Proteasome inhibition: an early or late event in nitric oxide-induced neuronal death?

Nitric oxide (NO), ubiquitously expressed in the central nervous system, has been perceived to be a potential neuromodulator. Employing cultured murine primary cortical neurons, NO resulted in an inhibition of the ubiquitin-proteasome system (UPS) with a dose- and time-dependent decrease in cell viability. This is consistent with a previous study that reported a dysfunction of UPS with consequential apoptotic death in macrophage cell with NO treatment. However, it cannot be unclear if the drop in UPS efficiency is directly imposed on by NO. Therefore by using microarray analysis, our study revealed an early down-regulation or non-significant differential expression of genes encoding UPS proteins in NOC-18 (NO donor)-treated neurons as compared to an observed elevation of corresponding gene expression genes in lactacystin (classical proteasome inhibitor)-treated neurons (conducted earlier). Furthermore, time-course analysis of proteasome activity in NOC-18-treated neurons demonstrated a late onset of reduction. This is intriguing as it is well established that in an exclusive proteasome dysfunction-induced cell death, a compensatory feedback mechanism will be activated with an initial and concerted up-regulation of genes encoding proteins involved in UPS as seen when neurons were treated with lactacystin. Thus, it is highly suggestive that NO-triggered neuronal death takes on a different signaling cascade from that of a classical proteasome inhibitor, and that the late reduction of proteasome activity is a downstream event following the activation of apoptotic cellular signaling cascade. In intracellular condition, the proteasome is not NO preferred primary target responsible for the trigger of the cell death machinery. In conclusion, we presented novel findings that shed light into NO-induced cell death signaling cascade, which would be important in understanding the pathogenesis of neurodegenerative disorders such as Parkinson's disease.     

Cell cycle events mediate lactacystin‐induced apoptotic death of neuronal PC12 cells

Dysfunction of the UPS (ubiquitin—proteasome system) has been implicated in dopaminergic neuronal death in PD (Parkinson's disease). Recent studies suggest that unregulated cell cycle events play a key role in neuronal death. In this study, the effects of UPS dysfunction on cell cycle events in neuronal differentiated PC12 cells were analysed using a specific inhibitor of proteasome, lactacystin. Lactacystin induced apoptosis, G₂/M cell cycle arrest and sustained the phosphorylation of the pRB (retinoblastoma protein), the key molecular process of G₁/S transition, in neuronal PC12 cells. Furthermore, inhibition of cell cycle progression protected against lactacystin‐induced cell apoptosis. Finally, we determined that lactacystin activated the ERK signalling pathway. Inhibition of ERK1/2 activation by MEK‐1 inhibitor PD98059 decreased cell cycle aberrant and prevented apoptosis induced by lactacystin. These results indicate that aberrant cell cycle events contribute to apoptotic death induced by UPS dysfunction.     

Estrogen receptor α promotes Cav1.2 ubiquitination and degradation in neuronal cells and in APP/PS1 mice

Cav1.2 is the pore‐forming subunit of L‐type voltage‐gated calcium channel (LTCC) that plays an important role in calcium overload and cell death in Alzheimer's disease. LTCC activity can be regulated by estrogen, a sex steroid hormone that is neuroprotective. Here, we investigated the potential mechanisms in estrogen‐mediated regulation of Cav1.2 protein. We found that in cultured primary neurons, 17β‐estradiol (E2) reduced Cav1.2 protein through estrogen receptor α (ERα). This effect was offset by a proteasomal inhibitor MG132, indicating that ubiquitin–proteasome system was involved. Consistently, the ubiquitin (UB) mutant at lysine 29 (K29R) or the K29‐deubiquitinating enzyme TRAF‐binding protein domain (TRABID) attenuated the effect of ERα on Cav1.2. We further identified that the E3 ligase Mdm2 (double minute 2 protein) and the PEST sequence in Cav1.2 protein played a role, as Mdm2 overexpression and the membrane‐permeable PEST peptides prevented ERα‐mediated Cav1.2 reduction, and Mdm2 overexpression led to the reduced Cav1.2 protein and the increased colocalization of Cav1.2 with ubiquitin in cortical neurons in vivo. In ovariectomized (OVX) APP/PS1 mice, administration of ERα agonist PPT reduced cerebral Cav1.2 protein, increased Cav1.2 ubiquitination, and improved cognitive performances. Taken together, ERα‐induced Cav1.2 degradation involved K29‐linked UB chains and the E3 ligase Mdm2, which might play a role in cognitive improvement in OVX APP/PS1 mice.     

alpha-synuclein is required for the fibrillar nature of ubiquitinated inclusions induced by proteasomal inhibition in primary neurons

Proteasomal dysfunction may underlie certain neuro-degenerative conditions such as Parkinson disease. We have shown that pharmacological inhibition of the proteasome in cultured neuronal cells leads to apoptotic death and formation of cytoplasmic ubiquitinated inclusions. These inclusions stain for alpha-synuclein and assume a fibrillar structure, as assessed by thioflavine S staining, and therefore resemble Lewy bodies. alpha-Synuclein is thought to be a central component of Lewy bodies. Whether alpha-synuclein is required for inclusion formation or apoptotic death has not been formally assessed. The present study examines whether alpha-synuclein deficiency in neurons alters their sensitivity to proteasomal inhibition-induced apoptosis or inclusion formation. Cortical neurons derived from alpha-synuclein-null mice showed a similar sensitivity to death induced by the proteasomal inhibitor lactacystin compared with neurons derived from wild-type mice. Furthermore, the absence of alpha-synuclein did not influence the percentage of lactacystin-treated neurons harboring cytoplasmic ubiquitinated inclusions or alter the solubility of such inclusions. In contrast, however, ubiquitinated inclusions in alpha-synuclein-deficient neurons lacked amyloid-like fibrillization, as determined by thioflavine S staining. This indicates that although alpha-synuclein deficiency does not affect the formation of ubiquitinated inclusions, it does significantly alter their structure. The lack of effect on survival in alpha-synuclein knock-out cultures further suggests that the fibrillar nature of the inclusions does not contribute to neuronal degeneration in this model.     

The octadecaneuropeptide ODN prevents 6‐hydroxydopamine‐induced apoptosis of cerebellar granule neurons through a PKC‐MAPK‐dependent pathway

Oxidative stress, induced by various neurodegenerative diseases, initiates a cascade of events leading to apoptosis, and thus plays a critical role in neuronal injury. In this study, we have investigated the potential neuroprotective effect of the octadecaneuropeptide (ODN) on 6‐hydroxydopamine (6‐OHDA)‐induced oxidative stress and apoptosis in cerebellar granule neurons (CGN). ODN, which is produced by astrocytes, is an endogenous ligand for both central‐type benzodiazepine receptors (CBR) and a metabotropic receptor. Incubation of neurons with subnanomolar concentrations of ODN (10^?18 to 10^?12 M) inhibited 6‐OHDA‐evoked cell death in a concentration‐dependent manner. The effect of ODN on neuronal survival was abrogated by the metabotropic receptor antagonist, cyclo_1–8[DLeu⁵]OP, but not by a CBR antagonist. ODN stimulated polyphosphoinositide turnover and ERK phosphorylation in CGN. The protective effect of ODN against 6‐OHDA toxicity involved the phospholipase C/ERK MAPK transduction cascade. 6‐OHDA treatment induced an accumulation of reactive oxygen species, an increase of the expression of the pro‐apoptotic gene Bax, a drop of the mitochondrial membrane potential and a stimulation of caspase‐3 activity. Exposure of 6‐OHDA‐treated cells to ODN blocked all the deleterious effects of the toxin. Taken together, these data demonstrate for the first time that ODN is a neuroprotective agent that prevents 6‐OHDA‐induced oxidative stress and apoptotic cell death.     

Proteasomal inhibition and apoptosis regulatory changes in human isolated lymphocytes: the synergistic role of dopamine

Abnormal deposition of protein aggregates and increased susceptibility to apoptotic cell death may result from defects in the activity of the ubiquitin-proteasome system (UPS); neurotoxicity related to UPS defects seems to require dopamine to be fully expressed. The aim of this study was to investigate the pro-apoptotic effects caused by proteasomal activity inhibition, as well as the synergistic effect of dopaminergic stimulation in human lymphocytes isolated from healthy volunteers. Cells were incubated 20 h at 37 degrees C, with: (1) lactacystin, (2) increasing concentrations of dopamine or (3) mixture of dopamine and lactacystin. Activities of proteasome 20S and pro-apoptotic caspases-3 and -9 and levels of anti-apoptotic Bcl-2 were measured with fluorimetric or immunochemical assays, while a "DNA diffusion" assay was used to determine the apoptosis. Incubation of lymphocytes with lactacystin, which caused reduction of proteasomal activity, was associated with activation of caspases. A clear, dose-dependent reduction of proteasomal activity was also seen in the presence of increasing doses of dopamine, which was accompanied by a slight dose-dependent increase of caspases activities and Bcl-2 levels. Both effects on proteasome and caspase activities were enhanced when cells were simultaneously exposed to lactacystin and elevated concentrations of dopamine. Apoptosis was detected in all treated samples, but not in controls, without significant differences among the treatment groups; however, the association of dopamine and lactacystin induced a clear reduction in the number of cells being analyzed, pointing to marked cytotoxicity. Our data confirm the potentiation of cytotoxicity related to proteasome inhibition, in the presence of dopaminergic stimulation.     

Endoplasmic reticulum stress-induced cell death mediated by the proteasome

Cells exposed to sustained endoplasmic reticulum (ER) stress undergo programmed cell death and display features typical of apoptosis, such as cysteine aspartyl protease (caspase) activation, cytochrome c release, and DNA fragmentation. Here, we show that the execution of cell death induced by ER stress is mediated via the proteasome. Inhibition of the proteasome by lactacystin prevented ER stress-induced degradation of Bcl-2, release of cytochrome c, processing of effector caspase-3, and exposure of phosphatidylserine. Owing to the ability of lactacystin to inhibit cytochrome c release, we propose that the pro-apoptotic activity of the proteasome lies upstream of mitochondrial activation. Thus, the proteasome serves as a principal mediator of ER stress-induced cell death in this system.     

Resistin protects against 6‐hydroxydopamine‐induced cell death in dopaminergic‐like MES23.5 cells

Resistin is originally reported as an adipose tissue‐specific hormone and is thought to represent a link between obesity and insulin‐resistant diabetes. Adipokines exert energy‐regulation and has been reported to have neuroprotective effect like leptin, adiponectin, and ghrelin. However, the role of resistin in neuroprotective effect has not been explored. 6‐hydroxydopamine (6‐OHDA), one of the most investigated Parkinson's disease neurotoxins, is widely used to study mechanisms of cell death in dopaminergic neurons. In the present study, our results show that treatment of resistin protects 6‐OHDA‐induced cell death in dopaminergic‐like MES23.5 cells. Resistin also antagonizes 6‐OHDA‐induced apoptotic cell death measured by fluorescence‐activated cell sorter (FACS) analysis and Hochest 33342 staining. Furthermore, treatment of resistin also dramatically reduces 6‐OHDA‐mediated ROS production and mitochondria transmembrane potential dissipation. Moreover, expression of 6‐OHDA‐induced apoptotic markers, such as Bcl‐2 degradation, Bax expression, PARP degradation and caspase 3 activity increase, are all attenuated by resistin treatment. Our results also show that resistin induces up‐regulation of heat shock protein (Hsp) 32 (heme oxygenase‐1, HO‐1) and Hsc (heat shock cognate) 70. The protective effect of resistin on 6‐OHDA‐induced cell death is abolished by HO‐1 inhibitor zinc protoporphyrin IX and HSP inhibitor KNK437. These results suggest the neuroprotective effects of resistin against 6‐OHDA‐induced cell death with the underlying mechanisms of inhibiting oxidative stress and apoptosis. Therefore, we suggest that resistin may provide a useful therapeutic strategy for neurodegenerative diseases such as Parkinson's disease. J. Cell. Physiol. 228: 563–571, 2013. © 2012 Wiley Periodicals, Inc.     

Proteasome inhibition by lactacystin in primary neuronal cells induces both potentially neuroprotective and pro-apoptotic transcriptional responses: a microarray analysis

Although inhibition of the ubiquitin proteasome system has been postulated to play a key role in the pathogenesis of neurodegenerative diseases, studies have also shown that proteasome inhibition can induce increased expression of neuroprotective heat-shock proteins (HSPs). The global gene expression of primary neurons in response to treatment with the proteasome inhibitor lactacystin was studied to identify the widest range of possible pathways affected. Our results showed changes in mRNA abundance, both at different time points after lactacystin treatment and at different lactacystin concentrations. Genes that were differentially up-regulated at the early time point but not when most cells were undergoing apoptosis might be involved in an attempt to reverse proteasome inhibitor-mediated apoptosis and include HSP70, HSP22 and cell cycle inhibitors. The up-regulation of HSP70 and HSP22 appeared specific towards proteasome inhibitor-mediated cell death. Overexpression of HSP22 was found to protect against proteasome inhibitor-mediated loss of viability by up to 25%. Genes involved in oxidative stress and the inflammatory response were also up-regulated. These data suggest an initial neuroprotective pathway involving HSPs, antioxidants and cell cycle inhibitors, followed by a pro-apoptotic response possibly mediated by inflammation, oxidative stress and aberrant activation of cell cycle proteins.     

Over‐expression of HSP70 attenuates caspase‐dependent and caspase‐independent pathways and inhibits neuronal apoptosis

HSP70 is a member of the family of heat‐shock proteins that are known to be up‐regulated in neurons following injury and/or stress. HSP70 over‐expression has been linked to neuroprotection in multiple models, including neurodegenerative disorders. In contrast, less is known about the neuroprotective effects of HSP70 in neuronal apoptosis and with regard to modulation of programmed cell death (PCD) mechanisms in neurons. We examined the effects of HSP70 over‐expression by transfection with HSP70‐expression plasmids in primary cortical neurons and the SH‐SY5Y neuronal cell line using four independent models of apoptosis: etoposide, staurosporine, C2‐ceramide, and β‐Amyloid. In these apoptotic models, neurons transfected with the HSP70 construct showed significantly reduced induction of nuclear apoptotic markers and/or cell death. Furthermore, we demonstrated that HSP70 binds and potentially inactivates Apoptotic protease‐activating factor 1, as well as apoptosis‐inducing factor, key molecules involved in development of caspase‐dependent and caspase‐independent PCD, respectively. Markers of caspase‐dependent PCD, including active caspase‐3, caspase‐9, and cleaved PARP were attenuated in neurons over‐expressing HSP70. These data indicate that HSP70 protects against neuronal apoptosis and suggest that these effects reflect, at least in part, to inhibition of both caspase‐dependent and caspase‐independent PCD pathways.     

Estrogen‐regulated developmental neuronal apoptosis is determined by estrogen receptor subtype and the Fas/Fas ligand system

Adult sexual dimorphism in neuronal cell number is controlled by estrogen exposure during a tightly defined period of rat brain development. The mechanisms of estrogen's effect are unknown; one possibility is regulation of programmed cell death (apoptosis). In this study we have shown that estradiol can function as a neuroprotective agent or an inducer of apoptosis, depending on the estrogen receptor‐subtype present in the cell. Thus, ERα has a neuroprotective effect, while ERβ mediates the induction of apoptosis in neuronal cells. Moreover, we show that estrogen‐induced apoptosis through ER‐β requires the expression of Fas‐ and Fas ligand (FasL) proteins, since the absence of FasL in neurons prevents this effect. Furthermore, we demonstrate that microglia‐secreted products induce the expression of FasL necessary to mediate estradiol–ERβ apoptotic effect. These findings may explain the dichotomous effect of fetal estradiol on the adult neuronal number. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 64–78, 2000     

Prostaglandin J2 alters pro-survival and pro-death gene expression patterns and 26 S proteasome assembly in human neuroblastoma cells

Many neurodegenerative disorders are characterized by two pathological hallmarks: progressive loss of neurons and occurrence of inclusion bodies containing ubiquitinated proteins. Inflammation may be critical to neurodegeneration associated with ubiquitin-protein aggregates. We previously showed that prostaglandin J2 (PGJ2), one of the endogenous products of inflammation, induces neuronal death and the accumulation of ubiquitinated proteins into distinct aggregates. We now report that temporal microarray analysis of human neuroblastoma SK-N-SH revealed that PGJ2 triggered a "repair" response including increased expression of heat shock, protein folding, stress response, detoxification and cysteine metabolism genes. PGJ2 also decreased expression of cell growth/maintenance genes and increased expression of apoptotic genes. Over time pro-death responses prevailed over pro-survival responses, leading to cellular demise. Furthermore, PGJ2 increased the expression of proteasome and other ubiquitin-proteasome pathway genes. This increase failed to overcome PGJ2 inhibition of 26 S proteasome activity. Ubiquitinated proteins are degraded by the 26 S proteasome, shown here to be the most active proteasomal form in SK-N-SH cells. We demonstrate that PGJ2 impairs 26 S proteasome assembly, which is an ATP-dependent process. PGJ2 perturbs mitochondrial function, which could be critical to the observed 26 S proteasome disassembly, suggesting a cross-talk between mitochondrial and proteasomal impairment. In conclusion neurotoxic products of inflammation, such as PGJ2, may play a role in neurodegenerative disorders associated with the aggregation of ubiquitinated proteins by impairing 26 S proteasome activity and inducing a chain of events that culminates in neuronal cell death. Temporal characterization of these events is relevant to understanding the underlying mechanisms and to identifying potential early biomarkers.     

Application of proteasomal inhibitors to mouse sympathetic neurons activates the intrinsic apoptotic pathway

Proteasomal dysfunction may play a role in a number of neurodegenerative conditions, and in particular Parkinson's disease (PD) and related Lewy body (LB) diseases. Application of proteasomal inhibitors to neuronal cell culture systems is associated with survival-promoting effects or with cell death depending on the model system. We have applied pharmacological proteasomal inhibitors to cultured neonatal mouse sympathetic neurons in order to investigate whether these catecholaminergic neurons, which are affected in PD, are sensitive to proteasomal inhibition and, if so, which cell death pathway is activated. We report here that proteasomal inhibition leads to apoptotic death of mouse sympathetic neurons. This death is accompanied by caspase 3 activation and cytochrome c release from the mitochondria and is abrogated by caspase inhibition. Bax deletion prevented both cytochrome c release and caspase 3 activation, and also provided complete protection against proteasomal inhibition-induced death. Bcl-2 overexpression achieved a similar survival-promoting effect. There was no change in Bax levels following proteasomal inhibition, suggesting that Bax itself is not regulated by the proteasome in this cell culture system, and that a primary increase in Bax is unlikely to account for death. In contrast, levels of the BH3-only protein, Bim, increased with proteasomal inhibition. We conclude that proteasomal inhibition of mouse sympathetic neurons activates the intrinsic apoptotic pathway involving bcl-2 family members and the mitochondria.     

Proteasome inhibitors suppress formation of polyglutamine-induced nuclear inclusions in cultured postmitotic neurons

At least nine neurodegenerative disorders are caused by expansion of polyglutamine repeats in various genes. This expansion induces the formation of nuclear inclusions (NI) within various cell types. In this study, we developed a model for polyglutamine diseases using primary cultures of sympathetic neurons from the superior cervical ganglia of prenatal rat pups. Transfection with a plasmid encoding 127 glutamine repeats causes NI to develop in approximately 70% of the sympathetic neurons within 6 days. In addition, it causes somatic atrophy and inhibits dendritic growth. The NIs contain ubiquitinated proteins and sequester the molecular chaperone heat shock protein 70 (Hsp70). We found that two specific proteasome inhibitors, lactacystin and CEP1612, suppress thezformation of polyglutamine-induced NI. In addition, lactacystin treatment induced the removal of preexisting NI. Western blotting and immunocytochemistry revealed that lactacystin and CEP1612 strongly induce the expression of Hsp70, whereas less specific proteasome inhibitor such as N-acetyl-Leu-Leu-Norleucinal does not. Coexpression of 127 glutamines with a plasmid encoding wild-type Hsp70 gene resulted in a marked reduction of the percentage of neurons containing NI. In addition, transfection with plasmids encoding mutant Hsp70 blocked the effects of lactacystin. These findings further implicate Hsp70 as a neuroprotective molecule and they suggest the potential utility of certain proteasome inhibitors in the treatment of polyglutamine diseases.     

Parkin deficiency increases the resistance of midbrain neurons and glia to mild proteasome inhibition: the role of autophagy and glutathione homeostasis

Parkin mutations in humans produce parkinsonism whose pathogenesis is related to impaired protein degradation, increased free radicals and abnormal neurotransmitter release. In this study, we have investigated whether partial proteasomal inhibition by epoxomicin, an ubiquitin proteasomal system (UPS) irreversible inhibitor, further aggravates the cellular effects of parkin suppression in midbrain neurons and glia. We observed that parkin null (PK‐KO) midbrain neuronal cultures are resistant to epoxomicin‐induced cell death. This resistance is due to increased GSH and DJ‐1 protein levels in PK‐KO mice. The treatment with epoxomicin increases, in wild type (WT) cultures, the pro‐apoptotic Bax/Bcl‐2 ratio, the phosphorylation of tau, and the levels of chaperones heat‐shock protein 70 and C‐terminal Hsc‐interacting protein, but none of these effects took place in epoxomicin‐treated PK‐KO cultures. Poly‐ubiquitinated proteins increased more in WT than in PK‐KO‐treated neuronal cultures. Parkin accumulated in WT neuronal cultures treated with epoxomicin. Markers of autophagy, such as LC3II/I, were increased in naïve PK‐KO cultures, and further increased after treatment with epoxomicin, implying that the blockade of the proteasome in PK‐KO neurons triggers the enhancement of autophagy. The treatment with l ‐buthionine‐S,R‐sulfoximine and the inhibition of autophagy, however, reverted the increase resistance to epoxomicin of the PK‐KO cultures. We also found that PK‐KO glial cells, stressed by growth in defined medium and depleted of GSH, were more susceptible to epoxomicin induced cell death than WT glia treated similarly. This susceptibility was linked to reduced GSH levels and less heat‐shock protein 70 response, and to activation of p‐serine/threonine kinase protein signaling pathway as well as to increased poly‐ubiquitinated proteins. These data suggest that mild UPS inhibition is compensated by other mechanisms in PK‐KO midbrain neurons. However the depletion of GSH, as happens in stressed glia, suppresses the protection against UPS inhibition‐induced cell death. Furthermore, GSH inhibition regulated differentially UPS activity and in old PK‐KO mice, which have depletion of GSH, UPS activity is decreased in comparison with that of old‐WT.     

Cancer‐upregulated gene 2 (CUG2) overexpression induces apoptosis in SKOV‐3 cells

Cancer‐upregulated gene 2 (CUG2) was originally identified as a potential oncogene commonly up‐regulated in various human cancers. Recently, CUG2 was also identified as a new member of a centromere protein complex, important in the formation of a functional kinetochore complex. Presently, we report the pro‐apoptotic effect of CUG2 when this gene was overexpressed in the SKOV‐3 human ovarian cancer cell line. Apoptotic cell death mediated by CUG2 overexpression was independently demonstrated using cell viability determination, flow cytometry analysis, chromosome fragmentation assay, and the cleavage of the death substrate poly(ADP‐ribose) polymerase. Moreover, activation of caspase‐3 and ‐8 and the cytoplasmic translocation of mitochondrial cytochrome c were evident upon CUG2 expression. Apoptotic cell death was also observed during early development of zebrafish when CUG2 was overexpressed in zebrafish embryo. We propose that high expression of CUG2 induces apoptotic cell death. Copyright © 2010 John Wiley & Sons, Ltd.