesis in vitro, we investigated the protective effects of VKs (phylloquinone, vitamin K(1); menaquinone-4, vitamin K(2) ) on methylmercury-induced death in primary cultured neurons from the cerebella of rat pups. As expected, VKs inhibited the death of the primary cultured neurons. It has been reported that the mechanisms underlying methylmercury toxicity involve a decrement of intracellular glutathione (GSH). Actually, treatment with GSH and a GSH inducer, N-acetyl cysteine, inhibited methylmercury-induced neuronal death in the present study. Thus, we investigated whether VKs also have protective effects against GSH-depletion-induced cell death by employing two GSH reducers, L-buthionine sulfoximine (BSO) and diethyl maleate (DEM), in primary cultured neurons and human neuroblastoma IMR-32 cells. Treatment with VKs affected BSO- and DEM-induced cell death in both cultures. On the other hand, the intracellular GSH assay showed that VK(2), menaquinone-4, did not restore the reduced GSH amount induced by methylmercury or BSO treatments. These results indicate that VKs have the potential to protect neurons against the cytotoxicity of methylmercury and agents that deplete GSH, without increasing intracellular GSH levels. The protective effect of VKs may lead to the development of treatments for neural diseases involving GSH depletion.