Improvement of embryonic dopaminergic neurone survival in culture and after grafting into the striatum of hemiparkinsonian rats by CEP-1347

Transplantation of embryonic nigral tissue ameliorates functional deficiencies in Parkinson's disease (PD). A main constraint of neural grafting is the poor survival of dopaminergic neurones grafted into patients. Studies in rats indicated that many grafted neurones die by apoptosis. CEP-1347 is a mixed-lineage-kinase (MLK) inhibitor with neuroprotective action in several in vitro and in vivo models of neuronal apoptosis. We studied the effect of CEP-1347 on the survival of embryonic rat dopaminergic neurones in culture, and after transplantation in hemiparkinsonian rats. CEP-1347 and the alternative MLK inhibitor CEP-11004 significantly increased the survival of dopaminergic neurones in primary cultures from rat ventral mesencephalon and in Mn2+-exposed PC12 cells, a surrogate model of dopaminergic lethal stress. Moreover, combined treatment of the grafting cell suspension and the host animal with CEP-1347 significantly improved the long-term survival of rat dopaminergic neurones transplanted into the striatum of hemiparkinsonian rats. Also, the protective effect of CEP-1347 resulted in an increase in total graft size and in enhanced fibre outgrowth. Thus, treatment with CEP-1347 improved dopaminergic cell survival under severe stress and might be useful to improve the positive outcome of transplantation therapy in PD and reduce the amount of human tissue required.     

Control of death receptor and mitochondrial-dependent apoptosis by c-Jun N-terminal kinase in hippocampal CA1 neurones following global transient ischaemia

c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, is activated in response to a number of extracellular stimuli, including inflammatory cytokines, UV irradiation and ischaemia. A large body of evidence supports a role for JNK signalling in stress-induced apoptosis. It has been hypothesized that JNK may contribute to the apoptotic response by regulating the intrinsic cell death pathway involving the mitochondria. Here, we examined the role of the JNK signalling pathway in hippocampal CA1 apoptotic neurones following transient ischaemia in gerbils. We showed early activation of death receptor-dependent apoptosis (caspase-8 activation 2 days after ischaemia) and a biphasic activation of caspase-3 and caspase-9 after ischaemia. Activation of the mitochondrial pathway, as measured by cytochrome c release, appeared as a late event (5-7 days after ischaemia). AS601245, a novel JNK inhibitor, antagonized activation of both pathways and significantly protected CA1 neurones from cell death. Our results suggest a key role of JNK in the control of death receptor and mitochondrial-dependent apoptosis after transient ischaemia.     

Apoptosis is not an invariable component of in vitro models of cortical cerebral ischaemia

Characterising the mechanisms of cell death following focal cerebral ischaemia has been hampered by a lack of an in vitro assay emulating both the apoptotic and necrotic features observed in vivo. The present study systematically characterised oxygen-glucose-deprivation (OGD) in primary rat cortical neurones to establish a reproducible model with components of both cell-death endpoints. OGD induced a time-dependent reduction in cell viability, with 80% cell death occurring 24 h after 3 h exposure to 0% O2 and 0.5 mM glucose. Indicative of a necrotic component to OGD-induced cell death, N-methyl-D-aspartate (NMDA) receptor inhibition with MK-801 attenuated neuronal loss by 60%.The lack of protection by the caspase inhibitors DEVD-CHO and z-VAD-fmk suggested that under these conditions neurones did not die by an apoptotic mechanism. Moderating the severity of the insult by decreasing OGD exposure to 60 min did not reduce the amount of necrosis, but did induce a small degree of apoptosis (a slight reduction in cell death was observed in the presence of 10 μM DEVD-CHO). In separate experiments purported to enhance the apoptotic component, cells were gradually deprived of 02, exposed to 4% 02 (as opposed to 0%) during the OGD period, or maintained in serum-containing media throughout. While NMDA receptor antagonism significantly reduced cortical cell death under all conditions, a caspase-inhibitor sensitive component of cell death was not uncovered. These studies suggest that OGD of cultured cortical cells models the excitotoxic, but not the apoptotic component of cell death observed in vivo.     

Activation and possible involvement of calpain, a calcium-activated cysteine protease, in down-regulation of apoptosis of human monoblast U937 cells

An active form of calpain mu, a low-Ca(2+)-requiring intracellular cysteine protease, was detected using a cleavage site-directed antibody in apoptotic human monoblast U937 cells treated with tumor necrosis factor-alpha and interferon-gamma. Membrane-permeable calpain inhibitors accelerated apoptosis of U937 cells thus induced and suppressed the activation of procalpain. These findings suggest that calpain down-regulates apoptosis by shutting off the intracellular signals for cell death.     

Calpain 2 activity increases at the time of implantation in rat uterine luminal epithelial cells and administration of calpain inhibitor significantly reduces implantation sites

The present study investigated the role of calpain 2 in rat uterine luminal epithelial cells during early pregnancy. Calpain 2 is an intracellular calcium-dependent proteolytic enzyme which cleaves numerous focal adhesion proteins. Calpain 2 was concentrated along the basal cell surface of uterine luminal epithelial cells at the predicted site of focal adhesions on day 1 of pregnancy and remained unchanged at the time of implantation as observed by immunofluorescence microscopy. However, Western blotting analysis showed a marked increase in the active form and a significant decrease in the latent form of calpain 2 at the time of implantation. The increase in calpain 2 activity coincides with the disassembly of focal adhesion proteins, talin, paxillin, integrin β1 and β3 from the site of focal adhesions. Intraperitoneal injection of calpain inhibitor, calpain inhibitor l (ALLN), significantly reduced the number of implantation sites, implying that calpain 2 plays an important role in implantation. The present study suggests a role for calpain 2 in the disassembly of focal adhesions, which has been previously shown to play a key role in uterine receptivity for implantation.     

Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis

Cross-talk between calpain and caspase proteolytic systems has complicated efforts to determine their distinct roles in apoptotic cell death. This study examined the effect of overexpressing calpastatin, the specific endogenous calpain inhibitor, on the activity of the two proteolytic systems following an apoptotic stimulus. Human SH-SY5Y neuroblastoma cells were stably transfected with full-length human calpastatin cDNA resulting in 20-fold overexpression based on Western blot and 5-fold greater calpain inhibitory activity in cell extracts. Wild type and calpastatin overexpressing (CST1) cells were neuronally differentiated and apoptosis-induced with staurosporine (0.1-1.0 microm). Calpastatin overexpression decreased calpain activation, increased caspase-3-like activity, and accelerated the appearance of apoptotic nuclear morphology. Following 0.1-0.2 microm staurosporine, plasma membrane integrity based on calcein-acetoxymethyl fluorescence was significantly greater at 24 h in differentiated CST1 compared with differentiated wild type cells. However, this protective effect was lost at higher staurosporine doses (0.5-1.0 microm), which resulted in pronounced caspase-mediated degradation of the overexpressed calpastatin. These results suggest a dual role for calpains during neuronal apoptosis. In the early execution phase, calpain down-regulates caspase-3-like activity and slows progression of apoptotic nuclear morphology. Subsequent calpain activity, facilitated by caspase-mediated degradation of calpastatin, contributes to plasma membrane disruption and secondary necrosis.     

Detection and localization of calpain 3-like protease in a neuronal cell line: possible regulation of apoptotic cell death through degradation of nuclear IkappaBalpha

Calpains are a family of calcium-dependent cysteine proteases involved in major cellular processes including cell death. Their intracellular localization is essential to the understanding of their biological functions. In a previous confocal microscopy study, we observed the presence of a calpain 3-like protein in the mammalian brain. We thus first identified and confirmed the presence of a calpain 3-like protease in a neuronal cell model (NGF-differentiated PC12 cells). The goal of this study was to determine, for the first time in non-muscular cells, the relation between the subcellular localization, activation and function of this protease. We thus investigated its ability to regulate nuclear IkappaBalpha and therefore NF-kappaB activation after cell death stimulation. The IkappaBalpha/NF-kappaB signalling pathway indeed influences the neurodegenerative process by directly affecting gene expression in neurons. In the present study, we found that calpain 3 is present in the cytoplasm and nucleus of neuron-like PC12 cells and could be activated through autolysis in the nuclei of cells undergoing apoptosis after ionomycin treatment. Moreover, in these conditions, we demonstrated formation of the IkappaBalpha/calpain 3 complex and an increase in calpain-dependent IkappaBalpha cleavage products in cell nuclei. Stimulation of calpain-dependent cell death in neuron activated nuclear calpain 3-like protease and IkappaBalpha proteolysis resulted in the regulation of NF-kappaB activation. These data suggest a new mechanism by which calpain 3 activation is able to regulate the IkappaBalpha/NF-kappaB pathway and thus neurodegenerative processes.     

Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UVr-1 cells derived from ultraviolet-sensitive human RSa cells

Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14 - 18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CTheta, Src, Bax and mu-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated mu-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, mu-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells.     

Calpain activation in apoptosis

Programmed cell death is an active process wherein the cell initiates a sequence of events culminating in the fragmentation of its DNA, nuclear collapse, and disintegration of the cell into small, membrane-bound apoptotic bodies. Examination of the death program in various models has shown common themes, including a rise in cytoplasmic calcium, cytoskeletal changes, and redistribution of membrane lipids. The calcium-dependent neutral protease calpain has putative roles in cytoskeletal and membrane changes in other cellular processes; this fact led us to test the role of calpain in a well-known model of apoptotic cell death, that of thymocytes after treatment with dexamethasone. Assays for calcium-dependent proteolysis in thymocyte extracts reveal a rise in activity with a peak at about 1 hr of incubation with dexamethasone, falling to background at approximately 2 hr. Western blots indicate autolytic cleavage of the proenzyme precursor to the calpain I isozyme, providing additional evidence for calpain activation. We have also found that apoptosis in thymocytes, whether induced by dexamethasone or by low-level irradiation, is blocked by specific inhibitors of calpain. Apoptosis of metamyelocytes incubated with cycloheximide is also blocked by calpain inhibitors. These studies suggest a required role for calpain in both “induction” and “release” models of apoptotic cell death. © 1994 wiley-Liss, Inc.     

Caspases cleave the amino-terminal calpain inhibitory unit of calpastatin during apoptosis in human Jurkat T cells

We have previously reported the activation of procalpain mu (precursor for low-calcium-requiring calpain) in apoptotic cells using a cleavage-site-directed antibody specific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full-length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp-233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino-terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin.     

Human Umbilical Cord-Derived Schwann-Like Cell Transplantation Combined with Neurotrophin-3 Administration in Dyskinesia of Rats with Spinal Cord Injury

Mesenchymal stem cells are capable of differentiating into Schwann-like cells. In this study, we induced human umbilical-cord mesenchymal stem cells (HUMSCs) in vitro into neurospheres constituted by neural stem-like cells, and further into cells bearing strong morphological, phenotypic and functional resemblances with Schwann-like cells. These HUMSC-derived Schwann-like cells, after grafting into the injured area of the rats’ spinal cord injury (SCI), showed a partial therapeutic effect in terms of improving the motor function. Neurotrophin-3 (NT-3) was reported to improve the local microenvironment of the grafted cells, and we, therefore, further tested the effect of Schwann-like cell grafting combined with NT-3 administration at the site of cell transplantation. The results showed that NT-3 administration significantly promoted the survival of the grafted cells in the host-injured area. Significant improvement in rats treated by Schwann-like cell grafting combined with NT-3 administration was demonstrated in the behavioral test as compared with that in animal models received the cell grafting only. These results suggest that transplantation of the Schwann-like cells combined with NT-3 administration may represent a new strategy of stem cell therapy for spinal cord injury.     

Hypoxia-induced cell death of HepG2 cells involves a necrotic cell death mediated by calpain

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of μ-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially μ-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.     

Programmed Myocyte Cell Death Affects the Viable Myocardium after Infarction in Rats

To determine whether apoptotic and necrotic myocyte cell death occur acutely and chronically after infarction, the formation of DNA strand breaks and the localization of myosin monoclonal antibody labeling were analyzed in the surviving myocardium from 20 min to 1 month. DNA strand breaks in myocyte nuclei were detected as early as 3 h following coronary artery occlusion and were still present at 1 month. This cellular process was characterized biochemically by internucleosomal DNA fragmentation which produced DNA laddering on agarose gel electrophoresis. Quantitatively, 155 myocyte nuclei per 10⁶cells exhibited DNA strand breaks in the portion adjacent to the infarcted tissue at 3–12 h. This parameter increased to 704 at 1–2 days and subsequently decreased to 364 at 7 days, 188 at 14 days, and 204 at 1 month. In the remote myocardium, the number of myocyte nuclei with DNA strand breaks was 84 per 10⁶at 3–12 h and remained essentially constant up to 1 month. Programmed myocyte cell death was accompanied by a decrease in the expression of bcl-2 and an increase in the expression of bax. The changes in the expression of these genes were present at 1 and 7 days after coronary artery occlusion. In conclusion, the mechanical load produced by myocardial infarction and ventricular failure may affect the regulation of bcl-2 and bax in the viable myocytes, triggering programmed cell death and the remodeling of the ventricular wall.     

In Vivo Measurement Using Microdialysis of the Release and Metabolism of 5-Hydroxytryptamine in Raphe Neurones Grafted to the Rat Hippocampus

The overflow and metabolism of serotonin (5-hydroxytryptamine; 5-HT) from transplants of embryonic medullary and mesencephalic raphe neurones in the previously 5-HT-denervated hippocampus have been analyzed in vivo using intracerebral dialysis. The average density of 5-HT-immunoreactive fibres in the grafted hippocampus was less than in nonlesioned hippocampus. Nonetheless, both basal and potassium-stimulated levels of 5-HT in the dialysates were restored to approximately normal after transplantation of medullary raphe cells, whereas mesencephalic implants resulted in over twice the 5-HT output observed in control hippocampus. However, 5-hydroxyindoleacetic acid (5-HIAA) overflow was increased only after grafting of mesencephalic raphe and then only to normal levels; medullary implants, by contrast, failed to enhance 5-HIAA output above that from lesion-only hippocampus. The evidence of a relative hyperactivity of the grafted neurones may explain the disproportionate improvements in various lesion-induced behavioural deficits after grafting of nervous tissue. In addition, differences in the presynaptic regulation of 5-HT release and metabolism are also apparent in the transplants; these variations are dependent on the precise origin of the serotoninergic cells.     

A Peptide to Reduce Pulmonary Edema in a Rat Model of Lung Transplantation

Background

Despite significant advances in organ preservation, surgical techniques and perioperative care, primary graft dysfunction is a serious medical problem in transplantation medicine in general and a specific problem in patients undergoing lung transplantation. As a result, patients develop lung edema, causing reduced tissue oxygenation capacity, reduced lung compliance and increased requirements for mechanical ventilatory support. Yet, there is no effective strategy available to protect the grafted organ from stress reactions induced by ischemia/reperfusion and by the surgical procedure itself.

Methods

We assessed the effect of a cingulin-derived peptide, XIB13 or a random peptide in an established rat model of allogeneic lung transplantation. Donor lungs and recipients received therapeutic peptide at the time of transplantation and outcome was analyzed 100min and 28 days post grafting.

Results

XIB13 improved blood oxygenation and reduced vascular leak 100min post grafting. Even after 28 days, lung edema was significantly reduced by XIB13 and lungs had reduced fibrotic or necrotic zones. Moreover, the induction of an allogeneic T cell response was delayed indicating a reduced antigen exchange between the donor and the host.

Conclusions

In summary, we provide a new tool to strengthen endothelial barrier function thereby improving outcomes in lung transplantation.     

Spontaneous regeneration of older dystrophic muscle does not reflect its regenerative capacity

Young dystrophic (dy) murine muscle is capable of "spontaneous" regeneration (i.e., regeneration in the absence of external trauma); however, by the time the mice are 8 weeks old, this regeneration ceases. It has been suggested that the cessation of regeneration in dystrophic muscle may be due to exhaustion of the mitotic capability of myosatellite cells during the early stages of the disease. To test this hypothesis, orthotopic transplantation of bupivacaine treated, whole extensor digitorum longus muscles has been performed on 14 to 16-week-old 129 ReJ/++ and 129 ReJ/dydy mice. The grafted dystrophic muscle is able to produce and maintain for 100 days post-transplantation 356 +/- 22 myofibers, a number similar to that found in age-matched dystrophic muscle. The ability of old dystrophic muscle to regenerate subsequent to extreme trauma indicates that the cessation of "spontaneous" regeneration is due to factor(s) other than the exhaustion of mitotic capability of myosatellite cells. Moreover, there is no significant difference in myosatellite cell frequencies between grafted normal and dystrophic muscles (100 days post-transplantation). Myosatellite cell frequencies in grafted muscles are similar to those in age-matched, untraumatized muscles. While grafting of young dystrophic muscle modifies the phenotypic expression of histopathological changes usually associated with murine dystrophy, grafts of older dystrophic muscle show extensive connective-tissue infiltration and significantly fewer myofibers than do grafts of age-matched normal muscle. As early as 14 days post-transplantation, it is possible to distinguish between grafts of old, normal and dystrophic muscles. It is suggested that the connective tissue stroma, present in the dystrophic muscle at the time of transplantation, may survive the grafting procedure.     

Relationships of apoptotic signaling mediated by ceramide and TNF-alpha in U937 cells

It is commonly assumed that ceramide is a second messenger that transduces signaling leading to apoptosis. We tested this hypothesis by investigating the role of ceramide in TNF-alpha-initiated apoptotic signaling using the histiocytic lymphoma cell line U937. We found considerable differences between cell killing by TNF-alpha and by ceramide. U937 cells treated with TNF-alpha are committed early and irreversibly to the apoptotic pathway and start to die 90 min after treatment. U937 cells treated with ceramide start to die 12 h after the initial treatment. The cell death signaling initiated by TNF-alpha is transduced within minutes of exposure to TNF-alpha and it is irreversible. Exogenous ceramide increases the intracellular level of ceramide rapidly, significantly, and well above the physiological levels, within minutes, but cellular commitment to death does not occur until after the first 6 h of incubation. Furthermore, the endogenous ceramide in U937 cells treated with TNF-alpha increases well after the commitment to the apoptotic pathway. The differences between ceramide and TNF-alpha in the kinetics and the commitment to the apoptotic pathway suggest that, (a) ceramide is not a second messenger in the apoptotic signaling of TNF-alpha, (b) ceramide elevations, in TNF-alpha treated cells, are a consequence rather than a cause of apoptosis and (c) exogenously added ceramide and TNF-alpha kill cells via different pathways.     

Morphological evidence of the continuum between necrosis and apoptosis in model of anoxia in vitro

Growing evidence suggests that two modes of cell death, known as apoptosis and necrosis, are involved in postanoxic injury. The current opinion on these two types of cell death is that apoptosis and necrosis are not always the uniform and distinct events. The aim of this study was to determine ultrastructural criteria of postanoxic neuronal changes in model of anoxia in vitro. The organotypic cultures of rat hippocampus exposed to 10‐ and 20‐min of anoxic insult revealed the morphological features classic for both necrotic and apoptotic neuronal cell injury. Some neurones exhibited the typical necrotic lysis whereas others clearly reflected an active apoptotic form of cell death consisting of nuclear condensation with early preservation of cell membranes. However, numerous damaged cells shared both apoptotic and necrotic ultrastructural characteristics. These results evidenced the morphological continuum between apoptosis and necrosis under anoxia in vitro.     

Real-time visualization of cytoplasmic calpain activation and calcium deregulation in acute glutamate excitotoxicity

Although calpain (EC 3.4.22) protease activation was suggested to contribute to excitotoxic delayed calcium deregulation (DCD) via proteolysis of Na⁺/Ca²⁺ exchanger 3 (NCX3), cytoplasmic calpain activation in relation to DCD has never been visualized in real-time. We employed a calpain fluorescence resonance energy transfer substrate to simultaneously image calpain activation and calcium deregulation in live cortical neurons. A calpain inhibitor-sensitive decline in fluorescence resonance energy transfer was observed at 39 ± 5 min after the occurrence of DCD in neurons exposed to continuous glutamate (100 μM). Inhibition of calpain by calpeptin did not delay the onset of DCD, recovery from DCD-like reversible calcium elevations, or cell death despite inhibiting α-spectrin processing by > 90%. NCXs reversed during glutamate exposure, the NCX antagonist KB-R7943 prolonged the time to DCD, and significant NCX3 cleavage following 90 min of glutamate exposure was not observed. Our findings suggest that robust calpain activation associated with acute glutamate toxicity occurs only after a sustained loss in calcium homeostasis. Processing of NCX3 or other calpain substrates is unlikely to be the primary cause of acute excitotoxicity in cortical neurons. However, a role for calpain as a contributing factor or in response to milder glutamate insults is not excluded.