ABSTRACT
BACKGROUND AND OBJECTIVES: Autoimmune nodopathies with antibodies against the paranodal proteins show a distinct phenotype of a severe sensorimotor neuropathy. In some patients, complete remission can be achieved after treatment with rituximab whereas others show a chronic course. For optimal planning of treatment, predicting the course of disease and therapeutic response is crucial. METHODS: We stimulated peripheral blood mononuclear cells in vitro to find out whether secretion of specific autoantibodies may be a predictor of the course of disease and response to rituximab. RESULTS: Three patterns could be identified: In most patients with anti-Neurofascin-155-, anti-Contactin-1-, and anti-Caspr1-IgG4 autoantibodies, in vitro production of autoantibodies was detected, indicating autoantigen-specific memory B cells and short-lived plasma cells/plasmablasts as the major source of autoantibodies. These patients generally showed a good response to rituximab. In a subgroup of patients with anti-Neurofascin-155-IgG4 autoantibodies and insufficient response to rituximab, no in vitro autoantibody production was found despite high serum titers, indicating autoantibody secretion by long-lived plasma cells outside the peripheral blood. In the patients with anti-pan-Neurofascin autoantibodies-all with a monophasic course of disease-no in vitro autoantibody production could be measured, suggesting a lack of autoantigen-specific memory B cells. In some of them, autoantibody production by unstimulated cells was detectable, presumably corresponding to high amounts of autoantigen-specific plasmablasts-well in line with a severe but monophasic course of disease. DISCUSSION: Our data suggest that different B-cell responses may occur in autoimmune nodopathies and may serve as markers of courses of disease and response to rituximab.
Subject(s)
Autoantibodies , Leukocytes, Mononuclear , Rituximab , Humans , Autoantibodies/blood , Autoantibodies/immunology , Female , Male , Adult , Leukocytes, Mononuclear/immunology , Rituximab/pharmacology , Middle Aged , Nerve Growth Factors/immunology , Young Adult , Contactin 1/immunology , Aged , Autoimmune Diseases of the Nervous System/immunology , Autoimmune Diseases of the Nervous System/drug therapy , Immunoglobulin G/blood , Immunoglobulin G/immunology , Adolescent , Cell Adhesion Molecules/immunologyABSTRACT
The phosphate rock mineral is the main source of P-fertilizer production. It is estimated to become depleted in next century. Thus, the recovery of phosphorus from waste streams has attracted great interest. The cellulosic ethanol production is seen as more and more important in future. During the production of cellulosic ethanol, the phosphorus element is released from lignocellulosic biomasses and ends up dissolved as phosphate ions in the stillage stream. In this study, the struvite (MgNH4PO4 · 6 H2O) recovery from the concentrated cellulosic ethanol stillage (ES) was conducted under room conditions with an initial pH at 7-9. The effect of Mg2+, PO43-, NH4+ and Ca2+ during struvite precipitation tests was investigated. The optimized pH value for struvite recovery is estimated at 8.5, by which 85% of PO43- and 46% of Mg2+ are removed from the liquid stream. The mass fraction of struvite in recovered crystal sample reaches 82 wt.%. The economic evaluation of struvite recovery from ES was also investigated. This work proves that the struvite is potentially to be recovered with high purity from the concentrated cellulosic ethanol stillage.
Subject(s)
Ethanol , Phosphates , Fertilizers , Phosphorus , StruviteABSTRACT
While fibrillar deposits of hyperphosphorylated protein tau are a key hallmark of several neurodegenerative diseases such as Alzheimer's disease, small oligomers have been speculated to be the key toxic aggregate species. Trivalent metal ions were shown to promote tau oligomer formation in vitro. However, little is known about potential intercellular spreading mechanisms or toxic modes of action of such oligomers. We investigated interactions of tau monomers and Fe3+/Al3+-induced oligomers with small unilamellar vesicles derived from 1-palmitoyl-2-oleoyl-phosphatidylcholine (neutral, liquid-crystalline phase) and dipalmitoyl-phosphatidylcholine (neutral, gel-phase). We further evaluated the influence of glycogen synthase kinase 3ß (GSK-3ß)-mediated tau phosphorylation applying the single-particle fluorescence spectroscopy techniques fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, and scanning for intensely fluorescent targets. In these experiments, no binding to neutral lipid surfaces was observed for tau monomers. In contrast, metal-ion-induced tau oligomers showed a gain of function in binding to neutral lipid surfaces. Of note, tau phosphorylation by GSK-3ß increased both oligomer formation and membrane affinity of the resulting oligomers. In conclusion, our data imply a pathological gain of function of metal-ion-induced oligomers of hyperphosphorylated tau, enabling membrane binding irrespective of surface charge even at nanomolar protein concentrations.
Subject(s)
Glycogen Synthase Kinase 3 beta , Metals , tau Proteins , Alzheimer Disease/metabolism , Ions , Lipids , Phosphorylation , tau Proteins/metabolismABSTRACT
BACKGROUND: The continuation of life-sustaining therapy in critical care patients with anoxic-ischemic disorders of consciousness (AI-DOC) depends on prognostic tests such as serum neuron-specific enolase (NSE) concentration levels. OBJECTIVES: To apply predictive models using machine learning methods to examine, one year after onset, the prognostic power of serial measurements of NSE in patients with AI-DOC. To compare the discriminative accuracy of this method to both standard single-day, absolute, and difference-between-days, relative NSE levels. METHODS: Classification algorithms were implemented and K-nearest neighbours (KNN) imputation was used to avoid complete case elimination of patients with missing NSE values. Non-imputed measurements from Day 0 to Day 6 were used for single day and difference-between-days. RESULTS: The naive Bayes classifier on imputed serial NSE measurements returned an AUC of (0.81±0.07) for n=126 patients (100 poor outcome). This was greater than logistic regression (0.73±0.08) and all other classifiers. Naive Bayes gave a specificity and sensitivity of 96% and 49%, respectively, for an (uncalibrated) probability decision threshold of 90%. The maximum AUC for a single day was Day 3 (0.75) for a subset of n=79 (61 poor outcome) patients, and for differences between Day 1 and Day 4 (0.81) for a subset of n=46 (39 poor outcome) patients. CONCLUSION: Imputation avoided the elimination of patients with missing data and naive Bayes outperformed all other classifiers. Machine learning algorithms could detect automatically discriminatory features and the overall predictive power increased from standard methods due to the larger data set. CODE AVAILABILITY: Data analysis code is available under GNU at: https://github.com/emilymuller1991/outcome_prediction_nse.
Subject(s)
Consciousness Disorders , Hypoxia-Ischemia, Brain , Machine Learning , Phosphopyruvate Hydratase/blood , Aged , Algorithms , Bayes Theorem , Biomarkers/blood , Consciousness Disorders/complications , Consciousness Disorders/diagnosis , Consciousness Disorders/epidemiology , Consciousness Disorders/therapy , Critical Care , Female , Humans , Hypoxia-Ischemia, Brain/complications , Hypoxia-Ischemia, Brain/diagnosis , Hypoxia-Ischemia, Brain/epidemiology , Hypoxia-Ischemia, Brain/therapy , Male , Middle Aged , Prognosis , Treatment OutcomeABSTRACT
Synucleinopathies such as dementia with Lewy bodies or Parkinson's disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregation process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation.
Subject(s)
Biopolymers/metabolism , HSP70 Heat-Shock Proteins/metabolism , Spectrum Analysis/methods , alpha-Synuclein/metabolism , Cell Line, Tumor , Humans , Luminescent Proteins/metabolismABSTRACT
OBJECTIVE: We tested the hypotheses (i) that autoimmune encephalitis is associated with new-onset headache, and (ii) that the occurrence of headache is associated with the presence of anti-N-methyl-D-aspartate (NMDA)-receptor antibodies. BACKGROUND: Autoimmune encephalitis presents with cognitive dysfunction as well as neuro-psychiatric symptoms. Its pathophysiology might involve antibody-mediated dysfunction of the glutamatergic system as indicated by the presence of anti-NMDA-receptor antibodies in some patients. METHODS: In this cross-sectional study, patients with autoimmune encephalitis were assessed with a standardized interview for previous headache and headache associated with autoimmune encephalitis. Headache was classified according to the International Classification of Headache Disorders, second edition. Clinical and paraclinical findings were correlated with the occurrence of headache. RESULTS: Of 40 patients with autoimmune encephalitis, 19 did not have a history of headache. Of those, nine suffered from encephalitis-associated headache. Seven of these nine had anti-NMDA-receptor antibodies in contrast to only two among the remaining 10 patients without new-onset headache (P = .023, odds ratio: 14, 95% confidence interval: 1.5; 127). In most patients headache occurred in attacks on more than 15 days/month, was severe, and of short duration (less than 4 hours). International Headache Society criteria for migraine were met in three patients. CONCLUSIONS: New-onset headache is a relevant symptom in patients with autoimmune encephalitis who have no history of previous headache, especially in the subgroup with anti-NMDA-receptor antibodies. This indicates a thorough investigation for secondary headaches including anti-NMDA-R antibodies for patients with new-onset headache and neuropsychiatric findings. Glutamatergic dysfunction might be important for the generation of head pain but may only occasionally be sufficient to trigger migraine-like attacks in nonmigraineurs.
Subject(s)
Anti-N-Methyl-D-Aspartate Receptor Encephalitis/complications , Encephalitis/complications , Encephalitis/immunology , Hashimoto Disease/complications , Hashimoto Disease/immunology , Headache/complications , Adult , Antibodies/blood , Female , Humans , Male , Middle Aged , Receptors, N-Methyl-D-Aspartate/immunology , Statistics, NonparametricABSTRACT
We present histological, MRI, and clinical features of an adult patient with relapsing encephalomyelitis and antibodies against myelin oligodendrocyte glycoprotein (MOG). Furthermore, we report molecular details of the recognized epitope that is specific for human MOG. A brain biopsy revealed multiple sclerosis (MS)-type II pathology. Some features overlapped with both MS and neuromyelitis optica spectrum disorders (NMOSD), whereas others were distinct from both MS and NMOSD. Immunoadsorption and rituximab induced clinical stabilization. This case contributes a new, so far missing link in the emerging spectrum of MOG-antibody-associated encephalomyelitis.
ABSTRACT
BACKGROUND: In several neurodegenerative diseases, hyperphosphorylation at position Ser129 is found in fibrillar deposits of alpha-synuclein (asyn), implying a pathophysiological role of asyn phosphorylation in neurodegeneration. However, recent animal models applying asyn phosphorylation mimics demonstrated a protective effect of phosphorylation. Since metal-ion induced asyn oligomers were identified as a potential neurotoxic aggregate species with membrane pore-forming abilities, the current study was undertaken to determine effects of asyn phosphorylation on oligomer membrane binding. METHODS: We investigated the influence of S129 phosphorylation on interactions of metal-ion induced asyn oligomers with small unilamellar lipid vesicles (SUV) composed of POPC and DPPC applying the phosphorylation mimic asyn129E. Confocal single-particle fluorescence techniques were used to monitor membrane binding at the single-particle level. RESULTS: Binding of asyn129E monomers to gel-state membranes (DPPC-SUV) is slightly reduced compared to wild-type asyn, while no interactions with membranes in the liquid-crystalline state (POPC-SUV) are seen for both asyn and asyn129E. Conversely, metal-ion induced oligomer formation is markedly increased in asyn129E. Surprisingly, membrane binding to POPC-SUV is nearly absent in Fe(3+) induced asyn129E oligomers and markedly reduced in Al(3+) induced oligomers. CONCLUSION: The protective effect of pseudophosphorylation seen in animal models may be due to impeded oligomer membrane binding. Phosphorylation at Ser129 may thus have a protective effect against neurotoxic asyn oligomers by preventing oligomer membrane binding and disruption of the cellular electrophysiological equilibrium. Importantly, these findings put a new complexion on experimental pharmaceutical interventions against POLO-2 kinase.
Subject(s)
Cell Membrane/metabolism , Protein Multimerization , Serine/metabolism , Unilamellar Liposomes/metabolism , alpha-Synuclein/chemistry , alpha-Synuclein/metabolism , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Humans , Mutation , Phosphatidylcholines/metabolism , Phosphorylation , Porosity , Protein Binding , Protein Structure, Quaternary , Temperature , Unilamellar Liposomes/chemistry , alpha-Synuclein/geneticsSubject(s)
Gliosis/congenital , Leukoencephalopathies , Receptor, Macrophage Colony-Stimulating Factor/genetics , Biopsy , Gliosis/diagnosis , Gliosis/genetics , Gliosis/pathology , Humans , Leukoencephalopathies/diagnosis , Leukoencephalopathies/genetics , Leukoencephalopathies/pathology , Male , Middle AgedABSTRACT
Despite antibiotic therapy, acute and long-term complications are still frequent in pneumococcal meningitis. One important trigger of these complications is oxidative stress, and adjunctive antioxidant treatment with N-acetyl-l-cysteine was suggested to be protective in experimental pneumococcal meningitis. However, studies of effects on neurological long-term sequelae are limited. Here, we investigated the impact of adjunctive N-acetyl-l-cysteine on long-term neurological deficits in a mouse model of meningitis. C57BL/6 mice were intracisternally infected with Streptococcus pneumoniae. Eighteen hours after infection, mice were treated with a combination of ceftriaxone and placebo or ceftriaxone and N-acetyl-l-cysteine, respectively. Two weeks after infection, neurologic deficits were assessed using a clinical score, an open field test (explorative activity), a t-maze test (memory function), and auditory brain stem responses (hearing loss). Furthermore, cochlear histomorphological correlates of hearing loss were assessed. Adjunctive N-acetyl-l-cysteine reduced hearing loss after pneumococcal meningitis, but the effect was minor. There was no significant benefit of adjunctive N-acetyl-l-cysteine treatment in regard to other long-term complications of pneumococcal meningitis. Cochlear morphological correlates of meningitis-associated hearing loss were not reduced by adjunctive N-acetyl-l-cysteine. In conclusion, adjunctive therapy with N-acetyl-l-cysteine at a dosage of 300 mg/kg of body weight intraperitoneally for 4 days reduced hearing loss but not other neurologic deficits after pneumococcal meningitis in mice. These results make a clinical therapeutic benefit of N-acetyl-l-cysteine in the treatment of patients with pneumococcal meningitis questionable.
Subject(s)
Acetylcysteine/chemistry , Acetylcysteine/therapeutic use , Anti-Bacterial Agents/therapeutic use , Meningitis, Pneumococcal/drug therapy , Animals , Anti-Bacterial Agents/chemistry , Ceftriaxone/therapeutic use , Cochlea/microbiology , Disease Models, Animal , Hearing Loss/drug therapy , Male , Meningitis, Pneumococcal/microbiology , Mice , Mice, Inbred C57BLABSTRACT
Alzheimer's disease and Parkinson's disease are neurodegenerative disorders characterised by the misfolding of proteins into soluble prefibrillar aggregates. These aggregate complexes disrupt mitochondrial function, initiating a pathophysiological cascade leading to synaptic and neuronal degeneration. In order to explore the interaction of amyloid aggregates with mitochondrial membranes, we made use of two in vitro model systems, namely: (i) lipid vesicles with defined membrane compositions that mimic those of mitochondrial membranes, and (ii) respiring mitochondria isolated from neuronal SH-SY5Y cells. External application of soluble prefibrillar forms, but not monomers, of amyloid-beta (Aß42 peptide), wild-type α-synuclein (α-syn), mutant α-syn (A30P and A53T) and tau-441 proteins induced a robust permeabilisation of mitochondrial-like vesicles, and triggered cytochrome c release (CCR) from isolated mitochondrial organelles. Importantly, the effect on mitochondria was shown to be dependent upon cardiolipin, an anionic phospholipid unique to mitochondria and a well-known key player in mitochondrial apoptosis. Pharmacological modulators of mitochondrial ion channels failed to inhibit CCR. Thus, we propose a generic mechanism of thrilling mitochondria in which soluble amyloid aggregates have the intrinsic capacity to permeabilise mitochondrial membranes, without the need of any other protein. Finally, six small-molecule compounds and black tea extract were tested for their ability to inhibit permeation of mitochondrial membranes by Aß42, α-syn and tau aggregate complexes. We found that black tea extract and rosmarinic acid were the most potent mito-protectants, and may thus represent important drug leads to alleviate mitochondrial dysfunction in neurodegenerative diseases.
Subject(s)
Amyloid beta-Peptides/metabolism , Mitochondrial Membranes/metabolism , Polyphenols/pharmacology , Cell Line, Tumor , Humans , Mitochondrial Membranes/drug effects , Permeability , Recombinant Proteins/metabolism , alpha-Synuclein/metabolism , tau Proteins/metabolismABSTRACT
Mitochondrial dysfunction has been strongly implicated in the pathogenesis of Parkinson's disease (PD) and Alzheimer's disease (AD), but its relation to protein aggregation is unclear. PD is characterized by synuclein aggregation (i.e., Lewy body [LB] formation). In AD, the abnormal accumulation of tau protein forms neurofibrillary tangles. In this study, we laser-dissected LB-positive and -negative neurons from the substantia nigra of postmortem PD brains, and tau-positive and -negative hippocampal neurons from AD brains. We quantified mitochondrial DNA deletions in relation to the cellular phenotype and in comparison with age-matched controls. Deletion levels were highest in LB-positive neurons of PD brains (40.5 ± 16.8%), followed by LB-negative neurons of PD cases (31.8 ± 14.4%) and control subjects (25.6 ± 17.5%; analysis of variance p < 0.005). In hippocampal neurons, deletion levels were 25%-30%, independent of disease status and neurofibrillary tangles. The presented findings imply increased mitochondrial DNA damage in LB-positive midbrain neurons, but do not support a direct causative link of respiratory chain dysfunction and protein aggregation.
Subject(s)
DNA Damage , DNA, Mitochondrial/genetics , Lewy Bodies/genetics , Lewy Bodies/pathology , Parkinson Disease/genetics , Parkinson Disease/pathology , Aged , Aged, 80 and over , Female , Humans , Male , Mesencephalon/cytology , Neurons/cytology , Neurons/metabolism , Oxidative Stress/genetics , Oxidative Stress/physiology , Reactive Oxygen Species/metabolism , alpha-Synuclein/metabolismABSTRACT
Cumulative evidence now suggests that the abnormal aggregation of the protein α-synuclein (αS) is a critical factor in triggering neurodegeneration in Parkinson's disease (PD). In particular, a fundamental pathogenetic mechanism appears to involve targeting of neuronal membranes by soluble oligomeric intermediates of αS, leading to their disruption or permeabilisation. Therefore, a model assay was developed in which fluorophore-loaded unilamellar vesicles were permeabilised by soluble oligomers, the latter formed by aggregation of human recombinant αS protein. The αS oligomers induced an impairment of membrane integrity similar to that of the pore-forming bacterial peptide gramicidin. The lipid vesicle permeabilisation assay was then utilised to screen 11 natural polyphenolic compounds, 8 synthetic N'-benzylidene-benzohydrazide compounds and black tea extract for protection against membrane damage by wild-type and mutant (A30P, A53T) synuclein aggregates. A select group of potent inhibitory compounds included apigenin, baicalein, morin, nordihydroguaiaretic acid, and black tea extract. Structure-activity analysis further suggests that a 5,7-dihydroxy-chromen-4-one moiety appears to be favourable for the inhibition reaction. In conclusion, we have identified a group of polyphenols that can effectively hinder membrane damage by αS aggregates. These may serve as a viable source of lead compounds for the development and design of novel therapeutic agents in PD.
Subject(s)
Membrane Lipids/metabolism , Polyphenols/pharmacology , alpha-Synuclein/toxicity , Cell Membrane Permeability , In Vitro Techniques , Kinetics , Liposomes , Spectrometry, Fluorescence , Structure-Activity RelationshipSubject(s)
Hypernatremia/complications , Myelinolysis, Central Pontine/pathology , Aged , Anti-Bacterial Agents/therapeutic use , Clostridium Infections/complications , Female , Humans , Magnetic Resonance Imaging , Myelinolysis, Central Pontine/etiology , Nervous System Diseases/etiology , Nervous System Diseases/psychology , Parenteral Nutrition, Home/adverse effects , Pneumonia/complications , Sodium/blood , Tomography, X-Ray ComputedABSTRACT
Aggregation of α-synuclein is involved in the pathogenesis of Parkinson's disease (PD). Studies of in vitro aggregation of α-synuclein are rendered complex because of the formation of a heterogeneous population of oligomers. With the use of confocal single-molecule fluorescence techniques, we demonstrate that small aggregates (oligomers) of α-synuclein formed from unbound monomeric species in the presence of organic solvent (DMSO) and iron (Fe(3+)) ions have a high affinity to bind to model membranes, regardless of the lipid-composition or membrane curvature. This binding mode contrasts with the well-established membrane binding of α-synuclein monomers, which is accompanied with α-helix formation and requires membranes with high curvature, defects in the lipid packing, and/or negatively charged lipids. Additionally, we demonstrate that membrane-bound α-synuclein monomers are protected from aggregation. Finally, we identified compounds that potently dissolved vesicle-bound α-synuclein oligomers into monomers, leaving the lipid vesicles intact. As it is commonly believed that formation of oligomers is related PD progression, such compounds may provide a promising strategy for the design of novel therapeutic drugs in Parkinson's disease.
Subject(s)
Protein Multimerization , Spectrometry, Fluorescence/methods , Unilamellar Liposomes/metabolism , alpha-Synuclein/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , Dimethyl Sulfoxide/chemistry , Fluorescence Resonance Energy Transfer , Parkinson Disease/metabolism , Phospholipids/chemistry , Phospholipids/metabolism , Protein Binding , Protein Structure, Quaternary , Unilamellar Liposomes/chemistry , alpha-Synuclein/chemistryABSTRACT
Cell outgrowth and migration in the developing nervous system result from guidance cues, whose molecular bases and clinical correlates are only partly known. We describe a patient with brain stem malformation, paroxysmal left sided lacrimation when eating ("crocodile tears") and mirror movements in addition to Wildervanck's cervico-oculo-acusticus (COA) syndrome, which encompasses Klippel-Feil anomaly, congenital hearing loss and Duane's syndrome. The unique symptom constellation has not been reported in that combination before and can be discussed in the context of congenital disordered axonal migration based on dysfunction of signalling pathways. However, mutations in some recently discovered genes, associated with single findings also present in our patient, were not found. Therefore, we suppose that the disturbance of an as yet unknown regulatory factor may explain the congenital malformation syndrome of our patient. In general, only a few human disorders have yet been found to result from defects in axon guidance. Nevertheless, disorders of axon guidance can certainly be regarded as a new category of neurodevelopmental disorders.
Subject(s)
Abnormalities, Multiple/pathology , Axons/pathology , Duane Retraction Syndrome/pathology , Hearing Loss/congenital , Klippel-Feil Syndrome/pathology , Movement Disorders/pathology , Abnormalities, Multiple/physiopathology , Adult , Cell Movement , Duane Retraction Syndrome/physiopathology , Hearing Loss/pathology , Hearing Loss/physiopathology , Heart Defects, Congenital , Heart Septal Defects, Atrial , Humans , Klippel-Feil Syndrome/physiopathology , Lower Extremity Deformities, Congenital , Male , Movement Disorders/physiopathology , Neurogenesis , Neurons/pathology , Upper Extremity Deformities, CongenitalABSTRACT
Aggregation of alpha-synuclein (αS) into oligomers is critically involved in the pathogenesis of Parkinson's disease (PD). Using confocal single-molecule fluorescence spectroscopy, we have studied the effects of 14 naturally-occurring polyphenolic compounds and black tea extract on αS oligomer formation. We found that a selected group of polyphenols exhibited potent dose-dependent inhibitory activity on αS aggregation. Moreover, they were also capable of robustly disaggregating pre-formed αS oligomers. Based upon structure-activity analysis, we propose that the key molecular scaffold most effective in inhibiting and destabilizing self-assembly by αS requires: (i) aromatic elements for binding to the αS monomer/oligomer and (ii) vicinal hydroxyl groups present on a single phenyl ring. These findings may guide the design of novel therapeutic drugs in PD.
Subject(s)
Flavonoids/pharmacology , Phenols/pharmacology , Protein Multimerization/drug effects , alpha-Synuclein/chemistry , Acetylcysteine/pharmacology , Antioxidants/pharmacology , Apigenin/chemistry , Apigenin/pharmacology , Ascorbic Acid/pharmacology , Deferoxamine/pharmacology , Dose-Response Relationship, Drug , Flavanones/chemistry , Flavanones/pharmacology , Flavonoids/chemistry , Humans , Iron Chelating Agents/pharmacology , Microscopy, Confocal , Microscopy, Fluorescence , Molecular Structure , Mutation , Parkinson Disease/metabolism , Parkinson Disease/pathology , Phenols/chemistry , Polyphenols , Protein Structure, Quaternary/drug effects , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Fluorescence/methods , Structure-Activity Relationship , alpha-Synuclein/genetics , alpha-Synuclein/metabolismABSTRACT
Aggregated α-synuclein (α-syn) is a characteristic pathological finding in Parkinson's disease and related disorders, such as dementia with Lewy bodies. Recent evidence suggests that α-syn oligomers represent the principal neurotoxic species; however, the pathophysiological mechanisms are still not well understood. Here, we studied the neurophysiological effects of various biophysically-characterized preparations of α-syn aggregates on excitatory synaptic transmission in autaptic neuronal cultures. Nanomolar concentrations of large α-syn oligomers, generated by incubation with organic solvent and Fe(3+) ions, were found to selectivity enhance evoked α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor, but not NMDA-receptor, mediated synaptic transmission within minutes. Moreover, the analysis of spontaneous AMPA-receptor-mediated miniature synaptic currents revealed an augmented frequency. These results collectively indicate that large α-syn oligomers alter both pre- and post-synaptic mechanisms of AMPA-receptor-mediated synaptic transmission. The augmented excitatory synaptic transmission may directly contribute to nerve cell death in synucleinopathies. Indeed, already low micromolar glutamate concentrations were found to be toxic in primary cultured neurons incubated with large α-syn oligomers. In conclusion, large α-syn oligomers enhance both pre- and post-synaptic AMPA-receptor-mediated synaptic transmission, thereby aggravating intracellular calcium dyshomeostasis and contributing to excitotoxic nerve cell death in synucleinopathies.
Subject(s)
Iron/pharmacology , Receptors, AMPA/physiology , Synaptic Transmission/physiology , alpha-Synuclein/pharmacology , Animals , Astrocytes/metabolism , Cell Death/drug effects , Cell Survival/drug effects , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Electrophoresis, Polyacrylamide Gel , Electrophysiological Phenomena , Excitatory Postsynaptic Potentials/physiology , Fluorescent Dyes , Hippocampus/cytology , Hippocampus/drug effects , Hippocampus/metabolism , Humans , Mice , Microscopy, Confocal , Patch-Clamp Techniques , Receptors, AMPA/drug effects , Recombinant Proteins/pharmacology , Synaptic Transmission/drug effects , Tetrazolium Salts , Thiazoles , alpha-Synuclein/biosynthesisABSTRACT
BACKGROUND: Synucleinopathies such as Parkinson's disease are characterized by the deposition of aggregated α-synuclein in affected brain areas. As genes involved in mitochondrial function, mitochondrial toxins, and age-related mitochondrial impairment have been implicated in Parkinson's disease pathogenesis, an increase in reactive oxygen species resulting from mitochondrial dysfunction has been speculated to induce α-synuclein aggregation. In vitro, pore-forming, SDS-resistant α-synuclein oligomers are formed in presence of ferric iron and may represent an important toxic particle species. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the interplay of reactive oxygen species, antioxidants and iron oxidation state in regard to α-synuclein aggregation using confocal single particle fluorescence spectroscopy, Phenanthroline spectrometry and thiobarbituric acid reactive substances assay. We found that the formation of α-synuclein oligomers in presence of Fe³âº is due to a direct interaction. In contrast, oxidizing agents and hydroxyl radicals generated in the Fenton reaction did not directly affect α-synuclein oligomerization. However, reactive oxygen species could enhance aggregation via oxidation of ferrous to ferric iron when iron ions were present. CONCLUSIONS/SIGNIFICANCE: Our data thus indicate that oxidative stress affects α-synuclein aggregation via oxidation of iron to the ferric state. This provides a new perspective on the role of mitochondrial toxins and mitochondrial dysfunction in the pathogenesis of Parkinson's disease.