Evidence that Glyceraldehyde-3-Phosphate Dehydrogenase Is Involved in Age-Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.     

Adenovirus-Mediated Gene Transfer of Inhibitors of Apoptosis Proteins Delays Apoptosis in Cerebellar Granule Neurons

Abstract : The inhibitor of apoptosis (IAP) family of anti-apoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 m M potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 m M potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N -acetly-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, not at later time points suggesting that IAPS delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 μ M or 1 m M glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

ERK activation promotes neuronal degeneration predominantly through plasma membrane damage and independently of caspase-3

Our recent studies have shown that extracellular-regulated protein kinase (ERK) promotes cell death in cerebellar granule neurons (CGN) cultured in low potassium. Here we report that the "death" phenotypes of CGN after potassium withdrawal are heterogeneous, allowing the distinction between plasma membrane (PM)-, DNA-, and PM/DNA-damaged populations. These damaged neurons display nuclear condensation that precedes PM or DNA damage. Inhibition of ERK activation either by U0126 or by dominant-negative mitogen-activated protein kinase/ERK kinase (MEK) overexpression results in a dramatic reduction of PM damaged neurons and nuclear condensation. In contrast, overexpression of constitutively active MEK potentiates PM damage and nuclear condensation. ERK-promoted cellular damage is independent of caspase-3. Persistent active ERK translocates to the nucleus, whereas caspase-3 remains in the cytoplasm. Antioxidants that reduced ERK activation and PM damage showed no effect on caspase-3 activation or DNA damage. These data identify ERK as an important executor of neuronal damage involving a caspase-3-independent mechanism.     

Apoptosis-inducing and -preventing signal transduction pathways in cultured cerebellar granule neurons

1. Cultured cerebellar granule neurons maintained in medium containing 26 mM potassium (high K+ or HK+) undergo cell death when switched to medium with 5 mM potassium (low K+ or LK+). This low K(+)-induced cell death has typical features of apoptosis. The intracellular signaling pathway of low K(+)-induced apoptosis has been investigated. 2. Cerebellar granule neurons become committed to undergo apoptosis between 2 and 5 h after K+ deprivation, judging from the inability of high K+ to rescue them after this time. Although the levels of most mRNAs decrease markedly concomitant with commitment, expression of c-jun mRNA increases 2-3 h after K+ deprivation. Among the family of caspases, a caspase-3-like protease is activated within 4 h of lowering the K+ concentration. A caspase-1-like protease is also activated within 2 h of K+ deprivation. 3. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity by LY294002 or wortmannin also induces apoptosis in cerebellar granule neurons. The intracellular signaling pathway of LY294002-induced apoptosis has been investigated. The activity of c-Jun N-terminal kinase (JNK) increases 8 h after addition of LY294002 to high K+ medium or low K+ medium containing BDNF. Expression of c-Jun protein also increases almost simultaneously. 4. The low K(+)-induced apoptosis of cerebellar granule neurons is prevented by high K+ (membrane depolarization by high K+), BDNF, IGF-1, bFGF or cAMP. The intracellular signaling pathways by which these agents prevent low K(+)-induced apoptosis have been investigated. Agents other than cAMP prevent apoptosis through PI3-K and a Ser/Thr kinase, Akt/PKB. The survival-promoting effect of cAMP does not depend on the PI3-K-Akt pathway.     

Alix, a protein regulating endosomal trafficking, is involved in neuronal death

Alix/AIP1 is a cytoplasmic protein, which was first characterized as an interactor of ALG-2, a calcium-binding protein necessary for cell death. Alix has also recently been defined as a regulator of the endo-lysosomal system. Here we have used post-mitotic cerebellar neurons to test Alix function in caspase-dependent and -independent cell death. Indeed, these neurons survived when cultured in 25 mm potassium-containing medium but underwent apoptosis soon after the extracellular potassium was lowered to 5 mm. In agreement with other studies, we show that caspases are activated after K+ deprivation, but that inhibition of these proteases, using the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone, has no effect on cell survival. Transfection experiments demonstrated that Alix overexpression is sufficient to induce caspase activation, whereas overexpression of its C-terminal half, Alix-CT, blocks caspase activation and cell death after K+ deprivation. We also define a 12-amino acid PXY repeat of the C-terminal proline-rich domain necessary for binding ALG-2. Deletion of this domain in Alix or in Alix-CT abolished the effects of the overexpressed proteins on neuronal survival, demonstrating that the ALG-2-binding region is crucial for the death-modulating function of Alix. Overall, these findings define the Alix/ALG-2 complex as a regulator of cell death controlling both caspase-dependent and -independent pathways. They also suggest a molecular link between the endo-lysosomal system and the effectors of the cell death machinery.     

Roles of carbonic anhydrase 8 in neuronal cells and zebrafish

Background

Carbonic anhydrase 8 (CA8) is an isozyme of α-carbonic anhydrases (CAs). Previous studies showed that CA8 can be detected in human adult brain, with more intense expression in the cerebellum. Single mutations in CA8 were reported to cause novel syndromes like ataxia, mild mental retardation or the predisposition to quadrupedal gait.

Methods

In the present study, we examine the functions of CA8 in neuronal cell lines, mouse cerebellar granule neurons and zebrafish.

Results and conclusions

We demonstrated that overexpression of CA8 in neuronal cells significantly decreased cell death under staurosporine treatment. Moreover, CA8 overexpression significantly increased cell migration and invasion ability in neuronal cells and in mouse cerebellar granule neurons, implicating that CA8 may be involved in neuron motility and oncogenesis. By using zebrafish as an animal model, motor reflection of 3 dpf zebrafish embryos was significantly affected after the down-regulation of CA8 through ca8 morpholino.

Conclusions

We concluded that CA8 overexpression desensitizes neuronal cells to STS induced apoptotic stress and increases cell migration and invasion ability in neuronal cells. In addition, down-regulated CA8 decreases neuron mobility in neuronal cells and leads to abnormal calcium release in cerebellar granule neurons. Knockdown of the ca8 gene results in an abnormal movement pattern in zebrafish.

General significance

Our findings provide evidence to support that the impaired protective function of CA8 contributes to human neuropathology, and to suggest that zebrafish can be used as an animal model to study the biological functions of human CA8 in vivo.     

Identification of a novel kinase-like gene induced during neuronal cell death.

When deprived of neurotrophic factors, neuronal cells undergo a form of programmed cell death that involves a cascade of gene expression. To better understand this cascade, we screened the genes induced during programmed cell death evoked in neuronal PC6-3 cells by NGF-depletion and discovered a novel gene, NIPK (Neuronal cell death Inducible Putative Kinase), that contains a kinase-like domain. Expression of NIPK was also induced in cultured sympathetic neurons by NGF deprivation and in cortical neurons exposed to the Ca2+ ionophore, A23187. In contrast, NIPK was not induced during non-neuronal cell death evoked by serum or growth factor deprivation, or by treatment with methyl methanesulfonate, an agent that causes cell death by damaging DNA. Taken together, these findings suggest that NIPK is involved in programmed cell death via a pathway that is present in neurons but is absent in non-neurons.     

Genomic profiling of cortical neurons following exposure to beta-amyloid

In vitro and in vivo studies have shown that beta-amyloid peptide induces neuronal cell death. To explore the molecular basis underlying beta-amyloid-induced toxicity, we analyzed gene expression profiles of cultured rat cortical neurons treated for 24 and 48 h with synthetic beta-amyloid peptide. From the 8740 genes interrogated by oligonucleotide microarray analysis, 241 genes were found to be differentially expressed and segregated into distinct clusters. Functional clustering based on gene ontologies showed coordinated expression of genes with common biological functions and metabolic pathways. The comparison with genes differentially expressed in cerebellar granule neurons following serum and potassium deprivation indicates the existence of common regulatory mechanisms underlying neuronal cell death. Our results offer a genomic view of the changes that accompany beta-amyloid-induced neurodegeneration.     

Excitotoxic and apoptotic neuronal death induce different patterns of glial activation in vitro

We have studied glial activation in rat cerebellar neuronal-glial cultures after inducing neuronal death using various stimuli. Cultures were exposed to 100 microm glutamate for 20 min, which induces excitotoxic neuronal death, or to potassium/serum deprivation, which induces apoptosis of granule neurons. We evaluated alterations in several parameters related to glial activation: nuclear factor-kappaB activation, nitric oxide and tumour necrosis factor-alpha production, which are associated with a pro-inflammatory response, glial proliferation and phagocytic activity. Although the two experimental models of neuronal damage resulted in the death of most neuronal cells within 24 h, differences were observed in the response of the various glial parameters evaluated. While nitric oxide production was not detected in any case, tumour necrosis factor-alpha production, nuclear factor-kappaB activation and glial proliferation were only induced in the presence of excitotoxic neuronal death. However, phagocytosis was induced in both cases, although earlier in the case of apoptotic neuronal death. These results show that glial cells respond to excitotoxic neuronal death with an inflammatory response associated with proliferation and phagocytosis. In contrast, whilst glial cells do not produce pro-inflammatory molecules in the presence of apoptotic neuronal death, phagocytic activity is rapidly induced.     

Molecular morphology of neuronal apoptosis: Analysis of caspase 3 activation during postnatal development of mouse cerebellar cortex

We have used the mammalian post-natal cerebellar cortex as a model to dissect out the molecular morphology of neuronal apoptosis in a well-defined population of central neurons: the cerebellar granule cells. By immunocytochemistry, in situ labeling of apoptotic cells, and analysis of cerebellar slices following particle-mediated gene transfer (biolistics), we have studied the relationship of cell death and cleavage of caspase 3, a key molecule in the execution of apoptosis, and monitored caspase 3 activation in living cells. Our results demonstrate the existence of caspase dependent and independent apoptotic pathways affecting the cerebellar granule cells at different stages of their life. Apoptosis of proliferating precursors and young pre-migratory cells occurs in the absence of caspase 3 cleavage, whereas cell death of post-mitotic post-migratory neurons is directly linked to caspase 3 activation. Data obtained from cerebellar cortex can be generalized to outline a more comprehensive picture of the cellular and molecular mechanisms of neuronal death not only in development, but also in a number of pathological conditions leading to neuronal loss.     

Microglial Response Factor (MRF)-1: Constitutive Expression in Ramified Microglia and Upregulation upon Neuronal Death Induced by Ischemia or Glutamate Exposure

We have isolated a new microglial gene, mrf-1, which is upregulated on microglia in response to apoptosis of granule neurons in cerebellar cell cultures. We examined whether or not upregulation of MRF-1 is observed in response to necrotic neuronal death both in vivo and in vitro. Though MRF-1 was detected on ramified/resting microglia in the brain of normal adult rats, activated microglia in the region of the brain where neuronal damage was induced by ischemia were strongly immunostained with anti-MRF-1 anti-body. In the in vitro system, we confirmed, with immunocytochemistry or RT-PCR, that MRF-1 or mrf-1 mRNA were constitutively expressed in ramified microglia at significant but lower levels than in amoeboid one. Moreover, by Northern blot, it was ascertained that expression level of mrf-1 mRNA on microglia was markedly upregulated in response to glutamate-induced death of granule cells in a cerebellar cell culture. These results indicate the following: 1) expression of mrf-1 in microglia may be markedly enhanced upon not only apoptotic but also necrotic neuronal death, and 2) MRF-1 is, thus, an useful marker for identifying all types of microglia in vivo and in vitro.     

Choline release and inhibition of phosphatidylcholine synthesis precede excitotoxic neuronal death but not neurotoxicity induced by serum deprivation

N-Methyl-d-aspartate (NMDA) receptor overactivation has been proposed to induce excitotoxic neuronal death by enhancing membrane phospholipid degradation. In previous studies, we have shown that NMDA releases choline and reduces membrane phosphatidylcholine in vivo. We now observed that glutamate and NMDA induce choline release in primary neuronal cortical cell cultures. This effect is Ca(2+)-dependent and is blocked by MK-801 ((+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate). In cortical neurons, the NMDA receptor-mediated choline release precedes excitotoxic cell death but not neuronal death induced by either osmotic lysis or serum deprivation. Glutamate, at concentrations that release arachidonic acid, does not release choline in cerebellar granule cells, unless these cells are rendered susceptible to excitotoxic death by energy deprivation. The NMDA-evoked release of choline is not mediated by phospholipases A(2) or C. Moreover, NMDA does not activate phospholipase D in cortical cells. However, NMDA inhibits incorporation of [methyl-(3)H]choline into both membrane phosphatidylcholine and sphingomyelin. These results show that the increase in extracellular choline induced by NMDA receptor activation is directly related with excitotoxic cell death and indicate that choline release is an early event of the excitotoxic process produced by inhibition of phosphatidylcholine synthesis and not by activation of membrane phospholipid degradation.