Calpain-mediated Hsp70.1 cleavage in hippocampal CA1 neuronal death

Necrotic neuronal death is recently known to be mediated by the calpain-cathepsin cascade from simpler organisms to primates. The main event of this cascade is calpain-mediated lysosomal rupture and the resultant release of lysosomal cathepsins into the cytoplasm. However, the in-vivo substrate of calpain for inducing lysosomal destabilization still remains completely unknown. The recent proteomics data using the post-ischemic hippocampal CA1 tissues and glaucoma-suffered retina from the primates suggested that heat shock protein (Hsp) 70.1 might be the in-vivo substrate of activated μ-calpain at the lysosomal membrane of neurons. Hsp70.1 is known to stabilize lysosomal membrane by recycling damaged proteins and protect cells from oxidative stresses. Here, we studied the molecular interaction between activated μ-calpain and the lysosomal Hsp70.1 in the monkey hippocampal CA1 neurons after the ischemia-reperfusion insult. Immunofluorescence histochemistry showed a colocalization of the activated μ-calpain and upregulated Hsp70.1 at the lysosomal membrane of the post-ischemic CA1 neurons. In-vitro cleavage assay of hippocampal Hsp70.1 by Western blotting demonstrated that Hsp70.1 in the CA1 tissue is an in-vivo substrate of activated μ-calpain, and that carbonylated Hsp70.1 in the CA1 tissue by artificial oxidative stressors such as hydroxynonenal (HNE) or hydrogen peroxide is much more vulnerable to the calpain cleavage. These data altogether suggested that Hsp70.1 can become a target of the carbonylation by HNE, and Hsp70.1 is a modulator of calpain-mediated lysosomal rupture/permeabilization after the ischemia-reperfusion injury.     

Macrophage colony stimulating factor prevents NMDA-induced neuronal death in hippocampal organotypic cultures

Macrophage colony stimulating factor (M-CSF) and its receptor are up-regulated in the brain in Alzheimer's disease (AD), in transgenic mouse models for AD, and experimental models for traumatic and ischemic brain injury. M-CSF induces activation and proliferation of microglial cells and expression of proinflammatory cytokines. We examined the role of M-CSF in excitotoxic neuronal cell death in organotypic hippocampal cultures. NMDA treatment induced neuronal apoptosis and caspase-3 activation in organotypic hippocampal cultures, whereas treatment with M-CSF protected hippocampal neurons from NMDA-induced apoptosis. Caspase-3 activation was inhibited by M-CSF treatment to the same degree as with the caspase inhibitor Z-VAD-FMK. These results suggest that M-CSF has neuroprotective properties through inhibition of caspase-3 that could promote neuronal survival after excitotoxic insult. The role of M-CSF in neurological disease should be reevaluated as a microglial activator with potentially neuroprotective effects.     

Mechanisms of kainate-induced region-specific neuronal death in immature organotypic hippocampal slice cultures

Excessive activation of excitatory amino acid receptors has been implicated in neuronal death in a number of central nervous system insults. We have here investigated, the time course and mechanisms of kainate (KA)- induced neuronal death in immature organotypic hippocampal slice cultures (OHCs) using Fluoro-Jade B (FJB) staining as a marker of cell death, and immunoblotting, immunocytochemistry, and electron microscopy as methods to clarify the mechanisms. After 6 KA treatment (5 microM), no significant neuronal death was detected in any hippocampal subregion, whereas the treatment of 12, 24, and 48 h resulted in neuronal death in the CA3 regions, but not in CA1. The 48 h resting period in normal medium after KA-treatment did not rescue the cells but further increased the number of dead neurons in CA3 as compared to the corresponding acute phase. In Western blotting, the expression levels of the active, 17 kDa form of caspase-3, and the 84-85 kDa cleaved fragment of poly(ADP ribose)polymerase (PARP) were not altered from the control levels. Moreover, no active caspase-3 labelled cells were detected in immunocytochemical study 24 h after KA treatment either in the acute or resting groups. Electron microscopy showed non-apoptotic injury in the CA3a/b pyramidal neurons in KA-treated slices. Our results suggest that KA-induced neuronal death in immature OHCs is a strictly region-specific, irreversible, necrotic process.     

Kynurenine 3-mono-oxygenase inhibitors attenuate post-ischemic neuronal death in organotypic hippocampal slice cultures

Kynurenine 3-mono-oxygenase (KMO) inhibitors reduce 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN) neosynthesis and facilitate kynurenine metabolism towards kynurenic acid (KYNA) formation. They also reduce tissue damage in models of focal or transient global cerebral ischemia in vivo. We used organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) to investigate KMO mechanism(s) of neuroprotective activity. Exposure of the slices to 30 min of OGD caused CA1 pyramidal cell death and significantly decreased the amount of KYNA released in the incubation medium. The KMO inhibitors (m-nitrobenzoyl)-alanine (30-100 micro m) or 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (1-10 micro m) reduced post-ischemic neuronal death and increased KYNA concentrations in slice incubation media. The maximal concentration of KYNA detected in the incubation media of slices treated with KMO inhibitors was approximately 50 nm and was too low to efficiently interact with alpha7 nicotinic acetylcholine receptors or with the glycineb site of N-methyl-d-aspartate (NMDA) receptors. On the other hand, the addition of either 3-HK or QUIN (1-10 micro m) to OGD-exposed hippocampal slices prevented the neuroprotective activity of KMO inhibitors. Our results suggest that KMO inhibitors reduce the neuronal death found in the CA1 region of organotypic hippocampal slices exposed to 30 min of OGD by decreasing the local synthesis of 3-HK and QUIN.     

β-Amyloid activates nitric oxide synthesis and causes neuronal death in hippocampal astrocytes

Digital fluorescence imaging was used to study the effects of the amyloid-beta peptide βA (full-length peptide, βA 1–42) and its neurotoxic fragment (βA 25–35) on nitric oxide (NO) synthesis and cell viability in mixed cultures of rat hippocampal neurons and astrocytes. It was found that both βA 1–42 and βA 25–35 stimulated NO synthesis in astrocytes, but not in neurons. L-NAME, an inhibitor of the inducible NO synthase, blocked the effect of βA on NO production almost completely, reduced βA-induced mitochondrial depolarization in astrocytes, and partly prevented neuronal death. The rate of NO synthesis was decreased in Ca²⁺-free medium, increased in the presence of antioxidants and the NADPH oxidase inhibitor, and decreased in the presence of the SH-reagent thimerosal.     

Programmed death in a unicellular organism has species-specific fitness effects

Programmed cell death (PCD) is an ancient phenomenon and its origin and maintenance in unicellular life is unclear. We report that programmed death provides differential fitness effects that are species specific in the model organism Chlamydomonas reinhardtii. Remarkably, PCD in this organism not only benefits others of the same species, but also has an inhibitory effect on the growth of other species. These data reveal that the fitness effects of PCD can depend upon genetic relatedness.     

S-adenosyl-L-methionine protects the hippocampal CA1 neurons from the ischemic neuronal death in rat

We investigated the effect of S-adenosyl-L-methionine (SAMe) on the prevention of the delayed neuronal death in rats subjected to transient and brief forebrain ischemia. As the results, SAMe dose-dependently protected the hippocampal CA1 neurons from degeneration and necrosis, whose effect was suppressed by simultaneous administration of S-adenosyl-L-homocysteine, a potent inhibitor in transmethylation. No protective effect was observed in CDP-choline, phosphatidylcholine and L-methionine. Therefore, it is necessary for the prevention of the delayed neuronal death to enhance cerebral SAMe level and to activate transmethylation using SAMe as a methyl donor in postischemic brain.     

Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells

Rat calvaria osteoblasts derived from 21-day-old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al: Biochem. Biophis. Res. Commun. 148:1129, 1987) and is similar to that observed in cultures of subcultivated 16-day-old embryonic chick calvaria-derived osteoblasts (COB) (Gerstenfeld, et.al: Dev. Biol. 122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of beta-glycerol-phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200-fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/maturation in vitro.     

Preweaning enrichment has no lasting effects on adult hippocampal neurogenesis in four-month-old mice

Since both living in an enriched environment and physical activity stimulate hippocampal neurogenesis in adult mice, we endeavored to examine whether preweaning enrichment, a sensory enrichment paradigm with very limited physical activity, had similar effects on neurogenesis later in life. Mice were removed from the dams for periods of increasing length from postnatal day 7 to 21, and exposed to a variety of sensory stimuli. At the age of 4 months, significant differences could be found between previously enriched and non-enriched animals when spontaneous activity was monitored. Enriched mice moved longer distances, and spent more time in a defined center zone of the open field. Adult neurogenesis was examined by labeling proliferating cells in the dentate gyrus with bromodeoxyuridine (BrdU). Cell proliferation, survival of the newborn cells, and net neurogenesis were similar in both groups. Volumetric measurements and stereological assessment of total granule cell counts revealed no difference in size of the dentate gyrus between both groups. Thus, in contrast to postweaning enrichment, preweaning enrichment had no lasting measurable effect on adult neurogenesis. One of the parameters responsible for this effect might be the lack of physical activity in preweaning enrichment. As physical activity is an integral part of postweaning enrichment, it might be a necessary factor to elicit a neurogenic response to environmental stimuli. The result could also imply that baseline adult hippocampal neurogenesis is independent of the changes induced by preweaning enrichment and might not contribute to the sustained types of plasticity seen in enriched animals.     

Identification of the major proteins that promote neuronal process outgrowth on Schwann cells in vitro

Schwann cells have a unique role in regulating the growth of axons during regeneration and presumably during development. Here we show that Schwann cells are the best substrate yet identified for promoting process growth in vitro by peripheral motor neurons. To determine the molecular interactions responsible for Schwann cell regulation of axon growth, we have examined the effects of specific antibodies on process growth in vitro, and have identified three glycoproteins that play major roles. These are the Ca2+-independent cell adhesion molecule (CAM), L1/Ng-CAM; the Ca2+-dependent CAM, N-cadherin; and members of the integrin extracellular matrix receptor superfamily. Two other CAMs present on neurons and/or Schwann cells-N-CAM and myelin-associated glycoprotein-do not appear to be important in regulating process growth. Our results imply that neuronal growth cones use integrin-class extracellular matrix receptors and at least two CAMs--N-cadherin and L1/Ng-CAM-for growth on Schwann cells in vitro and establish each of these glycoproteins as a strong candidate for regulating axon growth and guidance in vivo.     

Comparative study of endoplasmic reticulum stress-induced neuronal death in rat cultured hippocampal and cerebellar granule neurons

In this study, experiments were performed to characterize further the pathways responsible for neuronal death induced by endoplasmic reticulum (ER) stress in cultured hippocampal neurons (HPN) and cerebellar granule neurons (CGN) using tunicamycin (TM) and amyloid beta-peptide (Abeta). Exposure of HPN to Abeta or TM resulted in a time-dependent increase in the expression of 78-kDa glucose-regulated protein (GRP78) and caspase-12, an ER-resident caspase. In contrast, in CGN, although a drastic increase in the expression of GRP78 was found as was the case in HPN, no up-regulation of caspase-12 was detected. These results were consistent with immunohistochemical results that there were far lower number of caspase-12-positive cells in the cerebellum than in the cerebral cortex and hippocampus, and that caspase-12-positive cells were not identified in the external granule cell layer of the cerebellum of P7 rats. In CGN, a significant increase in the expression of C/EBP homologous protein (CHOP) protein was detected after exposure to Abeta or TM, whereas no such an increase in the protein expression was observed in HPN. In addition, S-allyl-L-cysteine (SAC), an organosulfur compound purified from aged garlic extract, protected neurons against TM-induced neurotoxicity in HPN but not in CGN, as in the case of Abeta-induced neurotoxicity. These results suggest that the pathway responsible for neuronal death induced by Abeta and TM in HPN differs from that in CGN, and that a caspase-12-dependent pathway is involved in HPN while a CHOP-dependent pathway is involved in CGN in ER stress-induced neuronal death.     

促白木香结香真菌的快速筛选

以海南省澄迈县海南科大林业公司所有的白木香人工林地为研究对象,采用前期已分离的菌株对白木香白木进行固体催化,利用GC-MS技术对具有催化效果(白木化学成分在催化前后变化显著)的菌株进行筛选,将筛选出的菌株接种至白木香树体中,6个月后对沉香物质挥发油以及化学成分进行测定。对白木固体催化产物的提取物分析,鉴定出其乙醚提取物共24种化学成分,其中共有成分5种、芳香族类化合物6种、其他类化合物18种。Trichoderma sp.、Neurospora sp.和Melanotus flavolivens这3种菌株使白木香白木的化学成分发生显著变化。对结香处理6个月后已结香木材的提取物分析,鉴定出其乙醚提取物共84种化学成分,其中共有成分30种,芳香族类化合物17种、倍半萜类化合物34种和其他类化合物33种。Trichoderma sp.、Neurospora sp.、Melanotus flavolivens和CK样品乙醚提取物得率分别为3.62%、4.04%、3.97%和1.94%。其中芳香族类成分相对含量之和分别为15.15%、17.29%、12.13%和7.95%。倍半萜类成分相对含量之和分别为10.61%、11.19%、11.4%和0%。在6个月内Trichoderma sp.、Neurospora sp.和Melanotus flavolivens这3种经过白木香白木固体催化筛选所得的菌株能够有效诱导白木香结香。     

Rosiglitazone treatment reduces hippocampal neuronal damage possibly through alleviating oxidative stress in chronic cerebral hypoperfusion

Oxygen free radicals and lipid peroxidation may play significant roles in the progress of injury induced by chronic cerebral hypoperfusion of the central nervous system. Rosiglitazone, a well known activator of PPARγ, has neuroprotective properties in various animal models of acute central nervous system damage. In the present study, we evaluate the possible impact of rosiglitazone on chronic cerebral hypoperfused-rats in regard to the levels of oxidative stress, reduced glutathione, and hippocampal neuronal damage. Chronic cerebral hypoperfusion was generated by permanent ligation of both common carotid arteries of Wistar rats for one month. Animals in treatment group were given rosiglitazone orally at doses of 1.5, 3, or 6mg/kg per day of the 1month duration. The treatment significantly lowered the levels of both malondialdehyde and neuronal damage, while elevated the reduced glutathione level markedly. These findings suggest that the beneficial effect of rosiglitazone on hypoperfusion-induced hippocampal neuronal damage might be the result of inhibition of oxidative insult.     

Conjugated linoleic acid exhibits stimulatory and inhibitory effects on prostanoid production in human endothelial cells and platelets

In addition to their reported antitumorigenic properties, various conjugated linoleic acid (CLA) isomers have also been shown to decrease prostanoid synthesis as a result of inhibiting the cyclooxygenase (COX) enzyme. We have previously reported that several CLA isomers inhibited both platelet aggregation and formation of thromboxane A(2) (TXA(2)), a proaggregatory and vasoconstrictive agent. Since the interaction between platelets and vascular endothelial cells is essential to maintaining vascular homeostasis, we decided to investigate the effects of various CLA isomers on the production of endothelial prostacyclin (PGI(2)), a potent vasodilator and inhibitor of platelet function. Using interleukin 1-beta (IL1-beta)-stimulated human umbilical vein endothelial cells (HUVECs), we initially established that HUVECs of passage #2 should be used since these cells were most responsive to thrombin-induced conversion of endogenous arachidonic acid to PGI(2), as monitored by the formation of its stable, inactive metabolite, 6-ketoPGF(1alpha). In the first part of the study, the effects of CLA isomers in the free fatty acid form were tested. The 10(E), 12(Z)- and 9(Z), 11(E)-CLA isomers inhibited thrombin-induced 6-ketoPGF(1alpha) formation with I(50)'s of 2.6 and 5.5 microM, whereas the 9(Z), 11(Z)- and 9(E), 11(E)-CLA were ineffective at concentrations up to 60 microM. The inhibitory effect of the 10(E), 12(Z)-CLA was irreversible. Next, the effects of CLA incorporation into HUVECs on PGI(2) generation was determined. An average 8-fold stimulation of 6-ketoPGF(1alpha) formation was obtained with quiescent IL1-beta-exposed HUVECs pretreated for 18 h with 25 microM 9(Z), 11(Z)-CLA, whereas cells preincubated with the 10(E), 12(Z) isomer enhanced this eicosanoid 3-fold. Such IL1-beta-treated HUVECs prelabeled with 25 microM 9(Z), 11(Z)-CLA became refractory to thrombin stimulation, as measured by 6-ketoPGF(1alpha) production, whereas a small, statistically insignificant, inhibition was observed upon thrombin treatment of HUVECs prelabeled with the 10(E), 12(Z) isomer. Qualitative similar results were obtained with resting or thrombin-stimulated platelets containing these esterified CLA isomers indicating that these effects occur with cells that contain either the COX-1 or COX-2 isozymes. The results of this in vitro study indicate that the effects of CLA on cellular prostanoid formation in endothelial cells and platelets can be either inhibitory or stimulatory, and this seems to depend not only on the specific CLA isomer and whether or not the CLA is in the free fatty acid form or esterified into cellular lipids, but also whether cells are in the resting or stimulated state. These findings suggest that in vivo, CLA might have multiple, complex effects on vascular homeostasis.     

Increased NADPH oxidase-derived superoxide is involved in the neuronal cell death induced by hypoxia-ischemia in neonatal hippocampal slice cultures

Neonatal brain hypoxia-ischemia (HI) results in neuronal cell death. Previous studies indicate that reactive oxygen species, such as superoxide, play a key role in this process. However, the cellular sources have not been established. In this study we examine the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in neonatal HI brain injury and elucidate its mechanism of activation. Rat hippocampal slices were exposed to oxygen glucose deprivation (OGD) to mimic the conditions seen in HI. Initial studies confirmed an important role for NADPH oxidase-derived superoxide in the oxidative stress associated with OGD. Further, the OGD-mediated increase in apoptotic cell death was inhibited by the NADPH oxidase inhibitor apocynin. The activation of NADPH oxidase was found to be dependent on the p38 mitogen-activated protein kinase-mediated phosphorylation and activation of the p47(phox) subunit. Using an adeno-associated virus antisense construct to selectively decrease p47(phox) expression in neurons showed that this led to inhibition of both the increase in superoxide and the neuronal cell death associated with OGD. We also found that NADPH oxidase inhibition in a neonatal rat model of HI or scavenging hydrogen peroxide reduced brain injury. Thus, we conclude that activation of the NADPH oxidase complex contributes to the oxidative stress during HI and that therapies targeted against this complex could provide neuroprotection against the brain injury associated with neonatal HI.     

Dissociation of progressive dopaminergic neuronal death and behavioral impairments by Bax deletion in a mouse model of Parkinson's diseases

Parkinson's disease (PD) is a common, late-onset movement disorder with selective degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Although the neurotoxin 6-hydroxydopamine (6-OHDA) has been used to induce progressive degeneration of DA neurons in various animal models of PD, the precise molecular pathway and the impact of anti-apoptotic treatment on this neurodegeneration are less understood. Following a striatal injection of 6-OHDA, we observed atrophy and progressive death of DA neurons in wild-type mice. These degenerating DA neurons never exhibited signs of apoptosis (i.e., caspase-3 activation and cytoplasmic release of cytochrome C), but rather show nuclear translocation of apoptosis-inducing factor (AIF), a hallmark of regulated necrosis. However, mice with genetic deletion of the proapoptotic gene Bax (Bax-KO) exhibited a complete absence of 6-OHDA-induced DA neuron death and nuclear translocation of AIF, indicating that 6-OHDA-induced DA neuronal death is mediated by Bax-dependent AIF activation. On the other hand, DA neurons that survived in Bax-KO mice exhibited marked neuronal atrophy, without significant improvement of PD-related behavioral deficits. These findings suggest that anti-apoptotic therapy may not be sufficient for PD treatment, and the prevention of Bax-independent neuronal atrophy may be an important therapeutic target.     

HSP27 but not HSP70 has a potent protective effect against alpha-synuclein-induced cell death in mammalian neuronal cells

alpha-Synuclein is a neuronally expressed protein which is mutated in familial Parkinson's disease. We have previously shown that disease-associated mutants of alpha-synuclein cause enhanced neuronal cell death in response to a variety of stimuli, whereas wild-type alpha-synuclein has a protective effect against some stimuli, whilst enhancing the death response to others. We demonstrate, for the first time, that over-expression of the heat shock protein HSP27 has a potent protective anti-apoptotic effect against the damaging effects of wild-type and particularly of mutant alpha-synuclein. In contrast, HSP70 has some protective effect against the damaging effect of the wild-type protein, but has no effect against the mutant proteins, whilst HSP56 has no protective effect in this system. Our results indicate that disease-associated mutants of alpha-synuclein enhance its death-inducing properties and lead to increased apoptosis, which can be mitigated by either the use of specific caspase inhibitors or HSP27 over-expression. This potent protective effect of HSP27 against the mutant and wild-type proteins may be of potential therapeutic importance.     

Inhibitory effects of actinomycin D and cycloheximide on neuronal death in adult Manduca sexta

A decline in circulating 20-hydroxyecdysone permits the emergence of the adult Manduca sexta moth; this endocrine signal also triggers the death of approximately half of the neurons in the unfused abdominal ganglia of the moth central nervous system. This programmed death of neurons was markedly reduced by treatment with either actinomysin D (an RNA synthesis inhibitor) or cycloheximide (a protein synthesis inhibitor). Similar results were found after addition of these agents to ventral nerve cord cultures. The effectiveness of these treatments in delaying or blocking neuronal death depended upon their time of administration relative to the normal time of post-emergence death in the particular motoneuron under study: late-dying neurons, for example, could still be saved by these treatments even after early-dying neurons had already initiated degeneration. In both intact moths and cultured ventral nerve cords, the ability of actinomycin D to prevent neuronal death waned at the same time at which replacement of the steroid hormone could no longer block neuronal death. This suggests that the steroid commitment point represents the time at which the genes that mediate cell death are transcribed. Cycloheximide remained effective in delaying or blocking neuronal death until shortly before the onset of degeneration, suggesting that ongoing protein synthesis is essential for the initiation of the degeneration response. 1994 John Wiley & Sons, Inc.