alpha-synuclein and lactacystin

Histone deacetylase 6 (HDAC6) has been shown to control major cell response pathways to the cytotoxic ubiquitinated aggregates in some protein aggregation diseases. However, it is not well known whether HDAC6 affects the aggregation process of α-synuclein (α-syn) in Parkinson's disease (PD). Previously, we demonstrated that HDAC6 inhibition exacerbated the nigrostriatal dopamine neurodegeneration and up-regulated α-syn oligomers in a heat shock protein 90 (Hsp90)-dependent manner in PD mouse model. Here, we further showed that HDAC6 overexpression partly improved the behavior deficits of the PD model and alleviated the nigrostriatal dopamine (DA) neurons injury. Furthermore, HDAC6 was found to regulate α-syn oligomers levels through activation of chaperone-mediated autophagy (CMA). During this process, Hsp90 deacetylation mediated the crosstalk between HDAC6 and lysosome-associated membrane protein type 2A. Liquid chromatography-tandem mass spectrometry and mutational analysis showed that acetylation status Hsp90 at the K489 site was a strong determinant for HDAC6-induced CMA activation, α-syn oligomers levels, and cell survival in the cell model of PD. Therefore, our findings uncovered the mechanism of HDAC6 in the PD model that HDAC6 regulated α-syn oligomers levels and DA neurons survival partly through modulating CMA, and Hsp90 deacetylation at the K489 site mediated the crosstalk between HDAC6 and CMA. HDAC6 and its downstream effectors appear as key modulators of the cytotoxic α-syn aggregates, which deserve further investigations to evaluate their values as potential therapeutic targets in PD.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Chaperone-Mediated Autophagy; Histone Deacetylase 6; HSP90 Heat-Shock Proteins; Humans; Male; Mice; Mice, Inbred C57BL; Parkinsonian Disorders; Protein Aggregates

Ubiquitin proteasome system (UPS) impairment, excessive cellular oxidative stress, and iron dyshomeostasis are key to substantia nigra dopaminergic neuronal degeneration in Parkinson's disease (PD); however, a link between these features remains unconfirmed. Using the proteasome inhibitor lactacystin we confirm that nigral injury via UPS impairment disrupts iron homeostasis, in turn increasing oxidative stress and promoting protein aggregation. We demonstrate the neuroprotective potential of two novel 1-hydroxy-2(1H)-pyridinone (1,2-HOPO) iron chelators, compounds C6 and C9, against lactacystin-induced cell death. We demonstrate that this cellular preservation relates to the compounds' iron chelating capabilities and subsequent reduced capacity of iron to form reactive oxygen species (ROS), where we also show that the ligands act as antioxidant agents. Our results also demonstrate the ability of C6 and C9 to reduce intracellular lactacystin-induced α-synuclein burden. Stability constant measurements confirmed a high affinity of C6 and C9 for Fe

Topics: Acetylcysteine; alpha-Synuclein; Animals; Dopamine; Dopaminergic Neurons; Humans; Iron; Iron Chelating Agents; Neuroprotective Agents; Parkinson Disease; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Substantia Nigra; Ubiquitin

Lewy bodies, the histopathological hallmarks of Parkinson's disease (PD), contain insoluble and aggregated α-synuclein (aSyn) and many other proteins, proposing a role for failure in protein degradation system in the PD pathogenesis. Proteasomal dysfunction has indeed been linked to PD and aSyn oligomers have been shown to inhibit proteasomes and autophagy. Our recent studies have shown that inhibitors of prolyl oligopeptidase (PREP) can prevent the aggregation and enhance the clearance of accumulated aSyn, and therefore, we wanted to study if PREP inhibition can overcome the aSyn aggregation and toxicity induced by lactacystin, a proteasomal inhibitor. The cells overexpressing human A30P or A53T mutated aSyn were incubated with lactacystin and a PREP inhibitor, KYP-2047, for 48h. Theafter, the cells were fractioned, and the effects of lactacystin with/without 1μM KYP-2047 on aSyn aggregation and ubiquitin accumulation, cell viability and on autophagic markers (p62, Beclin1 and LC3BII) were studied. We found that KYP-2047 attenuated lactacystin-induced cell death in mutant aSyn overexpressing cells but not in non-overexpressing control cells. KYP-2047 reduced significantly SDS-insoluble high-molecular-weight aSyn oligomers that were in line with the cell viability results. In addition, significant reduction in protein accumulation marker, p62, was seen in SDS fraction while LC3BII, a marker for autophagosome formation, was increased, indicating to enhanced autophagy. Our results further streghten the possibilities for PREP inhibitors as a potential drug therapy against synucleinopathies and other protein aggregating diseases.

Topics: Acetylcysteine; alpha-Synuclein; Autophagy; Cell Line, Tumor; Cell Survival; Humans; Mutation; Proline; Prolyl Oligopeptidases; Proteasome Inhibitors; Protein Aggregates; Serine Endopeptidases; Serine Proteinase Inhibitors

We previously reported a loss of cholinergic neurons within the pedunculopontine tegmental nucleus (PPTg) in rats that had been intra-nigrally lesioned with the proteasomal inhibitor lactacystin, with levels of neuronal loss corresponding to that seen in the post-mortem pedunculopontine nucleus (PPN) of advanced Parkinson's disease (PD) patients. Here we reveal lower expression values of the acetylcholine synthesising enzyme, choline acetyltransferase, within the remaining PPTg cholinergic neurons of lesioned rats compared to sham controls. We further characterise this animal model entailing dopaminergic- and non-dopaminergic neurodegeneration by reporting on stereological counts of non-cholinergic neurons, to determine whether the toxin is neuro-type specific. Cell counts between lesioned and sham-lesioned rats were analysed in terms of the topological distribution pattern across the rostro-caudal extent of the PPTg. The study also reports somatic hypotrophy in the remaining non-cholinergic neurons, particularly on the side closest to the nigral lesion. The cytotoxicity affecting the PPTg in this rat model of PD involves overexpression and accumulation of alpha-synuclein (αSYN), affecting cholinergic and non-cholinergic neurons as well as microglia on the lesioned hemispheric side. We ascertained that microglia within the PPTg become fully activated due to the extensive neuronal damage and neuronal death resulting from a lactacystin nigral lesion, displaying a distinct rostro-caudal distribution profile which correlates with PPTg neuronal loss, with the added implication that lactacystin-induced αSYN aggregation might trigger neuronophagia for promoting PPTg cell loss. The data provide critical insights into the mechanisms underlying the lactacystin rat model of PD, for studying the PPTg in health and when modelling neurodegenerative disease.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Cell Count; Choline O-Acetyltransferase; Cholinergic Neurons; Disease Models, Animal; Dopaminergic Neurons; Male; Microglia; Neurons; Parkinson Disease; Parkinsonian Disorders; Pars Compacta; Pedunculopontine Tegmental Nucleus; Protein Aggregation, Pathological; Rats; Rats, Sprague-Dawley; Tyrosine 3-Monooxygenase

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide and recent studies implicate a central role for ubiquitin-proteasome system (UPS) impairment in the etiopathogenesis of PD. To explore the possible role of UPS dysfunction in PD and the proteins involved, PC12 cells were treated with 10μM lactacystin, a 20S proteasome inhibitor, for 24h. Lactacystin induced cell death and α-synuclein-positive inclusions in cytoplasm. Following two-dimensional difference in-gel electrophoresis (2-D DIGE) which was used to separate the cellular proteins, the proteins that were significantly altered were analyzed and identified. Proteomic study identified 6 differentially expressed proteins between lactacystin-treated and control cells in this study. Four proteins (heat shock 70kDa protein 8, 78kDa glucose-regulated protein, serine proteinase inhibitor clade B member 6 and aldehyde reductase) were increased and 2 proteins (peripherin and tyrosine hydroxylase) were decreased following proteasomal inhibition. The results revealed that PC12 cells treated with 10μM lactacystin for 24h could be used as a cellular model of PD. The proteins identified in the present indicate not only the damage of proteasomal inhibition to the cells but also the possible responses of the cells. These data show that proteomic study may provide information relevant to biological basis for PD and potential new treatment targets.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Cell Death; Cytoplasm; Heat-Shock Proteins; Inclusion Bodies; Parkinson Disease; PC12 Cells; Proteasome Inhibitors; Proteome; Rats; Tyrosine 3-Monooxygenase

This study has shown that purified recombinant human α-synuclein (20 μM) causes membrane depolarization and loss of phosphorylation capacity of isolated purified rat brain mitochondria by activating permeability transition pore complex. In intact SHSY5Y (human neuroblastoma cell line) cells, lactacystin (5 μM), a proteasomal inhibitor, causes an accumulation of α-synuclein with concomitant mitochondrial dysfunction and cell death. The effects of lactacystin on intact SHSY5Y cells are, however, prevented by knocking down α-synuclein expression by specific siRNA. Furthermore, in wild-type (non-transfected) SHSY5Y cells, the effects of lactacystin on mitochondrial function and cell viability are also prevented by cyclosporin A (1 μM) which blocks the activity of the mitochondrial permeability transition pore. Likewise, in wild-type SHSY5Y cells, typical mitochondrial poison like antimycin A (50 nM) produces loss of cell viability comparable to that of lactacystin (5 μM). These data, in combination with those from isolated brain mitochondria, strongly suggest that intracellularly accumulated α-synuclein can interact with mitochondria in intact SHSY5Y cells causing dysfunction of the organelle which drives the cell death under our experimental conditions. The results have clear implications in the pathogenesis of sporadic Parkinson's disease. α-Synuclein is shown to cause mitochondrial impairment through interaction with permeability transition pore complex in isolated preparations. Intracellular accumulation of α-synuclein in SHSY5Y cells following proteasomal inhibition leads to mitochondrial impairment and cell death which could be prevented by knocking down α-synuclein gene. The results link mitochondrial dysfunction and α-synuclein accumulation, two key pathogenic mechanisms of Parkinson's disease, in a common damage pathway.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Cell Death; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Parkinson Disease; Rats, Wistar

A dysfunctional ubiquitin proteasome system may be a mediating factor of disease progression in Lewy body dementia (LBD). The effects of proteasome inhibition using lactacystin and epoxomicin in primary neuronal culture were studied to assess the validity of this model to reflect the cortical pathology of LBD. Treatment of primary cortical neurons with 5 μM lactacystin for 24 h led to a 38 % reduction in the levels of β-III-tubulin (p < 0.05), a 48 % reduction in the levels of synaptophysin (p < 0.05) and a 74 % reduction in the levels of drebrin (p < 0.01), when compared to controls. Results for epoxomicin were similar. The loss of neuronal protein occurred prior to any loss of mitochondrial activity or cell death. The results are reflective of the loss of synapses and the synaptic changes observed in LBD, which may be an early event in the neurodegeneration of LBD. The similarities with the pathological changes in LBD highlight the possibility that this model can potentially provide a platform to test novel treatments.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Blotting, Western; Cells, Cultured; Cysteine Proteinase Inhibitors; Immunohistochemistry; Lewy Body Disease; Neurons; Neuropeptides; Oligopeptides; Proteasome Endopeptidase Complex; Rats; Rats, Wistar; Synapses; Synaptophysin; Tubulin; Ubiquitination

Predominantly, magnetic resonance imaging (MRI) studies in animal models of Parkinson's disease (PD) have focused on alterations in T2 water 1H relaxation or 1H MR spectroscopy (MRS), whilst potential morphological changes and their relationship to histological or behavioural outcomes have not been appropriately addressed. Therefore, in this study we have utilised MRI to scan in vivo brains from rodents bearing a nigrostriatal lesion induced by intranigral injection of the proteasome inhibitor lactacystin.. Lactacystin induced parkinsonian-like behaviour, characterised by impaired contralateral forelimb grip strength and increased contralateral circling in response to apomorphine. T2-weighted MRI, 3-weeks post-lesion, revealed significant morphological changes in PD-relevant brain areas, including the striatum and ventral midbrain in addition to a decrease in T2 water 1H relaxation in the substantia nigra (SN), but not the striatum. Post-mortem histological analyses revealed extensive dopaminergic neuronal degeneration and alpha-synuclein aggregation in the SN. However, extensive neuronal loss could also be observed in extra-nigral areas, suggesting non-specific toxicity of lactacystin. Iron accumulation could also be observed throughout the midbrain reflecting changes in T2. Importantly, morphological, but not T2 relaxivity changes, were significantly associated with both behavioural and histological outcomes in this model.. A pattern of morphological changes in lactacystin-lesioned animals has been identified, as well as alterations in nigral T2 relaxivity. The significant relationship of morphological changes with behavioural and histological outcomes in this model raises the possibility that these may be useful non-invasive surrogate markers of nigrostriatal degeneration in vivo.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Apomorphine; Corpus Striatum; Cysteine Proteinase Inhibitors; Disease Models, Animal; Dopamine; Dopamine Agonists; Iron; Magnetic Resonance Imaging; Male; Mesencephalon; Motor Activity; Neurons; Parkinsonian Disorders; Random Allocation; Rats; Rats, Sprague-Dawley; Substantia Nigra

α-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t(½) = 54.9 ± 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Cell Line, Tumor; Cerebral Cortex; Cycloheximide; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Lewy Bodies; Neurons; Okadaic Acid; Parkinson Disease; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Phosphatase 1; Protein Phosphatase 2; Protein Synthesis Inhibitors; Rabbits; Ubiquitin

We were interested in studying nigral degeneration with inclusion body formation and behavioral changes in rats after proteasomal inhibition.. Observation of progressive behavioral and pathological changes in rats following a unilateral nigral injection of lactacystin, a selective proteasome inhibitor.. After administration at a concentration of 10 microg (2 microl) of lactacystin, when tyrosine hydroxylase (TH) immunostaining decreased gradually in the substantia nigra pars compacta (SNc) and corpus striatum, alpha-synuclein-immunopositive inclusion appeared extensively in the surviving neurons. We also observed the degeneration of diverse cellular organelles by transmission electron microscopy. The effect of cellular organelle degeneration on behavior, a clinical index, was striking and was statistically significant. Over the 3 weeks following the administration of lactacystin, a highly significant decrease in TH immunostaining was observed and alpha-synuclein-immunopositive inclusions gradually appeared. Interestingly, there was a strong correlation in behavioral changes and the increase in alpha-synuclein-immunopositive inclusions whereas the decrease in TH immunostaining did not seem to induce any behavioral changes.. Our results reveal that unilateral nigral proteasome inhibition induces degeneration in the SNc and corpus striatum as well as behavioral changes demonstrating strong time dependence. Behavioral changes were driven by the formation of alpha-synuclein inclusions, but not by decreased TH neurons.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Behavior, Animal; Corpus Striatum; Cysteine Proteinase Inhibitors; Disease Models, Animal; Hypokinesia; Inclusion Bodies; Male; Microscopy, Electron, Transmission; Motor Activity; Neurodegenerative Diseases; Neurons; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley; Substantia Nigra; Tyrosine 3-Monooxygenase

Protein accumulation and aggregation are signatures of several major neurodegenerative diseases. Proteasomal- and lysosomal-mediated protein degradation pathways are the two major pathways for intracellular protein degradation. Cross-regulation between these two pathways may be important for protein homeostasis. Pharmacological inhibition of proteasomal activities has been shown to up-regulate the levels of lysosomal enzymes. To determine whether the reverse regulatory mechanism also occurs in the cell, we investigated the effects of inhibition of lysosomal function on proteasomal activities. We found that rather than up-regulating proteasomal activities in response to lysosomal disruptors, reduced lysosomal function reduces proteasomal functions, indicating a lack of compensatory up-regulation of proteasomal functions. Inhibition of lysosomal or proteasomal activities led to higher levels of chaperone heat shock cognate protein Hsc70, suggesting an attempt to compensate protein degradation deficiency by enhancing chaperone-mediated autophagy.

Topics: Acetylcysteine; alpha-Synuclein; Ammonium Chloride; Autophagy; Blotting, Western; Cell Line, Tumor; Chloroquine; Enzyme Inhibitors; HSC70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Hydrogen-Ion Concentration; Lysosomes; Neurons; Proteasome Endopeptidase Complex

α-synuclein (α-SN) has been postulated to play a pivotal role in the pathogenesis of Parkinson's disease (PD). However, the physiological functions of α-SN and the molecular and cellular mechanisms underlying neuronal loss remain unclear. Recent studies suggest that α-SN plays dual roles of neuroprotection and neurotoxicity depending on its concentration or level of expression. In the present study, we explored the potential mechanisms for α-SN to regulate neuronal survival. α-SN at different concentrations (0.1 to 40 mumol/L) with or without 50 mumol/L 6-hydroxydopamine (6-OHDA) were added into the culture medium of the SH-SY5Y dopaminergic neural cells. The cell viability was measured on post-treatment day 1, 2 and 3. The activity of proteasome inhibited by α-SN was tested by a proteasome activity assay system after 2 h of α-SN treatment. According to the activity of proteasome inhibited by α-SN, the correlative dose of proteasome inhibitor--lactacystin (10 nmol/L to 5 mumol/L) with or without 50 mumol/L 6-OHDA were used and the cell viability was assayed on post-treatment day 1, 2 and 3. The results showed that α-SN played dual roles of neuroprotection and neurotoxicity depending on its concentration. At low concentration (0.1 to 5 mumol/L), α-SN promoted the proliferation and protected neurons against the neurotoxicity of 6-OHDA; in contrast, at high concentration (10 to 40 mumol/L), α-SN possessed cytotoxicity. The results of lactacystin treatment implied that the dual roles of α-SN were related to the moderate and strong inhibition of proteasome activity. The MEK1/2 specific inhibitor PD98059 completely blocked the protection of both α-SN and lactacystin, suggesting that MAPK pathway might be involved in the neuroprotection of α-SN.

Topics: Acetylcysteine; alpha-Synuclein; Apoptosis; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Humans; Neurons; Neuroprotective Agents; Neurotoxins; Oxidopamine; Parkinson Disease

Intracellular toxic effects of the dequalinium-induced protofibrils of alpha-synuclein have been investigated with the yeast system expressing alpha-synuclein-GFP fusion protein in single copy, which appears in the green halo around the plasma membrane. Intracellular responses of the green fluorescent protein were analyzed as the cells were treated with dequalinium (DQ) and lactacystin. Yeast cells expressing alpha-synuclein-GFP were susceptible to both compounds in alpha-synuclein-dependent manner. Upon DQ treatment, the green halo became smeared throughout the cytoplasm while lactacystin induced a few discrete green dots, reflecting intracellular formation of the protofibrils and the protein inclusions, respectively. The DQ-treated yeast cells were intensely stained with the nucleic acid stains of cell-permeable Hoechst 33342 and cell-impermeable propidium imidione, indicating that nucleus has been disrupted in addition to plasma membrane destabilization. Those DQ-treated yeast cells, however, still contained active mitochondria identified with MitoTracker Red. Therefore, the DQ-induced protofibrillar state of alpha-synuclein-GFP has been suggested to cause the nuclear damage either independently or in combination with the membrane destabilization without affecting mitochondria.

Topics: Acetylcysteine; alpha-Synuclein; Anti-Infective Agents, Local; Benzimidazoles; Blotting, Western; Dequalinium; Fluorescent Dyes; Green Fluorescent Proteins; Organic Chemicals; Plasmids; Proteasome Inhibitors; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Yeasts

Dementia with Lewy bodies (DLB) accounts for 15-20% of the millions of people worldwide with dementia. In the current work we investigate the association between proteasome dysfunction and the development of cortical Lewy body pathology. Analysis of post-mortem cortical tissue indicated levels of the alpha-subunit of the 20S proteasome were significantly reduced in DLB cortex, but not Alzheimer's, in comparison to control and this reduction correlated with both the severity and duration of dementia. Application of proteasome inhibitors to rodent cortical primary neurones in vitro and by direct injection onto rodent cholinergic forebrain neurons in vivo gave rise to dose dependent neuronal death and in rodent cortex -- marked cholinergic deficits accompanied by the accumulation of inclusions that stained positive for alpha-synuclein and ubiquitin. These findings suggest that proteasomal abnormalities are present within cortical Lewy body disease and the experimental inhibition of proteasomal function mirrors the neuropathological changes seen within the disorder.

Topics: Acetylcholine; Acetylcysteine; Aged; Aged, 80 and over; alpha-Synuclein; Animals; Basal Nucleus of Meynert; Cell Death; Cells, Cultured; Cerebral Cortex; Cholinergic Fibers; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Humans; Lewy Bodies; Lewy Body Disease; Male; Nerve Degeneration; Nerve Tissue Proteins; Neural Pathways; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley; Ubiquitin

Dysfunction of the ubiquitin-dependent protein degradation system, either at the level of the proteasome itself, or at the level of ubiquitination, may play a role in the pathogenesis of Parkinson's disease (PD) and other related neurodegenerative disorders. We have employed a cellular model of this dysfunction in which lactacystin or epoxomicin, selective pharmacological inhibitors of the proteasome, are applied to primary cultures of embryonic rat ventral midbrain. Proteasomal inhibition with either agent led to apoptotic death specifically within phenotypically defined tyrosine hydroxylase (TH)-positive dopaminergic neurons, with little or no apoptotic death induced in GABAergic neurons. Inhibition of the proteasome also led to the formation of ubiquitin and alpha-synuclein-positive cytoplasmic inclusions in TH-positive and TH-negative neurons. Inclusions were observed in viable as well as apoptotic neurons, and required new or ongoing transcription. Tyrosine hydroxylase immunolabeling was often present within the inclusions. Such mislocalization may lead to dysfunction of dopamine biosynthesis. Interestingly, dopaminergic neurons, unlike other neurons within these cultures or cultured cortical neurons, failed to induce the chaperone Hsp70 in response to proteasomal inhibition. This failure may explain in part the increased sensitivity of these neurons to proteasomal inhibitors.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Apoptosis; Cells, Cultured; Dopamine; HSP70 Heat-Shock Proteins; Inclusion Bodies; Mesencephalon; Nerve Tissue Proteins; Neurons; Oligopeptides; Proteasome Inhibitors; Rats; Synucleins; Transcription, Genetic; Ubiquitin; Up-Regulation

Parkinson's disease is characterized by dopaminergic neuronal death and the presence of Lewy bodies. alpha-Synuclein is a major component of Lewy bodies, but the process of its accumulation and its relationship to dopaminergic neuronal death has not been resolved. Although the pathogenesis has not been clarified, mitochondrial complex I is suppressed, and caspase-3 is activated in the affected midbrain. Here we report that a combination of 1-methyl-4-phenylpyridinium ion (MPP(+)) or rotenone and proteasome inhibition causes the appearance of alpha-synuclein-positive inclusion bodies. Unexpectedly, however, proteasome inhibition blocked MPP(+)- or rotenone-induced dopaminergic neuronal death. MPP(+) elevated proteasome activity, dephosphorylated mitogen-activating protein kinase (MAPK), and activated caspase-3. Proteasome inhibition reversed the MAPK dephosphorylation and blocked caspase-3 activation; the neuroprotection was blocked by a p42 and p44 MAPK kinase inhibitor. Thus, the proteasome plays an important role in both inclusion body formation and dopaminergic neuronal death but these processes form opposite sides on the proteasome regulation in this model.

Topics: 1-Methyl-4-phenylpyridinium; Acetylcysteine; alpha-Synuclein; Animals; Brain; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Endopeptidases; Dopamine; Dopamine Agents; Dose-Response Relationship, Drug; Enzyme Inhibitors; Herbicides; Immunoblotting; Immunohistochemistry; Ions; L-Lactate Dehydrogenase; Leupeptins; Male; MAP Kinase Signaling System; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Nerve Tissue Proteins; Neurons; Phosphorylation; Proteasome Endopeptidase Complex; Rats; Rats, Wistar; Rotenone; Synucleins; Time Factors; Ubiquitin

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.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Apoptosis; Benzothiazoles; Blotting, Western; Cells, Cultured; Cytoplasm; Genotype; Lewy Bodies; Mice; Mice, Knockout; Microscopy, Fluorescence; Nerve Tissue Proteins; Neurons; Protease Inhibitors; Proteasome Inhibitors; Rats; Synucleins; Thiazoles; Time Factors; Ubiquitin

Synphilin-1 is linked to the pathogenesis of Parkinson's disease (PD) based on its identification as an alpha-synuclein (PARK1) and parkin (PARK2) interacting protein. Moreover, synphilin-1 is a component of Lewy bodies (LB) in brains of sporadic PD patients. Therefore, we performed a detailed mutation analysis of the synphilin-1 gene in 328 German familial and sporadic PD patients. In two apparently sporadic PD patients we deciphered a novel C to T transition in position 1861 of the coding sequence leading to an amino acid substitution from arginine to cysteine in position 621 (R621C). This mutation was absent in a total of 702 chromosomes of healthy German controls. To define a possible role of mutant synphilin-1 in the pathogenesis of PD we performed functional analyses in SH-SY5Y cells. We found synphilin-1 capable of producing cytoplasmic inclusions in transfected cells. Moreover we observed a significantly reduced number of inclusions in cells expressing C621 synphilin-1 compared with cells expressing wild-type (wt) synphilin-1, when subjected to proteasomal inhibition. C621 synphilin-1 transfected cells were more susceptible to staurosporine-induced cell death than cells expressing wt synphilin-1. Our findings argue in favour of a causative role of the R621C mutation in the synphilin-1 gene in PD and suggest that the formation of intracellular inclusions may be beneficial to cells and that a mutation in synphilin-1 that reduces this ability may sensitize neurons to cellular stress.

Topics: Acetylcysteine; Adult; Aged; alpha-Synuclein; Amino Acid Sequence; Carrier Proteins; Case-Control Studies; Cell Death; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Female; Green Fluorescent Proteins; Humans; Luminescent Proteins; Male; Middle Aged; Molecular Sequence Data; Multienzyme Complexes; Mutation; Nerve Tissue Proteins; Neurons; Parkinson Disease; Proteasome Endopeptidase Complex; Recombinant Fusion Proteins; Staurosporine; Synucleins; Ubiquitin-Protein Ligases

Mutations in Cu,Zn-superoxide dismutase (SOD-1) are associated with some familial cases of amyotrophic lateral sclerosis (ALS), but it is not known how they result in cell death. We examined effects of overexpression of wild-type SOD-1 or the G37R or G85R mutations on the accumulation of ubiquitinated and nitrated proteins, and on loss of cell viability induced by the proteasome inhibitor, lactacystin. Wild-type SOD-1 had no effect on proteasomal activity, but the mutants decreased it somewhat. Treatment with lactacystin (1 micro m) caused only limited cell viability loss, even though it induced a marked inhibition of proteasomal activities. However, viability loss due to apoptosis was substantial in response to lactacystin when cells were overexpressing a mutant SOD-1. The frequency of cells showing immunoreactivity against ubiquitinated- or nitrated-proteins was enhanced when wild-type and mutant SOD-1 s were overexpressed. Ubiquitinated or nitrated alpha-tubulin, SOD-1, alpha-synuclein and 68K neurofilaments were observed in the aggregates. Similar aggregates were observed in cells overexpressing mutant parkin (Del3-5, T240R and Q311'X). The nitric oxide synthase inhibitor, l-NAME, decreased viability loss and aggregation, suggesting that nitration of proteins may play an important role in aggregation and in the cell death accompanying it.

Topics: Acetylcysteine; alpha-Synuclein; Cell Line; Cell Survival; Cysteine Endopeptidases; Enzyme Inhibitors; Gene Transfer Techniques; Humans; Ligases; Macromolecular Substances; Multienzyme Complexes; Nerve Tissue Proteins; Neurofilament Proteins; Nitrates; Nitric Oxide Synthase; Proteasome Endopeptidase Complex; Proteins; Superoxide Dismutase; Superoxide Dismutase-1; Synucleins; Tubulin; Ubiquitin; Ubiquitin-Protein Ligases

Mutations in the alpha-synuclein gene (A30P and A53T) are reported to cause familial Parkinson's disease (PD), but it is not known how they result in selective dopaminergic cell death. Here we report on effects of mutant alpha-synucleins on dopamine transporter (DAT)-mediated toxicity of the selective dopaminergic neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+) in vitro. We established human embryonic kidney HEK-293 cell lines stably co-expressing each alpha-synuclein isoform and the human DAT. We demonstrate that expression of all alpha-synuclein isoforms enhances toxicity of general complex I inhibition (rotenone), but only the expression of mutant alpha-synucleins induces significant increased DAT-dependent toxicity of very low concentrations of MPP+ compared to wild-type protein. Proteasomal inhibition by lactacystin does not alter MPP+-toxicity in all cell lines. Our data suggest a new mechanism of MPP+-induced dopaminergic toxicity by an interaction between mutant alpha-synucleins and the DAT, which is independent of the function of the proteasome.

Topics: 1-Methyl-4-phenylpyridinium; Acetylcysteine; alpha-Synuclein; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dopamine Plasma Membrane Transport Proteins; Gene Expression; Herbicides; Humans; In Vitro Techniques; Kidney; Membrane Glycoproteins; Membrane Transport Proteins; Multienzyme Complexes; Mutagenesis; Nerve Tissue Proteins; Proteasome Endopeptidase Complex; Rotenone; Synucleins; Transfection; Uncoupling Agents

Parkinson's disease is characterized by loss of nigral dopaminergic neurons and the presence of cytoplasmic inclusions known as Lewy bodies. alpha-Synuclein and its interacting partner synphilin-1 are among constituent proteins in these aggregates. The presence of ubiquitin and proteasome subunits in these inclusions supports a role for this protein degradation pathway in the processing of proteins involved in this disease. To begin elucidating the kinetics of synphilin-1 in cells, we studied its degradation pathway in HEK293 cells that had been engineered to stably express FLAG-tagged synphilin-1. Pulse-chase experiments revealed that this protein is relatively stable with a half-life of about 16 h. Treatment with proteasome inhibitors resulted in attenuation of degradation and the accumulation of high molecular weight ubiquitinated synphilin-1 in immunoprecipitation/immunoblot experiments. Additionally, proteasome inhibitors stimulated the formation of peri-nuclear inclusions which were immunoreactive for synphilin-1, ubiquitin and alpha-synuclein. Cell viability studies revealed increased susceptibility of synphilin-1 over-expressing cells to proteasomal dysfunction. These observations indicate that synphilin-1 is ubiquitinated and degraded by the proteasome. Accumulation of ubiquitinated synphilin-1 due to impaired clearance results in its aggregation as peri-nuclear inclusions and in poor cell survival.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Blotting, Western; Carrier Proteins; Cell Line; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dimethyl Sulfoxide; Humans; Inclusion Bodies; Intracellular Signaling Peptides and Proteins; Kidney; Leupeptins; Macromolecular Substances; Mice; Multienzyme Complexes; Nerve Tissue Proteins; Neuroblastoma; Precipitin Tests; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Synucleins; Transfection; Ubiquitin

Parkinson's disease (PD) is a common progressive neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic neural cell death occurs remains unknown. Proteins encoded by two other genes in which mutations cause familial PD, parkin and UCH-L1, are involved in regulation of the ubiquitin-proteasome pathway, suggesting that dysregulation of the ubiquitin-proteasome pathway is involved in the mechanism by which these mutations cause PD. We established inducible PC12 cell lines in which wild-type or mutant alpha-synuclein can be de-repressed by removing doxycycline. Differentiated PC12 cell lines expressing mutant alpha-synuclein showed decreased activity of proteasomes without direct toxicity. Cells expressing mutant alpha-synuclein showed increased sensitivity to apoptotic cell death when treated with sub-toxic concentrations of an exogenous proteasome inhibitor. Apoptosis was accompanied by mitochondrial depolarization and elevation of caspase-3 and -9, and was blocked by cyclosporin A. These data suggest that expression of mutant alpha-synuclein results in sensitivity to impairment of proteasome activity, leading to mitochondrial abnormalities and neuronal cell death.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Apoptosis; Blotting, Western; Caspase 3; Caspase 9; Caspases; Cyclosporine; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Gene Expression Regulation; Immunoenzyme Techniques; Membrane Potentials; Mitochondria; Multienzyme Complexes; Mutation; Nerve Tissue Proteins; Parkinson Disease; PC12 Cells; Proteasome Endopeptidase Complex; Rats; Synucleins; Tetracycline; Transfection

The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin-proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14-3-3, and alpha-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin-proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.

Topics: 14-3-3 Proteins; Acetylcysteine; alpha-Synuclein; Carrier Proteins; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum Chaperone BiP; Exons; Heat-Shock Proteins; Humans; Huntingtin Protein; Huntington Disease; Immunoblotting; Inclusion Bodies; Membrane Proteins; Microscopy, Fluorescence; Models, Biological; Molecular Chaperones; Multienzyme Complexes; Mutation; Nerve Tissue Proteins; Nuclear Proteins; Peptide Fragments; Poly(A)-Binding Proteins; Proteasome Endopeptidase Complex; Proteins; Recombinant Fusion Proteins; RNA-Binding Proteins; Synucleins; T-Cell Intracellular Antigen-1; Transgenes; Tyrosine 3-Monooxygenase; Vimentin

Proteasomal dysfunction has been recently implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease and diffuse Lewy body disease. We have developed an in vitro model of proteasomal dysfunction by applying pharmacological inhibitors of the proteasome, lactacystin or ZIE[O-tBu]-A-leucinal (PSI), to dopaminergic PC12 cells. Proteasomal inhibition caused a dose-dependent increase in death of both naive and neuronally differentiated PC12 cells, which could be prevented by caspase inhibition or CPT-cAMP. A percentage of the surviving cells contained discrete cytoplasmic ubiquitinated inclusions, some of which also contained synuclein-1, the rat homologue of human alpha-synuclein. However the total level of synuclein-1 was not altered by proteasomal inhibition. The ubiquitinated inclusions were present only within surviving cells, and their number was increased if cell death was prevented. We have thus replicated, in this model system, the two cardinal pathological features of Lewy body diseases, neuronal death and the formation of cytoplasmic ubiquitinated inclusions. Our findings suggest that inclusion body formation and cell death may be dissociated from one another.

Topics: Acetylcysteine; alpha-Synuclein; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Cell Differentiation; Cyclic AMP; Cysteine Endopeptidases; Immunoblotting; Immunohistochemistry; Inclusion Bodies; Lewy Body Disease; Multienzyme Complexes; Nerve Tissue Proteins; Neuroprotective Agents; Oligopeptides; Parkinson Disease; PC12 Cells; Protease Inhibitors; Proteasome Endopeptidase Complex; Rats; Synucleins; Ubiquitins