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Inhibition of ischaemic hippocampal neuronal death in primates with cathepsin B inhibitor CA-074: a novel strategy for neuroprotection based on 'calpain-cathepsin hypothesis'
Abstract
Although Cornu Ammonis (CA) 1 neurons of the hippocampus are known to be vulnerable to transient ischaemia, the mechanism of ischaemic neuronal death is still unknown, and there are very few strategies to prevent neuronal death at present. In a previous report we demonstrated micro-calpain activation at the disrupted lysosomal membrane of postischaemic CA1 neurons in the monkey undergoing a complete 20 min whole brain ischaemia. Using the same experimental paradigm, we observed that the enzyme activity of the lysosomal protease cathepsin B increased throughout the hippocampus on days 3-5 after the transient ischaemia. Furthermore, by immunocytochemistry cathepsin B showed presence of extralysosomal immunoreactivity with specific localization to the cytoplasm of CA1 neurons and the neuropil of the vulnerable CA1 sector. When a specific inhibitor of cathepsin B, the epoxysuccinyl peptide CA-074 (C18H29N3O6) was intravenously administered immediately after the ischaemic insult, approximately 67% of CA1 neurons were saved from delayed neuronal death on day 5 in eight monkeys undergoing 20 min brain ischaemia: the extent of inhibition was excellent in three of eight and good in five of eight monkeys. The surviving neurons rescued by blockade of lysosomal activity, showed mild central chromatolysis and were associated with the decreased immunoreactivity for cathepsin B. These observations indicate that calpain-induced cathepsin B release is crucial for the development of the ischaemic neuronal death, and that a specific inhibitor of cathepsin B is of potential therapeutic utility in ischaemic injuries to the human CNS.
... Here, we discuss such a common cascade as 'oxidative stress-generation of hydroxynonenal-calpain activation-Hsp70.1 carbonylation-cleavage of Hsp70.1-lysosomal membrane disintegrity-cathepsin release-cell death' Yamashima and Oikawa, 2009) which leads to disorders of the brain, pancreas, and liver. The authors are convinced that this is exactly the first review discussing that the above three lifestyle-related diseases may occur by the same culprit, 'hydroxynonenal'. ...
... The lysosomal membrane destabilization was thought to be responsible for the oxidative stress-induced cell damage, since ROS were well known to induce leakage of the lysosomal content (Zdolsek and Svensson, 1993;Antunes et al., 2001;Dare et al., 2001;Persson et al., 2003). However, implication of ROS for the programmed cell death in diseases was not elucidated in detail until the formulation of the 'calpain-cathepsin hypothesis' by the authors (Yamashima et al., 1998;Yamashima, 2000;Oikawa et al., 2009;Yamashima and Oikawa, 2009). Although extremely rare to encounter in the advanced stage of Alzheimer's disease, Yamashima. ...
... The lysosomal membrane destabilization was thought to be responsible for the oxidative stress-induced cell damage, since ROS were well known to induce leakage of the lysosomal content (Zdolsek and Svensson, 1993;Antunes et al., 2001;Dare et al., 2001;Persson et al., 2003). However, implication of ROS for the programmed cell death in diseases was not elucidated in detail until the formulation of the 'calpain-cathepsin hypothesis' by the authors (Yamashima et al., 1998;Yamashima, 2000;Oikawa et al., 2009;Yamashima and Oikawa, 2009). Although extremely rare to encounter in the advanced stage of Alzheimer's disease, Yamashima. ...
Alzheimer’s disease, type 2 diabetes, and non-alcoholic steatohepatitis (NASH) constitute increasingly prevalent disorders. Individuals with type 2 diabetes are well-known to be susceptible to Alzheimer’s disease. Although the pathogenesis of each disorder is multifactorial and the causal relation remains poorly understood, reactive oxygen species (ROS)-induced lipid and protein oxidation conceivably plays a common role. Lipid peroxidation product was recently reported to be a key factor also for non-alcoholic steatohepatitis, because of inducing hepatocyte degeneration/death. Here, we focus on implication of the representative lipid-peroxidation product ‘hydroxynonenal’ for the cell degeneration/death of brain, pancreas, and liver. Since Hsp70.1 has dual roles as a chaperone and lysosomal membrane stabilizer, hydroxynonenal-mediated oxidative injury (carbonylation) of Hsp70.1 was highlighted. After intake of high-fat diets, oxidation of free fatty acids in mitochondria generates ROS which enhance oxidation of ω-6 polyunsaturated fatty acids (PUFA) involved within biomembranes and generate hydroxynonenal. In addition, hydroxynonenal is generated during cooking deep-fried foods with vegetable oils especially containing linoleic acids. These intrinsic and exogenous hydroxynonenal synergically causes an increase in its serum and organ levels to induce Hsp70.1 oxidation. As it is amphiphilic; being water-soluble but displays strong lipophilic characteristics, hydroxynonenal can diffuse within the cells and react with targets like senile and/or atheromatous plaques outside the cells. Hydroxynonenal can deepen and expand lysosomal injuries by facilitating ‘calpain-mediated cleavage of the carbonylated Hsp70.1’. Despite the unique anatomical, physiological, and biochemical characteristics of each organ for its specific disease, there should be a common cascade of the cell degeneration/death which is caused by hydroxynonenal. This review aims to implicate hydroxynonenal-mediated Hsp70.1 carbonylation for lysosomal membrane permeabilization/rupture and the resultant cathepsin leakage for inducing cell degeneration/death. Given the tremendous number of worldwide people suffering various lifestyle-related diseases, it is valuable to consider how ω-6 PUFA-rich vegetable oils is implicated for the organ disorder.
... The extra lysosomal immunoreactivity of CTPB has been implicated as an early event in neuronal death following in vivo and in vitro transient cerebral ischemia (Kilinc et al. 2010;Wang et al. 2011;Windelborn and Lipton 2008;Yamashima et al. 1998). Increased expression of CTPB was observed from 2 h to 7 d following reperfusion except for 1 d after I/R which temporarily decreased (Seyfried et al. 1997). ...
... Similar results were obtained from the proteomics study that showed many lysosomal hydrolases, including CTPD and CTPB, dramatically upregulated at 2 and 7 d, while slightly decreased at 1 d following cerebral I/R (Wen et al. 2019). Two possibilities emerge to the extralysosomal localization of CTPB: (1) they release from the degenerated lysosomes, (2) newly synthesized CTBPs are not transported into lysosomes (Yamashima et al. 1998). The presence of LAMP-1, a lysosomal membrane protein, which is spilled into the cytoplasm along with the release of CTPB, suggests that lysosomes after cerebral ischemia become leaky (Kilinc et al. 2010), a phenomenon called lysosomal membrane permeabilization (LMP), which is an important mechanism implicated in cellular damage (Nixon and Cataldo 1993). ...
... apoptosis) have been reported following the LMP in cerebral ischemia. Yamashima et al. demonstrated that leakage of CTPB into the cytoplasm caused necrosis in the monkey hippocampal CA1 neurons, which disappeared after treatment with a specific CTP inhibitor, and led to decreased death of neurons (Yamashima et al. 1998). They also revealed that µ-calpain, a cytosolic protease activated by Ca 2+ influx, translocated to the lysosomal membrane and contributed to the rupture of the lysosomes that subsequently resulted in eosinophilic coagulation necrosis and plasma membrane disruption, without formation of the apoptotic body. ...
Ischemic stroke is the second leading cause of mortality and disability globally. Neuronal damage following ischemic stroke is rapid and irreversible, and eventually results in neuronal death. In addition to activation of cell death signaling, neuroinflammation is also considered as another pathogenesis that can occur within hours after cerebral ischemia. Under physiological conditions, subcellular organelles play a substantial role in neuronal functionality and viability. However, their functions can be remarkably perturbed under neurological disorders, particularly cerebral ischemia. Therefore, their biochemical and structural response has a determining role in the sequel of neuronal cells and the progression of disease. However, their effects on cell death and neuroinflammation, as major underlying mechanisms of ischemic stroke, are still not understood. This review aims to provide a comprehensive overview of the contribution of each organelle on these pathological processes after ischemic stroke.
... This was largely because the ability of caspase inhibitors, such as zVAD-fmk, to inhibit cell death was considered as proof of not "necrotic" but "apoptotic" cell death, and previous researchers had failed to confirm evidence of lysosomal membrane rupture by electron microscopy (120,121). However, Yamashima et al. (122) ultrastructurally confirmed evidence of lysosomal membrane rupture in postischemic CA1 neurons after transient brain ischemia. Surprisingly, in the cortical neurons of an Alzheimer's patient, double-membrane structures presumably indicating lysosomal membrane permeabilization/rupture ( Figure 3C) can be, although extremely difficult to observe, confirmed by electron microscopy under highmagnification observation. ...
... As Hsp70.1 is involved in maintaining lysosomal membrane integrity, reactive oxygen species-induced Hsp70.1 carbonylation and calpain-mediated cleavage, in combination, cause lysosomal membrane disintegrity. The "calpain-cathepsin hypothesis" (9,21,122) can explain how impairments in proteolysis contribute to the development of neuronal dysfunction, degeneration, and death from low-species animals to primates. The pathogenic synergism of calpain activation and Hsp70.1 carbonylation works in concert to destabilize lysosomal membranes via BMP downregulation. ...
... According to the "calpain-cathepsin hypothesis" (Figure 2) (9,21,122), enzymatically active calpains destabilize the lysosome by cleaving Hsp70.1, which normally works as a molecular chaperone and protects the integrity of the lysosomal membrane. After oxidative stress-induced carbonylation, Hsp70.1 becomes extremely vulnerable to calpain-mediated cleavage (84,23). ...
Although excessive consumption of deep-fried foods is regarded as 1 of the most important epidemiological factors of lifestyle diseases such as Alzheimer's disease, type 2 diabetes, and obesity, the exact mechanism remains unknown. This review aims to discuss whether heated cooking oil-derived peroxidation products cause cell degeneration/death for the occurrence of lifestyle diseases. Deep-fried foods cooked in ω-6 PUFA-rich vegetable oils such as rapeseed (canola), soybean, sunflower, and corn oils, already contain or intrinsically generate "hydroxynonenal" by peroxidation. As demonstrated previously, hydroxynonenal promotes carbonylation of heat-shock protein 70.1 (Hsp70.1), with the resultant impaired ability of cells to recycle damaged proteins and stabilize the lysosomal membrane. Until now, the implication of lysosomal/autophagy failure due to the daily consumption of ω-6 PUFA-rich vegetable oils in the progression of cell degeneration/death has not been reported. Since the "calpain-cathepsin hypothesis" was formulated as a cause of ischemic neuronal death in 1998, its relevance to Alzheimer's neuronal death has been suggested with particular attention to hydroxynonenal. However, its relevance to cell death of the hypothalamus, liver, and pancreas, especially related to appetite/energy control, is unknown. The hypothalamus senses information from both adipocyte-derived leptin and circulating free fatty acids. Concentrations of circulating fatty acid and its oxidized form, especially hydroxynonenal, are increased in obese and/or aged subjects. As overactivation of the fatty acid receptor G-protein coupled receptor 40 (GPR40) in response to excessive or oxidized fatty acids in these subjects may lead to the disruption of Ca2+ homeostasis, it should be evaluated whether GPR40 overactivation contributes to diverse cell death. Here, we describe the molecular implication of ω-6 PUFA-rich vegetable oil-derived hydroxynonenal in lysosomal destabilization leading to cell death. By oxidizing Hsp70.1, both the dietary PUFA- (exogenous) and the membrane phospholipid- (intrinsic) peroxidation product "hydroxynonenal," when combined, may play crucial roles in the occurrence of diverse lifestyle diseases including Alzheimer's disease.
... Recently, it has been reported that partial rupture or permeabilization of the lysosomal membrane induces apoptosis through mitochondrial transmembrane potential loss or caspase activation, while severe lysosomal rupture induces necrosis in cancer cells (6,9). The leakage of cathepsins caused by lysosomal membrane permeabilization (LMP) has been observed in the brain tissues of various rodents and non-human primate stroke models; for example, it was reported 1 h after global ischemia in monkey brain (10,11), after transient focal ischemia in mouse brain (12) and after a 5-min oxygen glucose deprivation in the rat hippocampal slices (13). However, it remains unclear whether LMP is involved in glutamate excitotoxic cascades, which are regarded as the leading cause of neuronal death. ...
... This suggested that the LMP-cathepsin activation mediated by ROS was an early irreversible injury to neural cells following glutamate excitotoxicity, which was consistent with the observations of a strong protective effect of cathepsin B inhibitors in monkey (10) and mouse (12) ischemia models. These findings, along with those of previous studies (10)(11)(12), provide further evidence that LMP may be a promising target for neuronal protection. ...
... In order to determine the detailed molecular mechanisms underlying ROS-mediated LMP caused by glutamate further investigation is required. Increasing evidence suggests that LMP may be governed by several distinct mechanisms in a stimulus-and cell-type-dependent manner (6,9,10,12,13,16,18). Different from other studies which demonstrated that calpain promoted lysosomal membrane destabilization during neuronal death with different stimuli, such as transient focal ischemia ,oxygen glucose deprivation or global ischemia (10,13,28,29), the results of the present study revealed that glutamate-induced LMP was not mediated by calpain. ...
Glutamate is the principal neurotransmitter in the central nervous system. Glutamate-mediated excitotoxicity is the predominant cause of cerebral damage. Recent studies have shown that lysosomal membrane permeabilization (LMP) is involved in ischemia‑associated neuronal death in non‑human primates. This study was designed to investigate the effect of glutamate on lysosomal stability in primary cultured cortical neurons. Glutamate treatment for 30 min induced the permeabilization of lysosomal membranes as assessed by acridine orange redistribution and immunofluorescence of cathepsin B in the cytoplasm. Inhibition of glutamate excitotoxicity by the NMDA receptor antagonist MK‑801 and the calcium chelator ethylene glycol‑bis (2‑aminoethylether)‑N, N, N', N'‑tetraacetic acid, rescued lysosomes from permeabilization. The role of calpain and reactive oxygen species (ROS) in inducing LMP was also investigated. Ca2+ overload following glutamate treatment induced the activation of calpain and the production of ROS, which are two major contributors to neuronal death. It has been reported that lysosomal‑associated membrane protein 2 (LAMP2) and heat shock protein (HSP)70 are two calpain substrates that promote LMP in cancer cells; however, it was found that calpains were activated by glutamate, but only LAMP2 was subsequently degraded. Furthermore, LMP was not alleviated by treatment with the calpain inhibitors calpeptin and SJA6017, which blocked the cleavage of the calpain substrate α‑fodrin. It was demonstrated that LMP was significantly alleviated by treatment with the antioxidant N‑Acetyl‑L‑cysteine, indicating that LMP involvement in early glutamate excitotoxicity may be mediated partly by ROS rather than calpain activation. Overall, these data shed light on the role of ROS-mediated LMP in early glutamate excitotoxicity.
... Ischemia and inflammatory brain injuries in animal models also resulted in elevated CTSB. For example, increased CTSB of 2-to 3-fold above controls occurred in brains of acute and chronic rat and nonhuman primate ischemic models (Seyfried et al., 1997;Yamashima et al., 1998;Tsuchiya et al., 1999;Tsubokawa et al., 2006). Inflammation due to bacterial meningitis (Hoegen et al., 2011), sepsis (Ruff and Secrist, 1984;Hummel et al., 1988), neuroinflammation (Terada et al., 2010;Wu et al., 2017;Ni et al., 2019), and inflammatory pain resulted in elevation of brain CTSB that was 50% to sixfold above controls. ...
... Indeed, chemical inhibition of CTSB for improvement of behavioral deficits and neuropathology of brain disorders has been investigated in the field for Alzheimer's disease (Hook et al., , 2007(Hook et al., , 2008b(Hook et al., , 2011(Hook et al., 2014b, TBI and brain trauma (Knoblach et al., 2004;Sun et al., 2013;Luo et al., 2010;Hook et al., 2014a;Ni et al., 2012), ischemia (Inuzuka et al., 1990;Yamashima et al., 1998;Tsuchiya et al., 1999;Seyfried et al., 2001;Yoshida et al., Cathepsin B in Neurologic Diseases 2002;Tsubokawa et al., 2006), pain Nakanishi, 2020), meningitis (Ruff and Secrist, 1984), and other neurologic and neurodegenerative disease animal models (reviewed in Hook et al., 2020;Sharma et al., 2022). These studies have used (a) the selective CTSB inhibitor, CA-074 Towatari et al., 1991), administered in vivo to animal models as the prodrug form of CA-074Me (Buttle et al., 1992), (b) the pancysteine protease inhibitor E64c (Hashida et al., 1980;Tamai et al., 1986), administered as its prodrug form of E64d, and (c) other inhibitors of CTSB, such as K11777 (Turk et al., 2012), Z-Phe-Arg-FMK , and other related inhibitors. ...
Cathepsin B (CTSB) is a powerful lysosomal protease. This review evaluated CTSB gene knockout (KO) outcomes for amelioration of brain dysfunctions in neurologic diseases and aging animal models. Deletion of the CTSB gene resulted in significant improvements in behavioral deficits, neuropathology, and/or biomarkers in traumatic brain injury, ischemia, inflammatory pain, opiate tolerance, epilepsy, aging, transgenic Alzheimer's disease (AD), and periodontitis AD models as shown in 12 studies. One study found beneficial effects for double CTSB and cathepsin S KO mice in a multiple sclerosis model. Transgenic AD models using amyloid precursor protein (APP) mimicking common sporadic AD in three studies showed that CTSB KO improved memory, neuropathology, and biomarkers; two studies used APP representing rare familial AD and found no CTSB KO effect, and two studies used highly engineered APP constructs and reported slight increases in a biomarker. In clinical studies, all reports found that CTSB enzyme was upregulated in diverse neurologic disorders, including AD in which elevated CTSB was positively correlated with cognitive dysfunction. In a wide range of neurologic animal models, CTSB was also upregulated and not downregulated. Further, human genetic mutation data provided precedence for CTSB upregulation causing disease. Thus, the consilience of data is that CTSB gene KO results in improved brain dysfunction and reduced pathology through blockade of CTSB enzyme upregulation that causes human neurologic disease phenotypes. The overall findings provide strong support for CTSB as a rational drug target and for CTSB inhibitors as therapeutic candidates for a wide range of neurologic disorders. SIGNIFICANCE STATEMENT: This review provides a comprehensive compilation of the extensive data on the effects of deleting the cathepsin B (CTSB) gene in neurological and aging mouse models of brain disorders. Mice lacking the CTSB gene display improved neurobehavioral deficits, reduced neuropathology, and amelioration of neuronal cell death and inflammatory biomarkers. The significance of the compelling CTSB evidence is that the data consilience validates CTSB as a drug target for discovery of CTSB inhibitors as potential therapeutics for treating numerous neurological diseases.
... These molecular events are grossly similar to the neurodegeneration of hypothalamic arcuate nucleus occurring after the intake of high-fat diets [12] or hippocampal CA1 sector exposed to the oxidative stress during reperfusion after transient ischemia [3]. In 1998, Yamashima and his colleagues formulated the 'calpain-cathepsin hypothesis' as a mechanism of ischemic neuronal death [14,15]. Thereafter, they suggested role of lysosomal rupture due to calpain-mediated cleavage of oxidized (carbonylated) heat-shock protein 70.1 (Hsp70.1; ...
... Concerning the molecular mechanism of ischemic neuronal death, Yamashima et al. formulated the 'calpain-cathepsin hypothesis' in 1998 [14]. Thereafter, concerning the mechanism of Alzheimer's neuronal death, they suggested that Hsp70.1 with dual functions of molecular chaperone and lysosomal stabilizer becomes vulnerable to the cleavage by activated μ-calpain, which is facilitated after Hsp70.1 carbonylation by hydroxynonenal [33,69,70,71]. ...
Background For their functions of insulin biosynthesis and glucose- and fatty acid- induced insulin secretion, the Langerhans β-cells require an intracellular milieu rich in oxygen. This requirement makes β-cells, with their constitutively low antioxidative defense, susceptible to the oxidative stress. Although much progress has been made in identifying its molecular basis in the experimental systems, whether the oxidative stress due to excessive fatty acids plays a crucial role in the Langerhans degeneration in primates is still debated. Methods Focusing on Hsp70.1, which has dual functions as a molecular chaperone and lysosomal stabilizer, the mechanism of lipotoxicity to the Langerhans islet cells was studied using Japanese macaque monkeys (Macaca fuscata) after the consecutive injections of the lipid peroxidation product hydroxynonenal. Based on the ‘calpain-cathepsin hypothesis’ of ischemic neuronal death formulated in 1998, calpain activation, Hsp70.1 cleavage, and lysosomal integrity were studied by immunofluorescence histochemistry, electron microscopy and Western blotting. Results Light microscopy showed higher vacuole formation in the treated islet cells than in the control cells. Electron microscopy showed that vacuolar changes that were identified as enlarged rough endoplasmic reticula occurred mainly in β-cells followed by δ-cells. Intriguingly, both cell types showed a marked decrease in insulin and somatostatin granules. Furthermore, they exhibited marked increases in peroxisomes, autophagosomes/autolysosomes, lysosomal and peroxisomal membrane rupture/permeabilization, and mitochondrial degeneration. Disrupted peroxisomes were often localized in the close vicinity of degenerating mitochondria or autolysosomes. Immunofluorescence histochemical analysis showed an increased colocalization of activated µ-calpain and Hsp70.1 with the extralysosomal release of cathepsin B. Western blotting showed increases in µ-calpain activation, Hsp70.1 cleavage, and hydroxynonenal receptor GPR109A expression. Conclusions Taken together, these data implicate hydroxynonenal in both the carbonylation of Hsp70.1 and the activation of µ-calpain. The calpain-mediated cleavage of the carbonyl group on Hsp70.1 after the hydroxynonenal-mediated carbonylation of Hsp70.1, may cause lysosomal membrane rupture/permeabilization. The low defense of primate Langerhans cells against exogenous hydroxynonenal and peroxisomally-generated hydrogen peroxide, was presumably overwhelmed to facilitate cell degeneration.
... In post-ischemic neuronal necrosis, lysosomal rupture is induced in two different pathways at different times 150 . For one thing, in 9 previous study of ischemic brain model of monkey, Yamashima et al. 33,151,152 formulated the "calpains-cathepsin" hypothesis to illustrate the execution of programmed neuronal necrosis affecting lysosomal membrane. μ-Calpains at the lysosomal membrane of cornu ammonis 1 (CA1) neurons gets activated when Ca 2+ homeostasis is broken during transient brain ischemia. ...
... μ-Calpains at the lysosomal membrane of cornu ammonis 1 (CA1) neurons gets activated when Ca 2+ homeostasis is broken during transient brain ischemia. The activated form of μ-calpains was localized at the vacuolated or disrupted membrane of lysosome causing lysosomal membrane disruption and ensuing spillage of cathepsins from lysosome 33,151 . For another, during reperfusion, ROS oxidizes membrane fatty acids (e.g., linoleic and arachidonic acid) to generates 4-hydroxy-2-nonenal which can cause carbonylation of HSP70 150 . ...
Lysosome is a ubiquitous acidic organelle fundamental for the turnover of unwanted cellular molecules, particles, and organelles. Currently, the pivotal role of lysosome in regulating cell death is drawing great attention. Over the past decades, we largely focused on how lysosome influences apoptosis and autophagic cell death. However, extensive studies showed that lysosome is also prerequisite for the execution of regulated necrosis (RN). Different types of RN have been uncovered, among which, necroptosis, ferroptosis, and pyroptosis are under the most intensive investigation. It becomes a hot topic nowadays to target RN as a therapeutic intervention, since it is important in many patho/physiological settings and contributing to numerous diseases. It is promising to target lysosome to control the occurrence of RN thus altering the outcomes of diseases. Therefore, we aim to give an introduction about the common factors influencing lysosomal stability and then summarize the current knowledge on the role of lysosome in the execution of RN, especially in that of necroptosis, ferroptosis, and pyroptosis.
... Excitotoxicity is initiated by over-stimulation of ionotropic glutamate receptors (iGluRs), especially the N-methyl-D-aspartate (NMDA) receptors (Choi, 1988;Olney, 1969;Simon et al., 1984), which permit excessive influx of extracellular calcium (Ca 2+ ) into the cytosol to over-activate proteases (Ginet et al., 2014;Lankiewicz et al., 2000;Wang et al., 1996;Yamashima et al., 1998), neuronal nitric oxide synthase (nNOS) (Sattler et al., 1999) and NADPH oxidase 2 (NOX2) (Brennan et al., 2009). The excitotoxicity-activated proteases cleave specific neuronal proteins to modulate their activities, biological functions and stability (Tominaga et al., 1998;Wang et al., 1996). ...
... Enhanced proteolysis of neuronal proteins is a key cellular event directing neuronal death in excitotoxicity (Brorson et al., 1995;Hossain et al., 2013;Yamashima et al., 1998). Using the TAILS method (Kleifeld et al., 2011), we identified and quantified over 5000 N-terminal peptides derived from neuronal proteins in all experimental conditions. ...
Excitotoxicity, a neuronal death process in neurological disorders, is initiated by over-stimulation of neuronal ionotropic glutamate receptors. The over-stimulated receptors dysregulate proteases, protein kinases and phosphatases, which in turn modify target neuronal proteins to induce cell death. To decipher this cell death mechanism, we used quantitative proteomics, phosphoproteomics and N-terminomics to identify modified proteins in excitotoxic neurons. Data, available in ProteomeXchange (identifiers: PXD019527 and PXD019211), enabled us to identify over one thousand such proteins with calpains, cathepsins and over twenty protein kinases as their major modifiers. These protein modification events can potentially perturb signalling pathways governing cell survival, synaptogenesis, axonal guidance and mRNA processing. Importantly, blocking the modification of Src protein kinase, a signalling hub in excitotoxic neurons, protected against neuronal loss in vivo in a rat model of neurotoxicity. Besides offering new insights into excitotoxic neuronal death mechanism, our findings suggest potential neuroprotective therapeutic targets for treating neurological disorders.
Graphical abstract
Highlights
Multi-dimensional proteomic analysis identified proteins modified by proteolysis and altered phosphorylation in neurons undergoing excitotoxic cell death.
Calpains, cathepsins and over twenty protein kinases are major modifiers of these proteins.
These protein modification events are predicted to impact cell survival, axonal guidance, synaptogenesis and mRNA processing.
Blocking modification of an identified protein Src, which acts as a major signalling hub in neurons, was protective against excitotoxic injury in vivo .
In Brief
Using multidimensional proteomic approaches, Ameen, et al . mapped the changes of proteome, phosphoproteome and N-terminome of cultured primary neurons during excitotoxicity, a crucial neuronal death process in neurological disorders. These proteomic changes document new excitotoxicity-associated molecular events, and offer insights into how these events are organized to induce neuronal death. Potential therapeutic relevance of these molecular events is illustrated by the demonstration that in vivo blockade of one of these events could protect against excitotoxic neuronal loss.
... We operated ischemia-reperfusion according to the procedure previously described. (22,23) Briefly, after removal of the sternum, the innominate and left subclavian arteries were exposed in the mediastinum and were clipped for 20 min, then reperfusion was done. The effectiveness of clipping was demonstrated by an almost complete absence of cerebral blood flow, which was monitored by laser Doppler (Vasamedics, St. Paul, MN). ...
... Hippocampal DG and CA1 tissues were resected from both the sham-operated controls (n = 3), postischemic day 3 (n = 3), day 5 (n = 3) and day 7 (n = 3) monkeys at indicated time points after the ischemic insult under the general anaesthesia. (23) The control monkeys were dissected on the same day of sham operation. Dissected fresh samples were immediately put into the liquid nitrogen and stored at -80°C until use. ...
It is well-known that the cornu Ammonis 1 (CA1) sector of hippocampus is vulnerable for the ischemic insult, whereas the dentate gyrus (DG) is resistant. Here, to elucidate its underlying mechanism, alternations of protein oxidation and expression of DG in the monkey hippocampus after ischemia-reperfusion by the proteomic analysis were studied by comparing CA1 data. Oxidative damage to proteins such as protein carbonylation interrupt the protein function. Carbonyl modification of molecular chaperone, heat shock 70 kDa protein 1 (Hsp70.1) was increased remarkably in CA1, but slightly in DG. In addition, expression levels of nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase sirtuin-2 (SIRT2) was significantly increased in DG after ischemia, but decreased in CA1. Accordingly, it is likely that SIRT2 upregulation and negligible changes of carbonylation of Hsp70.1 exert its neuroprotective effect in DG. On the contrary, carbonylation level of dihydropyrimidinase related protein 2 (DRP-2) and l-lactate dehydrogenase B chain (LDHB) were slightly increased in CA1 as shown previously, but remarkably increased in DG after ischemia. It is considered that DRP-2 and LDHB are specific targets of oxidative stress by ischemia insult and high carbonylation levels of DRP-2 may play an important role in modulating ischemic neuronal death.
... Interestingly, inhibition of cathepsins not only inhibited necrosis but also further lysosomal destabilization [95]. LMP was shown to be an early event during cerebral and cardiac ischemia [208][209][210][211] and in H 2 O 2 induced necrosis [78]. Studies in rodent and primate models of cerebral ischemia showed lysosomal rupture and relocation of proteases such as cathepsin B and D into the cytosol resulting in neuronal cell death. ...
... As a result of increased mobilization of intracellular Ca 2+ during ischemia, calpains are activated and play a critical role in the execution of neuronal necrosis after ischemia by inducing LMP. The "Calpain-Cathepsin hypothesis" was postulated based on the observations by Yamashima et al. [210,214], that calpain mediates LMP leading to release of cathepsins into the cytosol and eventually necrosis in hippocampal CA1 neurons after ischemia. This hypothesis was confirmed in various experimental models ranging from nematodes to rodents and observations in postmortem human brains [4,[215][216][217]. ...
Lysosomes are small specialized organelles containing a variety of different hydrolase enzymes that are responsible for degradation of all macromolecules, entering the cells through the endosomal system or originated from the internal sources. This allows for transport and recycling of nutrients and internalization of surface proteins for antigen presentation as well as maintaining cellular homeostasis. Lysosomes are also important storage compartments for metal ions and nutrients. The integrity of lysosomal membrane is central to maintaining their normal function, but like other cellular membranes, lysosomal membrane is subject to damage mediated by reactive oxygen species. This results in spillage of lysosomal enzymes into the cytoplasm, leading to proteolytic damage to cellular systems and organelles. Several forms of lysosomal dependent cell death have been identified in diseases. Examination of these events are important for finding treatment strategies relevant to cancer or neurodegenerative diseases as well as autoimmune deficiencies. In this review, we have examined the current literature on involvement of lysosomes in induction of programed cell death and have provided an extensive list of therapeutic approaches that can modulate cell death. Exploitation of these mechanisms can lead to novel therapies for neurodegenerative diseases.
... Lysosome-dependent cell death is characterized by the destabilization of its limiting membrane (Fig. 5) followed by the leakage of cathepsins from the lysosomal lumen into the cytoplasm [167,[213][214][215][216][217][218]. Using the monkey experimental systems of transient brain ischemia, Yamashima et al. [219][220][221] formulated the 'calpain-cathepsin hypothesis' as a mechanism of programmed neuronal necrosis. They demonstrated that the lysosomal membrane of hippocampal CA1 neurons is disrupted by the activated μ-calpain after transient ischemia, which causes the release of lysosomal cathepsins B and L. Thereafter, the 'calpain-cathepsin hypothesis' has been confirmed, using a variety of experimental paradigms from C. elegans to rodents [222][223][224][225]. ...
... 'Hydroxynonenal-induced Hsp70 carbonylation' (Fig. 6C) followed by 'calpain-mediated cleavage of carbonylated Hsp70' (Fig. 6D) may be crucial for the execution of both ischemic and degenerative neuronal death. Calpain activation and Hsp70 disorder, combined together, at the lysosomal membranes may bring about programmed neuronal death by releasing hydrolytic cathepsin enzymes [219][220][221]. The pathway of cerebral ischemia and/or oxidative stresses, either acute (in case of stroke) or chronic (in case of degeneration) could result the following sequence of μ-calpain activation → excessive intake of ω-6 vegetable oils → increase of hydroxynonenal in the brain → hydroxynonenal-mediated Hsp70 carbonylation → activated μ-calpainmediated cleavage of carbonylated Hsp70 → lysosomal membrane destabilization → cathepsin release → breakdown of the cell constitutive proteins, which may in turn represent a central role not only for ischemic neuronal death [221,236,244] but also for Alzheimer neuronal death [237,245]. ...
... Neurons are not the only cells affected by excitotoxicity, as astrocytes and endothelial cells in the brain tissue are also targets for degradation of cytoplasmic macromolecules and organelles via the lysosomal system ( Xu and Zhang, 2011). In addition, in other models of focal ischemia (in rodents and primates) an increase of cathepsins in the cytosol was found as a result of lysosomal permeabilization ( Yamashima et al., 1998Yamashima et al., , 2003. In this work we found an increase in the intermediate and mature forms of Cat D in the cytosolic fraction of Cx (Fig. 1B,C) and HIP (Fig. 2B,C) in HI rats. ...
... Many studies have shown that different excitotoxic stimuli can lead to an increase in the activity of calpains, calcium-dependent proteases, and to the generation of reactive oxygen species that promote the lysosomal membrane destabilization during neuronal damage. Examples of such stimuli may be transient focal ischemia, glucose and oxygen deprivation, or global ischemia ( Yamashima et al., 1996Yamashima et al., , 1998Yap et al., 2006). ...
Neuronal excitotoxicity induced by glutamatergic receptor overstimulation contributes to brain damage. Recent studies have shown that lysosomal membrane permeabilization (LMP) is involved in ischemia-associated neuronal death. In this study we evaluated the effect of neonatal hypoxia-ischemia (HI), as a model of excitotoxicity, on the lysosomal integrity throughout the distribution of the lysosomal proteins cathepsin D and prosaposin. Rat pups (7 days old) of the Wistar Kyoto strain were submitted to HI and they were euthanized 4 days after treatment and the cerebral cortex (Cx) and hippocampus (HIP) were processed for immunohistochemistry or immunoblotting. Treatment induced an increase of gliosis and also a redistribution of both prosaposin and cathepsin D (as intermediate and mature forms), into the cytosol of the HIP and Cx. In addition, HI induced a decrease of LAMP-1 in the membranous fraction and the appearance of a reactive band to anti-LAMP-1 in the cytosolic fraction, suggesting a cleavage of this protein. From these results, we propose that the abnormal release of Cat D and PSAP to the cytosol is triggered as a result of LAMP-1 cleavage in HI animals, which leads to cell damage. This could be a common mechanism in pathological conditions that compromises neuronal survival and brain function.
... Calpain is Ca 2+ -regulated cysteine protease, playing an important role in the regulation of cell death [22]. The 'calpaincathepsin hypothesis' corroborated the role of lysosomal rupture as an executor of programmed neuronal necrosis after transient brain ischemia in the non-human primates [12,14,[37][38][39][40][41][42][43][44]. During ischemia, excessive Ca 2+ mobilization occurs specifically in the CA1 neuron, and μ-calpain is remarkably activated. ...
... Then, carbonylated Hsp70.1 is efficiently cleaved by activated μ-calpain, and this leads to the lysosomal membrane destabilization/rupture. Since calpain was found to be activated at the lysosomal membranes [38,41], both calpain activation and Hsp70.1 carbonylation may occur at the same place simultaneously. Consequently, release of hydrolytic enzyme cathepsins from the lysosomal lumen occurs to induce programmed CA1 neuronal necrosis within the CA1 sector [12]. ...
... Ten monkeys were divided into control animals (n=4) undergoing sham-operation and ischemic animals (n=3 for each post-ischemic days 7 and 15). Under general anesthesia, transient global brain ischemia was carried out as described previously [24][25][26]. Briefly, by the mediastinal approach after resecting the sternum, both the innominate and left subclavian arteries were clamped for 20 min under general anesthesia, according to the surgical procedure previously described [24,25]. ...
... Under general anesthesia, transient global brain ischemia was carried out as described previously [24][25][26]. Briefly, by the mediastinal approach after resecting the sternum, both the innominate and left subclavian arteries were clamped for 20 min under general anesthesia, according to the surgical procedure previously described [24,25]. A laser Doppler (Vasamedics, St. Paul MN) was used to demonstrate the effectiveness of clamping. ...
Background: Polyunsaturated fatty acids (PUFA) are known to be crucial for learning and memory. However, the detailed mechanism of PUFA effects upon neuronal functions remains almost unknown except for the possible facilitation of membrane fluidity. G-protein coupled receptor 40 (GPR40) was found to induce Ca2+ mobilization in response to diverse PUFA. Thereafter, the authors found GPR40 expression in the newborn neurons of the monkey hippocampus after ischemia. This suggested implications of PUFA-mediated GPR40 signaling for adult neurogenesis underlying learning and memory.
Objective: This study aims at evaluating whether PUFA-mediated GPR40 activation can affect synthesis of brain-derived neurotrophic factor (BDNF) with the aid of its proteolytic enzyme furin.
Methods: Monkeys underwent 20 min transient whole brain ischemia by clamping both the innominate and left subclavian arteries. On days 7 and 15 after ischemia/reperfusion, when adult neurogenesis was shown to be maximal previously by the authors, the brain samples were resected. By the Western blotting analysis of mature-BDNF (m-BDNF), pro-BDNF and furin, syntheses of BDNF in response to two GPR40 agonists as well as selective GPR40 antagonist GW1100, were studied using normal and post-ischemic monkey dentate gyrus (DG) tissue extracts.
Results: Both up-regulation of m-BDNF synthesis in response to two GPR40 agonists; fish oil PUFA and docosahexaenoic acid (DHA) and its down-regulation in response to GW1100, were observed. GPR40 antagonist inhibited m-BDNF synthesis, whereas two GPR40 agonists stimulated m-BDNF synthesis conceivably via furin activation. Cleavage of p-BDNF to m-BDNF by furin as well as syntheses of m-BDNF and furin in the DG tissues, occurred immediately after incubation with fish oil PUFA or DHA. Dynamic changes of GPR40, m-BDNF synthesis, and furin occurred simultaneously.
Conclusions: These data, although correlative, suggested that m-BDNF may be synthesized by the cleavage of pre-stocked pro-BDNF and/or released from the cell store in response to PUFA. By activating GPR40 and furin, PUFA may be related to adult neurogenesis and the concomitant synaptic plasticity for learning and memory. To the best of our knowledge, this is the first report suggesting a role of GPR40 in PUFA-mediated m-BDNF synthesis.
... Cathepsins are key regulators of apoptosis induced by a variety of different stimuli, including TNF-α (Guicciardi et al., 2000;Foghsgaard et al., 2001), TRAIL (Vigneswaran et al., 2005;Nagaraj et al., 2006), Fas (Wille et al., 2004), STS (paper I) (Bidère et al., 2003), UV irradiation (Bivik et al., 2006), ischemia (Yamashima et al., 1998), and oxidative stress (Roberg and Öllinger, 1998a;Öllinger, 2000;Kågedal et al., 2001a). The relative importance of different cathepsins seems to vary with the death stimulus and cell type. ...
... Cell death induced by ischaemia or HOCl exposure was associated with an increase in cytosolic Ca 2+ , activation of calpains, and LMP. Immuno cytochemical studies showed that activated µ-calpain located to the lysosomal membrane prior to release of cathepsin B to the cytosol, indicating its possible involvement in LMP (Yamashima et al., 1996;Yamashima et al., 1998;Yamashima et al., 2003). Furthermore, HOCl-induced permeabilization of the lysosomal membrane was abrogated by pharmacological inhibition of calpains (Yap et al., 2006). ...
... It has been reported that inhibition of Ca 2+ uptake by mitochondria can suppress necrotic cell death, as increased Ca 2+ levels in the cytoplasm lead to more controlled cell death pathways [ 24 ] . Moreover, the lysosomal damage and the calpain-cathepsin liberation must play essential roles in necrotic neuronal cell death, as lysosomal protease inhibitors have been shown to protect against delayed neuronal death produced by global ischemia [ 25,26 ] . Under some specifi c circumstances, necrosis may also be well regulated and is described as being "programmed necrosis" (also known as Type III PCD), a type of PCD. ...
... The spreading of hydrolytic enzymes into the cytoplasm through injury or rupture of the lysosomal membrane was confi rmed in ischemic brain injuries. This is the basis of the "calpain-cathepsin hypothesis" [ 26,142 ] , a hypothesis encompassing calpain and cathepsin as the key mediators of this process. For example, recent experiments on mice in which the endogenous calpain inhibitor calpastatin has been overexpressed or knocked out underscore the importance of calpains as an activator of lethal MOMP in neuronal cell death [ 143 ] . ...
Delayed neuronal death in the penumbral region of a stroke is largely responsible for many negative implications seen in stroke victims. This type of neuronal death occurs in many forms, including apoptosis, necrosis, and alternative mechanisms. Although caspases are usually associated with apoptosis, there are several morphologically and biochemically distinct types of cell death that are independent of caspase activation. Downstream effectors and processes of mitochondrial damage, such as AIF, endonuclease G, BNIP3, mitophagy, mitochondrial biogenesis, chaperone-mediated autophagy, reactive oxygen species production as well as parallel endoplasmic reticular stress and lysosomal dysfunction, have all been shown to play a role in post-stroke delayed neuronal cell death. In this chapter, we attempt to summarize these caspase-independent events and their potential therapeutic applications as targets for intervention.
... [185] Moreover, the lysosomal damage and the calpain-cathepsin liberation must play essential roles in necrotic neuronal cell death, as lysosomal protease inhibitors have been shown to protect against delayed neuronal death produced by global ischemia. [186,187] Under some specific circumstances, necrosis may also be well regulated and is described as being "programmed necrosis" (also known as Type III PCD), a type of PCD. Necrosis is actually usually the opposite of controlled and regulated cell death; that is, spontaneous, quick, and disordered destruction/lysis of the cell. ...
... The spreading of hydrolytic enzymes into the cytoplasm through injury or rupture of the lysosomal membrane was confirmed in ischemic brain injuries. This is the basis of the "calpaincathepsin hypothesis",[187,310] a hypothesis encompassing calpain and cathepsin as the key mediators of this process. For example, recent experiments on mice in which the endogenous calpain inhibitor calpastatin has been overexpressed or knocked out underscore the importance of calpains as an activator of lethal MOMP in neuronal cell death.[311] ...
Autophagy is a physiological process by which the cell eliminates damaged organelles, toxic agents, and long-lived proteins by degradation through lysosomal system. Mitophagy, the specific autophagic elimination of mitochondria, regulates mitochondrial number to match metabolic demand and is a core machinery of quality control to remove damaged mitochondria. A neuroprotective role of physiological autophagy/mitophagy has been discovered. However, recent studies suggested that highly accelerated autophagy/mitophagy might contribute to neuronal death in various pathological situations including cerebral ischemia. In this study, we aimed to investigate the activation of excessive autophagy, particularly, the more specific mitophagy, in neuronal tissues and its contribution to ischemia/hypoxia (I/H)-induced delayed neuronal death. I/H injury was induced by oxygen and glucose deprivation (OGD) followed by reperfusion (RP) on primary cortical neurons in vitro. Cerebral ischemia was induced by unilateral common carotid artery occlusion and hypoxia in neonatal mice in vivo.
In order to determine the extent to which autophagy contributes to neuronal death in cerebral ischemia, we performed multiple methods and found that in both primary cortical neurons and SH-SY5Y cells exposed to OGD for 6 h and RP for 24, 48, and 72 h, respectively, an increase of autophagy was observed as determined by the increased ratio of LC3-II to LC3-I and Beclin 1 expression. Using Fluoro-Jade C and monodansylcadaverine double-staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3-methyladenine. The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3-7 days postinjury from a rat model of neonatal cerebral I/H as shown by increased punctate LC3 staining and Beclin-1 expression. We thus obtained the conclusion that excessive activation of autophagy contributes to neuronal death in cerebral ischemia.
BNIP3 (Bcl-2/adenovirus E19 kD interacting protein 3), a member of a unique subfamily of death-inducing mitochondrial proteins, is highly associated with mitochondrial dysfunction and delayed neuronal death in stroke. It is known that BNIP3-induced neuronal death is caspase-independent and characterized by early mitochondrial damage. Recent evidence suggested that the BNIP3 family of proteins might be important regulators of mitophagy. Here, using both stroke models, we found that homodimer (60 kD) of BNIP3/NIX (BNIP3L) were highly expressed in a ‘delayed’ manner. Particularly, significant mitophagic activation was confirmed by electron microscopy. In contrast, both neonatal mitophagy and apoptosis were significantly inhibited in the BNIP3 knockout (KO) mice after I/H, which was also accompanied by a significantly increased autophagic response. In addition, the infarct volume in the BNIP3 KO mice was significantly reduced as compared to wild-type (WT) mice after 7 or 28 days recovery, showing a prominent neuroprotection of BNIP3 gene silencing. A protein-to-protein interaction of mitochondria-localized BNIP3 (60 kD) with the autophagosome marker, LC3, was confirmed by co-ip, immunocytochemistry and further quantified by ELISA, indicating BNIP3 was an effective LC3-binding target on damaged mitochondria. These data demonstrated a novel role of BNIP3 in regulating neuronal mitophagy and cell death during ischemic stroke.
... The X-ray crystal structure of cathepsin B protein in complex with CA-074 was downloaded in PDB format from the protein data bank using PDB ID: 1QDQ. This PDB was chosen because of its resolution of 2.18 Å, R-value of 0.152, and the co-crystallized ligand, CA-074 (IC 50 = 44 nM), that has been highly explored for neurodegenerative diseases as reported in the literature ( Fig. 4) [10,22,40,41]. The protein structure was visualized using Discovery Studio. ...
Cathepsin B is a cysteine protease lysosomal enzyme involved in several physiological functions. Overexpression of the enzyme enhances its proteolytic activity and causes the breakdown of amyloid precursor protein (APP) into neurotoxic amyloid β (Aβ), a characteristic hallmark of Alzheimer’s disease (AD). Therefore, inhibition of the enzyme is a crucial therapeutic aspect for treating the disease. Combined structure and ligand-based drug design strategies were employed in the current study to identify the novel potential cathepsin B inhibitors. Five different pharmacophore models were developed and used for the screening of the ZINC-15 database. The obtained hits were analyzed for the presence of duplicates, interfering PAINS moieties, and structural similarities based on Tanimoto’s coefficient. The molecular docking study was performed to screen hits with better target binding affinity. The top seven hits were selected and were further evaluated based on their predicted ADME properties. The resulting best hits, ZINC827855702, ZINC123282431, and ZINC95386847, were finally subjected to molecular dynamics simulation studies to determine the stability of the protein–ligand complex during the run. ZINC123282431 was obtained as the virtual lead compound for cathepsin B inhibition and may be a promising novel anti-Alzheimer agent.
Graphical abstract
The methodology utilized for the identification of novel cathepsin B inhibitors through combined structure and ligand-based drug design approach:
... Current studies of CA-074me have confirmed its treatment effect on fibrotic diseases, Alzheimer's disease, traumatic brain injury, ischemia, and inflammatory pain in animal models [66,67]. Yamashima et al. applied CA-074-me to adult monkeys to treat cerebral ischemia [68]. However, due to ethical issues and the difficulty of obtaining human samples of injured spinal cords, current studies haven't verified it in human tissue. ...
Hemorrhage and immune cell infiltration are the main pathological features of spinal cord injury (SCI). Excessive iron deposition is caused by leaking hemosiderin which may over-activate ferroptosis pathways, resulting in lipid peroxidation and mitochondrial dysfunction in cells. Inhibiting ferroptosis after SCI has been shown to aid functional recovery. However, the essential genes involved in cellular ferroptosis following SCI are still unknown. Here we show that Ctsb is a statistical significance gene by collecting multiple transcriptomic profiles and identifying differentially expressed ferroptosis-related genes, which are abundantly expressed in myeloid cells after SCI and widely distributed at the epicenter of the injury. The expression score of ferroptosis, calculated by ferroptosis driver/suppressor genes, was high in macrophages. Furthermore, we discovered that inhibiting cathepsin B (CTSB), specifically with a small-molecule drug, CA-074-methyl ester (CA-074-me), reduced lipid peroxidation and mitochondrial dysfunction in macrophages. We also found that alternatively activated M2-polarized macrophages are more susceptible to hemin-induced ferroptosis. Consequently, CA-074-me could reduce ferroptosis, induce M2 macrophage polarization, and promote the neurological function recovery of mice after SCI. Our study comprehensively analyzed the ferroptosis after SCI from the perspective of multiple transcriptomes and provided a novel molecular target for SCI treatment.
... C 18 H 29 N 3 O 6 methyl ester (CA-074me) and aloxistatin (E64d) can suppress the release of CTSB after cerebral ischemia and relieve brain injury (Yamashima et al., 1998;Xu et al., 2016). However, studies have shown that CA-074me and E64d are not specific CTSB inhibitors (Montaser et al., 2002;Mihalik et al., 2004;Ryu et al., 2014). ...
Cerebral ischemia is a serious disease that triggers sequential pathological mechanisms, leading to significant morbidity and mortality. Although most studies to date have typically focused on the lysosome, a single organelle, current evidence supports that the function of lysosomes cannot be separated from that of the endolysosomal system as a whole. The associated membrane fusion functions of this system play a crucial role in the biodegradation of cerebral ischemia-related products. Here, we review the regulation of and the changes that occur in the endolysosomal system after cerebral ischemia, focusing on the latest research progress on membrane fusion function. Numerous proteins, including N-ethylmaleimide-sensitive factor and lysosomal potassium channel transmembrane protein 175, regulate the function of this system. However, these proteins are abnormally expressed after cerebral ischemic injury, which disrupts the normal fusion function of membranes within the endolysosomal system and that between autophagosomes and lysosomes. This results in impaired "maturation" of the endolysosomal system and the collapse of energy metabolism balance and protein homeostasis maintained by the autophagy-lysosomal pathway. Autophagy is the final step in the endolysosomal pathway and contributes to maintaining the dynamic balance of the system. The process of autophagosome-lysosome fusion is a necessary part of autophagy and plays a crucial role in maintaining energy homeostasis and clearing aging proteins. We believe that, in cerebral ischemic injury, the endolysosomal system should be considered as a whole rather than focusing on the lysosome. Understanding how this dynamic system is regulated will provide new ideas for the treatment of cerebral ischemia.
... Excitotoxicity is initiated by over-stimulation of ionotropic glutamate receptors (iGluRs), especially N-methyl-D-aspartate (NMDA) receptors (Choi, 1988, Olney, 1969, Simon, Swan et al., 1984, which permit excessive influx of extracellular calcium (Ca 2+ ) into the cytosol to hyperactivate proteases (Ginet, Spiehlmann et al., 2014, Lankiewicz, Marc Luetjens et al., 2000, Wang, Nath et al., 1996, Yamashima, Kohda et al., 1998, neuronal nitric oxide synthase (nNOS) (Sattler, Xiong et al., 1999) and NADPH oxidase 2 (NOX2) (Brennan, Suh et al., 2009). The excitotoxicity-activated proteases cleave specific neuronal proteins to dysregulate their activities, biological functions and stability (Tominaga, Nakanishi et al., 1998, Wang et al., 1996, thereby contributing to neuronal cell death. ...
Excitotoxicity is a neuronal death process initiated by over-stimulation of ionotropic glutamate receptors. Although dysregulation of proteolysis and protein phosphorylation signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remains unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. Our investigation led to the discovery of proteins that, upon proteolysis by calpains, perturb synaptic organization and function. These included key synaptic regulatory proteins including CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ), which we found to undergo calpain-catalyzed proteolytic processing to generate stable truncated fragments with altered activities. We further show that blockade of proteolysis of Src by calpains could protect against neuronal loss in a rat model of neurotoxicity, and that CaMKIIβ and its isoform CaMKIIα undergo differential processing by calpains in mouse brains under physiological conditions and during ischemic stroke. Our findings thus reveal new insights into excitotoxic neuronal death mechanisms and suggest potential therapeutic targets for neurological disorders.
One Sentence Summary
Proteolytic events important for excitotoxic neuronal death as potential novel therapeutic targets.
Graphical Abstract
In Brief
Ameen, et al. used an N-terminomic method to identify neuronal proteins and the exact cleavage sites proteolyzed by calpains during excitotoxicity. Upon proteolysis, these proteins generate stable truncated fragments, which potentially induce synaptic dysfunction and loss, eventually leading to neuronal death. As such, some of these proteins such as protein kinases Src and CaMKII are potential targets for neuroprotection.
Highlights
Over 300 neuronal proteins are cleaved by calpains to form stable truncated fragments during excitotoxicity.
The calpain cleavage sites of these proteins unveil for the first time the preferred cleavage sequences of calpains in neurons.
These pathological proteolytic events potentially induce synaptic dysfunction and loss, which leads to excitotoxic neuronal death.
Some of the neuronal proteins proteolyzed by calpains are potential targets of neuroprotection.
... The release of cathepsin-B into cytosol leads to neuronal death in different models of cerebral ischemia (Kilinc et al., 2010;Tsubokawa et al., 2006). Cathepsin B inhibitor CA-074, a novel strategy for neuroprotection, protects against hippocampal neuronal death (Yamashima et al., 1998). The administration of CA074 is neuroprotective against cerebral ischemia by blocking the truncated Bidmitochondrial apoptotic signaling pathway (Xu et al., 2014). ...
Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory infiltration in association with demyelination in the central nervous system. Among the factors involved in the immunological mechanisms of MS, Th1, Th17, and Th22 cells play a critical role. In the present study, we investigated the role of CA-074, a potent Cathepsin B inhibitor, in MS progression, using the SJL/J mouse model of experimental autoimmune encephalomyelitis (EAE). Following induction of EAE, mice were administered CA-074 (10 mg/kg) intraperitoneally each day, beginning on day 14 and continuing until day 28, and were evaluated for clinical signs. We further investigated the effect of CA-074 on Th1 (T-bet/STAT4), Th17 (IL-17A/RORγT), Th22 (TNF-α/IL-22), and regulatory T (Treg/Foxp3) cells in the spleen, using flow cytometry. We also analyzed the effect of CA-074 on T-bet, IL-17A, RORγT, IL-22, and mRNA and protein levels using RT-PCR and western blot analysis for brain tissues. Cathepsin B expression were also assessed by western blot in the brain tissues. The severity of clinical scores decreased significantly in CA-074-treated mice compared with that in EAE control mice. Moreover, the percentage of CD4⁺T-bet⁺, CXCR5⁺T-bet⁺, CD4⁺STAT4⁺, CD4⁺IL-17A⁺, CXCR5⁺IL-17A⁺, CD4⁺RORγT⁺, CCR6⁺RORγT⁺, CD4⁺TNF-α⁺, CD4⁺IL-22⁺, and CCR6⁺IL-22⁺ cells decreased while CD25⁺Foxp3⁺ increased in CA-074-treated EAE mice as compared to vehicle-treated EAE mice. Further, CA-074-treated EAE mice had downregulated Cathepsin B protein expression which was associated with decreased T-bet, IL-17A, RORγT, and IL-22 mRNA/protein expression. These results suggest that Cathepsin B could be a novel therapeutic candidate against for the treatment of MS.
... With the assumption that most of the pro-inhibitor is converted to CA-074, the high concentration of CA-074 would be sufficient to inhibit cathepsin B located in different subcellular pH conditions of acidic lysosomes and neutral pH locations including cytosol. In animal studies, CA-074Me concentrations in the approximate range of 4-10 mg/kg and higher have been administered (52)(53)(54)(55)(56), which may correspond to micromolar and greater levels of the inhibitor that would inhibit cathepsin B at acidic to neutral pH cellular locations. Measurements of CA-074 and CA-074Me in vivo levels and clearance in animal studies will be important in future studies to assess in vivo inhibitor concentrations. ...
CA-074 is a selective inhibitor of cathepsin B, a lysosomal cysteine protease. CA-074 has been utilized in numerous studies to demonstrate the role of this protease in cellular and physiological functions. Cathepsin B in numerous human disease mechanisms involves its translocation from acidic lysosomes of pH 4.6 to neutral pH 7.2 of cellular locations, including the cytosol and extracellular environment. To gain in-depth knowledge of CA-074 inhibition under these different pH conditions, this study evaluated the molecular features, potency, and selectivity of CA-074 for cathepsin B inhibition under acidic and neutral pH conditions. This study demonstrated that CA-074 is most effective at inhibiting cathepsin B at an acidic pH of 4.6 with nM potency, which was more than 100-fold more potent than its inhibition at a neutral pH of 7.2. The pH-dependent inhibition of CA-074 was abolished by methylation of its C-terminal proline, indicating the requirement for the free C-terminal carboxyl group for pH-dependent inhibition. Under these acidic and neutral pH conditions, CA-074 maintained its specificity for cathepsin B over other cysteine cathepsins, displayed irreversible inhibition, and inhibited diverse cleavages of peptide substrates of cathepsin B assessed by profiling mass spectrometry. Molecular docking suggested that pH-dependent ionic interactions of the C-terminal carboxylate of CA-074 occur with His110 and His111 residues in the S2' subsite of the enzyme at pH 4.6, but these interactions differ at pH 7.2. While high levels of CA-074 or CA-074Me (converted by cellular esterases to CA-074) are used in biological studies to inhibit cathepsin B at both acidic and neutral pH locations, it is possible that adjusted levels of CA-074 or CA-074Me may be explored to differentially affect cathepsin B activity at these different pH values. Overall, the results of this study demonstrate the molecular, kinetic, and protease specificity features of CA-074 pH-dependent inhibition of cathepsin B.
... Linkermann et al. [38] pointed out that calpain was involved in programmed cell necrosis in acute kidney injury. Yamashima et al. [39] put forward the hypothesis of "calpain-cathepsin." Ischemia and hypoxia activate calpain that, in turn, acts on lysosomes, changing or disrupting lysosomal membrane permeability, leading to cathepsin in lysosomes being released into the cytoplasm. ...
Background
Idiopathic membranous nephropathy (IMN) is a cause of nephrotic syndrome that is increasing in incidence but has unclear pathogenesis. Urinary peptidomics is a promising technology for elucidating molecular mechanisms underlying diseases. Dysregulation of the proteolytic system is implicated in various diseases. Here, we aimed to conduct urinary peptidomics to identify IMN-related proteases.
Results
Peptide fingerprints indicated differences in naturally produced urinary peptide components among 20 healthy individuals, 22 patients with IMN, and 15 patients with other kidney diseases. In total, 1,080 peptide-matched proteins were identified, 279 proteins differentially expressed in the urine of IMN patients were screened, and 32 proteases were predicted; 55 of the matched proteins were also differentially expressed in the kidney tissues of IMN patients, and these were mainly involved in the regulation of proteasome-, lysosome-, and actin cytoskeleton-related signaling pathways. The 32 predicted proteases showed abnormal expression in the glomeruli of IMN patients based on Gene Expression Omnibus databases. Western blot revealed abnormal expression of calpain, matrix metalloproteinase 14, and cathepsin S in kidney tissues of patients with IMN.
Conclusions
This work shown the calpain/matrix metalloproteinase/cathepsin axis might be dysregulated in IMN. Our study is the first to systematically explore the role of proteases in IMN by urinary peptidomics, which are expected to facilitate discovery of better biomarkers for IMN.
... Recently Zhang et al. found that PDCoV infection upregulated the expression of cathepsin L and B in the endosome/lysosome to facilitate virus infection . An increase in the intracellular Ca 2+ concentration is well known to cause calpian-mediated lysosomal disruption with subsequent release of cathepsin B and L (Giorgi et al., 2008;Yamashima et al., 1998). These results are consistent with our conclusion that Ca 2+ plays an important role in PDCoV infection. ...
Ionic calcium (Ca2+) is a versatile intracellular second messenger that plays important roles in cellular physiological and pathological processes. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that causes serious vomiting and diarrhea in suckling piglets. In this study, the role of Ca2+ to PDCoV infection was investigated. PDCoV infection was found to upregulate intracellular Ca2+ concentrations of IPI-2I cells. Chelating extracellular Ca2+ by EGTA inhibited PDCoV replication, and this inhibitory effect was overcome by replenishment with CaCl2. Treatment with Ca2+ channel blockers, particularly the L-type Ca2+ channel blocker diltiazem hydrochloride, inhibited PDCoV infection significantly. Mechanistically, diltiazem hydrochloride reduces PDCoV infection by inhibiting the replication step of the viral replication cycle. Additionally, knockdown of CACNA1S, the L-type Ca2+ voltage-gated channel subunit, inhibited PDCoV replication. The combined results demonstrate that PDCoV modulates calcium influx to favor its replication.
... For TGBI experiments the total number of 5-10 years old animals (27 monkeys) was divided into five experimental groups of monkeys for Western blotting (15 monkeys, n=3 for each group of the control and post-ischemic days 3,7,9,15), and four experimental groups for immunofluorescence histochemical analyses (12 monkeys, n=3 for each group of the control and post-ischemic days 4,9,15). TGBI was made under general anesthesia, by clamping the innominate and left subclavian arteries for 20 min, according to the procedure described previously [38,39], whereas the control monkeys underwent a sham-operation. To label newly generated cells in adult macaque monkeys 5-bromo-2-deoxyuridine (BrdU) (Sigma, St Louis, MO) was intravenously injected in the saphenous vein at a dose of 100 mg/kg for five consecutive days before euthanasia. ...
Objective: Sub-ventricular Zone (SVZ) of the anterior horn of lateral ventricle is a source of neural stem cells in the adult mammalian brain along with hippocampal Sub-granular Zone (SGZ). Previously, we demonstrated that transient global brain ischemia in adult monkeys increases the number of neuronal progenitors in the SGZ via G-protein-coupled Receptor 40 (GPR40) signaling. Sonic Hedgehog (SHH) is indispensable for ischemia-induced neural progenitor proliferation in rodents. Although GPR40 is expressed in the SVZ, GPR40 synergy with SHH remains unelucidated in adult SVZ neurogenesis. Here, we studied GPR40 implication in SVZ neurogenesis using monkey model.
Methods: Adult monkeys underwent 20 min transient global brain ischemia by clamping both the innominate and left subclavian arteries. On days 3, 4, 7, 9 and 15 after ischemia/reperfusion, when SVZ neurogenesis increases, as it was shown previously by the authors, the brain samples were resected and normal and post-ischemic monkey SVZ tissues were used for Western blot and immunofluorescence histochemistry analysis.
Results: Ischemia/reperfusion increased GPR40 protein levels, proliferation of GPR40-positive stem/progenitor cells, the number of GPR40/PSA-NCAM or GPR40/doublecortin co-expressing immature neurons and GPR40- positive microglial cells in the SVZ vascular niche. GPR40 up-regulation correlated with that of SHH, Notch1 and β1-integrin; GPR40 co-localized with stem/progenitor cell markers in post-ischemic SVZ. GPR40-positive microglia showed the highest SHH expression relative to other cell types. SHH binding to Patched1 in the cells that surrounded GPR40/SHH-expressing microglia occurred within the distance of SHH paracrine secretion.
Conclusion: These data suggest that GPR40 is related to post-ischemic neurogenesis in the primate SVZ, GPR40-positive microglia support SHH paracrine secretion in the SVZ, and the cross-talk between perivasucular microglia and neuronal progenitors may be crucial in the vascular niche to activate neurogenesis by SHH and Notch1 signaling.
... When the activity of calpain abnormally increases, the oxidized Hsp70.1 is destroyed, the lysosomal membrane collapses, the proteolytic enzyme cathepsin leaks out, the cellular framework is gradually destroyed, and the cell dies. This is the "Calpain-Cathepsin Hypothesis" I proposed in 1998 [3]. The calpain-cathepsin cascade can explain the mechanisms of not only ischemic neuronal death but also Alzheimer neuronal death [4][5][6]. ...
... Previous studies show that the chemical agents CA-074me and E64d might offer neuroprotection by inhibiting CTSB after brain ischemia [37,38]. However, these inhibitors are not ideal therapeutic agents for the following reasons. ...
Neurons require an extraordinarily high level of membrane trafficking activities because of enriched axonal terminals and dendritic branches. For that reason, defects in the membrane trafficking pathway are a hallmark of most, and may be all, neurodegenerative disorders. A major cellular membrane trafficking pathway is the Golgi apparatus (Golgi hereafter)–late endosome–lysosome axis for supplying lysosomal enzymes. This pathway is regulated by N-ethylmaleimide-sensitive factor (NSF) ATPase. This review article is to discuss a novel hypothesis that brain ischemia inactivates NSF ATPase, resulting in a cascade of events of disruption of the Golgi—endosome—lysosome pathway, release of cathepsin B (CTSB), and induction of mitochondrial outer membrane permeabilization (MOMP) during the postischemic phase. This hypothesis is supported by recent studies demonstrating that NSF is trapped into inactive protein aggregates in neurons destined to die after brain ischemia. Consequently, Golgi, transport vesicles (TVs), and late endosomes (LEs) are accumulated and damaged, which is followed by CTSB release from these damaged structures. Moderate release of CTSB cleaves Bax-like BH3 protein (Bid) to become active truncated Bid (tBid). Active tBid is then translocated to the mitochondrial outer membrane, resulting in oligomerization of BCL2-associated X protein (Bax) forming the mitochondrial outer membrane pores, and releasing mitochondrial intramembranous proteins. Extensive CTSB release, however, can digest cellular proteins indiscriminately to induce cell death. Based on these new observations, we propose a novel hypothesis, i.e., brain ischemia leads to NSF inactivation, resulting in a massive buildup of damaged Golgi, TVs and LEs, fatal release of CTSB, induction of MOMP, and eventually brain ischemia-reperfusion injury.
... Cathepsins are lysosomal proteases involved in autophagic degradation and also major effectors of LMP leading to apoptosis, 25 hence they participate in the crosstalk between autophagy and apoptosis. Pioneer studies showed an increase and re-localization of CTSB immunoreactivity towards the cytosol in post-ischemic neurons, 36,37 and CTSB release from lysosomes after calpain-mediated LMP, has been proposed as the main causative factor of ischemic neuronal death. 26 Here, we show that inhibition of CTSB and CTSD protected neurons against GD/GR-induced neuronal death and that the recovery of lysosomes and CSTB immunoreactive particles correlated with increased cell survival, suggesting that extralysosome activated cathepsins might contribute to neuronal damage. ...
Autophagy is triggered during nutrient and energy deprivation in a variety of cells as a homeostatic response to metabolic stress. In the CNS, deficient autophagy has been implicated in neurodegenerative diseases and ischemic brain injury. However, its role in hypoglycemic damage is poorly understood and the dynamics of autophagy during the hypoglycemic and the glucose reperfusion periods, has not been fully described. In the present study, we analyzed the changes in the content of the autophagy proteins BECN1, LC3-II and p62/SQSTM1 by western blot, and autophagosome formation was followed through time-lapse experiments, during glucose deprivation (GD) and glucose reintroduction (GR) in cortical cultures. According to the results, autophagosome formation rapidly increased during GD, and was followed by an active autophagic flux early after glucose replenishment. However, cells progressively died during GR and autophagy inhibition reduced neuronal death. Neurons undergoing apoptosis during GR did not form autophagosomes, while those surviving up to late GR showed autophagosomes. Calpain activity strongly increased during GR and remained elevated during progressive neuronal death. Its activation led to the cleavage of LAMP2 resulting in lysosome membrane permeabilization (LMP) and release of cathepsin B to the cytosol. Calpain inhibition prevented LMP and increased the number of neurons containing lysosomes and autophagosomes increasing cell viability. Taken together, the present results suggest that calpain-mediated lysosome dysfunction during GR turns an adaptive autophagy response to energy stress into a defective autophagy pathway, which contributes to neuronal death. In these conditions, autophagy inhibition results in the improvement of cell survival.
... In a mouse ischemic-hypoxic model reported by Adhami, it showed that the features of autophagic cell death in many damaged neurons (1). In addition, up-regulation of cathepsin B activity has also been found in cerebral ischemia-induced brain injury, and treatment with the cathepsin B inhibitor reduced ischemia-induced neuron death (3,51). Nevertheless, previous studies have led to the opposite conclusion that autophagy inhibition may aggregate ischemic insults (10,46). ...
We previously reported that hypoxic postconditioning (HPC) ameliorated hippocampal neuronal death induced by transient global cerebral ischemia (tGCI) in adult rats. However, the mechanism of HPC-induced neuroprotection is still elusive. Notably, heat shock protein 27 (Hsp27) has recently emerged as a potent neuroprotectant in cerebral ischemia. Although its robust protective effect on stroke has been recognized, the mechanism of Hsp27-mediated neuroprotection is largely unknown. Here, we investigated the potential molecular mechanism by which HPC modulates the posttranslational regulations of Hsp27 after tGCI. We found that HPC increased expression of Hsp27 in CA1 subregion after tGCI. Inhibition of Hsp27 expression with lentivirus-mediated short hairpin RNA (shRNA) abolished the neuroprotection induced by HPC in vivo. Furthermore, pretreatment with cycloheximide, a protein synthesis inhibitor, resulted in a significant decrease in the degradation rate of Hsp27 protein in postconditioned rats, suggesting that the increase in the expression of Hsp27 after HPC might result from its decreased degradation. Next, pretreatment with leupeptin, a lysosomal inhibitor, resulted in an accumulation of Hsp27 after tGCI, indicating that autophagic pathway may be responsible for the degradation of Hsp27. We further showed that the formation of LC3-II and autophagosomes increased after tGCI. Meanwhile, the degradation of Hsp27 was suppressed and neuronal damage was reduced when blocking autophagy with 3-Methyladenine, whereas activating autophagy with rapamycin showed an opposite tendency. Lastly, we confirmed that HPC increased the expression of phosphorylated MAPKAP kinase 2 (MK2) and Hsp27 after tGCI. Also, administration of SB203580, a p38 mitogen-activated protein kinase inhibitor, decreased the expressions of phosphorylated MK2 and Hsp27. Our results suggested that inhibition of Hsp27 degradation mediated by down-regulation of autophagy may induce ischemic tolerance after HPC. Additionally, phosphorylation of Hsp27 induced by MK2 might be associated with the neuroprotection of HPC. This article is protected by copyright. All rights reserved.
... Among the lysosomal cathepsins, B, L and D are abundant in neurons [15]. Although the activity of cathepsin L markedly increases soon after OGD [16], a few studies have shown that the activity of cathepsin B increases 2 h after OGD [13,17]. It is therefore possible that the production of oxygen radicals by the metabolism of arachidonic acid following OGD induces cathepsin L release from lysosomes, and this may lead to the irreversible depolarization. ...
Oxygen and glucose deprivation (OGD) elicits a rapid and irreversible depolarization with a latency of ∼5 min in intracellular recordings of hippocampal CA1 neurons in rat slice preparations. In the present study, we examined the role of cathepsin L in the OGD-induced depolarization. OGD-induced depolarizations were irreversible as no recovery of membrane potential was observed. The membrane potential reached 0 mV when oxygen and glucose were reintroduced immediately after the onset of the OGD-induced rapid depolarization. The OGD-induced depolarizations became reversible when the slice preparations were pre-incubated with cathepsin L inhibitors (types I and IV at 0.3–2 nM and 0.3–30 nM, respectively). Moreover, pre-incubation with these cathepsin inhibitors prevented the morphological changes, including swelling of the cell soma and fragmentation of dendrites, observed in control neurons after OGD. These findings suggest that the activation of cathepsin L contributes to the irreversible depolarization produced by OGD.
... The "calpain-cathepsin hypothesis" was formulated to provide a mechanism for neuronal death based upon experimental observations in the ischemic monkey paradigm. The hypothesis posits that calpain 1 hyperactivation compromises the lysosomal membranes and causes the release of cathepsins into the cytoplasm 179 . Calpain activation has been confirmed in the ischemic monkey brain 180 and in brains of AD patients 23,181 . ...
Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy.
... Therefore cellular death response triggered by cytotoxic compounds may not only involve caspases but proteases from other organelles which can act independently or in collaboration with each other. For example a "calpain-cathepsin cascade" has been reported, in which activated calpains induce release of cathepsins and subsequent cell death [34,35]. ...
Geranylated 4-phenylcoumarins, DMDP-1 & -2 isolated from Mesua elegans were investigated for anticancer potential against human prostate cancer cells. Treatment with DMDP-1 & -2 resulted in cell death in a time and dose dependent manner in an MTT assay on all cancer cell lines tested with the exception of lung adenocarcinoma cells. DMDP-1 showed highest cytotoxic efficacy in PC-3 cells while DMDP-2 was most potent in DU 145 cells. Flow cytometry indicated that both coumarins were successful to induce programmed cell death after 24 h treatment. Elucidation on the mode-of-action via protein arrays and western blotting demonstrated death induced without any significant expressions of caspases, Bcl-2 family proteins and cleaved PARP, thus suggesting the involvement of caspase-independent pathways. In identifying autophagy, analysis of GFP-LC3 showed increased punctate in PC-3 cells pre-treated with CQ and treated with DMDP-1. In these cells decreased expression of autophagosome protein, p62 and cathepsin B further confirmed autophagy. In contrary, the DU 145 cells pre-treated with CQ and treated with DMDP-2 has reduced GFP-LC3 punctate although the number of cells with obvious GFP-LC3 puncta was significantly increased in the inhibitor-treated cells. The increase level of p62 suggested leakage of cathepsin B into the cytosol to trigger potential downstream death mediators. This correlated with increased expression of cathepsin B and reduced expression after treatment with its inhibitor, CA074. Also auto-degradation of calpain-2 upon treatment with DMDP-1 &-2 and its inhibitor alone, calpeptin compared with the combination treatment, further confirmed involvement of calpain-2 in PC-3 and DU 145 cells. Treatment with DMDP-1 & -2 also showed up-regulation of total and phosphorylated p53 levels in a time dependent manner. Hence, DMDP-1 & -2 showed ability to activate multiple death pathways involving autophagy, lysosomal and endoplasmic reticulum death proteins which could potentially be manipulated to develop anti-cancer therapy in apoptosis resistant cells.
... Calpains can cleave and activate caspases (Sharma & Rohrer, 2004), which can potentially proteolyse and modulate the function of key cellular proteins in the neuronal death pathways. Besides calpain and caspases, cathepsins are also involved in neuronal death in neurological diseases (Yamashima et al., 1998;Kikuchi et al., 2003;Sun et al., 2010). We therefore need an unbiased systems biology approach to define how cellular proteins modified by the two calpains, caspases, and cathepsins interplay to direct excitotoxic neuronal death. ...
... This may have special relevance to inflammation. Hsp70 can be degraded by calpain (described earlier as the protease that cleaves pro-IL-1α), which has been shown to contribute to LMD by a variety of studies in neurons (199,257,(330)(331)(332)(333). Next, I will focus on one more mechanism for promoting LMD as it relates to the consequences of crosstalk between lysosomes and mitochondria in cell death. ...
Sterile particles underlie the pathogenesis of numerous inflammatory diseases. These diseases can often become chronic and debilitating. Moreover, they are common, and include silicosis (silica), asbestosis (asbestos), gout (monosodium urate), atherosclerosis (cholesterol crystals), and Alzeihmer’s disease (amyloid Aβ). Central to the pathology of these diseases is a repeating cycle of particle-induced cell death and inflammation. Macrophages are the key cellular mediators thought to drive this process, as they are especially sensitive to particle-induced cell death and they are also the dominant producers of the cytokine responsible for much of this inflammation, IL-1β. In response to cytokines or microbial cues, IL-1β is synthesized in an inactive form (pro-IL-1β) and requires an additional signal to be secreted as an active cytokine. Although a multimolecular complex, called the NLRP3 inflammasome, controls the activation/secretion of IL-1β (and has been thought to also control cell death) in response to particles in vitro, the in vivo inflammatory response to particles occurs independently of inflammasomes. Therefore, I sought to better understand the mechanisms governing IL-1β production and cell death in response to particles, focusing specifically on the role of lysosomal cathepsin proteases. Inhibitor studies have suggested that one of these proteases, cathepsin B, plays a role in promoting inflammasome activation subsequent to particle-induced lysosomal damage, however genetic models of cathepsin B deficiency have argued otherwise. Through the use of inhibitors, state-of-the-art biochemical tools, and multi-cathepsin-deficient genetic models, I found that multiple redundant cathepsins promote pro-IL-1β synthesis as well as particle-induced NLRP3 activation and cell death. Importantly, I also found that particle-induced cell death does not depend on inflammasomes, suggesting that this may be why inflammasomes do not contribute to particle-induced inflammation in vivo. Therefore, my observations suggest that cathepsins may be multifaceted therapeutic targets involved in the two key pathological aspects of particle-induced inflammatory disease, IL-1β production and cell death.
... The "calpain-cathepsin hypothesis" was formulated to provide a mechanism for neuronal death based upon experimental observations in the ischemic monkey paradigm. The hypothesis posits that calpain 1 hyperactivation compromises the lysosomal membranes and causes the release of cathepsins into the cytoplasm 179 . Calpain activation has been confirmed in the ischemic monkey brain 180 and in brains of AD patients 23,181 . ...
Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy.
... The calpain-cathepsin hypothesis posits calpain activation due to excitotoxic stimulus disrupts lysosomal membranes. This is ensued by the release of the lysosomal proteases, including cathepsins, the breakdown of cellular proteins and, ultimately,necrosis (Yamashima et al., 1998;Yamashima et al., 2003). In support of this model, the sequential activation of calpain, cathepsins and caspases occurs in a model of focal ischemia (Chaitanya and Babu, 2008), while the administration of E64d, a combined µcalpain-cathepsin B inhibitor, decreases infarct volume, edema and neurologic deficit following focal ischemia in rats (Tsubokawa et al., 2006a;Tsubokawa et al., 2006b). ...
Stroke is the third leading cause of death in the United States and the second leading cause of death in the world. Despite the epidemiological significance of this disease, there are few treatment options. The purpose of this dissertation is to expand the understanding of underlying mechanisms mediating neuronal death caused by stroke, or cerebral ischemia. Two major metabolic disturbances occur due to ischemia—persistent protein synthesis inhibition and secondary energy depletion. All ischemia-affected neurons experience protein synthesis inhibition. However, neurons that recover protein synthesis live, while neurons that fail to recover die. This makes protein synthesis a robust predictor of neuronal death. However, the underlying mechanisms of persistent protein synthesis inhibition remain unknown. The hypothesis of this dissertation is that persistent protein synthesis inhibition is caused by activation of the calcium-sensitive protease calpain, which degrades eukaryotic translation initiation factor (eIF) 4G. Inhibition of calpain or overexpression of eIF4G results in increased protein synthesis and increased neuronal viability following the in vitro model of ischemia oxygen glucose deprivation in rat primary cortical neurons. Importantly, the neuroprotective effect of preservation of eIF4G is only partly due to its restoration of protein synthesis. Potential protein synthesis-independent mechanisms eIF4G-mediated protection are discussed. Neurons subjected to ischemia suffer an initial loss of energy in the form of ATP, which returns to baseline within fifteen minutes of restoration of blood flow. However, ischemia-sensitive neurons undergo secondary energy depletion prior to delayed neuronal death. The cause of secondary energy failure is hypothesized to be due to DNA recognition enzyme poly(ADP)-ribose polymerase (PARP)-1 depletion of the energy substrate NAD+. Evidence is presented linking PARP-1 activation to mitochondrial calcium dysregulation with subsequent calpain activation and apoptosis-inducing factor release. The results of these two findings are discussed in depth and future experiments are outlined. The potential of role of eIF4G in mitochondrial biogenesis, inhibition of autophagy and prevention of secondary energy loss is postulated. The research presented in this dissertation provides a novel perspective regarding the mechanisms underlying delayed neuronal death and may eventually lead to the development of clinically applicable neuroprotective strategies.
... Cathepsin B has been implicated in ischemic injury of the rat hippocampus. 12 Cathepsin L is a liposomal/endosomal protease that is converted from its inactive pre-pro-enzyme to the active precursor pro-cathepsin L within the cell, then through cleavage of the 96 residue pro-region (between the N-terminus and signal sequence) in an autocatalytic manner to the active form. 13,14 Conversion of cathepsin B into its active form requires removal of its 62 residue pro-region, six residue COOH terminus, and residues 47 and 49 to generate the two chain form. ...
During focal cerebral ischemia, the degradation of microvessel basal lamina matrix occurs acutely and is associated with edema formation and microhemorrhage. These events have been attributed to matrix metalloproteinases (MMPs). However, both known protease generation and ligand specificities suggest other participants. Using cerebral tissues from a non-human primate focal ischemia model and primary murine brain endothelial cells, astrocytes, and microglia in culture, the effects of active cathepsin L have been defined. Within 2 hours of ischemia onset cathepsin L, but not cathepsin B, activity appears in the ischemic core, around microvessels, within regions of neuron injury and cathepsin L expression. In in vitro studies, cathepsin L activity is generated during experimental ischemia in microglia, but not astrocytes or endothelial cells. In the acidic ischemic core, cathepsin L release is significantly increased with time. A novel ex vivo assay showed that cathepsin L released from microglia during ischemia degrades microvessel matrix, and interacts with MMP activity. Hence, the loss of microvessel matrix during ischemia is explained by microglial cathepsin L release in the acidic core during injury evolution. The roles of cathepsin L and its interactions with specific MMP activities during ischemia are relevant to strategies to reduce microvessel injury and hemorrhage.Journal of Cerebral Blood Flow & Metabolism advance online publication, 22 July 2015; doi:10.1038/jcbfm.2015.170.
... To date, growing evidence exists supporting the contribution of nonconventional (caspase-independent) cell-death mechanisms to the progression of neurodegenerative diseases [19,20]. Activation of calpains (calcium-dependent proteases) and cathepsins (lysosomal proteases) in fact has been reported in degenerative processes [21,22]. Calpains are ubiquitously expressed and highly activated under stress conditions in response to an increased influx of ions through cGMP-gated cation channels. ...
Herein, we have investigated retinal cell-death pathways in response to the retina toxin sodium iodate (NaIO3) both in vivo and in vitro. C57/BL6 mice were treated with a single intravenous injection of NaIO3 (35 mg/kg). Morphological changes in the retina post NaIO3 injection in comparison to untreated controls were assessed using electron microscopy. Cell death was determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining. The activation of caspases and calpain was measured using immunohistochemistry. Additionally, cytotoxicity and apoptosis in retinal pigment epithelial (RPE) cells, primary retinal cells, and the cone photoreceptor (PRC) cell line 661W were assessed in vitro after NaIO3 treatment using the ApoToxGlo™ assay. The 7-AAD/Annexin-V staining was performed and necrostatin (Nec-1) was administered to the NaIO3-treated cells to confirm the results. In vivo, degenerating RPE cells displayed a rounded shape and retracted microvilli, whereas PRCs featured apoptotic nuclei. Caspase and calpain activity was significantly upregulated in retinal sections and protein samples from NaIO3-treated animals. In vitro, NaIO3 induced necrosis in RPE cells and apoptosis in PRCs. Furthermore, Nec-1 significantly decreased NaIO3-induced RPE cell death, but had no rescue effect on treated PRCs. In summary, several different cell-death pathways are activated in retinal cells as a result of NaIO3.
... Vor allem Cathepsin D, welches sich von den anderen Cathepsinen strukturell durch eine spezielle Aspartat-Bindung unterscheidet [125], scheint in pathologische Prozesse des ZNS involviert zu sein. DerPathomechanismus besteht in der Induktion des neuronalen Zelltodes oder einer Veränderung der proteolytischen Prozesse innerhalb der Nervenzellen[76,84]. Die anderen Cathepsine, wie z.B. auch Cathepsin B, wurden hauptsächlich im Zusammenhang mit der neuronalen Apoptose nach Ischämie[134,124] oder bei neurodegenerativen Prozessen nachgewiesen[69,47].In Anbetracht der bisherigen Arbeiten wäre somit im ERG am ehesten mit einer verminderten Signalamplitude oder einer Verzögerung der Latenzzeit bei der Ctsz-/-Maus zu rechnen gewesen. Die durchgängig leichte Signalerhöhung in allen Zellreihen, wie auch die signifikant erhöhte Zapfenantwort bei den Ctsz-/-Mutanten, lassen sich am ehesten im Sinne eines überschießenden Kompensationsmechanismus durch Cathepsin B erklären, haben in Anbetracht der unauffälligen Kurvenverläufe und der bisherigen Arbeiten aber wahrscheinlich nur geringen physiologischen Stellenwert. ...
In dieser Arbeit sollte die Lokalisation und Funktion der Cathepsine B und Z in der Maus Netzhaut erstmalig untersucht werden und eine mögliche Beteiligung an retinalen Angiogeneseprozessen evaluiert werden. Es zeigte sich ein ubiquitäres Vorkommen der Cathepsine B und Z in allen Netzhautschichten mit prominenter Expression in neuronalen Zellschichten und entlang der membrana limitans externa. Trotz ubiquitären Vorkommens wiesen Cathepsin Z-knockout, Cathepisn B-knockout und auch Cathepsin B/Z-Doppelknockout Mäuse eine normlae Netzhaut-, Chorioidea- und RPE Struktur auf. Auch im Maus-Elektroretingramm fanden sich keine signifikanten Einschränkungen. Im Laser-CNV.Modell konnte erstmalig eine Beteiligung der Cathepsine B und Z an retinalen Angiogeneseprozessen bewiesen werden. Während der Verlust eine Cathepsins durch Kompensationsmechanismen nicht zu signifikanten Veränderungen der Laser-CNV-Fläche führt, ezigt die Cathepsin B/Z-doppelknockut signifikant kleinere CNV-Flächen im Vergleich zum Wildtyp.
... primate global cerebral ischemia, the cathepsin B inhibitor CA-074 protects against neuronal death from lysosomal rupture and release of cathepsins into the cytosol of neurons in the hippocampus, which is the basis of their "calpain-cathepsin hypothesis"[62]. They subsequently found that hydroxynonenal (HNE) phosphorylates heat shock protein 1 (HSP-1), which is responsible for permeabilization of lysosomal membranes, allowing release of cathepsins into the cytosol[63] and DNase II into the nucleus[64]. ...
Excitotoxicity involves the excessive release of glutamate from presynaptic nerve terminals and from reversal of astrocytic glutamate uptake, when there is excessive neuronal depolarization. N-methyl-D-aspartate (NMDA) receptors, a subtype of glutamate receptor, are activated in postsynaptic neurons, opening their receptor-operated cation channels to allow Ca2 + influx. The Ca2 + influx activates two enzymes, calpain I and neuronal nitric oxide synthase (nNOS). Calpain I activation produces mitochondrial release of cytochrome c (cyt c), truncated apoptosis-inducing factor (tAIF) and endonuclease G (endoG), the lysosomal release of cathepsins B and D and DNase II, and inactivation of the plasma membrane Na+-Ca2 + exchanger, which adds to the buildup of intracellular Ca2 +. tAIF is involved in large-scale DNA cleavage and cyt c may be involved in chromatin condensation; endoG produces internucleosomal DNA cleavage. The nuclear actions of the other proteins have not been determined. nNOS forms nitric oxide (.NO), which reacts with superoxide (.O2-) to form peroxynitrite (ONOO-). These free radicals damage cellular membranes, intracellular proteins and DNA. DNA damage activates poly(ADP-ribose) polymerase-1 (PARP-1), which produces poly(ADP-ribose) (PAR) polymers that exit nuclei and translocate to mitochondrial membranes, also releasing AIF. Poly(ADP-ribose) glycohydrolase hydrolyzes PAR polymers into ADP-ribose molecules, which translocate to plasma membranes, activating melastatin-like transient receptor potential 2 (TRPM-2) channels, which open, allowing Ca2 + influx into neurons. NADPH oxidase (NOX1) transfers electrons across cellular membranes, producing .O2-. The result of these processes is neuronal necrosis, which is a programmed cell death that is the basis of all acute neuronal injury in the adult brain.
... As shown here, CatB inhibitors reduced both flAβ and pGlu-Aβ and, thus, will likely be effective if either of these Aβ species cause the disease. Moreover, CatB inhibitors are potent neuroprotectants in models of traumatic brain injury and ischemia, which are risk factors for AD [101,102]. And CatB knockout studies show that the absence of CatB prevents tumor necrosis factor (TNF) induced cell death and interleukin-1β (IL1β) inflammation, both of which occur in AD [103][104][105][106][107]. Thus, CatB inhibitor therapeutics would reduce flAβ and pGlu-Aβ, as well as provide neuroprotection and reduce apoptosis and inflammation. ...
Pyroglutamate amyloid-β peptides (pGlu-Aβ) are particularly pernicious forms of amyloid-β peptides (Aβ) present in Alzheimer's disease (AD) brains. pGlu-Aβ peptides are N-terminally truncated forms of full-length Aβ peptides (flAβ(1-40/42)) in which the N-terminal glutamate is cyclized to pyroglutamate to generate pGlu-Aβ(3-40/42). β-secretase cleavage of amyloid-β precursor protein (AβPP) produces flAβ(1-40/42), but it is not yet known whether the β-secretase BACE1 or the alternative β-secretase cathepsin B (CatB) participate in the production of pGlu-Aβ. Therefore, this study examined the effects of gene knockout of these proteases on brain pGlu-Aβ levels in transgenic AβPPLon mice, which express AβPP isoform 695 and have the wild-type (wt) β-secretase activity found in most AD patients. Knockout or overexpression of the CatB gene reduced or increased, respectively, pGlu-Aβ(3-40/42), flAβ(1-40/42), and pGlu-Aβ plaque load, but knockout of the BACE1 gene had no effect on those parameters in the transgenic mice. Treatment of AβPPLon mice with E64d, a cysteine protease inhibitor of CatB, also reduced brain pGlu-Aβ(3-42), flAβ(1-40/42), and pGlu-Aβ plaque load. Treatment of neuronal-like chromaffin cells with CA074Me, an inhibitor of CatB, resulted in reduced levels of pGlu-Aβ(3-40) released from the activity-dependent, regulated secretory pathway. Moreover, CatB knockout and E64d treatment has been previously shown to improve memory deficits in the AβPPLon mice. These data illustrate the role of CatB in producing pGlu-Aβ and flAβ that participate as key factors in the development of AD. The advantages of CatB inhibitors, especially E64d and its derivatives, as alternatives to BACE1 inhibitors in treating AD patients are discussed.
... Cathepsins have been implicated in CNS apoptosis following ischemia or during neurodegenerative processes. For instance, cathepsin B released from compromised lysosomes into the cytoplasm was crucial for the post-ischemic neuronal death in vivo (Seyfried et al., 1997: Yamashima et al., 1998, and in vitro studies suggested that this process was dependent on NMDA-mediated calcium influx and ROS production (Windelborn and Lipton, 2008). Cathepsin L was also identified as an important mediator of the ß-amyloid protein-induced apoptosis in cultured cortical neurons (Boland and Campbell, 2004). ...
Excitotoxicity is the underlying mechanism for all acute neuronal injury, from cerebral ischemia, status epilepticus, traumatic CNS injury, and hypoglycemia. It causes morphological neuronal necrosis, and it triggers a programmed cell death program. Excessive calcium entry through the NMDA‐receptor‐operated cation channel activates two key enzymes—calpain I and neuronal nitric oxide synthase (nNOS). Calpain I, a cytosolic enzyme, translocates to mitochondrial and lysosomal membranes, causing release of cytochrome c, endonuclease G, and apoptosis‐inducing factor (AIF) from mitochondria and DNase II and cathepsins B and D from lysosomes. These all translocate to neuronal nuclei, creating DNA damage, which activates poly(ADP) ribose polymerase‐1 (PARP‐1) to form excessive amounts of poly(ADP) ribose (PAR) polymers, which translocate to mitochondrial membranes, causing release of truncated AIF (tAIF). The free radicals that are released from mitochondria and peroxynitrite, formed from nitric oxide (NO) from nNOS catalysis of L‐arginine to L‐citrulline, damage mitochondrial and lysosomal membranes and DNA. The end result is the necrotic death of neurons. Another programmed necrotic pathway, necroptosis, occurs through a parallel pathway. As investigators of necroptosis do not recognize the excitotoxic pathway, it is unclear to what extent each contributes to programmed neuronal necrosis. We are studying the extent to which each contributes to acute neuronal necrosis and the extent of cross‐talk between these pathways.
Ischemia-reperfusion injuries produce reactive oxygen species that promote the peroxide lipid oxidation process resulting in the production of an endogenic lipid peroxide, 4-hydroxy-trans-2-nonenal (4-HNE), a highly cytotoxic aldehyde that induces cell death. We synthesized a novel 4-HNE scavenger – a carnosine-hydrazide derivative, l-carnosine hydrazide (CNN) – and examined its neuroprotective effect in a model of transient ischemia.
PC-12 cells were pre-incubated with various doses (0–50 mmol/L) of CNN for 30 min, followed by incubation with 4-HNE (250 μM). An MTT assay was performed 24 h later to examine cell survival. Transient ischemia was induced by bilateral common carotid artery occlusion (BCCO) in the Mongolian gerbil. Animals were assigned to sham-operated (n = 6), placebo-treated (n = 12), CNN pre-treated (20 mg/kg; n = 12), CNN post-treated (100 mg/kg; n = 11), and histidyl hydrazide (a previously known 4-HNE scavenger) post-treated (100 mg/kg; n = 7) groups. Heat shock protein 70 immunoreactivity in the hippocampal CA1 region was evaluated 24 h later, while delayed neuronal death using 4-HNE staining was evaluated 7 days later.
Pre-incubation with 30 mmol/L CNN completely inhibited 4-HNE-induced cell toxicity. CNN prevented delayed neuronal death by >60% in the pre-treated group (p < 0.001) and by >40% in the post-treated group (p < 0.01). Histidyl hydrazide post-treatment elicited no protective effect. CNN pre-treatment resulted in high heat shock protein 70 and low 4-HNE immunoreactivity in CA1 pyramidal neurons. Higher 4-HNE immunoreactivity was also found in the placebo-treated animals than in the CNN pre-treated animals.
Our novel compound, CNN, elicited highly effective 4-HNE scavenging activity in vitro. Furthermore, CNN administration both pre- and post-BCCO remarkably reduced delayed neuronal death in the hippocampal CA1 region via its induction of heat shock protein 70 and scavenging of 4-HNE.
Proteases are enzymes which catalyze the irreversible hydrolysis of peptide bonds in proteins. Cysteine cathepsins belonging to proteases have also been termed as papain-like proteases because they resemble the overall fold of papain. The present chapter aims to focus on the historical aspects, structure, cellular distribution, biosynthesis, mechanism of catalysis, its regulation, physiological functions, and its association with rheumatoid arthritis. As these enzymes are also new therapeutic drug targets, information on available assays of cysteine cathepsins and their inhibitors are also highlighted which will help in the development of therapies in various diseases.
A series of chalcone derivatives were synthesized and evaluated for their μ-calpain and cathepsin B inhibitory activities. Among the tested chalcone derivatives, two compounds, 7 and 11, showed potent inhibitory activities against μ-calpain and cathepsin B and were selected for further evaluation. Compounds 7 and 11 showed enzyme inhibitory activities at the cellular level and displayed neuroprotective effects against oxidative stress-induced apoptosis in SH-SY5Y cells, a human neuroblastoma cell line. Moreover, compounds 7 and 11 reduced p25 formation, tau phosphorylation and insoluble Aβ peptide formation. Enzyme kinetic experiments and docking studies revealed that compounds 7 and 11 competitively inhibited both μ-calpain and cathepsin B enzymes.
Autophagy is a process responsible for the turnover of unnecessary or dysfunctional organelles and proteins. It facilitates normal cell growth and development, and it is also a survival pathway, required during starvation or growth factor deprivation. Massive vacuolation as a result of uncontrolled autophagy leads to autophagic cell death. On the other hand, necroptosis is a regulated cell death and shares identical subcellular events with necrosis and secondary necrosis. It is widely postulated that various proteins and pathways related to autophagy and necroptosis signaling are deregulated during cancer development. This chapter highlights the signaling pathways of autophagy and necroptosis and the relevant therapeutic targets in cancer and summarizes the current state of development of novel therapeutics in various phases of clinical trials. In addition, crosstalks in apoptosis, autophagy, and necroptosis signaling pathways and future treatment strategies will be reviewed.
There is currently no therapeutic drug treatment for traumatic brain injury (TBI) despite decades of experimental clinical trials. This may be because the mechanistic pathways for improving TBI outcomes have yet to be identified and exploited. As such, there remains a need to seek out new molecular targets and their drug candidates to find new treatments for TBI. This review presents supporting evidence for cathepsin B, a cysteine protease, as a potentially important drug target for TBI. Cathepsin B expression is greatly up-regulated in TBI animal models, as well as in trauma patients. Importantly, knockout of the cathepsin B gene in TBI mice results in substantial improvements of TBI-caused deficits in behavior, pathology, and biomarkers, as well as improvements in related injury models. During the process of TBI-induced injury, cathepsin B likely escapes the lysosome, its normal subcellular location, into the cytoplasm or extracellular matrix (ECM) where the unleashed proteolytic power causes destruction via necrotic, apoptotic, autophagic, and activated glia-induced cell death, together with ECM breakdown and inflammation. Significantly, chemical inhibitors of cathepsin B are effective for improving deficits in TBI and related injuries including ischemia, cerebral bleeding, cerebral aneurysm, edema, pain, infection, rheumatoid arthritis, epilepsy, Huntington’s disease, multiple sclerosis, and Alzheimer’s disease. The inhibitor E64d is unique among cathepsin B inhibitors in being the only compound to have demonstrated oral efficacy in a TBI model and prior safe use in man and as such it is an excellent tool compound for preclinical testing and clinical compound development. These data support the conclusion that drug development of cathepsin B inhibitors for TBI treatment should be accelerated.
Neuroinflammation is a hallmark that leads to selective neuronal loss and/or dysfunction in neurodegenerative disorders. Microglia-derived lysosomal cathepsins are increasingly recognized as important inflammatory mediators to trigger signaling pathways that aggravate neuroinflammation. However, cathepsin H (Cat H), a cysteine protease, has been far less studied in neuroinflammation, compared to cathepsins B, D, L, and S. The expression patterns and functional roles of Cat H in the brain in neuroinflammation remain unknown.
C57BL/6J mice were intraperitoneally injected with either 0.9% saline or lipopolysaccharide (LPS, 5 mg/kg). Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to analyze expression and localization of Cat H in the brain. Nitrite assay was used to examine microglial activation in vitro; ELISA was used to determine the release of Cat H and proinflammatory cytokines (TNF-α, IL-1β, IL-6, IFN-γ). Cat H activity was analyzed by cellular Cat H assay kit. Flow cytometry and in situ cell death detection were used to investigate neuronal death. Data were evaluated for statistical significance with one-way ANOVA and t test.
Cat H mRNA was only present in perivascular microglia and non-parenchymal sites under normal conditions. After LPS injection, Cat H mRNA expression in activated microglia in different brain regions was increased. Twenty-four hours after LPS injection, Cat H mRNA expression was maximal in SNr; 72 h later, it peaked in cerebral cortex and hippocampus then decreased and maintained at a low level. The expression of Cat H protein exhibited the similar alterations after LPS injection. In vitro, inflammatory stimulation (LPS, TNF-α, IL-1β, IL-6, and IFN-γ) increased the release and activity of Cat H in microglia. Conversely, addition of Cat H to microglia promoted the production and release of NO, IL-1β, and IFN-γ which could be prevented by neutralizing antibody. Further, addition of Cat H to Neuro2a cells induced neuronal death.
Taken together, these data indicate that the up-regulated microglial Cat H expression, release, and activity in the brain lead to neuronal death in neuroinflammation. The functional link of Cat H with microglial activation might contribute to the initiation and maintenance of microglia-driven chronic neuroinflammation.
Intense proteolysis of cytoskeletal proteins occurs in brain within minutes of transient ischemia, possibly because of the activation of calcium-sensitive proteases (calpains). This proteolytic event precedes overt signs of neuronal degeneration, is most pronounced in regions of selective neuronal vulnerability, and could have significant consequences for the integrity of cellular function. The present studies demonstrate that (i) the early phase of enhanced proteolysis is a direct response to hypoxia rather than other actions of ischemia, (ii) it is possible to pharmacologically inhibit the in vivo proteolytic response to ischemia, (iii) inhibition of proteolysis is associated with a marked reduction in the extent of neuronal death, and (iv) protected neurons exhibit normal-appearing electrophysiological responses and retain their capacity for expressing long-term potentiation, a form of physiological plasticity thought to be involved in memory function. These observations indicate that calcium-activated proteolysis is an important component of the post-ischemic neurodegenerative response and that targeting this response may be a viable therapeutic strategy for preserving both the structure and function of vulnerable neurons.
During the past 100 years clinical studies of amnesia have linked memory impairment to damage of the hippocampus. Yet the damage in these cases has not usually been confined to the hippocampus, and the status of memory functions has often been based on incomplete neuropsychological information. Thus, the human cases have until now left some uncertainty as to whether lesions limited to the hippocampus are sufficient to cause amnesia. Here we report a case of amnesia in a patient (R.B.) who developed memory impairment following an ischemic episode. During the 5 years until his death, R.B. exhibited marked anterograde amnesia, little if any retrograde amnesia, and showed no signs of cognitive impairment other than memory. Thorough histological examination revealed a circumscribed bilateral lesion involving the entire CA1 field of the hippocampus. Minor pathology was found elsewhere in the brain (e.g., left globus pallidus, right postcentral gyrus, left internal capsule), but the only damage that could be reasonably associated with the memory defect was the lesion in the hippocampus. To our knowledge, this is the first reported case of amnesia following a lesion limited to the hippocampus in which extensive neuropsychological and neuropathological analyses have been carried out.
The CA1 pyramidal neurons in the hippocampus are selectively vulnerable to transient ischemic damage. In experimental animals, the CA1 pyramidal neurons undergo cell death several days after brief forebrain ischemia. It remains, however, unknown whether this delayed neuronal death is necrosis or apoptosis. To investigate the degenerating processes of the CA1 pyramidal neurons in gerbil hippocampus after brief ischemia, lysosomal and nuclear alterations in the cells were examined using immunocytochemistry, in situ nick-end labeling, and Southern blotting. By light and electron microscopy, immunoreactivity for cathepsins B, H, and L, representative lysosomal cysteine proteinases, increased in the CA1 pyramidal neurons 3 d after ischemic insult, which showed cell shrinkage. By morphometric analysis, the volume density of cathepsin B-positive lysosomes markedly increased 3 d after ischemic insult, while that of autophagic vacuole-like structures also increased at this stage, suggesting that cathepsin B-immunopositive lysosomes increasing in the neurons after ischemic insult are mostly autolysosomes. Nuclei of the CA1 neurons were nick-end labeled by biotinylated dUTP mediated by terminal deoxytransferase 3 and 4 d after ischemic insult, but not in the prior stages. Simultaneously, dense chromatin masses appeared in nuclei of the neurons. By Southern blotting, laddering of DNA occurred only in CA1 hippocampal tissues obtained 4 d after ischemic insult. Confocal laser scanning microscopy demonstrated that the fragmented DNA in the CA1 pyramidal layer was phagocytosed by microglial cells. The results suggest that delayed death of the CA1 pyramidal neurons after brief ischemia is not necrotic but apoptotic.
To clarify the mechanism of postischaemic delayed cornu Ammonis (CA)-1 neuronal death, we studied correlations among calpain activation and its subcellular localization, the immunoreactivity of phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ mobilization in the monkey hippocampus by two independent experimental approaches: in vivo transient brain ischaemia and in vitro hypoxia-hypoglycaemia of hippocampal acute slices. The CA-1 sector undergoing 20 min of ischaemia in vivo showed microscopically a small number of neuronal deaths on day 1 and almost global neuronal loss on day 5 after ischaemia. Immediately after ischaemia, CA-1 neurons ultrastructurally showed vacuolation and/or disruption of the lysosomes. Western blotting using antibodies against inactivated or activated μ-calpain demonstrated μ-calpain activation specifically in the CA-1 sector immediately after ischaemia. This finding was confirmed in the perikarya of CA-1 neurons by immunohistochemistry. CA-1 neurons on day 1 showed sustained activation of μ-calpain, and increased immunostaining for inactivated and activated forms of μ- and m-calpains and for PIP2. Activated μ-calpain and PIP2 were found to be localized at the vacuolated lysosomal membrane or endoplasmic reticulum and mitochondrial membrane respectively, by immunoelectron microscopy. Calcium imaging data using hippocampal acute slices showed that hypoxia-hypoglycaemia in vitro provoked intense Ca2+ mobilization with increased PIP2 immunostaining specifically in CA-1 neurons. These data suggest that transient brain ischaemia increases intracellular Ca2+ and PIP2 breakdown, which will activate calpain proteolytic activity. Therefore, we suggest that activated calpain at the lysosomal membrane, with the possible release of biodegrading enzyme, will cause postischaemic CA-1 neuronal death.
We carried out an immunohistochemical study to detect changes in phosphatidylinositol 4,5-bisphosphate (PIP2) in gerbil hippocampus at various times after transient ischemia, using an anti-PIP2 antibody. About 24 h after transient ischemia for 5 min, an increase in the immunoreactivity was observed which was restricted to the area of CA1 pyramidal neurons. On the other hand, after less severe ischemia lasting 2 min, which did not lead to neuronal death, a decrease in PIP2 immunoreactivity was observed at about 48 h. The results indicate that levels of PIP2 following ischemia reflect dynamic changes in phosphatidylinositol (PI) turnover which may be related to neuronal degeneration.
The ultrastructural changes in the pyramidal neurons of the CA1 region of the hippocampus were studied 6 h, 24 h, 48 h, and 72 h following a transient 10 min period of cerebral ischemia induced by common carotid occlusion combined with hypotension. The pyramidal neurons showed delayed neuronal death (DND), i.e. at 24 h and 48 h postischemia few structural alterations were noted in the light microscope, while at 72 h extensive neuronal degeneration was apparent. The most prominent early ultrastructural changes were polysome disaggregation, and the appearance of electron-dense fluffy dark material associated with tubular saccules. Mitochondria and nuclear elements appeared intact until frank neuronal degeneration. The dark material accumulated with extended periods of recirculation in soma and in the main trunks of proximal dendrites, often beneath the plasma membrane, less frequently in the distal dendrites and seldom in spines. Protein synthesis inhibitors (anisomycin, cycloheximide) and an RNA synthesis inhibitor (actinomycin D), administered by intrahippocampal injections or subcutaneously, did not mitigate neuronal damage. Therefore, DND is probably not apoptosis or a form of programmed cell death. We propose that the dark material accumulating in the postischemic period represents protein complexes, possibly aggregates of proteins or internalized plasma membrane fragments, which may disrupt vital cellular structure and functions, leading to cell death.
To investigate cerebral injury in the monkey due to transient ischemia, monkeys were each subjected to temporary occlusion of eight (bilateral common carotid, internal and external carotid, and vertebral) major arteries. After 0 (control), 5, 10, 13, 15, and 18 min occlusion, blood flow was restored. The monkeys were sacrificed by perfusion fixation 5 days after the operation, and all brain regions were then histologically examined for ischemic neuronal changes induced by the occlusion. The amplitude of EEG signals from skull and scalp became almost isoelectric within 1-6 min after the onset of occlusion. The EEG signals from the hippocampus were markedly attenuated within 1-4 min, although they did not become completely isoelectric. Blood pressure was significantly increased after 10-min ischemia. Five-min occlusion produced no ischemic neuronal changes except a slight increment of glial cells in the striatum and III, V, and VI layers of the neocortices. After 10- to 15-min occlusion, there were ischemic cell changes restricted exclusively to the CA1 subfield of the hippocampus. Eighteen-min occlusion produced more prominent ischemic neuronal damage in the CA1 subfield of the hippocampus, but ischemic neuronal damage was no longer confined to the hippocampus. These results suggest that only the CA1 subfield of the monkey hippocampus could be damaged by mild ischemic insult. We demonstrate that the limited lesion of the hippocampus, especially the CA1 subfield, after 10- to 15-min occlusion of eight arteries in the monkey, produces a model equivalent to human amnesia caused by transient ischemic insult.
Transient forebrain ischemia of 30 min duration was produced in anaesthetized rats by four-vessel occlusion. After survival periods of 3 h to three days brains were perfusion-fixed and sections through the mid-dorsal hippocampus were processed for conventional staining and immunohistochemical analysis. Neuronal damage in the hilus was manifested 3-8 h after ischemia; neurons in the CA1 and CA2 sector suffered delayed neuronal death after 48-72 h whereas the dentate gyrus and the CA3 sector were normal. Vasogenic edema formation was visualized using antibodies against rat serum-proteins, serum albumin and immunoglobulins. By 3 h after ischemia, only faint and diffuse serum-staining was detected. At 8 h survival, weak astrocytic-staining was present. After 24-72 h CA1-CA2 exhibited massive serum extravasation. The molecular layer of the dentate gyrus showed edema formation in the absence of granule cell damage. The glial reaction was studied using antibodies against glial fibrillary acidic protein, vimentin and S-100 protein. Glial fibrillary acidic protein and S-100 protein-staining increased in areas with either edema or neuronal damage. In contrast, changes in vimentin were only detected in areas with neuronal necrosis. The observations demonstrate that following 30 min of ischemia neuronal damage is accompanied by changes in blood-brain barrier function and reactive glial alterations. The dissociation between neuronal necrosis and astroglial hypertrophy and hyperplasia reflects differences in cellular responsiveness which constitute inherent features of postischemic hippocampal injury.
Breakdown products (BDPs) resulting from the partial proteolysis of spectrin were examined in hippocampal slices after periods of hypoxia lasting for 5 or 10 min. The concentration of a approximately 155 kDa BDP increased nearly twofold after 5 min of hypoxia; further increases were not seen with 10 min episodes or 10 min of hypoxia followed by reoxygenation. The hypoxia-induced proteolysis was blocked by prior infusion of a newly introduced inhibitor of calpain (calpain inhibitor I, 200 microM). Together with previously published data showing improved recovery of hippocampal slices from hypoxia in the presence of calpain inhibitors, these data suggest that activation of calpain may contribute significantly to the pathophysiology of ischemia.
New derivatives of E-64 (compound CA-030 and CA-074) were tested in vitro and in vivo for selective inhibition of cathepsin B. They exhibited 10,000-30,000 times greater inhibitory effects on purified rat cathepsin B than on cathepsin H and L: their initial Ki values for cathepsin B were about 2-5 nM, like that of E-64-c, whereas their initial Ki values for cathepsins H and L were about 40 200 microM. In in vivo conditions, such as intraperitoneal injection of compound CA-030 or CA-074 into rats, compound CA-074 is an especially potent selective inhibitor of cathepsin B, whereas compound CA-030 does not show selectivity for cathepsin B, although both compounds CA-030 and CA-074 show complete selectivity for cathepsin B in vitro.
A series of new epoxysuccinyl peptides were designed and synthesized to develop a specific inhibitor of cathepsin B. Of these compounds, N-(L-3-trans-ethoxycarbonyloxirane-2-carbonyl)-L-isoleucyl-L-proli ne (compound CA-030) and N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-prol ine (compound CA-074) were the most potent and specific inhibitors of cathepsin B in vitro. The carboxyl group of proline and the ethyl ester group or the n-propylamide group in the oxirane ring were necessary, the ethyl ester group or the n-propylamide group being particularly effective for distinguishing cathepsin B from other cysteine proteinases such as cathepsins L and H, and calpains.
We measured cerebral intracellular pH using in vivo phosphorus-31 nuclear magnetic resonance spectroscopy during 1 week after forebrain ischemia or sham operation in eight and seven rats, respectively. Mean maximum pH was significantly higher (p less than 0.003) in the ischemic group than in the sham-operated group (7.34 +/- 0.03 and 7.19 +/- 0.02, respectively). The difference between mean maximum pH and baseline pH (7.08 +/- 0.01 in each group) was significantly greater (p less than 0.02) in the ischemic group than in the sham-operated group. In the ischemic group, alkalosis occurred primarily after 48-72 hours of recirculation. We speculate that brain tissue alkalosis occurring chronically after ischemia is associated with delayed ischemic neuronal death.
The lysosomal thiol proteinase, cathepsin B, has been localized in different regions of aged human brain by use of the peroxidase-antiperoxidase technique. Cathepsin B-immunoreactive material was detected in multiple neurons of human hippocampus, neocortical area A 10, prefrontal gyrus and nuc. basalis of Meynert as well as in single white matter astrocytes. In brains of Alzheimer disease-affected subjects cathepsin B was revealed in neuritic plaques too. Possible functional consequences with regard to normal aging, neuropeptide metabolism and pathological changes are discussed.
Transient forebrain ischemia is followed within minutes by accelerated proteolysis of the cytoskeletal protein, spectrin. This effect is most pronounced in the selectively vulnerable CA1 region of hippocampus which also experiences a second proteolytic phase during the terminal stages of neuronal degeneration. Both proteolytic phases are suppressed by MK-801, an NMDA receptor antagonist. Cytoskeletal disruption, via NMDA receptor-linked proteolytic events, is suggested to predispose vulnerable neurons to delayed cell death.
This study explored (a) whether postischemic accumulation of calcium in hippocampal neurons precedes or occurs pari passu with light microscopical signs of delayed neuronal necrosis, and (b) whether calcium initially accumulates in dendritic domains, presumed to have a high density of agonist-operated calcium channels. Transient ischemia of 10-min duration was induced in rats, and the animals were studied after 1, 2, 3, and 4 days of recovery. We measured total calcium and potassium contents in the stratum oriens, pyramidale, radiatum, and moleculare of the CA1 and CA3 sectors, using particle induced x-ray emission (PIXE) in the proton microprobe mode. The results showed significant accumulation of calcium and loss of potassium after 3 and 4 days of recovery in the CA1 sector, which developed neuronal necrosis, but not in the CA3 sector, which showed only occasional damage. In a few animals, calcium accumulation (and loss of potassium) was observed with no or only mild visible damage, but in the majority of animals the accumulation of calcium correlated to signs of neuronal necrosis. Since calcium accumulation was similar in all strata examined, the results failed to reveal preferential accumulation in dendritic or somal regions. Based on our results and those of Dux et al., we emphasize the possibility that delayed neuronal death is, at least in part, caused by increased calcium cycling of plasma membranes and gradual calcium overload of mitochondria.
The study of the age-dependent change in lysosomal enzyme activities of the cerebral tissue showed the significant increase of cathepsin D in the aged rat brain, while those of beta-glucuronidase and acid phosphatase remained unchanged. The subcellular distribution study of cathepsin D and beta-glucuronidase revealed the increased activity of these enzymes in the cytosolic fraction from the aged brain. In vitro incubation of the lysosome fraction from the aged rat brain resulted in more leakage of these two enzymes, indicating the instability of the lysosome in the aged brain, which resembled the effect of L-Leu-methyl ester to the lysosome.
Experimental focal cerebral ischemia was produced in monkeys (Macaca radiata) by occlusion of the right middle cerebral artery (MCA). The release of the lysosomal glycosidases, beta-D-hexosaminidase, alpha-L-fucosidase and alpha-D-mannosidase into the soluble fraction in the right basal ganglia of the experimental animals was measured at different periods from 30 min to 12 hr after occlusion and compared with the corresponding sham operated control animals. There was a significant increase in the released lysosomal enzymes in the MCA occluded animals at all periods and particularly at 4 hr after occlusion. The CSF from the experimental animals also showed elevated levels of hexosaminidase and fucosidase. The free fatty acids (FFA) measured in the basal ganglia at 30 min and 2 hr after occlusion showed a 100 fold increase in the experimental animals. The predominant fatty acid released was linoleic acid (18:2) followed by arachidonic acid (20:4). Lipid peroxidation in the basal ganglia measured by the thiobarbituric acid (TBA) reaction in the presence or absence of ascorbic acid also showed a significant increase in the experimental animals at all periods with a maximum at 30 min to 2 hr after occlusion. In order to assess whether lipid peroxidation causes damage to the lysosomes and release of the enzymes, a lysosome enriched P2 fraction from the normal monkey basal ganglia was prepared and the effect of peroxidation studied. Maximum peroxidation in the P2 fraction was observed in the presence of arachidonic acid, ascorbic acid and Fe2+.(ABSTRACT TRUNCATED AT 250 WORDS)
The cellular localization and regional distribution of cathepsin B within rat CNS was revealed by immunohistochemistry using a monospecific antiserum. Cathepsin B protein was found to be widely but unevenly distributed throughout rat brain. Neurons were always cathepsin B immunoreactive. Glial elements were only occasionally immunostained. The distribution of the enzyme resembles largely that of cathepsin D.
Transient ischemia in animals produces delayed cell death in vulnerable hippocampal neurons. To see if this occurs in humans, we reexamined brain slides from all patients with anoxic-ischemic encephalopathy and a well-documented cardiorespiratory arrest. Eight patients dying 18 hours or less after cardiac arrest had minimal damage in hippocampus and moderate damage in cerebral cortex and putamen. Six patients living 24 hours or more had severe damage in all four regions. The increase in damage with time postarrest was significant only in the hippocampus. Delayed hippocampal injury now documented in humans provides a target for possible therapy that can be initiated after cardiopulmonary resuscitation.
The present study was designed to examine the temporal changes in the theta (theta) rhythm recorded from the dorsal hippocampal formation of the chronic rat following 20 min of cerebral ischemia. Recordings were made during both wakefulness and paradoxical sleep. The experimental results show that ischemia resulted in a drastic amplitude reduction in both superficial and deep theta. Amplitude reduction occurred between days 1 and 4 postischemia and was maintained across the subsequent survival period. This EEG alteration was associated with an excessive loss of pyramidal cells in the hippocampal CA1 area, as observed by light microscopy. The above data strongly support our previous conclusions that neural components of the CA1 area may be of fundamental importance for the appearance and the maintenance of not only superficial theta but also deep theta. Moreover, they suggest that the preparation used in the present study may be a useful model for investigations on the neuroanatomophysiological effects of transient cerebral ischemia.
A survey was carried out of potential biochemical indices of brain senility, in which specific proteins, gangliosides, lysosomal enzymes and polyunsaturated fatty acids have been analysed in the frontal grey matter of middle aged and elderly normal as well as demented subjects. A marked deficiency was found in the concentration of a neuronal type protein (neuronin S-6) in the brain of demented patients, while there was a slight increase in the amount of a glial protein (S-100) compared to controls. In addition, subcellular organelles were isolated from senile brain very shortly after death. The nuclear fraction from these preparations was found to be remarkably enriched in protein and the soluble cytoplasmic fraction to contain enhanced lysosomal enzyme activity. The significance of these biochemical observations to the pathology of senile dementia is discussed.
This chapter discusses the structures and functions of lysosomal thiol proteinases and their endogenous inhibitor. There is a very high concentration of cathepsin L in liver, which is similar to that of cathepsin B. When lysosomes are broken during homogenization of tissues, the thiol proteinase inhibitor binds to proteinases immediately. Various proteinase inhibitors of microbial origin are useful in elucidating the importance of lysosomal thiol proteinases in protein degradation; however, these inhibitors do not distinguish between cathepsin B, cathepsin L, and other thiol proteinases. Thus, specific substrates for use in the assay of individual thiol proteinases and specific inhibitors of respective proteinases must be developed. Addition of proteinase inhibitors to cells or their injection in vivo inhibits lysosomal proteinase activities, causes formation of autophagic vacuoles, and induces synthesis of hemoglobin-hydrolyzing thiol proteinase. Various blood proteins—such as asialoproteins—are degraded in liver via processes involving receptor-mediated binding, pinocytosis, fusion with primary lysosomes, and subsequent hydrolysis in secondary lysosomes.
Growing appreciation of the multiple functions of proteolytic enzymes in intracellular protein degradation and post-translational modification, in the release of biologically active macromolecules and peptides from precursors and in cellular protein regulation and quality control has stimulated interest in proteases in neurobiology and neuropathology. In this article, the proteinases and peptidases thus far studied in the human central nervous system are reviewed with respect to their enzymology, anatomical and cytological distributions and contributions to neurological and psychiatric disease states. Though information concerning brain proteases in man is fragmentary, it suffices to establish the importance of these complex systems for advancing knowledge of human cerebral function in health and disease.
Mechanisms involved in the postischemic delay in neuronal recovery or death in rat hippocampus were evaluated by light and electron microscopy at 3, 15, 30, and 120 min and 24, 36, 48, and 72 h following severe cerebral ischemia that was produced by permanent occlusion of the vertebral arteries and 30-min occlusion of the common carotid arteries. During the early postischemic period, neurons in the Ca1 and Ca3 regions both showed transient mitochondrial swelling followed by the disaggregation of polyribosomes, decrease in rough endoplasmic reticulum (RER), loss of Golgi apparatus (GA) cisterns, and decrease in GA vesicles . Recovery of these organelles in Ca3 neurons was first noted between 24 and 36 h and was accompanied by a marked proliferation of smooth endoplasmic reticulum (SER). Many Ca1 neurons initially recovered between 24 and 36 h, but subsequent cell death at 48-72 h was often preceded by peripheral chromatolysis, constriction and shrinkage of the proximal dendrites, and cytoplasmic dilatation that was continuous with focal expansion of RER cisterns. Because SER accumulates in resistant Ca3 neurons and proximal neuronal processes are damaged in vulnerable Ca1 neurons, we hypothesize that delayed cell recovery or death in vulnerable and resistant postischemic hippocampal neurons is related to abnormalities in neuronal processes.
In the CA1 subfield of the gerbil hippocampus, an unusual series of changes were noticed after ischemia. Mongolian gerbils were subjected to bilateral carotid occlusion for 5 min. Perfusion fixation was performed 3, 6 and 12 h or 1, 2, 4, 7 and 21 days afterwards. Specimens obtained from the dorsal hippocampus were processed for light and electron microscopy. Three different types of changes were observed in the CA4, CA2 and CA1 subfields. In CA4, the change was rapid and corresponded to ischemic cell change. The alteration in CA2 was relatively slow, and identical to what has been called reactive change. On the contrary, the change in the CA1 pyramidal cells was very slow, only becoming apparent by light microscopy 2 days following ischemia. The CA1 subfield was selected for electron microscopic observation. The lamellar alignment of proliferated cisterns of the endoplasmic reticulum was the most conspicuous finding in these cells. Four days following ischemia, almost all of the pyramidal cells in CA1 were destroyed. In the CA1 neuropil, numerous presynaptic terminals remained without being apposed to normal postsynaptic sites. These changes in CA1, called here 'delayed neuronal death', may differ from those thought to be typical of ischemic neuronal damage. It was unlikely that the disturbance of local blood vessels was the cause of these changes.
This study examined the temporal profile of ischemic neuronal damage following transient bilateral forebrain ischemia in the rat model of four-vessel occlusion. Wistar rats were subjected to transient but severe forebrain ischemia by permanently occluding the vertebral arteries and 24 hours later temporarily occluding the common carotid arteries for 10, 20, or 30 minutes. Carotid artery blood flow was restored and the rats were killed by perfusion-fixation after 3, 6, 24, and 72 hours. Rats with postischemic convulsions were discarded. Ischemic neuronal damage was graded in accordance with conventional neuropathological criteria. Ten minutes of four-vessel occlusion produced scattered ischemic cell change in the cerebral hemispheres of most rats. The time to onset of visible neuronal damage varied among brain regions and in some regions progressively worsened with time. After 30 minutes of ischemia, small to medium-sized striatal neurons were damaged early while the initiation of visible damage to hippocampal neurons in the h1 zone was delayed for 3 to 6 hours. The number of damaged neurons in neocortex (layer 3, layers 5 and 6, or both) and hippocampus (h1, h3-5, paramedian zone) increased significantly (p less than 0.01) between 24 and 72 hours. The unique delay in onset of ischemic cell change and the protracted increase in its incidence between 24 and 72 hours could reflect either delayed appearance of ischemic change in previously killed neurons or a delayed insult that continued to jeopardize compromised but otherwise viable neurons during the postischemic period.
Fodrin, a neuronal cytoskeleton protein, is proteolyzed by calpain after ischemic insult. We examined proteolysis of fodrin induced by global forebrain ischemia in gerbil hippocampus in spatial terms by using the antibody specific to the calpain-proteolyzed form of fodrin.
In gerbils, a 10-minute forebrain ischemia was produced by occlusion of both carotid arteries. After recirculation, the hippocampus was processed for immunohistochemical and immunoblot study with the antibody against the calpain-proteolyzed form of fodrin. Additionally, short-term ischemia was studied to find the threshold of fodrin proteolysis.
Three phases of fodrin proteolysis distinct in chronology and distribution arose: (1) an early predegeneration phase in the molecular layer and stratum oriens of the CA1 and CA3 sectors within the first 15 minutes, which lasted up to 4 hours; (2) a late predegeneration phase in the whole CA1 sector, except for the pyramidal cells, between 12 hours and 2 days; and (3) a postdegeneration phase in the cytoplasm of the CA1 neurons, which arose in 3 to 7 days. A 4-minute (not a 3-minute) forebrain ischemia induced the late predegeneration phase of fodrin proteolysis and delayed neuronal death in CA1. Immunoblotting showed that the primary product of calpain action was further proteolyzed by an unidentified protease.
Calpain induced proteolysis of fodrin in ischemic hippocampus, and the late predegeneration phase of the proteolysis was closely associated with the delayed neuronal death in the CA1 sector. Calpain and another protease may play a role in the development of neuronal death after transient forebrain ischemia.
We previously reported lesions confined specifically to the hippocampus when produced by occluding eight vessels (the bilateral vertebral, common, internal, and external carotid arteries), which supply blood to the brain. However, histopathological changes in the primate brain, caused by ischemic injury, have not previously been thoroughly investigated. In the present study, macaque monkeys were subjected to 5-18-min ischemia by occluding the eight vessels. After the brains were perfused and fixed 5 days after the occlusion, all regions were histologically investigated for ischemic cell changes. Ischemia for 5 min produced no ischemic cell change. Ischemia for 10-15 min produced cell death limited to the deeper portion of the pyramidal cell layer of the CA1 subfield in the hippocampus. In most monkeys, no cell death was observed in any brain region outside of the hippocampus after ischemia for up to 15 min. Ischemia for 18 min produced more widespread cell death in the CA1 subfield of the hippocampus, and cell death was no longer confined to the hippocampus, but was observed in layers III, V, and VI of the neocortices, the striatum, and some other regions. Brains that were perfused and fixed 1 year after 15-min ischemic insult revealed no ischemic cell morphological change in any region, but the number of pyramidal cells in the CA1 subfield was decreased to about half. The results indicate that the CA1 subfield of the monkey hippocampus is the precise region of the brain most susceptible to ischemic insult in the primate forebrain, and after a critical time (15-min ischemia in this procedure) ischemic cell changes occur suddenly and extensively. Ischemia due to occlusion of eight arteries for 10-15 min could produce a model of human amnesia caused by transient ischemic insult.
This chapter discusses the structure, properties, mechanisms, and assays of cysteine protease inhibitors with emphasis on cystatins and E-64 derivatives. Cystatins are a special group of proteins of low and high molecular weights that inhibit a group of cysteine proteinases. Cystatins cannot inhibit calpains, although they belong to the family of cysteine proteinases. They form a superfamily of sequentially homologous proteins divided into three families. The chapter discusses the various types of cystatins and discusses the characteristics of the mammalian cystatins. All cystatins inhibit the majority of cysteins of the papain superfamily. Inhibitory spectra of cystatins, however, are variable for cysteine proteinases. E-64 and CA-074 are the most useful cysteine protease inhibitors in vivo because of their properties: (1) specific potent inhibition, (2) effective permeability into cells and tissues, (3) low toxicity, and (4) ease of synthesis and stability.
Mobilization of [Ca2+]i in the monkey hippocampal slices during transient hypoxia-hypoglycemia and KCl-induced depolarization was analyzed by microfluorometric imaging and anti-PIP2 immunohistochemistry. Hypoxia-hypoglycemia provoked the largest [Ca2+]i mobilization of CA-1 temperature-dependently whereas [Ca2+]i mobilization by KCl-induced depolarization occurred independent of the temperature in CA-2. Immunohistochemical analysis of the hippocampus after hypoxia-hypoglycemia showed an increased PIP2 staining preferentially in the perikarya of CA-1 neurons. These data suggest that release of Ca2+ from intracellular stores caused by PIP2 breakdown may induce elevated [Ca2+]i.
This research was performed to determine whether a selective inhibitor of the calcium-dependent protease, calpain, could reduce ischemia-associated brain damage when peripherally administered after a vascular occlusion.
A variation of the rat middle cerebral artery occlusion model was used. A range of doses of AK295 (a novel calpain inhibitor synthesized for this purpose) was continuously infused through the internal carotid artery, beginning 1.25 hours from the initiation of the occlusion. Rats were killed at 21 hours, and the infarct volume was quantified.
Postocclusion (1.25-hour) infusion of the calpain inhibitor AK295 elicited a dose-dependent neuroprotective effect after focal ischemia. The highest dose tested (3 mg/kg per hour) afforded the maximum effect, illustrated by a 32% reduction in infarct volume 21 hours after the ischemia (vehicle, 81.7 +/- 4.7 mm3; AK295, 54.9 +/- 6.9 mm3; P < .007).
These data provide the first evidence that a peripherally administered calpain inhibitor can protect against ischemic brain damage. They offer further support for an important role of calpain proteolysis in the brain degeneration associated with cerebral ischemic events and suggest that selective calpain inhibitors provide a rational, novel, and viable means of treating such neurodegenerative problems.
Excessive elevation of intracellular calcium and uncontrolled activation of calcium-sensitive events are believed to play a central role in ischemic neuronal damage. Calcium-activated proteolysis by calpain is a candidate to participate in this form of pathology because it is activated under ischemic conditions and its activation results in the degradation of crucial cytoskeletal and regulatory proteins. The present studies examined the effects of a cell-penetrating inhibitor of calpain on the pathological outcome after transient focal ischemia in the brain.
Twenty-five male Sprague-Dawley rats were divided into four groups: a saline-treated group, a vehicle-treated group, and two calpain inhibitor-treated groups (Cbz-Val-Phe-H; 30-mg/kg and 60-mg/kg cumulative doses). Ischemia was induced by occluding the left middle cerebral artery and both common carotid arteries for 3 hours followed by reperfusion. Animals were killed 72 hours after surgery, and quantitative measurements of infarction volumes were performed using histological techniques. Eight additional rats were killed 30 minutes after ischemia and examined for the extent of proteolysis using immunoblot techniques. A final group of 12 animals was decapitated after injection of vehicle or calpain inhibitor, and the proteolytic response was measured after 60 minutes of total ischemia.
Rats treated with Cbz-Val-Phe-H exhibited significantly smaller volumes of cerebral infarction than saline-treated or vehicle-treated control animals. Intravenous injections of cumulative doses of 30 mg/kg or 60 mg/kg of Cbz-Val-Phe-H were effective in reducing infarction, edema, and calcium-activated proteolysis. The proteolytic response to postdecapitation ischemia was also reduced by the calpain inhibitor.
These results demonstrate the neuroprotective effect of a cell-penetrating calpain inhibitor when administered systemically. The findings suggest that targeting intracellular, calcium-activated mechanisms, such as proteolysis, represents a viable therapeutic strategy for limiting neurological damage after ischemia.
Altered cellular levels and localizations of four distinct intracellular proteinases, cathepsins D, E, B, and L, with aging were studied in various rat brain tissues by enzymatic and immunohistochemical methods using discriminative antibodies specific for each enzyme. With regard to two aspartic proteinases, cathepsin E was barely detectable in all the brain tissues of young adult rats, including the cerebral cortex, the hippocampus, the neostriatum, and the cerebellum, whereas cathepsin D was ubiquitously found in these tissues. Two cysteine proteinases, cathepsins B and L, also existed in these tissues of young rats at the relatively high levels of activities. In aged rats, the cathepsin D levels in all of the brain tissues examined were about twice those of young rats. Cathepsin E was markedly increased in the cerebral cortex and neostriatum of aged rats, but not in the other tissues. The levels of cathepsin B were also increased significantly in the neostriatum of aged rats, but not significantly in the other tissues. In contrast, the activity levels of cathepsin L were strikingly decreased in all the brain tissues of aged rats. At the light microscopic level, the increased immunoreactivity of cathepsins D and E in the brain tissues of aged rats was eminent in both the neurons and the glial cells. By double-immunostaining technique, the cathepsin D-positive glial cells were mainly associated with reactive astrocytes, whereas the cathepsin E-positive glial cells were largely reactive microglial cells. Western blot analyses revealed that the molecular forms of cathepsins D and E increasingly expressed in the cerebral cortex of aged rats were similar to those of the respective normal mature enzymes. The increased immunoreactivity of cathepsin B in the neostriatum of aged rats was also found in both the neurons and the glial cells. Despite the marked decrease of the cathepsin L activity in various brain tissues of aged rats, the immunostaining for this enzyme was not significantly changed, indicating the occurrence of the catalytically inactive form of the enzyme in these tissues. These results suggest that the increased levels of cathepsins D, E, and B and the decrease in cathepsin L activity in brain regions of aged rats are related to both the neuronal degeneration and the reactivation of glial cells during the normal aging process of the brain.
Transient ischemia-induced perturbations in calcium homeostasis have been proposed to lead to pathological activation of the cysteine protease calpain I and subsequent delayed neuronal death in the CA1 region of hippocampus. We report here on the design and characterization of antibodies selective for calpain-generated fragments of brain spectrin, and their use for immunoblot and immunohistochemical analyses of calpain activation following cerebral ischemia in the gerbil. Although spectrin was susceptible to degradation in vitro by many mammalian proteases, only calpain degraded spectrin to generate fragments immunoreactive with the antibodies. Following 5 min of global ischemia, immunoreactivity for calpain-degraded spectrin was rapidly (within 30 min) and markedly elevated in the perikarya and dendrites of several populations of forebrain neurons. The rapid calpain activation was completely prevented by the NMDA receptor antagonist MK-801. At later times postischemia, but prior to frank neuronal necrosis, calpain-degraded spectrin was restricted to hippocampal area CA1 pyramidal neurons. Silver impregnation histochemistry confirmed that neuronal damage was confined to area CA1. The results indicate that while nonpathological NMDA receptor stimulation can activate calpain, only those neurons showing sustained calpain activation are destined to die.
The time course of the decline in energy levels during an in vitro ischemia-like condition was compared with changes in intracellular Ca2+ concentration ([Ca2+]i) in subregions of the gerbil hippocampal slice [CA1, CA3, and the inner and outer portions of the dentate gyrus (DG)]. Hippocampal transverse slices were loaded with a fluorescent indicator, rhod-2. During the on-line monitoring of [Ca2+]i, the slices were perfused with an in vitro ischemia-like medium (33 degrees C). The slices were collected at several experimental time points, frozen, dried, and dissected into subregions. The contents of adenine nucleotides (ATP, ADP, and AMP) and phosphocreatine (PCr) were measured by HPLC methods. Region-specific and acute [Ca2+]i elevations were observed in CA1 approximately 4 min after onset of the in vitro ischemia-like condition and also in the inner portion of the DG with a delay of 10-40 s. The change in ATP levels was related to the increase in [Ca2+]i. ATP levels in all subregions gradually decreased before the acute [Ca2+]i elevation. Concomitant with the acute [Ca2+]i elevation in CA1 and the inner portion of the DG, ATP levels in the subregions rapidly decreased, whereas declines in levels of high-energy-charge phosphates were gradual in CA3 and the outer portion of the DG, in which the remarkable [Ca2+]i elevation was not observed. These results suggest that ATP depletion observed in CA1 and the inner portion of the DG is due to the region-specific increase in [Ca2+]i, which activates a Ca(2+)-ATP-driven pump and produces a subsequent fall in neuronal ATP content.
The accumulation and localization of cathepsins E and D in the rat hippocampus and neostriatum during the neurodegenerating process induced by transient forebrain ischemia were investigated by immunoprecipitation and by immunohistochemistry using discriminative antibodies specific for each enzyme. While significant amounts of cathepsin D were found in both the hippocampus and the neostriatum of normal rats, cathepsin E was barely detectable in these tissues. No significant change in their levels was found in these tissues of postischemic rats for up to 3 days after transient forebrain ischemia. After 7 days of the treatment, cathepsin E was markedly increased in both tissues. Although the cathepsin D content in these tissues was also increased at this stage, the rate of increase was much less than that of cathepsin E. At the light microscopic level, the increased immunoreactivity for each enzyme was mainly found in reactive glial cells and degenerating neurons in the hippocampal CA1 subfield at 7 days postischemia. In the neostriatal dorsolateral portion, cathepsin D immunoreactivity was also increased in both reactive glial cells and degenerating neurons, whereas increased immunoreactivity of cathepsin E was only identified in reactive glial cells at 7 days postischemia. It was also found by double-immunostaining technique that the cathepsin E-positive glial cells were largely reactive microglial cells, whereas the cathepsin D-positive glial cells were associated mainly with reactive astrocytes. These results suggest that the accumulation of both cathepsins E and D in the regions of selective neuronal vulnerability may be associated with the postischemic development of intense gliosis and also probably neurodegenerative responses.
Microfluorometry was used to investigate the origin of intracellular Ca2+ ([Ca2+]i) elevation in field CA1 of gerbil hippocampal slices perfused with a glucose-free physiological medium equilibrated with a 95% N2/5% CO2 gas mixture (standard in vitro ischemia-like condition). Large [Ca2+]i elevation was detected about 4 min after the beginning of standard in vitro ischemia-like condition, which was accompanied with a negative shift of extracellular DC potential. When slices were perfused with Ca(2+)-free in vitro ischemia-like medium, large [Ca2+]i elevation was observed about 3.5 min after the beginning of Ca(2+)-free in vitro ischemia-like condition, however, the increase in [Ca2+]i was more gradual and of a lesser extent compared with that detected in the slices perfused with the standard in vitro ischemia-like medium that contained Ca2+. When slices were perfused with the Ca(2+)-free in vitro ischemia-like medium that contained dantrolene (50 microM) which is known to prevent Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores, the increase in [Ca2+]i was more gradual and of a lesser extent compared with that detected in the slices perfused with the Ca(2+)-free in vitro ischemia-like medium that did not contain dantrolene. These results indicate that large [Ca2+]i elevation induced by in vitro ischemia-like condition in field CA1 of the hippocampus was caused by both Ca2+ influx from extracellular space and Ca2+ release from intracellular Ca2+ stores, and that a part of the Ca2+ release was due to Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores.
Enzymatic activities, expressions, and the immunohistochemical localization of lysosomal cystein proteases, cathepsins B and L, were analyzed in the monkey hippocampus after transient ischemia to clarify the mechanism of delayed cornu Ammonis (CA)-1 neuronal death. By enzymatic assay, the activity of cathepsin B increased in CA-1, 24 h after the ischemic insult, while that of cathepsin L decreased. On Western blotting, the protein contents of both cathepsins B and L increased immediately after ischemia. By immunohistochemistry, cathepsins B and L were stained as coarse granules in the perikarya of control CA-1 neurons, but in postischemic CA-1 neurons they were released from lysosome granules. In contrast, in CA-2 and the remaining sectors, enzymatic activities increased after ischemia, and immunoreactivities of cathepsins B and L increased only within lysosome granules. These results suggest that cathepsins B and L may play an important role in the breakdown of certain cell proteins in the postischemic CA-1 neurons.