Rong Han

Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways.

It has been reported that ischemic insult increases the formation of autophagosomes and activates autophagy. However, the role of autophagy in ischemic neuronal damage remains elusive. This study was taken to assess the role of autophagy in ischemic brain damage. Focal cerebral ischemia was introduced by permanent middle cerebral artery occlusion (pMCAO). Activation of autophagy was assessed by morphological and biochemical examinations. To determine the contribution of autophagy/lysosome to ischemic neuronal death, rats were pretreated with a single intracerebral ventricle injection of the autophagy inhibitors 3-methyl-adenine (3-MA) and bafliomycin A1 (BFA) or the cathepsin B inhibitor Z-FA-fmk after pMCAO. The effects of 3-MA and Z-FA-fmk on brain damage, expression of proteins involved in regulation of autophagy and apoptosis were assessed with 2,3,5-triphenyltetrazolium chloride (TTC) staining and immunoblotting. The results showed that pMACO increased the formation of autophagosomes and autolysosomes, the mRNA and protein levels of LC3-II and the protein levels of cathepsin B. 3-MA, BFA and Z-FA-fmk significantly reduced infarct volume, brain edema, and motor deficits. The neuroprotective effects of 3-MA and Z-FA-fmk were associated with an inhibition on ischemia-induced upregulation of LC3-II and cathepsin B and a partial reversion of ischemia-induced downregulation of cytoprotective Bcl-2. These results demonstrate that ischemic insult activates autophagy and an autophagic mechanism may contribute to ischemic neuronal injury. Thus, autophagy may be a potential target for developing a novel therapy for stroke.

Autophagy activation is associated with neuroprotection in a rat model of focal cerebral ischemic preconditioning

Several recent studies have showed that autophagy is involved in ischemic brain damage, but it may also play a pro-survival role in ischemic preconditioning. This study was taken to determine the role of autophagy in an animal model of cerebral ischemic preconditioning (IPC). Focal cerebral IPC was produced in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The rats were pretreated with intracerebral ventricle infusion of the autophagy inhibitors 3-methyladenine (3-MA) and bafliomycin A1 (Baf A1) or the autophagy inducer rapamycin to evaluate the contribution of autophagy to IPC-induced neuroprotection. The results from electron microscopic examinations and immunofluorescence showed that both IPC and PFI induced autophagy activation, but the extent and persistence of autophagy activation were varied. IPC treatment significantly reduced infarct volume, brain edema and motor deficits after subsequent PFI, whereas 3-MA and Baf A1 suppressed the neuroprotection induced by IPC. 3-MA pretreatment also significantly attenuated upregulation of LC3-II, beclin 1 and HSP70 and downregulation of p62. To further determine if autophagy induction is responsible for IPC-induced neuroprotection, rats were treated with rapamycin 24 h before the onset of PFI. The results showed that rapamycin reduced infarct volume, brain edema and motor deficits induced by PFI. Rapamycin pretreatment also increased the protein levels of LC3-II and beclin 1. These results demonstrate that autophagy activation during IPC offers a remarkable tolerance to a subsequent fatal ischemic insult, and IPCs neuroprotective effects can be mimicked by autophagy inducers.

An autophagic mechanism is involved in apoptotic death of rat striatal neurons induced by the non-N-methyl-D-aspartate receptor agonist kainic acid

Previous studies found that kainic acid (KA)-induced apoptosis involved the lysosomal enzyme cathepsin B, suggesting a possible mechanism of autophagy in excitotoxicity. The present study was sought to investigate activation and contribution of autophagy to excitotoxic neuronal injury mediated by KA receptors. The formation of autophagosomes was observed with transmission electron microscope after excitotoxin exposure. The contribution of autophagic mechanisms to KA-induced up-regulation of microtubule-associated protein 1A/1B light chain 3 (LC3), lysosome-associated membrane protein 2 (LAMP2) and cathepsin B, release of cytochrome c, activation of caspase-3, down-regulation of Bcl-2, up-regulation of Bax, p53, puma and apoptotic death of striatal neurons were assessed with co-administration of the autophagy inhibitor 3-methyladenine (3-MA). These studies showed that KA brought about an increase in the formation of autophagosomes and autolysosomes in the cytoplasm of striatal cells. KA-induced increases in the ratio of LC3-II/LC3-I, LAMP2, cathepsin B, release of cytochrome c and activation of caspase-3 were blocked by pre-treatment with 3-MA. 3-MA also reversed KA-induced down-regulation of Bcl-2 and up-regulation of Bax protein levels, LC3, p53 and puma mRNA levels in the striatum. KA-induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by 3-MA. These results suggest that over-stimulation of KA receptors can activate autophagy. The autophagic mechanism participates in programmed cell death through regulating the mitochondria-mediated apoptotic pathway.

Autophagy regulates endoplasmic reticulum stress in ischemic preconditioning.

Recent studies have suggested that autophagy plays a prosurvival role in ischemic preconditioning (IPC). This study was taken to assess the linkage between autophagy and endoplasmic reticulum (ER) stress during the process of IPC. The effects of IPC on ER stress and neuronal injury were determined by exposure of primary cultured murine cortical neurons to 30 min of OGD 24 h prior to a subsequent lethal OGD. The effects of IPC on ER stress and ischemic brain damage were evaluated in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The results showed that both IPC and lethal OGD increased the LC3-II expression and decreased p62 protein levels, but the extent of autophagy activation was varied. IPC treatment ameliorated OGD-induced cell damage in cultured cortical neurons, whereas 3-MA (5–20 mM) and bafilomycin A1 (75–150 nM) suppressed the neuroprotection induced by IPC. 3-MA, at the dose blocking autophagy, significantly inhibited IPC-induced HSP70, HSP60 and GRP78 upregulation; meanwhile, it also aggregated the ER stress and increased activated caspase-12, caspase-3 and CHOP protein levels both in vitro and in vivo models. The ER stress inhibitor Sal (75 pmol) recovered IPC-induced neuroprotection in the presence of 3-MA. Rapamycin 50–200 nM in vitro and 35 pmol in vivo 24 h before the onset of lethal ischemia reduced ER stress and ischemia-induced neuronal damage. These results demonstrated that pre-activation of autophagy by ischemic preconditioning can boost endogenous defense mechanisms to upregulate molecular chaperones, and hence reduce excessive ER stress during fatal ischemia.

p53 mediates mitochondria dysfunction-triggered autophagy activation and cell death in rat striatum

In vivo administration of the mitochondrial inhibitor 3-nitropropionic acid (3-NP) produces striatal pathology mimicking Huntington disease (HD). However, the mechanisms of cell death induced by metabolic impairment are not fully understood. The present study investigated contributions of p53 signaling pathway to autophagy activation and cell death induced by 3-NP. Rat striatum was intoxicated with 3-NP by stereotaxic injection. Morphological and biochemical analyses demonstrated activation of autophagy in striatal cells as evidenced by increased the formation of autophagosomes, the expression of active lysosomal cathepsin B and D, microtubule associate protein light chain 3 (LC3) and conversion of LC3-I to LC3-II. 3-NP upregulated the expression of tumor suppressor protein 53 (p53) and its target genes including Bax, p53-upregulated modulator of apoptosis (PUMA) and damage-regulated autophagy modulator (DRAM). 3-NP-induced elevations in pro-apoptotic proteins Bax and PUMA, autophagic proteins LC3-II and DRAM were significantly reduced by the p53 specific inhibitor pifithrin-α (PFT). PFT also significantly inhibited 3-NP-induced striatal damage. Similarly, 3-NP-induced DNA fragmentation and striatal cell death were robustly attenuated by the autophagy inhibitor 3-methyladenine (3-MA) and bafilomycin A1 (BFA). These results suggest that p53 plays roles in signaling both autophagy and apoptosis. Autophagy, at least partially, contributes to neurodegeneration induced by mitochondria dysfunction.

NF-κB contributes to 6-hydroxydopamine-induced apoptosis of nigral dopaminergic neurons through p53

To evaluate the contribution of NF-kappaB and the NF-kappaB target gene p53 to nigral dopaminergic neuron degeneration in rodent models of Parkinsons disease, time-course of dopaminergic neuron loss as well as changes in the expression of some NF-kappaB-regulated proapoptotic proteins were assayed after unilateral infusion of 6-hydroxydopamine into rat medial forebrain bundle. Substantial loss of tyrosine hydroxylase immunoreactivity in nigral was observed 24 h after 6-hydroxydopamine treatment. The degenerative processes began 12 h after 6-hydroxydopamine administration as evidenced by a positive silver staining. Apoptotic death of dopaminergic neurons was suggested by the appearance of TUNEL-positive nuclei in substantia nigra and internucleosomal DNA fragmentation as detected by agarose gel electrophoresis. NF-kappaB activation in dopaminergic neurons as revealed by immunohistochemistry and electrophoresis mobility shift assay, began at 12 h after 6-hydroxydopamine administration. Levels of c-Myc and p53 immunoreactivities increased after 6-hydroxydopamine treatment, mainly in dopaminergic neurons as indicated by co-localization with tyrosine hydroxylase immunoreactivity. Blockade of NF-kappaB nuclear translocation with recombinant cell-permeable peptide NF-kappaB SN50 inhibited NF-kappaB nuclear translocation and p53 induction. SN50 and the p53 antagonist pifithrin-alpha significantly reduced nigral dopaminergic neuron degeneration. These results suggest that NF-kappaB activation contributes, at least in part, to oxidative stress-induced degeneration of dopaminergic neurons through a NF-kappaB-dependent p53-signaling pathway.

A long-form α-neurotoxin from cobra venom produces potent opioid-independent analgesia

AbstractAim:In light of the antinociceptive activity of the short-chain neurotoxin, cobrotoxin, and other acetylcholine antagonists, the antinociceptive activity and mechanisms of cobratoxin (CTX), a long-chain postsynaptic α-neurotoxin, was investigated in rodent pain models.Methods:CTX was administered intraperitoneally (30, 45, 68 μg/kg), intra-cerebral ventricularly (4.5 μg/kg) or microinjected into periaqueductal gray (PAG; 4.5 μg/kg). The antinociceptive action was tested using the hot-plate and acetic acid writhing tests in mice and rats. The involvement of the cholinergic system and opioid system in CTX-induced analgesia was examined by pretreatment of animals with atropine (0.5 mg/kg, im; or 10 mg/kg, ip) or naloxone (1 and 5 mg/kg, ip). The effect of CTX on motor activity was tested using the Animex test.Results:CTX exhibited a dose-dependent analgesic action in mice as determined by both the hot-plate and acetic acid writhing tests. The peak effect of analgesia was seen 3 h after administration. In the mouse acetic acid writhing test, the intra-cerebral ventricular administration of CTX at 4.5 μg/kg (1/12th of a systemic dose)produced marked analgesic effects. Microinjection of CTX (4.5 μg/kg) into the PAG region did not elicit an analgesic action in rats in the hot-plate test. Atropine at 0.5 mg/kg (im) and naloxone at 1 and 5 mg/kg (ip) both failed to block the analgesic effects of CTX, but atropine at 10 mg/kg (ip) did antagonize the analgesia mediated by CTX in the mouse acetic acid writhing test. Acetylsalicylic acid (300 mg/kg) did not enhance the analgesic effects of CTX. At the highest effective dose of 68 μg/kg the neurotoxin did not change the spontaneous mobility of mice.Conclusion:CTX has analgesic effects, which are mediated in the central nervous system though not through the PAG. The central cholinergic system but not opioid system appears to be involved in the antinociceptive action of CTX.

The endoplasmic reticulum stress inhibitor salubrinal inhibits the activation of autophagy and neuroprotection induced by brain ischemic preconditioning

Aim:To investigate whether endoplasmic reticulum (ER) stress participates in the neuroprotective effects of ischemic preconditioning (IPC)-induced neuroprotection and autophagy activation in rat brains.Methods:The right middle cerebral artery in SD rats was occluded for 10 min to induce focal cerebral IPC, and was occluded permanently 24 h later to induce permanent focal ischemia (PFI). ER stress inhibitor salubrinal (SAL) was injected via intracerebral ventricle infusion 10 min before the onset of IPC. Infarct volume and motor behavior deficits were examined after the ischemic insult. The protein levels of LC3, p62, HSP70, glucose-regulated protein 78 (GRP 78), p-eIF2α and caspase-12 in the ipsilateral cortex were analyzed using immunoblotting. LC3 expression pattern in the sections of ipsilateral cortex was observed with immunofluorescence.Results:Pretreatment with SAL (150 pmol) abolished the neuroprotective effects of IPC, as evidenced by the significant increases in mortality, infarct volume and motor deficits after PFI. At the molecular levels, pretreatment with SAL (150 pmol) significantly increased p-eIF2α level, and decreased GRP78 level after PFI, suggesting that SAL effectively inhibited ER stress in the cortex. Furthermore, the pretreatment with SAL blocked the IPC-induced upregulation of LC3-II and downregulation of p62 in the cortex, thus inhibiting the activation of autophagy. Moreover,SAL blocked the upregulation of HSP70, but significantly increased the cleaved caspase-12 level, thus promoting ER stress-dependent apoptotic signaling in the cortex.Conclusion:ER stress-induced autophagy might contribute to the neuroprotective effect of brain ischemic preconditioning.

Enhancement of neuroprotection and heat shock protein induction by combined prostaglandin A1 and lithium in rodent models of focal ischemia.

Both prostaglandin A(1) (PGA(1)) and lithium have been reported to protect neurons against excitotoxic and ischemic injury. The present study was undertaken to examine the effects of lithium and PGA1 on heat shock proteins (HSP) and the growth arrest and DNA-damage-inducible gene (GADD153) and to evaluate if lithium could potentiate PGA(1)s neuroprotective effects against cerebral ischemia. Rats were pretreated with a subcutaneous injection of lithium for 2 days and a single intracerebral ventricle administration of PGA(1) 15 min before ischemic insult. Brain ischemia was induced by a permanent middle cerebral artery occlusion. The infarct volume, motor behavior deficits and brain edema were analyzed 24 h after ischemic insult. The result showed that PGA(1) significantly reduced infarct volume, neurological deficits and brain edema. Except for neurological deficit, lithium enhanced PGA(1)s neuroprotection. The neuroprotective effects of PGA(1) were associated with an up-regulation of cytoprotective heat shock proteins HSP70 and GRP78 in the ischemic brain hemisphere as determined by immunoblotting and immunofluorescence. The induction of HSP70 and GRP78 was enhanced by lithium. However, although the expression of GADD153 was enhanced significantly after pMCAO, it was not influenced by either PGA(1) or lithium or their combination. These studies suggest that lithium can potentiate PGA(1)s neuroprotective effects and thus may have potential clinical value for the treatment of stroke in combination with other neuroprotective agents.

Down-regulation of Bcl-2 enhances autophagy activation and cell death induced by mitochondrial dysfunction in rat striatum.

In vivo administration of the mitochondrial inhibitor 3‐nitropropionic acid (3‐NP) produces striatal pathology mimicking Huntingtons disease (HD). However, the mechanisms of cell death induced by metabolic impairment are not fully understood. Previous studies showed that 3‐NP triggered p53‐depedent autophagy activation and cell death. The present study investigated the contribution of the Bcl‐2 signaling pathway to autophagy activation and cell death induced by 3‐NP. Rat striatum was intoxicated with 3‐NP by stereotaxic injection. 3‐NP up‐regulated the expression of the autophagic protein beclin 1 but down‐regulated the expression of the antiapoptotic protein Bcl‐2. Pretreatment with the autophagy inhibitor 3‐methyladenine (3‐MA) significantly inhibited the 3‐NP‐induced alterations in beclin 1 and Bcl‐2 protein levels. Similarly, the 3‐NP‐induced decline in Bcl‐2 was also prevented by the lysosomal inhibitor E64, indicating degradation of Bcl‐2 by lysosomes. In agreement with the time course of 3‐NP‐induced cell death, an increase in the release of cytochrome c from mitochondria was observed. 3‐MA also attenuated the 3‐NP‐induced release of cytochrome c. On the other hand, 3‐NP‐induced elevations in proapoptotic protein Bax and autophagic protein beclin 1 and LC3‐II were significantly enhanced by the Bcl‐2‐specific inhibitor HA14‐1. Furthermore, HA14‐1 increased the release of cytochrome c and 3‐NP‐induced striatal damage. These results suggest that induction of autophagy leads to degradation of Bcl‐2. Meanwhile, down‐regulation of Bcl‐2 amplifies autophagy activation and apoptotic signaling. Bcl‐2 thus plays important roles in mitochondria dysfunction‐induced apoptotic death of stritatal neurons by modulating both autophagic and apoptotic processes.