Primary rat microglia stimulated with either ATP or 2-and 3-O-(4-benzoylbenzoyl)-ATP (BzATPActivated microglia have been observed in patients suffering from both acute (stroke) and chronic (Alzheimer's disease) neurological disorders (1, 2). Microglia are believed to contribute to the progression of Alzheimer's disease (AD) 1 because these cells can release pro-inflammatory substances known to induce neurotoxicity (3). Reactive oxygen intermediates (ROIs), one of several pro-inflammatory substances released by microglia (4), are likely to play a very important role in AD because hallmark modifications of ROI damage such as lipid peroxidation and nitrotyrosine conjugates are characteristic of post-mortem AD brains (3). Hence, pro-inflammatory stimuli that promote microglial ROI production might contribute to the pathogenesis of AD.ATP is an important messenger in the brain and can be released from cells by both lytic and non-lytic mechanisms (5). ATP evokes a variety of biological responses in microglia (6 -9).The effects of ATP are mediated through interactions with the P2 purinoceptors, broadly classified into P2Y metabotropic and P2X ionotropic receptors (10). The P2Y receptors are G proteincoupled and P2X receptors are ligand-gated ion channels (10). Whereas the P2Y receptors are responsible for Ca 2ϩ release predominantly from intracellular stores, P2X receptors are responsible for Ca 2ϩ influx from extracellular sources. Microglia possess both P2Y and P2X receptors (11-13). The P2X 7 receptor is highly expressed by cells of the macrophage lineage, such as dendritic cells, alveolar macrophages, and microglia. Activation of the P2X 7 receptor is unique in triggering the formation of large nonselective membrane pores, permeable to molecules up to 900 Da which ultimately results in death of the cell (9, 14). ATP and ATP analogs have been used to characterize the role of P2 receptors in microglial activation. Micromolar concentrations of ATP are required to activate the P2Y receptors, whereas millimolar (1-5 mM) concentrations of ATP are required to activate the P2X receptors. The ATP analog BzATP is a selective agonist at the P2X receptor and does not bind P2Y receptors (15,16). Oxidized ATP (oATP) is a specific antagonist of P2X 7 that binds irreversibly to the receptor and prevents its activation by ATP (17). In this study, these pharmacological tools were used to determine the purinergic receptors involved in O 2. production in microglia.The P2X 7 receptor plays a role in the generation of superoxide in microglia. Our studies elucidate a putative signal transduction pathway that mediates this response. These studies also demonstrate that BzATP-and ATP-activated microglia can mediate neurotoxicity. Finally, a distinct alteration was detected in the staining pattern for P2X 7 receptor in a transgenic mouse model of AD, suggesting that P2X 7 receptor activation could play a contributing role in AD. MATERIALS AND METHODSReagents-Reagents not specified otherwise were obtained from Sigma. PD98059, SB203580, LY2940...

The degradation of ␣II-and ␤II-spectrin during apoptosis in cultured human neuroblastoma SH-SY5Y cells was investigated. Immunofluorescent staining showed that the collapse of the cortical spectrin cytoskeleton is an early event following staurosporine challenge. This collapse correlated with the generation of a series of prominent spectrin breakdown products (BDPs) derived from both ␣II-and ␤II-subunits. Major C-terminal ␣II-spectrin BDPs were detected at Ϸ150, 145, and 120 kDa (␣II-BDP150, ␣II-BDP145, and ␣II-BDP120, respectively); major C-terminal ␤II-spectrin BDPs were at Ϸ110 and 85 kDa (␤II-BDP110 and ␤II-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that ␣II-BDP150 and ␣II-BDP120 were generated by cleavages at DETD 1185 *S 1186 and DSLD 1478 *S 1479 , respectively. For ␤II-spectrin, a major caspase site was detected at DEVD 1457 *S 1458, and both ␤II-BDP110 and ␤II-BDP85 shared a common N-terminal sequence starting with Ser 1458 . An additional cleavage site near the C terminus, at ETVD 2146 *S 2147 , was found to account for ␤II-BDP85. Studies using specific caspase or calpain inhibitors indicate that the pattern of spectrin breakdown during apoptosis differs from that during non-apoptotic cell death. We postulate that in concert with calpain, caspase rapidly targets critical sites in both ␣II-and ␤II-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis.The importance of proteases in the expression of mammalian apoptosis has been the subject of many recent studies. The mammalian interleukin-1␤-converting enzyme (ICE) 1 -like protease family (renamed caspase (1) (5), and its deletion by gene knockout blocks neuronal death during brain development with consequential lethality (6). Besides the caspases, a second family of proteases implicated in the initiation and control of apoptosis are the calpains (7, 8), especially in several hematopoietic and neuronal cells (9 -12). The relationship between these two protease families, the consequences of each on their respective substrates and on cellular physiology, or the conditions under which each is activated remain poorly understood. While many proteins are cleaved during apoptosis, a prominent target of both calpain and caspase action is ␣II-spectrin, the major component of the cortical membrane skeleton. In neurons, calcium-activated calpain cleavage of ␣II-spectrin (non-erythroid ␣-spectrin or ␣-fodrin) accompanies N-methyl-D-aspartic acid receptor activation (13), 2 does not directly cause neuronal toxicity (7,15), and is postulated to be necessary for synaptic and neuronal plasticity (16 -18). Indeed, ␣II-spectrin cleavage by calpain appears to be a molecular mechanism by which skeletal plasticity can be enhanced without complete dissolution of the spectrin skeleton since calpain-mediated cleavage of ␣II-spectrin bestows calmodulin regulation on oligomeric spectrin-actin complexes, but does not dissociate them (unless ␤II-spectrin is also c...

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Activity of calpains and caspase-3 inferred from proteolysis of the cytoskeletal protein alpha-spectrin into signature spectrin breakdown products (SBDPs) was used to provide the first systematic and simultaneous comparison of changes in activity of these two families of cysteine proteases after traumatic brain injury (TBI) in rats. Distinct regional and temporal patterns of calpain/caspase-3 processing of alpha-spectrin were observed in brain regions ipsilateral to the site of injury after TBI, including large increases of 145 kDa calpain-mediated SBDP in cortex (up to 30-fold), and enduring increases (up to 2 weeks) of 145 kDa SBDP in hippocampus and thalamus. By contrast, 120 kDa caspase-3-mediated SBDP was absent in cortex and showed up to a 2-fold increase in hippocampus and striatum at early (hours) after TBI. Future studies will clarify the pathological significance of large regional differences in activation of calpain and caspase-3 proteases after TBI.

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Much recent research has focused on the pathological significance of calcium accumulation in the central nervous system (CNS) following cerebral ischemia, spinal cord injury (SCI), and traumatic brain injury (TBI). Disturbances in neuronal calcium homeostasis may result in the activation of several calcium-sensitive enzymes, including lipases, kinases, phosphatases, and proteases. One potential pathogenic event in a number of acute CNS insults, including TBI, is the activation of the calpains, calcium-activated intracellular proteases. This article reviews new evidence indicating that overactivation of calpains plays a major role in the neurodegenerative cascade following TBI in vivo. Further, this article presents an overview from in vivo and in vitro models of CNS injuries suggesting that administration of calpain inhibitors during the initial 24-h period following injury can attenuate injury-induced derangements of neuronal structure and function. Lastly, this review addresses the potential contribution of other proteases to neuronal damage following TBI.

This study examined the effect of unilateral controlled cortical impact on the appearance of calpain-mediated alpha-spectrin breakdown products (BDPs) in the rat cortex and hippocampus at various times following injury. Coronal sections were taken from animals at 15 min, 1 h, 3 h, 6 h, and 24 h after injury and immunolabeled with an antibody that recognizes calpain-mediated BDPs to alpha-spectrin (Roberts-Lewis et al., 1994). Sections from a separate group of rats were also taken at the same times and stained with hematoxylin and eosin. Analyses of early time points (15 min, 1 h, 3 h, and 6 h following injury) revealed alpha-spectrin BDPs in structurally intact neuronal soma and dendrites in cortex ipsilateral to site of injury that was not present in tissue from sham-injured control rats. By 24 h after injury labeling was not restricted to clearly defined neuronal structures in ipsilateral cortex, although there was an increased extent of diffuse labeling. BDPs to alpha-spectrin in axons were not detected until 24 h after injury, in contrast to the more rapid accumulation of BDPs observed in neuronal soma and dendrites. The presence of BDPs to alpha-spectrin in the cortex at the site of impact, and in the rostral and contralateral cortex, coincided with morphopathology detected by hematoxylin and eosin. alpha-Spectrin BDPs were also observed in the hippocampus ipsilateral to the injury in the absence of overt cell death. This investigation provides further evidence that calpain is activated after controlled cortical impact and could contribute to necrosis at the site of injury. The appearance of calpain-mediated BDPs at sites distal to the contusion site and in the hippocampus also suggests that calpain activation may precede and/or occur in the absence of extensive morphopathological changes.

We have examined the effect of lateral cortical impact injury on the levels of axonal cytoskeletal proteins in adult rats. Traumatic brain injury (TBI) causes a significant decrease in the protein levels of two prominent neurofilament (NF) proteins, NF68 and NF200. We employed quantitative immunoreactivity measurements on Western blots to examine NF68 and NF200 levels in homogenates of hippocampal and cortical tissue taken at several intervals postinjury. Sham injury had no effect on NF protein levels. However, injury was associated with a significant loss of NF68, restricted to the cortex ipsilateral to the injury site. NF68 loss was detectable as early as 3 h and lasted at least 2 weeks postinjury. Similarly, TBI induced a decrease in NF200 protein, although losses were observed both ipsilateral and contralateral to the injury site. No loss of NF68 or NF200 protein was detected in hippocampal samples obtained from the same injured animals. An increase in the presence of lower molecular weight (MW) NF68 immunopositive bands was associated with the decrease of NF68 in the ipsilateral cortex. This NF68 antigenicity pattern suggests the production of NF68 breakdown products caused by the pathologic activation of neuronal proteases, such as calpain. Putative NF68 breakdown products increase significantly until 1 day postinjury, suggesting that NF degradation may be ongoing until that time and indicating that a potential therapeutic window may exist within the first 24 h postinjury. In summary, these data identify specific biochemical alterations of the neuronal cytoskeleton following TBI and lay a foundation for further investigation of postinjury cytoskeletal changes in neuronal processes.