Alexander G. Yakovlev

Role of Poly(ADP-ribose) Polymerase (PARP) Cleavage in Apoptosis CASPASE 3-RESISTANT PARP MUTANT INCREASES RATES OF APOPTOSIS IN TRANSFECTED CELLS

An early transient burst of poly(ADP-ribosyl)ation of nuclear proteins was recently shown to be required for apoptosis to proceed in various cell lines (Simbulan-Rosenthal, C., Rosenthal, D., Iyer, S., Boulares, H., and Smulson, M. (1998) J. Biol. Chem. 273, 13703–13712) followed by cleavage of poly(ADP-ribose) polymerase (PARP), catalyzed by caspase-3. This inactivation of PARP has been proposed to prevent depletion of NAD (a PARP substrate) and ATP, which are thought to be required for later events in apoptosis. The role of PARP cleavage in apoptosis has now been investigated in human osteosarcoma cells and PARP −/− fibroblasts stably transfected with a vector encoding a caspase-3-resistant PARP mutant. Expression of this mutant PARP increased the rate of staurosporine and tumor necrosis factor-α-induced apoptosis, at least in part by reducing the time interval required for the onset of caspase-3 activation and internucleosomal DNA fragmentation, as well as the generation of 50-kilobase pair DNA breaks, thought to be associated with early chromatin unfolding. Overexpression of wild-type PARP in osteosarcoma cells also accelerated the apoptotic process, although not to the same extent as that apparent in cells expressing the mutant PARP. These effects of the mutant and wild-type enzymes might be due to the early and transient poly(ADP-ribose) synthesis in response to DNA breaks, and the accompanying depletion of NAD apparent in the transfected cells. The accelerated NAD depletion did not seem to interfere with the later stages of apoptosis. These results indicate that PARP activation and subsequent cleavage have active and complex roles in apoptosis.

The tumor suppressor protein p53 is required for neurite outgrowth and axon regeneration

Axon regeneration is substantially regulated by gene expression and cytoskeleton remodeling. Here we show that the tumor suppressor protein p53 is required for neurite outgrowth in cultured cells including primary neurons as well as for axonal regeneration in mice. These effects are mediated by two newly identified p53 transcriptional targets, the actin‐binding protein Coronin 1b and the GTPase Rab13, both of which associate with the cytoskeleton and regulate neurite outgrowth. We also demonstrate that acetylation of lysine 320 (K320) of p53 is specifically involved in the promotion of neurite outgrowth and in the regulation of the expression of Coronin 1b and Rab13. Thus, in addition to its recognized role in neuronal apoptosis, surprisingly, p53 is required for neurite outgrowth and axonal regeneration, likely through a different post‐translational pathway. These observations may suggest a novel therapeutic target for promoting regenerative responses following peripheral or central nervous system injuries.

Caspase-dependent apoptotic pathways in CNS injury

Recent studies have suggested a role for neuronal apoptosis in cell loss following acute CNS injury as well as in chronic neurodegeneration. Caspases are a family of cysteine requiring aspartate proteases with sequence similarity to Ced-3 protein of Caenorhabditis elegans. These proteases have been found to contribute significantly to the morphological and biochemical manifestations of apoptotic cell death. Caspases are translated as inactive zymogens and become active after specific cleavage. Of the 14 identified caspases, caspase-3 appears to be the major effector of neuronal apoptosis induced by a variety of stimuli. A role for caspase-3 in injury-induced neuronal cell death has been established using semispecific peptide caspase inhibitors. This article reviews the current literature relating to pathways regulating caspase activation in apoptosis associated with acute and chronic neurodegeneration, and suggests that identification of critical upstream caspase regulatory mechanisms may permit more effective treatment of such disorders.

BOK and NOXA are essential mediators of p53-dependent apoptosis.

Cellular stress leads to DNA damage and activation of the intrinsic apoptotic pathway in which translocation of mitochondrial cytochrome c to the cytosol plays a critical role. Previous studies have suggested alternative mechanisms responsible for this process. We examined initiation mechanisms of the intrinsic apoptotic pathway using human neuroblastoma and breast cancer cells. Results indicated that translocation of cytochrome c does not require prior activation of caspases but rather depends on activation of specific BCL-2 family members, depending upon the type of death signal. Thus, DNA damage-induced apoptosis requires new protein synthesis, accumulation of p53 tumor suppressor protein, and p53-dependent induction of BOK and NOXA genes, while a role for BAX in this pathway is not essential. In contrast, apoptosis induced by staurosporine does not require protein synthesis but is characterized by translocation of BAX. Based on these findings, we propose a model of the intrinsic apoptotic cascade induced by DNA damage where proapoptotic BOK substitutes for a function of BAX.

Mechanisms of neural cell death: implications for development of neuroprotective treatment strategies.

SummaryIt has been increasingly recognized that cell death phenotypes and their molecular mechanisms are highly diverse. Necrosis is no longer considered a single entity, passively mediated by energy failure. Moreover, caspase-dependent apoptosis is not the only pathway involved in programmed cell death or even the only apoptotic mechanism. Recent experimental work emphasizes the diverse and interrelated nature of cell death mechanisms. Thus, there are both caspase-dependent and caspase-independent forms of apoptosis, which may differ morphologically as well as mechanistically. There are also necrotic-like phenotypes that requirede novo protein synthesis and are, therefore, forms of programmed cell death. In addition, forms of cell death showing certain morphological features of both necrosis and apoptosis have been identified, leading to the term aponecrosis. Considerable experimental evidence also shows that modulation of one form of cell death may lead to another. Together, these observations underscore the need to substantially revise our conceptions about neuroprotection strategies. Use of multiple treatments that target different cell death cascades, or single agents that moderate multiple cell death pathways, is likely to lead to more effective neuroprotection for clinical disorders.

Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway.

C(2)-ceramide, a cell-permeable analog of ceramide, caused cell death in cultured rat cortical neuronal cells. C(2)-ceramide-induced neuronal loss was accompanied by upregulation of caspase-3 activity, measured by cleavage of its fluorogenic substrate Ac-DEVD-AMC. Similar results were obtained when cortical neuronal cultures were treated with sphingomyelinase, an enzyme responsible for ceramide formation in the cell. Morphological evaluation of C(2)-ceramide-treated cortical neurons showed nuclear condensation and fragmentation as visualized by Hoechst 33258 staining. Co-administration of the selective caspase-3 inhibitor z-DEVD-fmk or caspase-9 inhibitor z-LEHD-fmk significantly reduced C(2)-ceramide-induced cell death, while co-application of the caspase-8, inhibitor z-IETD-fmk, was without effect. Immunoblot analysis of protein extracts from C(2)-ceramide-treated cortical neuronal cultures revealed upregulation of active caspase-9 and caspase-3 protein levels, whereas presence of active caspase-8 immunoreactivity was undetectable in this system. Administration of C(2)-ceramide to SH-SY5Y human neuroblastoma cells also caused apoptotic cell death. Moreover, ceramide-induced cell death was significantly decreased in caspase-9 dominant-negative SH-SY5Y cells, while both caspase-8 dominant-negative cultures and mock-transfected cells showed equally high levels of cell death following C(2)-ceramide treatment. Taken together, these data suggest that neuronal death induced by ceramide may be linked to the caspase-9/caspase-3 regulated intrinsic pathway of cellular apoptosis.

Neuronal plasticity after spinal cord injury: identification of a gene cluster driving neurite outgrowth

Functional recovery after spinal cord injury (SCI) may result in part from axon outgrowth and related plasticity through coordinated changes at the molecular level. We employed microarray analysis to identify a subset of genes the expression patterns of which were temporally coregulated and correlated to functional recovery after SCI. Steady‐state mRNA levels of this synchronously regulated gene cluster were depressed in both ventral and dorsal horn neurons within 24 h after injury, followed by strong re‐induction during the following 2 wk, which paralleled functional recovery. The identified cluster includes neuritin, attractin, microtubule‐ associated protein 1a, and myelin oligodendrocyte protein genes. Transcriptional and protein regulation of this novel gene cluster was also evaluated in spinal cord tissue and in single neurons and was shown to play a role in axonal plasticity. Finally, in vitro transfection experiments in primary dorsal root ganglion cells showed that cluster members act synergistically to drive neurite outgrowth.

Anandamide-induced cell death in primary neuronal cultures: role of calpain and caspase pathways

AbstractAnandamide (arachidonoylethanolamide or AEA) is an endocannabinoid that acts at vanilloid (VR1) as well as at cannabinoid (CB1/CB2) and NMDA receptors. Here, we show that AEA, in a dose-dependent manner, causes cell death in cultured rat cortical neurons and cerebellar granule cells. Inhibition of CB1, CB2, VR1 or NMDA receptors by selective antagonists did not reduce AEA neurotoxicity. Anandamide-induced neuronal cell loss was associated with increased intracellular Ca2+, nuclear condensation and fragmentation, decreases in mitochondrial membrane potential, translocation of cytochrome c, and upregulation of caspase-3-like activity. However, caspase-3, caspase-8 or caspase-9 inhibitors, or blockade of protein synthesis by cycloheximide did not alter anandamide-related cell death. Moreover, AEA caused cell death in caspase-3-deficient MCF-7 cell line and showed similar cytotoxic effects in caspase-9 dominant-negative, caspase-8 dominant-negative or mock-transfected SH-SY5Y neuroblastoma cells. Anandamide upregulated calpain activity in cortical neurons, as revealed by α-spectrin cleavage, which was attenuated by the calpain inhibitor calpastatin. Calpain inhibition significantly limited anandamide-induced neuronal loss and associated cytochrome c release. These data indicate that AEA neurotoxicity appears not to be mediated by CB1, CB2, VR1 or NMDA receptors and suggest that calpain activation, rather than intrinsic or extrinsic caspase pathways, may play a critical role in anandamide-induced cell death.

Brain-derived neurotrophic factor prevents the nigrostriatal degeneration induced by human immunodeficiency virus-1 glycoprotein 120 in vivo

Glycoprotein 120 (gp120) from the T‐tropic strain of the human immunodeficiency virus type 1 has been shown to cause neuronal apoptosis through activation of the chemokine receptor CXCR4. Therefore, reducing CXCR4 expression may prevent gp120‐mediated apoptosis. Brain‐derived neurotrophic factor (BDNF) is known to reduce both gp120 neurotoxicity and CXCR4 expression in vitro. The scope of this work is to establish whether BDNF is neuroprotective against gp120 in vivo and, if so, whether this effect correlates with its ability to down‐regulate CXCR4. Serotype 2 adeno‐associated viral vector encoding for BDNF (rAAV‐BDNF) or control vector was microinjected into the striata of adult rats. Two weeks later gp120 was injected into the same striatum, and apoptosis determined. Pretreatment with rAAV‐BDNF prior to gp120 microinjection prevented caspase‐3 activation as well as in situ terminal deoxynucleotidyl transferase biotin–dUTP nick end labelling in the striatum and substantia nigra. In addition, rAAV‐BDNF reversed the loss of tyrosine hydroxylase immunoreactivity induced by gp120 in both areas. CXCR4 expression was then determined by immunohistochemistry and RT‐PCR, and found to be decreased in striata of rAAV‐BDNF‐treated rats. Conversely, BDNF heterozygous mice exhibited an increase in CXCR4 mRNA levels compared to wild‐type littermates. Our data suggest that down‐regulation of CXCR4 expression may contribute to the neuroprotective activity of BDNF against gp120 toxicity in the basal ganglia.

Epigenetic regulation of caspase-3 gene expression in rat brain development.

The expression levels of caspase-3, a major contributor to the execution of neuronal apoptosis, markedly decrease in the process of brain maturation. We have previously cloned the rat caspase-3 gene promoter and identified its essential regulatory elements. In the present study, we extended previous findings by examining transcriptional regulation of caspase-3 expression in the rat brain of two different ages, corresponding to the immature and mature brain. In particular, we determined that the rate of transcription initiation substantially declines during brain maturation. Furthermore, we established that mRNA levels of Ets1, Ets2, and Sp1 do not change in the brain with maturation, suggesting that these transcription factors do not contribute to age-dependent caspase-3 down-regulation. Hence, we examined a role of DNA methylation and histone modification in this process. Utilizing bisulfite DNA sequencing, we determined the presence of age-dependent differentially methylated fragments within the caspase-3 promoter region. Strikingly, differentially methylated CpG sites correspond to the predicted binding sites for a number of transcription factors that have been previously shown to be involved in neuronal development and differentiation. Moreover, using chromatin immunoprecipitation, we found that mature brains displayed significantly lower levels of histone 3 acetylated Lys14 and histone 4 acetylated Lys5, 8, 12, and 16. This observation is consistent with the decreased level of expression of caspase-3 in the mature brain. Together with our observation that histone deacetylase inhibitor, trichostatin A, increased the level of caspase-3 mRNA in cortical neurons in vitro, these results further indicate an important role of epigenetic factors in the regulation of caspase-3 gene expression.