4-hydroxy-2-nonenal and Parkinson-Disease

Oxidative stress is involved in various and numerous pathological states including several age-related neurodegenerative diseases. Peroxidation of the membrane lipid bilayer is one of the major sources of free radical-mediated injury that directly damages neurons causing increased membrane rigidity, decreased activity of membrane-bound enzymes, impairment of membrane receptors and altered membrane permeability and eventual cell death. Moreover, the peroxidation of polyunsaturated fatty acids leads to the formation of aldehydes, which can act as toxic by-products. One of the most abundant and cytotoxic lipid -derived aldehydes is 4-hydroxy 2-nonenal (HNE). HNE toxicity is mainly due to the alterations of cell functions by the formation of covalent adducts of HNE with proteins. A key marker of lipid peroxidation, HNE-protein adducts, were found to be elevated in brain tissues and body fluids of Alzheimer disease, Parkinson disease, Huntington disease and amyotrophic lateral sclerosis subjects and/or models of the respective age-related neurodegenerative diseases. Although only a few proteins were identified as common targets of HNE modification across all these listed disorders, a high overlap of these proteins occurs concerning the alteration of common pathways, such as glucose metabolism or mitochondrial function that are known to contribute to cognitive decline. Within this context, despite the different etiological and pathological mechanisms that lead to the onset of different neurodegenerative diseases, the formation of HNE-protein adducts might represent the shared leit-motif, which aggravates brain damage contributing to disease specific clinical presentation and decline in cognitive performance observed in each case.

Topics: Aging; Aldehydes; Alzheimer Disease; Amyloid beta-Peptides; Amyotrophic Lateral Sclerosis; Apolipoproteins E; Fatty Acids, Unsaturated; Glucose; Humans; Huntington Disease; Lipid Peroxidation; Mitochondria; Nerve Tissue Proteins; Oxidative Stress; Parkinson Disease; Protein Processing, Post-Translational

4-hydroxy-2-nonenal (HNE) is considered to be a strong marker of oxidative stress; the interaction between HNE and cellular proteins leads to the formation of HNE-protein adducts able to alter cellular homeostasis and cause the development of a pathological state. By virtue of its high lipid concentration, oxygen utilization, and the presence of metal ions participating to redox reactions, the brain is highly susceptible to the formation of free radicals and HNE-related compounds. A variety of neuropsychiatric disorders have been associated with elevations of HNE concentration. For example, increased levels of HNE were found in the cortex of bipolar and schizophrenic patients, while HNE plasma concentrations resulted high in patients with major depression. On the same line, high brain concentrations of HNE were found associated with Huntington's inclusions. The incidence of high HNE levels is relevant also in the brain and cerebrospinal fluid of patients suffering from Parkinson's disease. Intriguingly, in this case the increase of HNE was associated with an accumulation of iron in the substantia nigra, a brain region highly affected by the pathology. In the present review we recapitulate the findings supporting the role of HNE in the pathogenesis of different neuropsychiatric disorders to highlight the pathogenic mechanisms ascribed to HNE accumulation. The aim of this review is to offer novel perspectives both for the understanding of etiopathogenetic mechanisms that remain still unclear and for the identification of new useful biological markers. We conclude suggesting that targeting HNE-driven cellular processes may represent a new more efficacious therapeutical intervention.

Topics: Aldehydes; Bipolar Disorder; Cerebral Cortex; Depressive Disorder, Major; Humans; Huntington Disease; Iron; Lipid Peroxidation; Mitochondria; Molecular Targeted Therapy; Neuroprotective Agents; Oxidative Stress; Parkinson Disease; Schizophrenia; Substantia Nigra

This review highlights the role of oxidative stress and imbalances in metal ion homeostasis in the neurodegenerative diseases Alzheimer's disease and Parkinson's disease and in the progressive demyelinating disease multiple sclerosis. The chemistry and biochemistry of oxidative stress-induced protein damage are first described, followed by the evidence for a pathological role of oxidative stress in these disease states. It is tempting to speculate that free radical oxygen chemistry contributes to pathogenesis in all these conditions, though it is as yet undetermined what types of oxidative changes occur early in the disease, and what types are secondary manifestations of neuronal degeneration.

Topics: Aldehydes; alpha-Synuclein; Alzheimer Disease; Animals; Cross-Linking Reagents; Encephalomyelitis, Autoimmune, Experimental; Free Radicals; Glycation End Products, Advanced; Humans; Lipid Peroxidation; Malondialdehyde; Metals; Mice; Multiple Sclerosis; Neurodegenerative Diseases; Oxidation-Reduction; Oxidative Stress; Parkinson Disease; Proteins; Rats; Reactive Oxygen Species

Oxidative stress contributes to the cascade leading to dopamine cell degeneration in Parkinson's disease (PD). However, oxidative stress is intimately linked to other components of the degenerative process, such as mitochondrial dysfunction, excitotoxicity, nitric oxide toxicity and inflammation. It is therefore difficult to determine whether oxidative stress leads to, or is a consequence of, these events. Oxidative damage to lipids, proteins, and DNA occurs in PD, and toxic products of oxidative damage, such as 4-hydroxynonenal (HNE), can react with proteins to impair cell viability. There is convincing evidence for the involvement of nitric oxide that reacts with superoxide to produce peroxynitrite and ultimately hydroxyl radical production. Recently, altered ubiquitination and degradation of proteins have been implicated as key to dopaminergic cell death in PD. Oxidative stress can impair these processes directly, and products of oxidative damage, such as HNE, can damage the 26S proteasome. Furthermore, impairment of proteasomal function leads to free radical generation and oxidative stress. Oxidative stress occurs in idiopathic PD and products of oxidative damage interfere with cellular function, but these form only part of a cascade, and it is not possible to separate them from other events involved in dopaminergic cell death.

Topics: Aldehydes; Apoptosis; Corpus Striatum; Dopamine; Free Radicals; Glutathione Disulfide; Humans; Iron; Lipid Peroxidation; Mitochondria; Nerve Degeneration; Oxidation-Reduction; Oxidative Stress; Parkinson Disease; Peptide Hydrolases; Proteasome Endopeptidase Complex; Substantia Nigra; Superoxide Dismutase

The aggregation of α-synuclein (αSN) and increased oxidative stress leading to lipid peroxidation are pathological characteristics of Parkinson's disease (PD). Here, we report that aggregation of αSN in the presence of lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) increases the stability and the yield of αSN oligomers (αSO). Further, we show that ONE is more efficient than HNE at inducing αSO. In addition, we demonstrate that the two αSO differ in both size and shape. ONE-αSO are smaller in size than HNE-αSO, except when they are formed at a high molar excess of aldehyde. In both monomeric and oligomeric αSN, His50 is the main target of HNE modification, and HNE-induced oligomerization is severely retarded in the mutant His50Ala αSN. In contrast, ONE-induced aggregation of His50Ala αSN occurs readily, demonstrating the different pathways for inducing αSN aggregation by HNE and ONE. Our results show different morphologies of the HNE-treated and ONE-treated αSO and different roles of His50 in their modification of αSN, but we also observe structural similarities between these αSO and the non-treated αSO, e.g., flexible C-terminus, a folded core composed of the N-terminal and NAC region. Furthermore, HNE-αSO show a similar deuterium uptake as a previously characterized oligomer formed by non-treated αSO, suggesting that the backbone conformational dynamics of their folded cores resemble one another.

Topics: Aldehydes; alpha-Synuclein; Cell Line, Tumor; Humans; Lipid Peroxidation; Nuclear Magnetic Resonance, Biomolecular; Parkinson Disease; Protein Aggregates; Recombinant Proteins; Scattering, Small Angle; X-Ray Diffraction

Parkinson's Disease (PD) is a neurodegenerative disease for which there currently is no cure. Aggregation of the pre-synaptic protein α-synuclein (aSN) into oligomers (αSOs) is believed to play a key role in PD pathology, but little is known about αSO formation in vivo and how they induce neurodegeneration. Both the naturally occurring polyunsaturated fatty acid docosahexaenoic acid (DHA) and the lipid peroxidation product 4-hydroxynonenal (HNE), strongly upregulated during ROS conditions, stimulate the formation of αSOs, highlighting a potential role in PD. Yet, insight into αSOs structure and biological effects is still limited as most oligomer preparations studied to date are heterogeneous in composition. Here we have aggregated aSN in the presence of HNE and DHA and purified the αSOs using size exclusion chromatography. Both compounds stimulate formation of spherical αSOs containing anti-parallel β-sheet structure which have the same shape as unmodified αSOs though ca. 2-fold larger. Furthermore, the yield and stabilities of these oligomers are significantly higher than for unmodified aSN. Both modified and unmodified αSOs permeabilize synthetic vesicles, show high co-localisation with glutamatergic synapses and decrease Long Term Potentiation (LTP), in line with the reported synaptotoxic effects of αSOs. We conclude that DHA- and HNE-αSOs are convenient models for pathogenic disease-associated αSOs in PD.

Topics: Aldehydes; alpha-Synuclein; Animals; Circular Dichroism; Docosahexaenoic Acids; Epitopes; Fatty Acids, Unsaturated; Fluoresceins; Glutamine; Hippocampus; Humans; Light; Lipid Peroxidation; Long-Term Potentiation; Male; Mice; Mice, Inbred C57BL; Microscopy, Atomic Force; Neurons; Parkinson Disease; Protein Binding; Protein Structure, Secondary; Rats; Scattering, Radiation; Spectroscopy, Fourier Transform Infrared; Synapses

Parkinson's disease (PD) is characterized by accumulations of toxic α-synuclein aggregates in vulnerable neuronal populations in the brainstem, midbrain, and cerebral cortex. Recent findings suggest that α-synuclein pathology can be propagated transneuronally, but the underlying molecular mechanisms are unknown. Advances in the genetics of rare early-onset familial PD indicate that increased production and/or reduced autophagic clearance of α-synuclein can cause PD. The cause of the most common late-onset PD is unclear, but may involve metabolic compromise and oxidative stress upstream of α-synuclein accumulation. As evidence, the lipid peroxidation product 4-hydroxynonenal (HNE) is elevated in the brain during normal aging and moreso in brain regions afflicted with α-synuclein pathology. Here, we report that HNE increases aggregation of endogenous α-synuclein in primary neurons and triggers the secretion of extracellular vesicles (EVs) containing cytotoxic oligomeric α-synuclein species. EVs released from HNE-treated neurons are internalized by healthy neurons which as a consequence degenerate. Levels of endogenously generated HNE are elevated in cultured cells overexpressing human α-synuclein, and EVs released from those cells are toxic to neurons. The EV-associated α-synuclein is located both inside the vesicles and on their surface, where it plays a role in EV internalization by neurons. On internalization, EVs harboring pathogenic α-synuclein are transported both anterogradely and retrogradely within axons. Focal injection of EVs containing α-synuclein into the striatum of wild-type mice results in spread of synuclein pathology to anatomically connected brain regions. Our findings suggest a scenario for late-onset PD in which lipid peroxidation promotes intracellular accumulation and then extrusion of EVs containing toxic α-synuclein species; the EVs are then internalized by adjacent neurons, so propagating the neurodegenerative process.

Topics: Aldehydes; alpha-Synuclein; Animals; Axons; Biological Transport; Brain; Cells, Cultured; Extracellular Vesicles; HEK293 Cells; Humans; Lipid Peroxidation; Mice, Transgenic; Neurons; Oxidative Stress; Parkinson Disease

Biomarkers of oxidative stress are relevant in the evaluation of the disease status and prooxidant-antioxidant balance, advanced oxidation protein products and lipid peroxidation products (malondialdehyde and 4-hydroxynonenal) are being extensively evaluated regarding their relationship with clinical presentation and disease severity.. The aim of this study was to evaluate the levels of the above-mentioned parameters in plasma of 39 men and 17 women with Parkinson's disease, originated from the Republic of Serbia and their relation to clinicopathological characteristics (gender, age at examination, duration of the disease, and Hoehn and Yahr score) and oxidative status.. The incidence of disease was 2:1 towards males. The investigated oxidative parameters were gender and Hoehn and Yahr related. Significant association of higher Hoehn and Yahr scores was observed for malondialdehyde (p = 0.01) and prooxidant-antioxidant balance (p = 0.02). Relation between oxidant-antioxidant status was further supported by observed positive correlation between 4-hydroxynonenal (p = 0.04) and prooxidant-antioxidant balance (p = 0.03). Finally, the multivariate analysis indicated that prooxidant-antioxidant balance and malondialdehyde were partially determined by gender (10.6% and 7.6%) and Hoehn and Yahr scores (13.6% and 18.8%), while Hoehn and Yahr scores contributed to the variance of advanced oxidation protein products with 13.2%.. Our results indicate the higher level of oxidative stress (oxidant-antioxidant imbalance) and possible relation of several markers with gender and disease stage in patients with Parkinson's disease. The analyzed markers could be used to specify the severity of oxidative stress; however, their potential value should be analyzed in further studies.

Topics: Adult; Advanced Oxidation Protein Products; Aged; Aged, 80 and over; Aldehydes; Antioxidants; Female; Humans; Lipid Peroxidation; Male; Malondialdehyde; Middle Aged; Oxidants; Parkinson Disease; Serbia; Severity of Illness Index; Statistics, Nonparametric

Alpha-synuclein oligomers are linked to the pathogenesis of Parkinson's disease and related neurodegenerative diseases. In this chapter, we present a method to generate kinetically stable α-synuclein oligomers by the addition of reactive aldehydes, 4-hydroxy-2-nonenal, and 4-oxo-2-nonenal. We also describe biochemical and immunological techniques to characterize the generated oligomers.

Topics: Aldehydes; alpha-Synuclein; Electrophoresis, Polyacrylamide Gel; Humans; Microscopy, Atomic Force; Parkinson Disease; Protein Multimerization; Protein Stability

Parkinson's disease (PD) and other synucleinopathies are characterized by accumulation of misfolded aggregates of α-synuclein (α-syn). The normal function of α-syn is still under investigation, but it has been generally linked to synaptic plasticity, neurotransmitter release and the maintenance of the synaptic pool. α-Syn localizes at synaptic terminals where it can bind to synaptic vesicles as well as to other cellular membranes. It has become clear that these interactions have an impact on both α-syn functional role and its propensity to aggregate. In this study, we investigated the aggregation process of α-syn covalently modified with 4-hydroxy-2-nonenal (HNE). HNE is a product of lipid peroxidation and has been implicated in the pathogenesis of different neurodegenerative diseases by modifying the kinetics of soluble toxic oligomers. Although HNE-modified α-syn has been reported to assemble into stable oligomers, we found that slightly acidic conditions promoted further protein aggregation. Lipid vesicles delayed the aggregation process in a concentration-dependent manner, an effect that was observed only when they were added at the beginning of the aggregation process. Co-aggregation of lipid vesicles with HNE-modified α-syn also induced cytotoxic effects on differentiated SHSY-5Y cells. Under conditions in which the aggregation process was delayed cell viability was reduced. By exploring the behavior and potential cytotoxic effects of HNE-α-syn under acidic conditions in relation to protein-lipid interactions our study gives a framework to examine a possible pathway leading from a physiological setting to the pathological outcome of PD.

Topics: Aldehydes; alpha-Synuclein; Cell Line, Tumor; Cell Survival; Humans; Hydrogen-Ion Concentration; Lipid Metabolism; Lipid Peroxidation; Liposomes; Microscopy, Electron, Transmission; Oxidative Stress; Parkinson Disease; Protein Aggregation, Pathological; Protein Multimerization; Recombinant Proteins; Synaptic Vesicles

Thrombin and membrane lipid peroxidation (MLP) have been implicated in various central nervous system (CNS) disorders from CNS trauma to stroke, Alzheimer's (AD) and Parkinson's (PD) diseases. Because thrombin also induces MLP in platelets and its involvement in neurodegenerative diseases we hypothesized that its deleterious effects might, in part, involve formation of MLP in neuronal cells. We previously showed that thrombin induced caspase-3 mediated apoptosis in motor neurons, via a proteinase-activated receptor (PAR1). We have now investigated thrombin's influence on the oxidative state of neurons leading to induction of MLP-protein adducts. Translational relevance of thrombin-induced MLP is supported by increased levels of 4-hydroxynonenal-protein adducts (HNEPA) in AD and PD brains. We now report for the first time that thrombin dose-dependently induces formation of HNEPA in NSC34 mouse motor neuron cells using anti-HNE and anti-acrolein monoclonal antibodies. The most prominent immunoreactive band, in SDS-PAGE, was at ∼54kDa. Membrane fractions displayed higher amounts of the protein-adduct than cytosolic fractions. Thrombin induced MLP was mediated, at least in part, through PAR1 since a PAR1 active peptide, PAR1AP, also elevated HNEPA levels. Of interest, glutamate and Fe2SO4 also increased the ∼54kDa HNEPA band in these cells but to a lesser extent. Taken together our results implicate the involvement of thrombin and MLP in neuronal cell loss observed in various CNS degenerative and traumatic pathologies.

Topics: Acrolein; Aldehydes; Alzheimer Disease; Animals; Cells, Cultured; Cytoplasm; Dose-Response Relationship, Drug; Humans; Lipid Peroxidation; Membrane Lipids; Mice; Motor Neurons; Parkinson Disease; Receptor, PAR-1; Thrombin

The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is detoxified mainly by aldehyde dehydrogenase (ALDH). We find that the fungicide benomyl potently and rapidly inhibits ALDH and builds up DOPAL in vivo in mouse striatum and in vitro in PC12 cells and human cultured fibroblasts and glial cells. The in vivo results resemble those noted previously with knockouts of the genes encoding ALDH1A1 and 2, a mouse model of aging-related Parkinson's disease (PD). Exposure to pesticides that inhibit ALDH may therefore increase PD risk via DOPAL buildup. This study lends support to the "catecholaldehyde hypothesis" that the autotoxic dopamine metabolite DOPAL plays a pathogenic role in PD.

Topics: 3,4-Dihydroxyphenylacetic Acid; Aldehyde Dehydrogenase; Aldehydes; Animals; Antifungal Agents; Benomyl; Cell Line; Enzyme Inhibitors; Humans; Lipid Peroxidation; Mice; Parkinson Disease; PC12 Cells; Rats

4-Hydroxy-2-nonenal (4HNE) and acrolein (ACR) are highly reactive neurotoxic products of lipid peroxidation that are implicated in the pathogenesis and progression of Alzheimer's and Parkinson's diseases. Conjugation with glutathione (GSH) initiates the 4HNE and ACR detoxification pathway, which generates the mercapturates of 4HNE and ACR that can be excreted. Prior work has shown that the efficiency of the GSH-dependent renal detoxification of haloalkene derived mercapturates is significantly decreased upon their deacetylation because of rapid transformation of the deacetylated products into toxic compounds mediated by β-lyase. The enzymes of the GSH-conjugation pathway and β-lyases are expressed in the brain, and we hypothesized that a similar toxicity mechanism may be initiated in the brain by the deacetylation of 4HNE- and ACR-mercapturate. The present study was performed to identify an enzyme(s) involved in 4HNE- and ACR-mercapturate deacetylation, characterize the brain expression of this enzyme and determine whether its inhibition decreases 4HNE and 4HNE-mercapturate neurotoxicity. We demonstrated that of two candidate deacetylases, aminoacylases 1 (AA1) and 3 (AA3), only AA3 efficiently deacetylates both 4HNE- and ACR-mercapturate. AA3 was further localized to neurons and blood vessels. Using a small molecule screen we generated high-affinity AA3 inhibitors. Two of them completely protected rat brain cortex neurons expressing AA3 from the toxicity of 4HNE-mercapturate. 4HNE-cysteine (4HNE-Cys) was also neurotoxic and its toxicity was mostly prevented by a β-lyase inhibitor, aminooxyacetate. The results suggest that the AA3 mediated deacetylation of 4HNE-mercapturate may be involved in the neurotoxicity of 4HNE.

Topics: Acetylation; Acetylcysteine; Acrolein; Aldehydes; Alzheimer Disease; Amidohydrolases; Aminooxyacetic Acid; Animals; Cerebral Cortex; Enzyme Inhibitors; Male; Neurons; Parkinson Disease; Rats; Rats, Wistar

Accumulated evidence has indicated that neuroinflammation is one of the important etiologic factors of Parkinson's disease (PD). Earlier studies have employed the inflammogen lipopolysaccharide (LPS) to induce inflammation of dopaminergic neurons. Methamphetamine (MA) dopaminergic toxicity similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity is frequently cited as a model of PD. In the present study, we examined whether striatal LPS exposure potentiates MA-induced dopaminergic toxicity. Combined treatment with LPS and MA significantly potentiates behavioral impairment and dopaminergic deficit. However, this combination did not significantly alter the other monoaminergic systems (e.g., serotonin, norepinephrine, and histamine). Consistently, microglial activation, labeled by F4/80 or Iba-1 in the nigrostriatal region was more pronounced with the combined treatment of LPS and MA compared to either treatment alone, but this combination did not significantly alter the microglial activation in other brain regions (e.g., hippocampus, dorsal raphe nuclei, and locus ceruleus). Furthermore, neuroinflammation, oxidative stress, and pro-apoptotic changes in the striatum were more accentuated with combined treatment of LPS and MA compared to either treatment alone. In addition, it is important that cytoplasmic accumulation of alpha-synuclein was observed in the substantia nigra of mice treated with LPS plus MA, and that L-Dopa treatment significantly attenuated behavioral changes and dopaminergic deficits induced by LPS plus MA. These results suggest that combined treatment of LPS with MA is a potential animal model for PD.

Topics: Aldehydes; Animals; Blotting, Western; Chromatography, High Pressure Liquid; Corpus Striatum; Drug Synergism; Lipopolysaccharides; Male; Malondialdehyde; Methamphetamine; Mice; Mice, Inbred C57BL; Motor Activity; Parkinson Disease

Lipoxidative damage of aldolase A, enolase 1, and glyceraldehyde dehydrogenase (GAPDH) was found in the frontal cortex in a percentage of aged controls by bidimensional gel electrophoresis, Western blot test, in-gel digestion, and mass spectrometry. Aldolase A and enolase 1 were altered in 12 of 19 cases, whereas oxidation of GAPDH was found in 6 of 19 controls. The three enzymes were oxidized in the frontal cortex in the majority of cases of incidental Parkinson's disease (iPD), PD, and dementia with Lewy bodies (DLB). Differences were statistically significant (chi(2) test) for GAPDH in PD and DLB. Densitometric studies have shown that the ratio of oxidized protein per spot is higher in iPD, PD, and DLB compared with controls. These findings show oxidation of three enzymes linked with glycolysis and energy metabolism in the adult human brain as well as increased oxidation of aldolase A, enolase 1, and GAPDH in the frontal cortex in Lewy body diseases. Modifications of these enzymes may result in decreased activity and may partly account for impaired metabolism and function of the frontal lobe in PD.

Topics: Aged; Aged, 80 and over; Aldehydes; Biomarkers, Tumor; Blotting, Western; Densitometry; DNA-Binding Proteins; Electrophoresis, Gel, Two-Dimensional; Energy Metabolism; Female; Frontal Lobe; Fructose-Bisphosphate Aldolase; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycolysis; Humans; Lewy Body Disease; Male; Mass Spectrometry; Middle Aged; Oxidation-Reduction; Parkinson Disease; Phosphopyruvate Hydratase; Tumor Suppressor Proteins

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. The I93M mutation in ubiquitin C-terminal hydrolase L1 (UCH-L1) is associated with familial PD, and we have previously shown that the I93M UCH-L1-transgenic mice exhibit dopaminergic cell loss. Over 90% of neurodegenerative diseases, including PD, occur sporadically. However, the molecular mechanisms underlying sporadic PD as well as PD associated with I93M UCH-L1 are largely unknown. UCH-L1 is abundant (1-5% of total soluble protein) in the brain and is a major target of oxidative/carbonyl damage associated with sporadic PD. As well, abnormal microtubule dynamics and tubulin polymerization are associated with several neurodegenerative diseases including frontotemporal dementia and parkinsonism linked to chromosome 17. Here we show that familial PD-associated mutant UCH-L1 and carbonyl-modified UCH-L1 display shared aberrant properties: compared with wild-type UCH-L1, they exhibit increased insolubility and elevated interactions with multiple proteins, which are characteristics of several neurodegenerative diseases-linked mutants. Circular dichroism analyses suggest similar structural changes in both UCH-L1 variants. We further report that one of the proteins interacting with UCH-L1 is tubulin, and that aberrant interaction of mutant or carbonyl-modified UCH-L1 with tubulin modulates tubulin polymerization. These findings may underlie the toxic gain of function by mutant UCH-L1 in familial PD. Our results also suggest that the carbonyl modification of UCH-L1 and subsequent abnormal interactions of carbonyl-modified UCH-L1 with multiple proteins, including tubulin, constitute one of the causes of sporadic PD.

Topics: Aldehydes; Animals; Cell Line; Circular Dichroism; Cysteine; Humans; Models, Molecular; Mutation, Missense; Parkinson Disease; Protein Binding; Protein Carbonylation; Protein Processing, Post-Translational; Tubulin; Ubiquitin Thiolesterase

Several observations have implicated oxidative stress and aggregation of the presynaptic protein alpha-synuclein in the pathogenesis of Parkinson disease. alpha-Synuclein has been shown to have affinity for unsaturated fatty acids and membranes enriched in polyunsaturated fatty acids, which are especially sensitive to oxidation under conditions of oxidative stress. One of the most important products of lipid oxidation is 4-hydroxy-2-nonenal (HNE), which has been implicated in the pathogenesis of Parkinson disease. Consequently, we investigated the effects of the interaction of HNE with alpha-synuclein. Incubation of HNE with alpha-synuclein at pH 7.4 and 37 degrees C resulted in covalent modification of the protein, with up to six HNE molecules incorporated as Michael addition products. Fourier transform infrared and CD spectra indicated that HNE modification of alpha-synuclein resulted in a major conformational change involving increased beta-sheet. HNE modification of alpha-synuclein led to inhibition of fibrillation in an HNE concentration-dependent manner. This inhibition of fibrillation was shown to be due to the formation of soluble oligomers based on size exclusion high pressure liquid chromatography and atomic force microscope data. Small angle x-ray scattering analysis indicated that the HNE-induced oligomers were compact and tightly packed. Treatment with guanidinium chloride demonstrated that the HNE-induced oligomers were very stable with an extremely slow rate of dissociation. Addition of 5 mum HNE-modified oligomers to primary mesencephalic cultures caused marked neurotoxicity because the integrity of dopaminergic and GABAergic neurons was reduced by 95 and 85%, respectively. Our observations indicate that HNE modification of alpha-synuclein prevents fibrillation but may result in toxic oligomers, which could therefore contribute to the demise of neurons subjected to oxidative damage.

Topics: Aldehydes; alpha-Synuclein; Amyloid; Animals; Cells, Cultured; Fatty Acids, Unsaturated; Humans; Hydrogen-Ion Concentration; Neurons; Oxidation-Reduction; Oxidative Stress; Parkinson Disease; Protein Processing, Post-Translational; Protein Structure, Quaternary; Protein Structure, Secondary; Rats; Rats, Sprague-Dawley

The formation and toxicity of trans-4-hydroxy-2-nonenal in the central nervous system is well documented. However, the metabolism of HNE in the central nervous system (CNS) is not clear. HNE metabolism in the CNS appears to be different from that in other tissues and organs and may be dependent on the cell type and subcellular environment. Our data show that HNE metabolism is affected by the stereocenter of HNE and that oxidation of HNE may be a primary route of metabolism. Further metabolic analysis of HNE disposition is needed to clarify which pathways are truly important in normal and pathological states in the CNS.

Topics: Aldehydes; Alzheimer Disease; Animals; Central Nervous System; Humans; Mitochondria, Heart; Models, Neurological; Oxidation-Reduction; Parkinson Disease

An early biochemical change in the Parkinsonian substantia nigra (SN) is reduction in total glutathione (GSH + GSSG) levels in affected dopaminergic neurons prior to depletion in mitochondrial complex I activity, dopamine loss, and cell death. We have demonstrated using dopaminergic PC12 cell lines genetically engineered to inducibly down-regulate glutathione synthesis that total glutathione depletion in these cells results in selective complex I inhibition via a reversible thiol oxidation event. Here, we demonstrate that inhibition of complex I may occur either by direct nitric oxide (NO) but not peroxinitrite-mediated inhibition of complex I or through H2O2-mediated inhibition of the tricarboxylic acid (TCA) cycle enzyme alpha-ketoglutarate dehydrogenase (KGDH) which supplies NADH as substrate to the complex; activity of both enzymes are reduced in PD. While glutathione depletion causes a reduction in spare KGDH enzymatic capacity, it produces a complete collapse of complex I reserves and significant effects on mitochondrial function. Our data suggest that NO is likely the primary agent involved in preferential complex I inhibition following acute glutathione depletion in dopaminergic cells. This may have major implications in terms of understanding mechanisms of dopamine cell death associated with PD especially as they relate to complex I inhibition.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Aldehydes; Animals; Anti-Bacterial Agents; Blotting, Western; Cell Differentiation; Citrate (si)-Synthase; Dopamine; Dose-Response Relationship, Drug; Doxycycline; Electron Transport Complex I; Glutathione; Hydrogen Peroxide; Ketoglutarate Dehydrogenase Complex; Mitochondria; Multienzyme Complexes; Nitric Oxide; Oxygen; Parkinson Disease; PC12 Cells; Rats; Signal Transduction

Oxidative stress has been well documented in the substantia nigra in Parkinson disease (PD), but little is known about oxidative damage, particularly lipoxidation, advanced glycation (AGE), and AGE receptors (RAGE) in other structures, including the cerebral cortex, in early stages of diseases with Lewy bodies. The present study was undertaken to analyze these parameters in the frontal cortex (area 8), amygdala, and substantia nigra in selected cases with no neurologic symptoms and with neuropathologically verified incidental Lewy body disease-related changes, comparing them with healthy age-matched individuals. Results of the present study have shown mass spectrometric and immunologic evidences of increased lipoxidative damage by the markers malondialdehyde-lysine (MDAL) and 4-hydroxynonenal-lysine (HNE), increased expression of AGE in the substantia nigra, amygdala, and frontal cortex, and increased and heterogeneous RAGE cellular expression in the substantia nigra and frontal cortex in cases with early stages of parkinsonian neuropathology. In addition, increased content of the highly peroxidizable docosahexaenoic acid in the amygdala and frontal cortex. These changes were not associated to alpha-synuclein aggregation in cortex, contrasting with aggregates found in SDS-soluble fractions of frontal cortex in dementia with Lewy bodies (DLB) cases. The pattern of lipidic abnormalities differed in DLB and incidental Lewy body disease. Furthermore, although AGE and RAGE expression were raised in DLB, no increase in the total amount of HNE and MDAL adducts was found in the cerebral cortex in DLB. Preliminary analyses have identified 2 proteins with lipoxidative damage, alpha-synuclein and manganese superoxide dismutase (SOD2), in incidentally Lewy body disease cortex. This study demonstrates abnormal fatty acid profiles, increased and selective lipoxidative damage, and increased AGE and RAGE expression in the frontal cortex in cases with early stages of parkinsonian neuropathology without treatment. These findings further support antioxidant therapy in the treatment of PD to reduce cortical damage associated with oxidative stress.

Topics: Aged; Aged, 80 and over; Aldehydes; alpha-Synuclein; Blotting, Western; Docosahexaenoic Acids; Electrophoresis, Gel, Two-Dimensional; Fatty Acids; Female; Glycation End Products, Advanced; Humans; Immunohistochemistry; Lewy Body Disease; Male; Malondialdehyde; Mass Spectrometry; Neocortex; Nerve Tissue Proteins; Oxidative Stress; Parkinson Disease; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Synucleins

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.

Topics: Aldehydes; Circular Dichroism; Coumarins; Humans; Kinetics; Mutation; Parkinson Disease; Protein Structure, Secondary; Recombinant Proteins; Thiolester Hydrolases; Ubiquitin Thiolesterase; Ubiquitins

Multiple lines of evidence indicate that oxidative stress is a critical pathogenic factor in Parkinson disease (PD) and diffuse Lewy body disease (DLBD). Previously, we demonstrated increased levels of redox-active iron in Lewy bodies, and that Lewy bodies accumulate advanced glycation end-products. To further characterize the role of oxidative stress in diseases with Lewy body formation, we examined immunocytochemically eight cases of PD and five cases of DLBD for adducts of the lipid peroxidation adduct 4-hydroxy-2-nonenal, and for N(epsilon)-(carboxymethyl)lysine (CML). Our findings demonstrate immunolocalization of 4-hydroxynonenal and CML to Lewy bodies in PD and DLBD. These findings not only support prior studies indicating that lipid peroxidation is increased in patients with PD and DLBD but that oxidative damage may play a critical role in Lewy body formation.

Topics: Adult; Aged; Aged, 80 and over; Aldehydes; Brain Stem; Humans; Immunohistochemistry; Lewy Body Disease; Lipid Peroxidation; Middle Aged; Neocortex; Neurons; Oxidative Stress; Parkinson Disease

(E)-4-hydroxy-2-nonenal (HNE) is a toxic end-product of the free radical-stimulated peroxidation of phospholoipid-bound arachidonic acid in cell membranes. There is a growing body of evidence to suggest that free radicals may play an important role in the pathology of Parkinson's disease. HNE is highly electrophilic and is conjugated to reduced glutathione (GSH) by glutathione S-transferase. The depletion of GSH in the substantia nigra of Parkinson's patients and in the brainstem of mice treated with the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) prompted this study on the concentrations of HNE in the cerebrospinal fluid (CSF) and plasma of Parkinson's patients and the brainstem of mice treated with MPTP. HNE was identified and quantitated by a highly specific and sensitive method based on the gas chromatography-negative-ion chemical ionisation mass spectrometry of the O-pentafluorobenzyl oxime derivative using 9D3-4-hydroxy-2-nonenal as an internal standard. The mean concentration of HNE in the CSF of patients with Parkinson's disease was 1.47+/-0.76 microM (mean+/-SD, n=10), while the concentration in the CSF of a group of control patients was 0.38+/-0.14 microM (n=10; p < .01). The mean concentration of HNE in the plasma of Parkinson's patients was 0.68+/-0.15 microM (n=20) and the concentration in the control group was 0.47+/-0 12 microM (n=20; p < .05). The mean peak concentration of HNE in the brainstem of mice after a single s.c. dose of MPTP (40 mg/kg) was 3.62+/-0.36 nM/g wet wt. (n=17) at 12 h while the control value was 0.45+/-0.05 nM/g wet wt. (n=20; p < .05). The GSH concentration in the brainstem of MPTP-treated mice at 24 h. was 0.65+/-0.03 microM/g wet wt. (n=14) and the control value was 1.25+/-0.03 microM/g wet wt. (n=20; p < .01). The corresponding concentration of GSH-HNE-conjugate at 24 h was 0.32+/-0.09 microM/g wet wt. (n=12) compared with a control value of 0.05+/-0.02 (n=16; p < .01). After treatment with alpha-tocopherol (2.35 g/kg s.c. daily x 3) the mean concentration of HNE 12 hr. after MPTP injection was 0.89+/-0.06 nM/g wet wt. (n=18). The HNE concentration in a group not treated with alpha-tocopherol prior to MPTP injection was 3.49+/-0.09 nM/g wet wt. (n=14; p < .05). The concentration of GSH in the mice pretreated with alpha-tocopherol before MPTP injection was 1.14+/-0.02 microM/g wet wt. (n=17) at 24 h compared to 0.61+/-0.02 microM/g wet wt. (n=14) in the untreated mice (p < .05). The direct injecti

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Adult; Aged; Aldehydes; Animals; Brain Stem; Cross-Linking Reagents; Drug Administration Routes; Glutathione; Humans; Injections, Subcutaneous; Male; Mice; Middle Aged; Parkinson Disease; Substantia Nigra; Time Factors; Vitamin E

(E)-4-Hydroxy-2-nonenal (HNE) is a highly reactive product of the free radical-stimulated lipid peroxidation of phospholipid-bound arachidonic acid in cellular membranes. We describe a sensitive and specific method for the determination of HNE in clinical samples. The method is based on the formation of the O-pentafluorobenzyl (O-PFB) oxime derivative of HNE, which is then extracted and cleaned up by solid-phase extraction. The HNE O-PFB oxime is then analysed without further derivatisation by capillary column gas chromatography-negative ion chemical ionisation mass spectrometry (GC-NICI-MS) using selected-ion monitoring. Concentrations down to the pmol range were achieved using deuterated HNE as an internal standard. The method was used to determine HNE in the cerebrospinal fluid and plasma of patients with Parkinson's disease, the plasma of patients with HIV-1 infection and AIDS and in inflamed mucosal biopsy specimens from patients with inflammatory bowel disease.

Topics: Acquired Immunodeficiency Syndrome; Adult; Aged; Aldehydes; Chromatography, Gas; Female; Humans; Hydroxylamines; Indicators and Reagents; Inflammatory Bowel Diseases; Intestinal Mucosa; Lipid Peroxidation; Male; Mass Spectrometry; Middle Aged; Parkinson Disease

There is growing evidence that oxidative stress and mitochondrial respiratory failure with attendant decrease in energy output are implicated in nigral neuronal death in Parkinson disease (PD). It is not known, however, which cellular elements (neurons or glial cells) are major targets of oxygen-mediated damage. 4-Hydroxy-2-nonenal (HNE) was shown earlier to react with proteins to form stable adducts that can be used as markers of oxidative stress-induced cellular damage. We report here results of immunochemical studies using polyclonal antibodies directed against HNE-protein conjugates to label the site of oxidative damage in control subjects (ages 18-99 years) and seven patients that died of PD (ages 57-78 years). All the nigral melanized neurons in one of the midbrain sections were counted and classified into three groups according to the intensity of immunostaining for HNE-modified proteins--i.e., no staining, weak staining, and intensely positive staining. On average, 58% of nigral neurons were positively stained for HNE-modified proteins in PD; in contrast only 9% of nigral neurons were positive in the control subjects; the difference was statistically significant (Mann-Whitney U test; P < 0.01). In contrast to the substantia nigra, the oculomotor neurons in the same midbrain sections showed no or only weak staining for HNE-modified proteins in both PD and control subjects; young control subjects did not show any immunostaining; however, aged control subjects showed weak staining in the oculomotor nucleus, suggesting age-related accumulation of HNE-modified proteins in the neuron. Our results indicate the presence of oxidative stress within nigral neurons in PD, and this oxidative stress may contribute to nigral cell death.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aldehydes; Autopsy; Cross-Linking Reagents; Female; Humans; Immunohistochemistry; Levodopa; Male; Mesencephalon; Middle Aged; Motor Neurons; Nerve Tissue Proteins; Neurons; Oxidative Stress; Parkinson Disease; Reference Values; Substantia Nigra