Proteasome inhibition results in increased poly-ADP-ribosylation: implications for neuron death

This study demonstrates the ability of proteasome inhibitors (lactacystin, MG 115, MG 132) adenosine diphosphate to induce a time- and dose-dependent increase in poly-ADP-ribosylation (PAR) in the neural PC6 cell line, a subclone of PC12 cells. Elevated levels of PAR contribute to the toxicity associated with impaired proteasome activity, based on the ability of PAR inhibitors to ameliorate the toxicity associated with the application of lactacystin, MG 115, and MG 132. Proteasome inhibitors induced the accumulation of PAR and neuron death in primary hippocampal neuron cultures, which were both ameliorated by treatment with PAR inhibitors. Together, these data indicate a role for increased PAR in the toxicity associated with proteasome inhibition, and suggest that inhibitors of PAR may provide neuroprotection in conditions where proteasome inhibition occurs.     

GDNF Contributes to Oestrogen‐Mediated Protection of Midbrain Dopaminergic Neurones

Parkinson’s disease (PD) is characterised by the preferential loss of dopaminergic neurones from the substantia nigra (SN) that leads to the hallmark motor disturbances. Animal and human studies suggest a beneficial effect of oestrogen to the nigrostriatal system, and the regulation of neurotrophic factor expression by oestrogens has been suggested as a possible mechanism contributing to that neuroprotective effect. The present study was designed to investigate whether the neuroprotection exerted by 17β‐oestradiol on nigrostriatal dopaminergic neurones is mediated through the regulation of glial cell line‐derived neurotrophic factor (GDNF) expression. Using an in vivo rat model of PD, we were able to confirm the relevance of 17β‐oestradiol in defending dopaminergic neurones against 6‐hydroxydopamine (6‐OHDA) toxicity. 17β‐oestradiol, released by micro‐osmotic pumps, implanted 10 days before intrastriatal 6‐OHDA injection, prevented the loss of dopaminergic neurones induced by 6‐OHDA. 17β‐oestradiol treatment also promoted an increase in GDNF protein levels both in the SN and striatum. To explore the relevance of GDNF increases to 17β‐oestradiol neuroprotection, we analysed, in SN neurone‐glia cultures, the effect of GDNF antibody neutralisation and RNA interference‐mediated GDNF knockdown. The results showed that both GDNF neutralisation and GDNF silencing abolished the dopaminergic protection provided by 17β‐oestradiol against 6‐OHDA toxicity. Taken together, these results strongly identify GDNF as an important player in 17β‐oestradiol‐mediated dopaminergic neuroprotection.     

A yeast two-hybrid screen reveals that osteopontin associates with MAP1A and MAP1B in addition to other proteins linked to microtubule stability, apoptosis and protein degradation in the human brain

Osteopontin (OPN) expression is reduced in surviving dopaminergic neurones in the substantia nigra (SN) in Parkinson’s disease (PD), and protects against MPP⁺‐induced cell death in primary mesencephalic cultures and 6‐OHDA‐induced cell loss in the rat, while inactivation of OPN aggravates cell death. OPN is thought to act through interactions with integrin receptors or CD44. However, the specific protein interactions involved in OPN‐mediated neuroprotection are unknown and are the focus of this study. The yeast two‐hybrid (YTH) technique was utilised to investigate OPN–protein interactions, using full‐length human OPN to screen a human foetal brain cDNA library. Proteins involved in apoptosis, protein degradation and microtubule stability were identified as OPN binding partners. These included: MAP1A and MAP1B, which regulate microtubule stability; RNF138, an E3 ubiquitin‐ligase; proteasome β1 subunit, a subunit of the 20S proteasome involved in the ubiquitin‐dependent cleavage of peptides; BAG6, SGTΑ and EF1A, proteins implicated in control of apoptosis; DnaJB1, a co‐chaperone of Hsp70s; and pleiotrophin, a growth factor. The use of site‐directed mutagenesis to modify known OPN protein binding sites outside the RGD integrin binding domain, specifically Y165A and D139E, inhibited some of these interactions. Further investigation using affinity pull‐down assays, co‐immunoprecipitation and immunohistochemistry confirmed that OPN associates with MAP1A and MAP1B in rat SN and striatum. These findings indicate a role for OPN in the regulation of microtubule dynamics, apoptosis and proteolysis in the brain, suggesting that OPN may act as an endogenous multifunctional protective protein in PD.     

How does conserved dopamine neurotrophic factor protect against and rescue neurodegeneration of PC12 cells?

Conserved dopamine neurotrophic factor protects and rescues dopaminergic neurodegeneration induced by 6-hydroxydopamine in vivo,but its potential value in treating Parkinson’s disease remains controversial.Here,we used the proteasome inhibitors lactacystin and MG132 to induce neurodegeneration of PC12 cells.Afterwards,conserved dopamine neurotrophic factor was administrated as a therapeutic factor,both pretreatment and posttreatment.Our results showed that(1)conserved dopamine neurotrophic factor enhanced lactacystin/MG132-induced cell viability and morphology,and attenuated alpha-synuclein accumulation in differentiated PC12 cells.(2)Enzyme linked immunosorbent assay showed up-regulated 26S proteasomal activity in MG132-induced PC12 cells after pre-and posttreatment with conserved dopamine neurotrophic factor.Similarly,26S proteasome activity was upregulated in lactacystin-induced PC12 cells pretreated with conserved dopamine neurotrophic factor.(3)With regard proteolytic enzymes(specifically,glutamyl peptide hydrolase,chymotrypsin,and trypsin),glutamyl peptide hydrolase activity was up-regulated in lactacystin/MG132-administered PC12 cells after pre-and posttreatment with conserved dopamine neurotrophic factor.However,upregulation of chymotrypsin activity was only observed in MG132-administered PC12 cells pretreated with conserved dopamine neurotrophic factor.There was no change in trypsin expression.We conclude that conserved dopamine neurotrophic factor develops its neurotrophic effects by modulating proteasomal activities,and thereby protects and rescues PC12 cells against neurodegeneration.     

Proteasome inhibition modulates kinase activation in neural cells: Relevence to ubiquitination,ribosomes, and survival

In this study we examined whether established signal transduction cascades, p44/42 mitogen‐activated protein kinase (ERK1/2) and Jun N‐terminal kinases (JNK) pathways, are altered in N2a neural cells in response to proteasome inhibition. Additionally, we sought to elucidate the relative contribution of these signal transduction pathways to the multiple downstream effects of proteasome inhibition. Our data indicate that ERK1/2 and JNK are activated in response to proteasome inhibition. Washout of proteasome inhibitor (MG132) results in an enhancement of ERK1/2 activation and amelioration of JNK activation. Treatment with an established MAPK inhibitor resulted in an increase in proteasome inhibitor toxicity, and incubation with JNK inhibitor was observed to attenuate proteasome inhibitor toxicity significantly. Subsequent studies demonstrated that inhibition of ERK1/2 and JNK activity does not alter the gross increase in ubiquitinated protein following proteasome inhibitor administration. Similarly, ERK1/2 and JNK activity do not appear to play a role in the disruption of polysomes following proteasome inhibitor administration in neural cells. Together these data indicate that ERK1/2 and JNK activation may play differential roles in modulating neurochemical disturbances and neurotoxicity induced by proteasome inhibition. © 2009 Wiley‐Liss, Inc.     

Prevention of muscle disuse atrophy by MG132 proteasome inhibitor

Introduction: Our goal was to determine whether in vivo administration of the proteasome inhibitor MG132 can prevent muscle atrophy caused by hindlimb unloading (HU). Methods: Twenty‐seven NMRI mice were assigned to a weight‐bearing control, a 6‐day HU, or a HU+MG132 (1 mg/kg/48 h) treatment group. Results: Gastrocnemius wasting was significantly less in HU+MG132 mice (?6.7 ± 2.0%) compared with HU animals (?12.6 ± 1.1%, P = 0.011). HU was also associated with an increased expression of MuRF‐1 (P = 0.006), MAFbx (P = 0.001), and USP28 (P = 0.027) mRNA, whereas Nedd4, E3α, USP19, and UBP45 mRNA did not change significantly. Increases in MuRF‐1, MAFbx, and USP28 mRNA were largely repressed after MG132 administration. β5 proteasome activity tended to increase in HU (+16.7 ± 6.1%, P = 0.086). Neither β1 and β2 proteasome activities nor ubiquitin‐conjugated proteins were changed by HU. Conclusions: Our results indicate that in vivo administration of MG132 partially prevents muscle atrophy associated with disuse and highlight an unexpected regulation of MG132 proteasome inhibitor on ubiquitin‐ligases. Muscle Nerve, 2011     

Simvastatin prevents neuroinflammation by inhibiting N‐methyl‐D‐aspartic acid receptor 1 in 6‐hydroxydopamine‐treated PC12 cells

This study investigates the impact of simvastatin on neuroinflammation in experimental parkinsonian cell models. 6‐Hydroxydopamine (6‐OHDA)‐treated pheochromocytoma‐12 (PC12) cells were used to investigate the neuroprotective nature of simvastatin. After incubation with 6‐OHDA, simvastatin, and/or N‐methyl‐D‐aspartic acid receptor 1 (NMDAR1) siRNA for 24 hr, test kits were used to detect the levels of lactate dehydrogenase (LDH) and glutamate released from PC12 cells exposed to different culture media. The mRNA levels of tumor necrosis factor (TNF)‐α, interleukin (IL)?1β, and IL‐6 were determined by RT‐PCR, and the protein levels were analyzed by Western blot. NMDAR1 were also determined by RT‐PCR and the protein levels were analyzed by Western blot. LDH and glutamate levels in 6‐OHDA‐incubated PC12 cells increased compared with those in the controls, and incubation with simvastatin inhibited this elevation. Silencing of NMDAR1 with siRNA inhibited the expression of LDH and glutamate to a degree similar to simvastatin. The expression levels of NMDAR1, TNF‐α, IL‐1β, and IL‐6 were significantly upregulated after treatment with 6‐OHDA. The 6‐OHDA‐stimulated mRNA and protein levels of the proinflammatory cytokines NMDAR1, TNF‐α, IL‐1β, and IL‐6 were reduced by simvastatin. Silencing of NMDAR1 with siRNA decreased the NMDAR1, TNF‐α, IL‐1β, and IL‐6 mRNA and protein expression levels in 6‐OHDA‐stimulated PC12 cells. Simvastatin could also inhibit the expression of NMDAR1 and cytokines to a degree similar to silencing of NMDAR1 with siRNA. Our results suggest that NMDAR1 modulation could explain the anti‐inflammatory mechanisms of simvastatin in experimental parkinsonian cell models. © 2014 Wiley Periodicals, Inc.     

C-terminal mechano-growth factor induces heme oxygenase-1-mediated neuroprotection of SH-SY5Y cells via the protein kinase Cϵ/Nrf2 pathway

Recently, a variant of insulin‐like growth factor‐1, mechano‐growth factor (MGF), has been discovered whose 24‐amino‐acid carboxy end is protective in models of stroke, nerve injury, and amyotrophic lateral sclerosis, suggesting broad‐spectrum neuroprotective properties. Moreover, we recently demonstrated in vitro and in vivo that a modified protease‐resistant 24‐amino‐acid MGF derivative (MGF24) protects dopaminergic neurons from oxidative stress‐induced apoptosis via induction of the stress response protein heme oxygenase‐1. However, the underlying mechanism by which MGF24 up‐regulates heme oxygenase‐1 expression is unknown. In this study, we demonstrate that MGF24‐induced heme oxygenase‐1 up‐regulation is dependent on activation of protein kinase Cϵ and NF‐E2‐related factor‐2 (Nrf2). MGF24 induces nuclear translocation of Nrf2, and siRNA knockdown of Nrf2 or of heme oxygenase‐1 prevents MGF24‐induced heme oxygenase‐1 up‐regulation and neuroprotection of SH‐SY5Y cells against 6‐hydroxydopamine‐induced cell death. Pharmacological inhibition of ERK, p38 MAPK, PI3K/Akt, or PKC signaling revealed that only PKC inhibition by GF109203X prevents MGF24's ability to protect against 6‐hydroxydopamine‐induced cell death. GF109203X also prevented MGF24‐induced Nrf2 nuclear translocation and heme oxygenase‐1 up‐regulation. siRNA knockdown of protein kinase Cϵ blocks MGF24‐induced Nfr2 nuclear translocation, heme oxygenase‐1 expression, and neuroprotection. Taken together, these results demonstrate that PKC activity is needed for MGF24's activation of Nrf2, which in turn increases heme oxygenase‐1 expression, a critical event in mediating MGF24's neuroprotection against 6‐hydroxydopamine‐induced apoptosis. Published 2011 Wiley‐Liss, Inc.     

Reductions of the components of the calreticulin/calnexin quality‐control system by proteasome inhibitors and their relevance in a rodent model of Parkinson's disease

Evidence indicates that the ubiquitin‐proteasome system and the endoplasmic retculum (ER) quality‐control system work in concert to ensure that proteins are correctly folded in the ER and that misfolded proteins are retrotransported to the cytosol for degradation by proteasomes. Dysfunction of either system results in developmental abnormalities and even death in animals. This study investigates whether and how proteasome inhibition impacts the components of the calreticulin (CRT)/calnexin (CNX) glycoprotein folding machinery, a typical ER protein quality‐control system, in the context of early neuronal injury. Here we report that proteasome inhibitor treatments, at nonlethal levels, reduced protein levels of CRT and ERp57 but not of CNX. These treatments increased protein levels of CRT in culture media, an effect blocked by brefeldin A, an inhibitor of protein trafficking; by contrast, ERp57 was not detected in culture media. Knockdown of CRT levels alone increased the vulnerability of SH‐SY5Y, a neuronal cell line, to 6‐hydroxydopamine (6‐OHDA) toxicity. In a rat model of Parkinson's disease, intrastriatal 6‐OHDA lesions resulted in decreased levels of CRT and ERp57 in the midbrain. These findings suggest that reduction of the components of CRT/CNX glycoprotein quality‐control system may play a role in neuronal injury in Parkinson's disease and other neurodegenerative disorders associated with dysfunction of the ubiquitin‐proteasome system. © 2014 Wiley Periodicals, Inc.     

3‐[(2,4‐dimethoxy)benzylidene]‐anabaseine dihydrochloride protects against 6‐hydroxydopamine‐induced parkinsonian neurodegeneration through α7 nicotinic acetylcholine receptor stimulation in rats

To explore a novel therapy against Parkinson's disease through enhancement of α7 nicotinic acetylcholine receptor (nAChR), we evaluated the neuroprotective effects of 3‐[(2,4‐dimethoxy)benzylidene]‐anabaseine dihydrochloride (DMXBA; GTS‐21), a functionally selective α7 nAChR agonist, in a rat 6‐hydroxydopamine (6‐OHDA)‐induced hemiparkinsonian model. Microinjection of 6‐OHDA into the nigrostriatal pathway of rats destroys dopaminergic neurons selectively. DMXBA dose dependently inhibited methamphetamine‐stimulated rotational behavior and dopaminergic neuronal loss induced by 6‐OHDA. The protective effects were abolished by methyllycaconitine citrate salt hydrate, an α7 nAChR antagonist. Immunohistochemical study confirmed abundant α7 nAChR expression in the cytoplasm of dopaminergic neurons. These results indicate that DMXBA prevented 6‐OHDA‐induced dopaminergic neuronal loss through stimulating α7 nAChR in dopaminergic neurons. Injection of 6‐OHDA elevated immunoreactivities to glial markers such as ionized calcium binding adaptor molecule 1, CD68, and glial fibrillary acidic protein in the substantia nigra pars compacta of rats. In contrast, these immunoreactivities were markedly inhibited by comicroinjection of DMXBA. Microglia also expressed α7 nAChR in both resting and activated states. Hence, we hypothesize that DMXBA simultaneously affects microglia and dopaminergic neurons and that both actions lead to dopaminergic neuroprotection. The findings that DMXBA attenuates 6‐OHDA‐induced dopaminergic neurodegeneration and glial activation in a rat model of Parkinson's disease raisethe possibility that DMXBA could be a novel therapeutic compound to prevent Parkinson's disease development. © 2012 Wiley Periodicals, Inc.     

Protective effects of heme-oxygenase expression in cyclosporine A--induced injury

Cyclosporine A (CsA) is the immunosuppressant of first choice in allotransplantation. Its use is associated with side effects of nephrotoxicity and neurotoxicity, which are among the most prominent. This study was undertaken to explore whether expression and activity of heme oxygenase (HO), the rate-limiting enzyme in heme degradation, is altered in a rat model of CsA-induced injury. Male Sprague Dawley rats were divided into four groups and treated for 21 days. Group I (control) was injected with olive oil (vehicle), group II with CsA (15 mg/kg/day), group III with CsA and the HO inhibitor stannous mesoporphyrin (SnMP) (30 micromol/kg/day) and group IV with one dose of the HO inducer cobalt protoporphyrin (CoPP) 5 mg/100 or heme (10 mg/kg body weight), three days after onset of CsA treatment. Renal tissue was processed for light microscopy, and for HO-1 enzyme activity, assay and for Western blot analysis. In CsA-treated rats there was histological evidence of tubulointerstitial scarring. HO-1 was undetectable in CsA-treated rats compared to control while there was no change in HO-2. In animals treated with a combination of CsA and SnMP, HO-1 activity was further reduced. In animals treated with a combination of CsA and CoPP, HO-1 protein levels were partially restored. These observations indicate that downregulation of HO-1 expression by CsA could be one mechanism underlying CsA-induced toxicity. The CsA-induced decrease in HO-1 expression is partial and restorable, and attempts to preserve HO levels may attenuate CsA toxicity.     

Regulation of Wallerian degeneration and nerve growth factor withdrawal-induced pruning of axons of sympathetic neurons by the proteasome and the MEK/Erk pathway

Treatment of transected distal axons of rat sympathetic neurons in compartmented cultures with MG132 (5 microM) and other inhibitors of proteasome activity, preserved axonal mitochondrial function, assessed by Mitotracker-Orange and MTT staining, for at least 24 h. MG132 similarly protected axons from undergoing branch elimination (pruning) in response to local NGF deprivation. Axons protected by MG132 displayed persistent phosphorylation of Erk1/2, and pharmacological inhibition of MEK activity with U0126 (50 microM) restored rapid axonal degeneration. Therefore, the proteasome does not appear to be necessary as a general effector of protein degradation during axonal degeneration. Rather, the proteasome functions in the regulation of signaling pathways that control axonal survival and degeneration. Specifically, the down-regulation of the MEK/Erk pathway by the proteasome plays roles in Wallerian degeneration of severed axons and axonal pruning in response to local NGF deprivation. Identification of the pathways that regulate axonal survival and degeneration will provide possible target sites for pharmacological treatments of neurodegenerative diseases and traumatic injury.     

Role of proteasomes in the formation of neurofilamentous inclusions in spinal motor neurons of aluminum‐treated rabbits

We examined the role of the 20S proteasome in pathologic changes, including abnormal aggregation of phosphorylated neurofilaments, of spinal motor nerve cells from aluminum‐treated rabbits. Immunohistochemistry for the 20S proteasome revealed that many lumbar spinal motor neurons without intracytoplasmic neurofilamentous inclusions or with small inclusions were more intensely stained in aluminum‐treated rabbits than in controls, whereas the immunoreactivity was greatly decreased in some enlarged neurons containing large neurofilamentous inclusions. Proteasome activity in whole spinal cord extracts was significantly increased in aluminum‐treated rabbits compared with controls. Furthermore, Western blot analysis indicated that the 20S proteasome degraded non‐phosphorylated high molecular weight neurofilament (neurofilament‐H) protein in vitro. These results suggest that aluminum does not inhibit 20S proteasome activity, and the 20S proteasome degrades neurofilament‐H protein. We propose that abnormal aggregation of phosphorylated neurofilaments is induced directly by aluminum, and is not induced by the proteasome inhibition in the aluminum‐treated rabbits. Proteasome activation might be involved in intracellular proteolysis, especially in the earlier stages of motor neuron degeneration in aluminum‐treated rabbits.     

Autophagy is activated by proteasomal inhibition and involved in aggresome clearance in cultured astrocytes

A common pathway underlying a variety of neurodegenerative disorders is the aggregation and deposition of misfolded proteins. Proteasomal inhibition has been demonstrated to promote the formation of intracellular inclusions. We have shown before that astrocytes respond to the treatment with the proteasome inhibitor MG‐132 by aggresome formation and cytoskeletal disturbances, but unlike oligodendrocytes do not die by apoptotic cell death and have the capability to recover. This study was undertaken to elucidate if the autophagy‐lysosomal pathway participates in the efficient recovery process in astrocytes and is modulated under conditions of proteasomal inhibition. The data show that the autophagic pathway was stimulated during a 24‐h treatment with the proteasome inhibitor MG‐132 in a time and concentration‐dependent manner. It remained at an elevated level throughout a 24‐h recovery period in the absence of MG‐132 and participates in the aggregate clearing process. In the presence of the specific inhibitor of macroautophagy, 3‐methyladenine, cell viability was impaired, aggregates were not as efficiently removed and HSP25, αB‐crystallin and ubiquitinated proteins remained in the insoluble protein fraction. LC3‐II positive puncta, indicative of autophagosomes, were formed abundantly in the cells after proteasome inhibition and were seen in close association with the aggregates. Hence, the ability of astrocytes to upregulate autophagic degradation might contribute to their resistance against proteasomal stress situations and act as a compensatory mechanism when the proteasome is impaired. © 2010 Wiley‐Liss, Inc.     

Differentiation-dependent sensitivity to cell death induced in the developing retina by inhibitors of the ubiquitin-proteasome proteolytic pathway

The effects of inhibitors of proteasome function were studied in the retina of developing rats. Explants from the retina of neonatal rats at postnatal day (P) 3 or P6 were incubated with various combinations of the proteasome inhibitor carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), the protein synthesis inhibitor anisomycin, or the adenylyl cyclase activator forskolin. MG132 induced cell death in a subset of cells within the neuroblastic (proliferative) layer of the retinal tissue. The cells sensitive to degeneration induced by either MG132 or anisomycin, were birthdated by bromodeoxyuridine injections. This showed that the MG132-sensitive population includes both proliferating cells most likely in their last round of cell division, and postmitotic undifferentiated cells, at a slightly earlier stage than the population, sensitive to anisomycin-induced cell death. The results show that sensitivity to cell death induced by proteasome inhibitors defines a window of development in the transition from the cell cycle to the differentiated state in retinal cells.     

Differential effect of calmodulin antagonists on MG132-induced mitochondrial dysfunction and cell death in PC12 cells

Defects in proteasome function have been suggested to be involved in the pathogenesis of neurodegenerative diseases. We examined the effect of calmodulin antagonists on proteasome inhibitor-induced mitochondrial dysfunction and cell viability loss in undifferentiated PC12 cells. Caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk) and antioxidants attenuated cell death and decrease in GSH contents in PC12 cells treated with 20 microM MG132, a proteasome inhibitor. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) had a differential inhibitory effect on the MG132-induced cell death and GSH depletion depending on concentration with a maximal inhibitory effect at 0.5-1 microM. Addition of trifluoperazine and W-7 reduced the MG132-induced nuclear damage, loss of the mitochondrial transmembrane potential followed by cytochrome c release, formation of reactive oxygen species and elevation of intracellular Ca(2+) levels in PC12 cells. Calmodulin antagonists at 5 microM exhibited a cytotoxic effect on PC12 cells but attenuated the cytotoxicity of MG132. The results suggest that the toxicity of MG132 on PC12 cells is mediated by activation of caspase-8, -9 and -3. Trifluoperazine and W-7 at the concentrations of 0.5-1 microM may attenuate the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering of the intracellular Ca(2+) levels as well as calmodulin inhibition.     

蛋白酶体抑制剂MG132对动脉粥样硬化的影响

目的观察蛋白酶体抑制剂MG132对动脉粥样硬化的影响。方法将新西兰白兔30只随机分成高脂组、MG132(1)组和MG132(2)组。3组兔普通饲料喂养1w后行颈动脉球囊损伤术,术后高脂饲料(含1%胆固醇、3%猪油和15%蛋黄)喂养;MG132(1)组在术后高脂饲料喂养的同时血管局部应用蛋白酶体抑制剂MG132;MG132(2)组在术后高脂饲料喂养4w后血管局部应用蛋白酶体抑制剂MG132;3组均喂养8w后取颈总动脉血管制成病理切片行HE染色。结果高脂组兔的右颈总动脉管壁呈动脉粥样硬化改变。MG132(1)组兔右颈动脉血管组织结构基本正常。MG132(2)组兔的右颈总动脉管腔介于二者之间。结论局部应用蛋白酶体抑制剂MG132能够抑制血管内膜增生及动脉粥样形成,同时可能具有稳定斑块的作用。     

Reciprocal Effects of α-Synuclein Overexpression and Proteasome Inhibition in Neuronal Cells and Tissue

Defects in the 20S/26S proteasome and conformational changes in α-synuclein (α-syn) are implicated in the development of sporadic and familial cases of PD. The objective of this study was to evaluate whether α-syn affects proteolysis by the proteasome and, reciprocally, whether proteasome inhibition affects α-syn solubility and localization. Although α-syn directly inhibited purified 20S proteasomes reversibly in vitro, its overexpression in neuroblastoma (SH-SY5Y and SK-N-BE), embryonic kidney (HEK293) cells, or mouse brain did not affect proteasome activity. Proteasome inhibition with MG132 and epoxomicin in SH-SY5Y cells failed to induce α-syn aggregation, although it increased membrane bound forms of endogenous and overexpressed wild-type, but not mutant, α-syn. Concomitantly this treatment generated cytoplasmic α-syn inclusions devoid of polyubiquitin in a small percentage of cells. The combination of proteasome inhibition with serum deprivation, which induced oxidative stress and autophagy, caused the appearance of high molecular weight α-syn species, such as those found in Lewy bodies. Our data suggest that high concentrations of α-syn do not affect proteasome function in vivo, whereas proteasome inhibition can modify synuclein solubility, most prominently under conditions of cell stress which occur during aging. These results have implications for the convergence of age-related oxidative stress and impaired protein degradation in neurodegeneration.