Non-apoptotic Functions of Caspase-3 in Nervous Tissue

Some enzymes that have been recognized as "apoptotic" so far may be involved in important cellular processes not necessarily related to cell death in nervous tissue. The activity of caspase-3, an "apoptotic" enzyme, can be measured in normally functioning neurons. The results reported by several groups point to the possibility that caspases may be involved in nervous tissue function as top enzymes in the regulatory proteolytic cascade. A concept on a new mechanism of synaptic plasticity modulation involving caspase-3 has been formulated postulating a specific role of caspase-3 in normal brain functioning. The idea of synaptic plasticity modulation by caspase-3 is in line with data reported recently. For example, caspase-3 is possibly involved in the long-term potentiation (LTP) phenomenon since proteins that are key players of molecular mechanisms of LTP induction and maintenance are caspase-3 substrates. Experimental results on blocking LTP by a caspase-3 inhibitor confirm this concept.     

Caspase-3-dependent and -independent apoptosis in focal brain ischemia

BACKGROUND: Although extensive caspase-3 activation has been demonstrated in experimental brain ischemia produced in neonatal rat, the role this caspase plays in the focal ischemia of adult brain is not clear, as the levels of caspase-3 in adult rat brain are extremely low. This raises the question whether caspase-3 synthesis and activation are essential for execution of the apoptotic program and DNA fragmentation in permanent brain ischemia, a condition that impairs cellular protein synthesis. MATERIALS AND METHODS: Rat middle cerebral artery was permanently occluded and histochemical detection of procaspase-3, active caspase-3 and DFF 40/CAD and apoptotic morphology analysis were performed at 6, 24, 48, and 72 hours after occlusion. RESULTS: Necrosis and two types of programmed cell death (PCD) are identified in this study of permanent focal brain ischemia. The first type of PCD is represented by active caspase-3 and DFF 40/CAD-positive cells. The second type of PCD is represented by caspase-3 and DFF40/CAD negative cells, which display morphological signs of apoptosis-like PCD: namely, nuclear chromatin condensation in lump masses and apoptotic body formation. The cells of the first type have a maximum number noted after 24 hours of ischemia. The cells of the second type are primarily seen after 48 and 72 hours of ischemia. Necrotic cells, which are also detected in the stroke, are caspase-3 negative, and have swollen nuclei, without chromatin condensation and apoptotic body formation. CONCLUSIONS: Our results indicate that in permanent brain ischemia in adult rats, PCD processes occur differently in various parts of ischemic zone. In conditions of severe energy depletion, the reactions of cellular disassembly and packaging into apoptotic bodies are accomplished without either caspase-3 expression or the activation of caspase-3-dependent deoxyribonuclease.     

Ama "zinc" link between TrkB transactivation and synaptic plasticity

While Trk receptors can be activated in a neurotrophin-independent manner through "transactivation" by GPCR ligands, its physiological significance in the brain remains unknown. Huang et al. have now identified a novel mechanism of TrkB transactivation. They show that zinc ions can transactivate TrkB independent of neurotrophins and that such a transactivation is important for mossy fiber long-term potentiation (LTP).     

Formation of high molecular weight caspase-3 complex in neonatal rat brain

Caspase-3 plays an essential role in normal brain development. Recently, a large protein complex known as apoptosome, which catalyzes the activation of caspase-3, has been reported. To investigate structural characteristics of caspase-3 in the developing brain, rat neonatal cortex extract was analysed by gel filtration chromatography. We show here the formation of high molecular complex including procaspase-3 in the extract. When the extract was activated by cytochrome c, caspase-3 recruitment to the apoptosome was not observed, although apoptotic protease activating factor-1 (Apaf-1), caspase-9, and X-linked inhibitor of apoptosis protein (XIAP) existed in the apoptosome. These results indicate that procaspase-3 exists as a high molecular weight complex during brain development.     

The Effects of Middle Cerebral Artery Occlusion on Central Nervous System Apoptotic Events in Normal and Diabetic Rats

Apoptosis and neural degeneration are characteristics of cerebral ischemia and brain damage. Diabetes is associated with worsening of brain damage following ischemic events. In this study, the authors characterize the influence of focal cerebral ischemia, induced by middle cerebral artery occlusion, on 2 indexes of apoptosis,TUNEL(terminal deoxynucleotidyl transferase–mediated deoxyuridine 5-triphosphate nick end-labeling) staining and caspase- 3 immunohistochemistry. Diabetes was induced in normal rats using streptozotocin and maintained for 5 to 6 weeks. The middle cerebral artery of both normal and diabetic rats was occluded and maintained from 24 or 48 hours. Sham-operated normal and diabetic animals served as controls. Following 24 to 48 hours of occlusion, the animals were sacrificed and the brains were removed, sectioned, and processed for TUNEL staining or caspase-3 immunohistochemistry. Middle cerebral artery occlusion in normal rats was associated with an increase in the number of both TUNEL-positive and caspase-3– positive cells in selected brain regions (hypothalamic preoptic area, piriform cortex, and parietal cortex) when compared to nonoccluded controls. Diabetic rats without occlusion showed significant increases in both TUNEL-positive and caspase-3–positive cells compared to normal controls. Middle cerebral artery occlusion in diabetic rats resulted in increases in TUNEL-positive as well as caspase-3–positive cells in selected regions, above those seen in nonoccluded diabetic rats. Both TUNEL staining and caspase-3 immunohistochemistry revealed that the number of apoptotic cells in diabetic animals tended to be greatest in the preoptic area and parietal cortex. The authors conclude that focal cerebral ischemia is associated with a significant increase in apoptosis in nondiabetic rats, and that diabetes alone or diabetes plus focal ischemia are associated with significant increases in apoptotic cells.     

Caspase-3 Mediates In Part Hippocampal Apoptosis in Sepsis

The brain is one of the first organs affected during sepsis development resulting in apoptosis for a short-term and cognitive impairment for a long-term. Despite its importance, the mechanisms of brain dysfunction during sepsis are not fully elucidated. Thus, we here, in an animal model of sepsis, evaluated apoptosis in the dentate gyrus cell layer of the hippocampus to document the involvement of caspase-3 in the pathogenesis of neuronal apoptosis. Wistar rats sham-operated or submitted to the cecal ligation and perforation (CLP) procedure were killed at 12, 24, 48 h, and 10 days after surgery for the determination of caspase-3 and apoptosis rate. In a separate cohort of animals, a caspase-3-specific inhibitor was administered and animals were killed at 12 h after sepsis. An increase in the number of apoptotic cells 12, 24, and 48 h by histopathological evaluations and an increase of caspase-3 apoptotic cells 12 and 24 h after sepsis induction were observed. The caspase-3 inhibitor decreases the number of apoptotic cells by histopathological evaluations but not by immunohistochemistry evaluations. Caspase-3 is involved in part in apoptosis in the dentate gyrus cell layer of the hippocampus in septic rats submitted by CLP.     

Mitochondrial Impairment in the Developing Brain After Hypoxia–Ischemia

The pattern of cell death in the immature brain differs from that seen in the adult CNS. During normal development, more than half of the neurons are removed through apoptosis, and mediators like caspase-3 are highly upregulated. The contribution of apoptotic mechanisms in cell death appears also to be substantial in the developing brain, with a marked activation of downstream caspases and signs of DNA fragmentation. Mitochondria are important regulators of cell death through their role in energy metabolism and calcium homeostasis, and their ability to release apoptogenic proteins and to produce reactive oxygen species. We find that secondary brain injury is preceded by impairment of mitochondrial respiration, signs of membrane permeability transition, intramitochondrial accumulation of calcium, changes in the Bcl-2 family proteins, release of proapoptotic proteins (cytochrome C, apoptosis inducing factor) and downstream activation of caspase-9 and caspase-3 after hypoxia-ischemia. These data support the involvement of mitochondria-related mechanisms in perinatal brain injury.     

Key apoptosis regulating proteins are down-regulated during postnatal tissue development

The intrinsic apoptotic pathway is essential for murine development. We have previously shown that key mediators of this pathway, such as Bim, Apaf-1 and caspase-3, are down-regulated during the postnatal development of the retina. In this study, we demonstrate that this expression pattern is a feature of other distinct tissues such as the brain, heart and skeletal muscle. Caspase-9 expression is also examined and is shown to follow a different pattern in each tissue. Interestingly, we show that peripheral cells of the internal granular layer of the cerebellum do not down-regulate the intrinsic apoptotic pathway proteins Bim, Apaf-1 or caspase-3. Furthermore, Bim expression is also detectable in the brain stem and the CA3 region of the hippocampus in the adult cerebrum. Finally, we demonstrate that the incidence of TUNEL positive cells in the selected tissues decreases during postnatal development in correlation with the general down-regulation of key apoptotic pathway proteins. In contrast, we also demonstrate that apoptosis persists in the adult thymus and that this tissue continues to express Bim, Apaf-1 and caspase-3 at the same levels as the neonate. In summary, this study shows that a selection of post-mitotic tissues down-regulate key apoptotic proteins, in contrast to the thymus, which requires apoptosis for normal function in early adulthood.     

Apoptotic cell death, long-term persistence, and neuronal differentiation of aneuploid cells generated in the adult brain of teleost fish

Aneuploidy, caused by segregation defects during mitosis, has previously been identified in adult-born cells of mammals and teleosts. In the present study, we have examined the fate of these cells in the brain of the teleost fish Apteronotus leptorhynchus. By immunostaining against active caspase-3, we have shown that both cells with normal nuclear morphology and cells with mitotic segregation defects undergo apoptosis, but the relative number of apoptotic cells is higher among cells of the latter category. Long-term survival of cells with mitotic segregation defects could be demonstrated by incorporation of 5-bromo-2'-deoxyuridine into newly synthesized DNA during the S-phase of mitosis, and by employment of postadministration survival times of up to 860 days. Moreover, by combining 5-bromo-2'-deoxyuridine immunolabeling with immunostaining against the neuron-specific marker protein Hu, we have shown that among the long-term persistent cells with mitotic segregation defects a similar portion develops into neurons as does among the long-term persistent cells without such defects. It is possible that aneuploid cells play a role in the regulation of gene expression by somatic genomic alterations during postnatal development.     

Traumatic Brain Injury Induces an Up-Regulation of Hs1-Associated Protein X-1 (Hax-1) in Rat Brain Cortex

HS1-associated protein X-1 (Hax-1) is an intracellular protein with anti-apoptotic properties that, in addition to suppressing cell death by inhibiting the activation of initiator caspase-9 and death caspase-3, is involved in an increasing number of signaling cascades. However, its expression and function in the central nervous system lesion are still unclear. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the dynamic changes of Hax-1 expression in the brain cortex. Western blot and immunohistochemistry analysis revealed that Hax-1 was present in normal brain. It gradually increased, reached a peak at day 3 after TBI, and then declined during the following days. Double immunofluorescence staining showed that Hax-1 immunoreactivity (IR) was found in neurons, but not astrocytes and microglia. Moreover, the 3rd day post injury was the apoptotic peak implied by the alteration of caspase-3, Bcl-2 and TUNEL. All these results suggested that Hax-1 may be involved in the pathophysiology of TBI and further research is needed to have a good understanding of its function and mechanism.     

Emerging roles of caspase-3 in apoptosis

Caspases are crucial mediators of programmed cell death (apoptosis). Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins. However, the specific requirements of this (or any other) caspase in apoptosis have remained largely unknown until now. Pathways to caspase-3 activation have been identified that are either dependent on or independent of mitochondrial cytochrome c release and caspase-9 function. Caspase-3 is essential for normal brain development and is important or essential in other apoptotic scenarios in a remarkable tissue-, cell type- or death stimulus-specific manner. Caspase-3 is also required for some typical hallmarks of apoptosis, and is indispensable for apoptotic chromatin condensation and DNA fragmentation in all cell types examined. Thus, caspase-3 is essential for certain processes associated with the dismantling of the cell and the formation of apoptotic bodies, but it may also function before or at the stage when commitment to loss of cell viability is made.     

Ethanol-induced neuronal apoptosis in vivo requires BAX in the developing mouse brain

A single episode of ethanol intoxication triggers widespread apoptotic neurodegeneration in the infant rat or mouse brain. The cell death process occurs over a 6-16 h period following ethanol administration, is accompanied by a robust display of caspase-3 enzyme activation, and meets ultrastructural criteria for apoptosis. Two apoptotic pathways (intrinsic and extrinsic) have been described, either of which may culminate in the activation of caspase-3. The intrinsic pathway is regulated by Bax and Bcl-XL and involves Bax-induced mitochondrial dysfunction and release of cytochrome c as antecedent events leading to caspase-3 activation. Activation of caspase-8 is a key event preceding caspase-3 activation in the extrinsic pathway. In the present study, following ethanol administration to infant mice, we found no change in activated caspase-8, which suggests that the extrinsic pathway is not involved in ethanol-induced apoptosis. We also found that ethanol triggers robust caspase-3 activation and apoptotic neurodegeneration in C57BL/6 wildtype mice, but induces neither phenomenon in homozygous Bax-deficient mice. Therefore, it appears that ethanol-induced neuroapoptosis is an intrinsic pathway-mediated phenomenon involving Bax-induced disruption of mitochondrial membranes and cytochrome c release as early events leading to caspase-3 activation.     

Caspase-3 contributes to ZO-1 and Cl-5 tight-junction disruption in rapid anoxic neurovascular unit damage

Background

Tight-junction (TJ) protein degradation is a decisive step in hypoxic blood-brain barrier (BBB) breakdown in stroke. In this study we elucidated the impact of acute cerebral ischemia on TJ protein arrangement and the role of the apoptotic effector protease caspase-3 in this context.

Methodology/Principal Findings

We used an in vitro model of the neurovascular unit and the guinea pig whole brain preparation to analyze with immunohistochemical methods the BBB properties and neurovascular integrity. In both methodological approaches we observed rapid TJ protein disruptions after 30 min of oxygen and glucose deprivation or middle cerebral artery occlusion, which were accompanied by strong caspase-3 activation in brain endothelial cells (BEC). Surprisingly only few DNA-fragmentations were detected with TUNEL stainings in BEC. Z-DEVD-fmk, an irreversible caspase-3 inhibitor, partly blocked TJ disruptions and was protective on trans-endothelial electrical resistance.

Conclusions/Significance

Our data provide evidence that caspase-3 is rapidly activated during acute cerebral ischemia predominantly without triggering DNA-fragmentation in BEC. Further we detected fast TJ protein disruptions which could be partly blocked by caspase-3 inhibition with Z-DEVD-fmk. We suggest that the basis for clinically relevant BBB breakdown in form of TJ disruptions is initiated within minutes during ischemia and that caspase-3 contributes to this process.     

Tissue plasminogen activator neurovascular toxicity is controlled by activated protein C

Although thrombolytic effects of tissue plasminogen activator (tPA) are beneficial, its neurotoxicity is problematic. Here, we report that tPA potentiates apoptosis in ischemic human brain endothelium and in mouse cortical neurons treated with N-methyl-D-aspartate (NMDA) by shifting the apoptotic pathways from caspase-9 to caspase-8, which directly activates caspase-3 without amplification through the Bid-mediated mitochondrial pathway. In vivo, tPA-induced cerebral ischemic injury in mice was reduced by intracerebroventricular administration of caspase-8 inhibitor, but not by caspase-9 inhibitor, in contrast to controls in which caspase-9 inhibitor, but not caspase-8 inhibitor, was protective. Activated protein C (APC), a serine protease with anticoagulant, anti-inflammatory and antiapoptotic activities, which is neuroprotective during transient ischemia and promotes activation of antiapoptotic mechanisms in brain cells by acting directly on endothelium and neurons, blocked tPA vascular and neuronal toxicities in vitro and in vivo. APC inhibited tPA-induced caspase-8 activation of caspase-3 in endothelium and caspase-3-dependent nuclear translocation of apoptosis-inducing factor in NMDA-treated neurons and reduced tPA-mediated cerebral ischemic injury in mice. Data suggest that tPA shifts the apoptotic signal in stressed brain cells from the intrinsic to the extrinsic pathway which requires caspase-8. APC blocks tPA's neurovascular toxicity and may add substantially to the effectiveness of tPA therapy for stroke.     

LTP and LTD: an embarrassment of riches

LTP and LTD, the long-term potentiation and depression of excitatory synaptic transmission, are widespread phenomena expressed at possibly every excitatory synapse in the mammalian brain. It is now clear that "LTP" and "LTD" are not unitary phenomena. Their mechanisms vary depending on the synapses and circuits in which they operate. Here we review those forms of LTP and LTD for which mechanisms have been most firmly established. Examples are provided that show how these mechanisms can contribute to experience-dependent modifications of brain function.     

BAX contributes to apoptotic-like death following neonatal hypoxia-ischemia: evidence for distinct apoptosis pathways.

BACKGROUND: Hypoxic-ischemic (H-I) injury to the neonatal brain has been shown to result in rapid cell death with features of acute excitotoxicity/necrosis as well as prominent delayed cell death with features of apoptosis such as marked caspase-3 activation. BAX, a pro-apoptotic molecule, has been shown to be required for apoptotic neuronal cell death during normal development but the contribution of endogenous BAX in cell death pathways following H-I injury to the developing or adult brain has not been studied. MATERIALS AND METHODS: Bax +/+, +/-, and -/- mice at post-natal day 7 were subjected to unilateral carotid ligation followed by exposure to 45 minutes of 8% oxygen. At different timepoints following H-I, brain tissue was studied by conventional histology, immunohistochemistry, immunofluorescence, Western blotting, and enzymatic assay to determine the extent and type of cell injury as well as the amount of caspase activation. RESULTS: We found that bax -/- mice had significantly less (38%) hippocampal tissue loss than mice expressing bax. Some of the remaining cell death in bax -/- mice, however, still had features of apoptosis including evidence of nuclear shrinkage and caspase-3 activation. Though bax -/- mice had significantly decreased caspase-3 activation as compared to bax expressing mice following H-I, the density of cells with activated caspase-8 in the CA3 region of the hippocampus did not differ between bax +/- and bax -/- mice. CONCLUSIONS: These findings demonstrate that endogenous BAX plays a role in regulating cell death in the central nervous system (CNS) following neonatal H-I, a model of cerebral palsy. In addition, while BAX appears to modulate the caspase-3 activation following neonatal H-I, caspase-8 which is linked to death receptor activation, may contribute to apoptotic-like neuronal death in a BAX-independent manner.     

Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization.

Huntington disease (HD) is a genetically dominant condition caused by expanded CAG repeats coding for glutamine in the HD gene product huntingtin. Although HD symptoms reflect preferential neuronal death in specific brain regions, huntingtin is expressed in almost all tissues, so abnormalities outside the brain might be expected. Although involvement of nuclei and mitochondria in HD pathophysiology has been suggested, specific intracellular defects that might elicit cell death have been unclear. Mitochondria dysfunction is reported in HD brains; mitochondria are organelles that regulates apoptotic cell death. We now report that lymphoblasts derived from HD patients showed increased stress-induced apoptotic cell death associated with caspase-3 activation. When subjected to stress, HD lymphoblasts also manifested a considerable increase in mitochondrial depolarization correlated with increased glutamine repeats.     

Region-Specific Interrelations between Apoptotic Proteins Expression and DNA Fragmentation in the Neonatal Rat Brain

DNA fragmentation, mRNA and protein levels of Bcl-XL, Bax and caspase-3 were determined to characterize interrelations between expression of these apoptotic markers in the neonatal brain regions. High DNA fragmentation intensity in the cortex was in consonance with the lowest Bcl-XL/Bax expression ratio, the highest procaspase-3 and active caspase-3 levels. Low and intermediate DNA fragmentation levels in the cerebellum and hippocampus respectively were also in a good agreement with apoptotic proteins expression in these structures. In the cortex, hippocampus and cerebellum DNA fragmentation intensity was proportional to the active caspase-3 level. In contrast to these structures, in the brainstem, the lowest level of this protease was accompanied by the highest intensity of DNA fragmentation among the brain regions studied. The data suggest that cell death normally occurring during early postnatal life could be realized in the developing brainstem via caspase-3-independent pathways in animals that express this protease.     

A possible mechanism of dopamine-induced synergistic disinhibition of thalamic cells via "direct" and "indirect" pathways in basal ganglia

On the basis of the mechanism of synaptic plasticity that we have earlier suggested for striatal spiny neurons and with regard to the known data about the predominance of dopamine-sensitive D1/D2 receptors on the striatonigral/striatopallidal cells it is hypothesized that the induction of the long-term potentiation/depression of the efficacy of excitatory cortical inputs to these cells can underlie the excitatory/inhibitory effect of dopamine on the activity of neurons that originate the "direct"/"indirect" pathways through the basal ganglia. Both these effects will lead to an enhancement of the activity of thalamic cells and activity of the efferent neocortical neurons excited by thalamic cells. The long-term potentiation of corticostriatal inputs to striosomal neurons, where, predominantly, D1 receptors are located, can also be induced by dopamine. This effect can be responsible of a rise of inhibition of dopaminergic cells and decrease in dopamine release by these cells. Such an event sequence can provide a stable dopamine concentration in the loop neocortex-basal ganglia-thalamus-neocortex.