Loss of the Xeroderma Pigmentosum Group A Gene (XPA) Enhances Apoptosis of Cultured Cerebellar Neurons Induced by UV but Not by Low-K+ Medium

Abstract: To study the involvement of the xeroderma pigmentosum group A gene ( XPA ) in neuronal apoptosis, we cultured cerebellar neurons from mice lacking XPA gene ( XPA ^−/−) and induced apoptosis by exposure to UV irradiation or medium containing a low concentration of potassium (low-K⁺ medium). When cerebellar neurons from postnatal days 15–16 wild-type mice were treated with UV irradiation, apoptotic neuronal death was observed after 24–48 h. About 60% of neurons survived 48 h after UV irradiation at a dose of 5 J/m². On the other hand, neurons from XPA ^−/− mice showed a significantly increased vulnerability to UV irradiation, and >90% of neurons died 48 h after UV irradiation at a dose of 5 J/m². In contrast, low-K⁺ medium induced apoptosis of neurons from mice of each genotype with the same kinetics. These results suggest that the XPA gene is involved in neuronal DNA repair and that it thereby influences apoptosis induced by DNA damage in cultured cerebellar neurons.     

c-Jun contributes to amyloid beta-induced neuronal apoptosis but is not necessary for amyloid beta-induced c-jun induction

The role of gene expression in neuronal apoptosis may be cell- and apoptotic stimulus-specific. Previously, we and others showed that amyloid beta (Abeta)-induced neuronal apoptosis is accompanied by c-jun induction. Moreover, c-Jun contributes to neuronal death in several apoptosis paradigms involving survival factor withdrawal. To evaluate the role of c-Jun in Abeta toxicity, we compared Abeta-induced apoptosis in neurons from murine fetal littermates that were deficient or wild-type with respect to c-Jun. We report that neurons deficient for c-jun are relatively resistant to Abeta toxicity, suggesting that c-Jun contributes to apoptosis in this model. When changes in gene expression were quantified in neurons treated in parallel, we found that Abeta treatment surprisingly led to an apparent activation of the c-jun promoter in both the c-jun-deficient and wild-type neurons, suggesting that c-Jun is not necessary for activation of the c-jun promoter. Indeed, several genes induced by Abeta in wild-type neurons were also induced in c-jun-deficient neurons, including c-fos, fosB, ngfi-B, and ikappaB. In summary, these results indicate that c-Jun contributes to Abeta-induced neuronal death but that c-Jun is not necessary for c-jun induction.     

Intracellular amyloid-beta 1-42, but not extracellular soluble amyloid-beta peptides, induces neuronal apoptosis

Alzheimer disease (AD), the most frequent cause of dementia, is characterized by an important neuronal loss. A typical histological hallmark of AD is the extracellular deposition of beta-amyloid peptide (A beta), which is produced by the cleavage of the amyloid precursor protein (APP). Most of the gene mutations that segregate with the inherited forms of AD result in increasing the ratio of A beta 42/A beta 40 production. A beta 42 also accumulates in neurons of AD patients. Altogether, these data strongly suggest that the neuronal production of A beta 42 is a critical event in AD, but the intraneuronal A beta 42 toxicity has never been demonstrated. Here, we report that the long term expression of human APP in rat cortical neurons induces apoptosis. Although APP processing leads to production of extracellular A beta 1-40 and soluble APP, these extracellular derivatives do not induce neuronal death. On the contrary, neurons undergo apoptosis as soon as they accumulate intracellular A beta 1-42 following the expression of full-length APP or a C-terminal deleted APP isoform. The inhibition of intraneuronal A beta 1-42 production by a functional gamma-secretase inhibitor increases neuronal survival. Therefore, the accumulation of intraneuronal A beta 1-42 is the key event in the neurodegenerative process that we observed.     

Induction of the Serotonin1A Receptor in Neuronal Cells During Prolonged Stress and Degeneration

Abstract: Neuronal migration in brain is followed by differentiation of committed neurons and simultaneous apoptosis of uncommitted preneuronal cells due to a limiting supply of trophic factors and nutrients. We have dissected differentiation and apoptosis by designing a simple in vitro model for this nutrient deprivation using engineered neuronal cell lines stably transfected with a promoterless segment (G-21) of the intronless human serotonin_1A receptor (5-HT_1A-R) gene. Despite the use of widely different heterologous promoters (cytomegalovirus and Rous sarcoma virus) for the stable expression of G-21, a dramatic increase in expression of the 5-HT_1A-R (five- to 15-fold) and its mRNA was always observed during degeneration and apoptosis of nutrient-deprived neuronal cells. Involvement in this induction of a 170-bp 5'-end untranslated sequence (5'-UT) (tail end of the 500-bp natural promoter) of G-21 was confirmed by stable transfection of neuronal cells with an SV-40 promoter-driven construct harboring the 5'-UT and the reporter chloramphenicol acetyltransferase (CAT) cDNA. Presence of the 5'-UT resulted in a threefold increase in CAT expression during nutrient deprivation in randomly chosen clones. The induction was also observed in the endogenous 5-HT_1A-R, expressed by embryonic day 16 mouse hippocampal neurons, subsequent to nutrient deprivation and onset of degeneration. A trophic role of the 5-HT_1A-R has been suggested in earlier studies. Considering the example of protective heat shock proteins, which are induced during various types of stress, our results suggest that stressed neuronal cells undergoing degeneration and apoptosis synthesize increased levels of 5-HT_1A-R as a final attempt to survive.     

Recruitment of active glycogen synthase kinase-3 into neuronal lipid rafts

Glycogen synthase kinase (GSK)-3beta has emerged as a key molecule that regulates neuronal apoptosis. To examine the molecular mechanism(s) through which GSK-3beta regulates this process, we studied the subcellular localization of GSK-3beta following exposure of the cells to well-characterized apoptotic stimuli. Here, we report that the induction of apoptosis by withdrawal of serum and potassium triggers dephosphorylation of GSK-3beta at serine 9 and subsequent translocation of these molecules into neuronal lipid raft microdomains. Inhibition of GSK-3beta by small molecule inhibitors blocks specific phosphorylation of lipid raft associated protein Tau. Consistent with the notion that the lipid raft domains may serve as a platform for the cellular signaling complexes, disruption of lipid rafts protected neurons from apoptosis induced by withdrawal of serum and potassium as well as by HIV-1 Tat. Our observations reveal novel interaction of GSK-3beta and raft domains, and suggest that such interaction could contribute to neuronal apoptosis.     

PKCδ mediates Nrf2-dependent protection of neuronal cells from NO-induced apoptosis

A chemical inhibitor library of 84 compounds was screened to investigate the signaling pathway(s) leading to activation of Nrf2 in response to nitric oxide (NO). We identified the protein kinase C delta (PKCδ) inhibitor rottlerin as the only compound that reduced NO-induced ARE-luciferase reporter activity and diminished NO-induced up-regulation of two Nrf2/ARE-regulated proteins - NAD(P)H:quinone oxidoreductase-1 (NQO1) and hemeoxygenase-1 (HO-1) in SH-Sy5y cells. Rottlerin also sensitized neuroblastoma cells and mouse primary cortical neurons to NO-induced apoptosis. Stable over-expression of PKCδ augmented NO-induced, ARE-dependent gene expression of HO-1 in SH-Sy5y cells, which were more protected from NO killing. Conversely, NO-induced ARE-dependent gene expression was reduced in PKCδ-knockdown SH-EP cells, which displayed greater sensitivity to apoptosis. PKCδ^−/− cortical neurons exhibited increased NO-induced apoptosis and less HO-1 mRNA and protein induction compared with wild type neurons. Hence, PKCδ is an important positive modulator of NO-induced Nrf2/ARE-dependent signaling that counteracts NO-mediated apoptosis in neuronal cells.     

West Nile virus-induced neuroinflammation: glial infection and capsid protein-mediated neurovirulence

West Nile virus (WNV) infection causes neurological disease at all levels of the neural axis, accompanied by neuroinflammation and neuronal loss, although the underlying mechanisms remain uncertain. Given the substantial activation of neuroinflammatory pathways observed in WNV infection, we hypothesized that WNV-mediated neuroinflammation and cell death occurred through WNV infection of both glia and neurons, which was driven in part by WNV capsid protein expression. Analysis of autopsied neural tissues from humans with WNV encephalomyelitis (WNVE) revealed WNV infection of both neurons and glia. Upregulation of proinflammatory genes, CXCL10, interleukin-1beta, and indolamine-2',3'-deoxygenase with concurrent suppression of the protective astrocyte-specific endoplasmic reticulum stress sensor gene, OASIS (for old astrocyte specifically induced substance), was evident in WNVE patients compared to non-WNVE controls. These findings were supported by increased ex vivo expression of these proinflammatory genes in glia infected by WNV-NY99. WNV infection caused endoplasmic reticulum stress gene induction and apoptosis in neurons but did not affect glial viability. WNV-infected astrocytic cells secreted cytotoxic factors, which caused neuronal apoptosis. The expression of the WNV-NY99 capsid protein in neurons and glia by a Sindbis virus-derived vector (SINrep5-WNVc) caused neuronal death and the release of neurotoxic factors by infected astrocytes, coupled with proinflammatory gene induction and suppression of OASIS. Striatal implantation of SINrep5-WNV(C) induced neuroinflammation in rats, together with the induction of CXCL10 and diminished OASIS expression, compared to controls. Moreover, magnetic resonance neuroimaging showed edema and tissue injury in the vicinity of the SINrep5-WNVc implantation site compared to controls, which was complemented by neurobehavioral abnormalities in the SINrep5-WNVc-implanted animals. These studies underscore the important interactions between the WNV capsid protein and neuroinflammation in the pathogenesis of WNV-induced neurological disorders.     

Knockdown of integrin beta4 in primary cultured mouse neurons blocks survival and induces apoptosis by elevating NADPH oxidase activity and reactive oxygen species level

Recently, the specific roles of integrin beta4 in the signaling networks that drive pathological angiogenesis and tumor progression have been revealed. Our previous study showed that integrin beta4 might be involved in neuron survival signal transduction. To further our study on the role of integrin beta4 in the survival and apoptosis of primary cultured mouse neurons, we inhibited the expression of integrin beta4 by its specific small interfering RNA. Viability of the cells remarkably declined, and neurons underwent apoptosis with down-regulation of integrin beta4. Next, we investigated the effect of siRNA-mediated down-regulation of integrin beta4 on the level of intracellular reactive oxygen species and the activities of NADPH oxidase and superoxide dismutase. The level of reactive oxygen species in the neurons was elevated significantly, the activities of manganese-dependent superoxide dismutase and copper/zinc-dependent superoxide dismutase were not altered, but the activity of NADPH oxidase was increased. Furthermore, inhibition of NADPH oxidase by its specific inhibitor dibenziodolium chloride attenuated the neuronal death induced by integrin beta4 knockdown. The data suggest that integrin beta4 is a key factor in neuron survival and apoptosis and indicate that this integrin subunit might perform its action through regulating NADPH oxidase and the level of reactive oxygen species in neuronal survival and apoptosis.     

IL-2 induction of IL-1 beta mRNA expression in monocytes. Regulation by agents that block second messenger pathways

We have previously shown that in mixed cultures of PBL incubation with human rIL-2 induces the rapid expression of IL-1 alpha and IL-1 beta mRNA. Because studies have demonstrated that IL-2R can be expressed on the surface of human peripheral blood monocytes, we chose to investigate whether IL-1 beta mRNA could be directly induced in purified human monocytes by treatment with Il-2 and, if so, to analyze the second messenger pathways by which it may be controlled. Human monocytes do not spontaneously express IL-1 beta mRNA, but can express the gene as soon as 1 h after treatment with IL-2. The level of IL-1 beta mRNA induced by IL-2 at 5 h in human monocytes was about one-fourth that induced by LPS. LPS induction of IL-1 beta mRNA in human monocytes can be blocked by either an inhibitor of protein kinase C (PKc) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine or an inhibitor of calcium/calmodulin (CaM) kinase N-(6-aminohexyl) 5-chloro-1-naphthalenesulfonamide, suggesting that both PKc and CaM kinase are involved in transducing signals initiated by LPS. In contrast, IL-2 induction of IL-1 beta mRNA expression is blocked only by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, suggesting that PKc, and not CaM kinase, is activated by IL-2. These data suggest that overlapping but distinct second messenger pathways are involved in the transduction of signals initiated by IL-2 and LPS.     

Lysophosphatidic Acid Induces Necrosis and Apoptosis in Hippocampal Neurons

Abstract: A diverse body of evidence indicates a role for the lipid biomediator lysophosphatidic acid (LPA) in the CNS. This study identifies and characterizes the induction of neuronal death by LPA. Treatment of cultured hippocampal neurons from embryonic rat brains with 50 µ M LPA resulted in neuronal necrosis, as determined morphologically and by the release of lactate dehydrogenase. A concentration of LPA as low as 10 µ M led to the release of lactate dehydrogenase. In contrast, treatment of neurons with 0.1 or 1.0 µ M LPA resulted in apoptosis, as determined by chromatin condensation. In addition, neuronal death induced by 1 µ M LPA was characterized as apoptotic on the basis of terminal dUTP nick end-labeling (TUNEL) staining, externalization of phosphatidylserine, and protection against chromatin condensation, TUNEL staining, and phosphatidylserine externalization by treatment with N -benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad-spectrum inhibitor of caspases, i.e., members of the interleukin-1β converting enzyme family. Studies with antagonists of ionotropic glutamate receptors did not indicate a significant role for these receptors in apoptosis induced by 1 µ M LPA. LPA (1 µ M ) also induced a decrease in mitochondrial membrane potential. Moreover, pretreatment of neurons with cyclosporin A protected against the LPA-induced decrease in mitochondrial membrane potential and neuronal apoptosis. Thus, LPA, at pathophysiological levels, can induce neuronal apoptosis and could thereby participate in neurodegenerative disorders.     

Expression of Cell Cycle-Related Genes During Neuronal Apoptosis: Is there a Distinct Pattern?

An emerging hypothesis considers the process of neuronal apoptosis as a consequence of unscheduled and unsynchronized induction of cell cycle mediators. Induction of several cell cycle genes precedes neuronal apoptosis and may be involved in determination of cell fate. We have now characterized changes in expression of cell cycle genes during apoptosis induced by oxidative stress in chick post-mitotic sympathetic neurons. Induction of cyclin B occurred prior to the commitment of neurons to both dopamine- and peroxide-triggered apoptosis. Both the neuronal death and the rise in cyclin B were inhibited by antioxidant treatment, suggesting a functional role for cyclin B induction during neuronal apoptosis. Induction of the cyclin dependent kinase CDK5 protein coincided with the time point when neurons were irreversibly committed to die. Expression of other cell cycle mediators such as cyclin D1 and the cyclin dependent kinases CDC2 and CDK2 was undetected and not induced by exposure to oxidative stress. Comparative analysis of the profile of cell cycle mediators induced during neuronal apoptosis of different neuronal cell populations revealed no distinct pattern of events. There are no cell cycle stage-specific mediators that are ultimately stimulated during neuronal apoptosis, suggesting that multiple pathways of re-activating the dormant cell-cycle, converge to determine entry into apoptosis. Nevertheless, the existence of some cell cycle mediators, that were not reported so far to be induced in post mitotic neurons during oxidative stress, substantiate them as part of the strong differentiating forces.     

Inflammation, antichymotrypsin, and lipid metabolism: autogenic etiology of Alzheimer's disease

Alzheimer's disease is a multifactor pathology, some of whose causes have been inferred from genetic studies, primarily of associated early-onset cases. Much evidence implicates the A beta amyloid peptide as a neurotoxic agent, with chronic inflammation as an accompanying physiological contributor to the disease. The two central questions of how A beta kills neurons and of the autogenic basis of disease remain unanswered. We hypothesize that specific interactions of A beta with the inflammatory serpin, alpha 1-antichymotrypsin, abolish the serpin proteinase inhibitor activity and stimulate formation of the neurotoxic fibrillar form of A beta. Further, the fibrillar A beta interacts with specific cell surface receptors, prompting its own biosynthesis and disrupting cellular cholesterol metabolism. These molecular and cellular interactions autogenically sustain the processes of A beta formation, fibrillization, and receptor interaction, the last of which culminates in neuronal death through disruption of cholesterol metabolism.     

Calcium and pituitary adenylate cyclase-activating polypeptide induced expression of circadian clock gene mPer1 in the mouse cerebellar granule cell culture

Mammalian circadian clock genes Per1 and Per2 are rhythmically expressed not only in the suprachiasmatic nucleus where the mammalian circadian clock exists, but also in other brain regions and peripheral tissues. The induced circadian oscillation of Per genes after treatment with high concentrations of serum or various drugs in cultured cells suggests the ubiquitous existence of the oscillatory mechanism. These treatments also result in a rapid surge of expression of Per1. It has been shown that multiple signaling pathways are involved in Per1 gene induction in culture cells. We used a dispersed primary cell culture made up of mouse cerebellar granule cells to examine the stimuli inducing the mPer genes and their signaling pathways in neuronal tissues expressing mPer genes. We demonstrated that mPer1, but not mPer2, mRNA expression was dependent on the depolarization state controlled by extracellular KCl concentration in the granule cell culture. Nifedipine treatment reduced mPer1 induction, suggesting that mPer1 mRNA expression depends on intracellular calcium concentration regulated through a voltage-dependent Ca2+ channel. Transient mPer1 mRNA induction was observed after elevating KCl concentration in the medium from 5 mM to 25 mM. This increased expression was suppressed by a calmodulin antagonist, or CaMKII/IV inhibitor, but not by MEK inhibitors. Addition of pituitary adenylate cyclase-activating polypeptide-38 to the medium also induced transient Per1 gene expression. This induction was mimicked by dibutyryl-cAMP and suppressed by a protein kinase A (PKA) inhibitor, but not by MEK inhibitors. These results suggest that Ca2+/calmodulin-dependent protein kinase II/IV- and PKA-dependent pathways are involved in high-KCl and PACAP-induced mPer1 induction, respectively, and neural tissues use multiple signaling pathways for mPer1 induction similar to culture cells.