To date, there is no accepted clinical diagnostic test for Parkinson disease (PD) that is based on biochemical analysis of blood or CSF. The discovery of mutations in the SNCA gene encoding ...α-synuclein in familial parkinsonism and the accumulation of α-synuclein in the PD brain suggested a critical role for this protein in PD etiology.
We investigated total and α-synuclein oligomers levels in CSF from patients clinically diagnosed with PD, progressive supranuclear palsy (PSP), or Alzheimer disease (AD), and age-matched controls, using ELISA developed in our laboratory.
The levels of α-synuclein oligomers and oligomers/total-α-synuclein ratio in CSF were higher in the PD group (n = 32; p < 0.0001, Mann-Whitney U test) compared to the control group (n = 28). The area under the receiver operating characteristic curve (AUC) indicated a sensitivity of 75.0% and a specificity of 87.5%, with an AUC of 0.859 for increased CSF α-synuclein oligomers in clinically diagnosed PD cases. However, when the CSF oligomers/total-α-synuclein ratio was analyzed, it provided an even greater sensitivity of 89.3% and specificity of 90.6%, with an AUC of 0.948. In another cross-sectional pilot study, we confirmed that the levels of CSF α-synuclein oligomers were higher in patients with PD (n = 25) compared to patients with PSP (n = 18; p < 0.05) or AD (n = 35; p < 0.001) or control subjects (n = 43; p < 0.05).
Our results demonstrate that levels of α-synuclein oligomers in CSF and the oligomers/total-α-synuclein ratio can be useful biomarkers for diagnosis and early detection of PD.
Aims
Lewy body diseases are neuropathologically characterized by the abnormal accumulation of α‐synuclein (α‐syn) protein within vulnerable neurons. Although studies have evaluated α‐syn in post ...mortem brain tissue, previous findings have been limited by typically employing pan‐α‐syn antibodies that may not recognize disease‐relevant forms of protein. We investigated the presence of α‐syn species present in post mortem brain tissues from Lewy body disease and Alzheimer's disease.
Methods
Soluble and insoluble/aggregated α‐syn from frontal cortex of post mortem brain tissues form Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease (AD) and aged control cases were sequentially extracted using buffers with increasing detergent concentrations. Enzyme‐linked immunosorbent assay (ELISA) was used to quantify the levels of total‐, oligomeric‐ and phosphorylated‐Ser129‐α‐syn (t‐, o‐ and pS129‐α‐syn). ELISA data were validated by western blot and compared to histological data from the same region of the contralateral hemisphere.
Results
There was no difference in t‐α‐syn levels between groups in the aqueous‐soluble, detergent‐soluble or urea‐soluble tissue fractions. However, aqueous‐soluble non‐phosphorylated o‐α‐syn was increased not only in PD and DLB but also in AD without neocortical Lewy bodies. In PD and AD, pS129‐α‐syn was increased in the detergent‐soluble tissue fragment and, in AD, this was positively correlated with the burden of tau pathology. Increased levels of urea‐soluble pS129‐α‐syn were demonstrated only in DLB tissue lysates but this did not correlate with Lewy body pathological burden.
Conclusions
Taken together, these findings suggest that DLB have elevated levels of insoluble pS129‐α‐syn, but that increased levels of aqueous‐soluble o‐α‐syn and detergent‐soluble pS129‐α‐syn are also observed in PD and AD, suggesting different changes to α‐syn across the spectrum of neurodegenerative proteopathies.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract S-allyl cysteine (SAC), a sulfur containing amino acid derived from garlic, has been reported to have antioxidant, anti-cancer, antihepatotoxic and neurotrophic activity. This study was ...designed to examine the pre-treatment effects of SAC on cognitive deficits and oxidative damage in the hippocampus of intracerebroventricular streptozotocin (ICV-STZ)-infused mice. Mice pre-treated with SAC (30 mg/kg) and vehicle (intraperitoneal; once daily for 15 days) were bilaterally injected with ICV-STZ (2.57 mg/kg body weight), whereas sham rats received the same volume of vehicle. The pre-treatment of this drug to Swiss albino mice has prevented the cognitive and neurobehavioral impairments. An increased latency and path length were observed in lesion, i.e. streptozotocin (STZ) group as compared to sham group and these were protected significantly in STZ group pre-treated with SAC. Levels of reduced glutathione (GSH) and its dependent enzymes (Glutathione peroxidase GPx and glutathione reductase GR) were decreased in STZ group as compared to sham group and pre-treatment of STZ group with SAC has protected their activities significantly. Conversely, the elevated level of thiobarbituric acid reactive substances (TBARS) in STZ group was attenuated significantly in SAC pre-treated group when compared with STZ lesioned group. Apoptotic parameters like DNA fragmentation, expression of Bcl2 and p53 were protected by the pre-treatment of SAC against STZ induced cognitive impairment. This study concludes that intervention of SAC could prevent free radicals associated deterioration of cognitive functions and neurobehavioral activities.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We aimed to characterize in vivo α-synuclein (α-syn) aggregates in skin nerves to ascertain: 1) the optimal marker to identify them; 2) possible differences between synucleinopathies that may justify ...the clinical variability. We studied multiple skin nerve α-syn deposits in 44 patients with synucleinopathy: 15 idiopathic Parkinson's disease (IPD), 12 dementia with Lewy Bodies (DLB), 5 pure autonomic failure (PAF) and 12 multiple system atrophy (MSA). Ten healthy subjects were used as controls. Antibodies against native α-syn, C-terminal α-syn epitopes such as phosphorylation at serine 129 (p-syn) and to conformation-specific for α-syn mature amyloid fibrils (syn-F1) were used. We found that p-syn showed the highest sensitivity and specificity in disclosing skin α-syn deposits. In MSA abnormal deposits were only found in somatic fibers mainly at distal sites differently from PAF, IPD and DLB displaying α-syn deposits in autonomic fibers mainly at proximal sites. PAF and DLB showed the highest p-syn load with a widespread involvement of autonomic skin nerve fibers.
1) p-syn in skin nerves was the optimal marker for the in vivo diagnosis of synucleinopathies; 2) the localization and load differences of aggregates may help to identify specific diagnostic traits and support a different pathogenesis among synucleinopathies.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Background
Parkinson's disease is a progressive neurodegenerative disorder that results in the widespread loss of select classes of neurons throughout the nervous system. The pathological hallmarks ...of Parkinson's disease are Lewy bodies and neurites, of which α‐synuclein fibrils are the major component. α‐Synuclein aggregation has been reported in the gut of Parkinson's disease patients, even up to a decade before motor symptoms, and similar observations have been made in animal models of disease. However, unlike the central nervous system, the nature of α‐synuclein species that form these aggregates and the classes of neurons affected in the gut are unclear. We have previously reported selective expression of α‐synuclein in cholinergic neurons in the gut (J Comp Neurol. 2013; 521:657), suggesting they may be particularly vulnerable to degeneration in Parkinson's disease.
Methods
In this study, we used immunohistochemistry to detect α‐synuclein oligomers and fibrils via conformation‐specific antibodies after rotenone treatment or prolonged exposure to high K+ in ex vivo segments of guinea‐pig ileum maintained in organotypic culture.
Key Results
Rotenone and prolonged raising of K+ caused accumulation of α‐synuclein fibrils in the axons of cholinergic enteric neurons. This took place in a time‐ and, in the case of rotenone, concentration‐dependent manner. Rotenone also caused selective necrosis, indicated by increased cellular autofluorescence, of cholinergic enteric neurons, labeled by ChAT‐immunoreactivity, also in a concentration‐dependent manner.
Conclusions & Inferences
To our knowledge, this is the first report of rotenone causing selective loss of a neurochemical class in the enteric nervous system. Cholinergic enteric neurons may be particularly susceptible to Lewy pathology and degeneration in Parkinson's disease.
Impairment of bioenergetic homeostatic mechanisms and misfolding of a‐synuclein are implicated in the pathogenesis of Parkinson's disease. Here, we show that rotenone and elevated extracellular potassium cause cell‐specific fibrillation of a‐synuclein in cholinergic enteric neurons in the guinea pig ileum. Like the central nervous system, the enteric nervous system shows selective vulnerability to stressors implicated in the pathogenesis of Parkinson's disease.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Protein aggregation is the major pathological hallmark seen in neurodegenerative disorders such as Parkinson's disease (PD). Alpha-synuclein (αS) is the main component of protein aggregates ...that form Lewy bodies (LBs) in PD and dementia with LBs. There have been several attempts to intervene in the process of expression, modification, clearance, and aggregation of αS as a therapeutic strategy toward neuroprotection. In this study, we have employed a novel, predictive, system level approach in silico to study four different strategies of anti-aggregation therapies: (a) reduction in αS modifications such as phosphorylation, nitration, or truncation in an approach called “seed clearance;” (b) “anti-oligomerization” approach through blocking the early oligomers formation; (c) “oligomers clearance” process by increasing its lysosomal degradation; and (d) “anti-aggregation” that involves prevention of aggregate formation at a later stage. These strategies were tested in a virtual dopaminergic neuronal system triggered by overexpression (OE) of mutant αS-A53T with or without rotenone (Rot)-induced oxidative stress. The results were compared by analyzing markers related to various end points such as oxidative stress, dopamine (DA) metabolism, proteasome function, survival and apoptosis. The experimental system and anti-oligomerization strategies were recapitulated in vitro in M17 dopaminergic cells overexpressing mutant αS-A53T triggered with Cu(II)-mediated oxidative stress, and the experimental data prospectively corroborated with the predictive results. Through this analysis, we found that intervention in the early part of the aggregation pathway by prevention of oligomer formation and increased clearance is indeed a good neuroprotective strategy, whereas anti-aggregation efforts to break up the aggregate at later stages has negative effects on the system.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Highlights • PAT is a synthetic peptide analog of thymulin. • PAT exerts anti-hyperalgesic effects in two animal models of local inflammation. • Treatment with PAT reduces the increase of cytokines ...in the mouse air pouch model. • PAT significantly potentiates the α7-nAchR expressed in Xenopus Oocytes. • PAT mediates, at least partially, its reported actions by potentiating the α7-nAChR.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Fibrillar deposits of alpha-synuclein occur in several neurodegenerative diseases. Two mutant forms of alpha-synuclein have been associated with early-onset Parkinson's disease, and a fragment has ...been identified as the non-amyloid-beta peptide component of Alzheimer's disease amyloid (NAC). Upon aging, solutions of alpha-synuclein and NAC change conformation to beta-sheet, detectable by CD spectroscopy, and form oligomers that deposit as amyloid-like fibrils, detectable by electron microscopy. These aged peptides are also neurotoxic. Experiments on fragments of NAC have enabled the region of NAC responsible for its aggregation and toxicity to be identified. NAC(8-18) is the smallest fragment that aggregates, as indicated by the concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. Fragments NAC(8-18) and NAC(8-16) are toxic, whereas NAC(12-18), NAC(9-16) and NAC(8-15) are not. Hence residues 8-16 of NAC comprise the region crucial for toxicity. Toxicity induced by alpha-synuclein, NAC and NAC(1-18) oligomers occurs via an apoptotic mechanism, possibly initiated by oxidative damage, since these peptides liberate hydroxyl radicals in the presence of iron. Molecules with anti-aggregational and/or antioxidant properties may therefore be potential therapeutic agents.
The deposition of abnormal protein fibrils is a prominent pathological feature of many different 'protein conformational' diseases, including some important neurodegenerative diseases such as ...Alzheimer's disease (AD), Parkinson's disease (PD), motor neurone disease and the 'prion' dementias. Some of the fibril-forming proteins or peptides associated with these diseases have been shown to be toxic to cells in culture. A clear understanding of the molecular mechanisms responsible for this toxicity should shed light on the probable link between protein deposition and cell loss in these diseases. In the case of the beta-amyloid (Abeta), which accumulates in the brain in AD, there is good evidence that the toxic mechanism involves the production of reactive oxygen species (ROS). By means of an electron spin resonance (ESR) spin-trapping method, we have shown recently that solutions of Abeta liberate readily detectable amounts of hydroxyl radicals upon incubation in vitro followed by the addition of small amounts of Fe(II). We have also obtained similar results with alpha-synuclein, which accumulates in Lewy bodies in PD. Our data suggest that hydrogen peroxide accumulates during Abeta or alpha-synuclein incubation and that this is subsequently converted to hydroxyl radicals, on addition of Fe (II), by Fenton's reaction. Consequently, we now support the idea that one of the fundamental molecular mechanisms underlying the pathogenesis of cell death in AD, PD, and possibly some other protein conformational diseases, could be the direct production of ROS during formation of the abnormal protein aggregates. This hypothesis suggests a novel approach to the therapy of this group of diseases.
Familial British dementia (FBD) is an autosomal dominant neurodegenerative disorder, with biochemical and pathological similarities to Alzheimer’s disease. FBD is associated with a point mutation in ...the stop codon of the BRI gene. The mutation extends the length of the wild-type protein by 11 amino acids, and following proteolytic cleavage, results in the production of a cyclic peptide (ABri) 11 amino acids longer than the wild-type (WT) peptide produced from the normal gene BRI. ABri was found to be the main component of amyloid deposits in FBD brains. However, pathological examination of FBD brains has shown the presence of ABri as non-fibrillar deposits as well as amyloid fibrils. Taken together, the genetic, pathological and biochemical data support the hypothesis that ABri deposits play a central role in the pathogenesis of FBD. Here we report that ABri, but not WT peptide, can oligomerise and form amyloid-like fibrils. We show for the first time that ABri induces apoptotic cell death, whereas WT is not toxic to cells. Moreover, we report the novel findings that non-fibrillar oligomeric species of ABri are more toxic than protofibrils and mature fibrils. These findings provide evidence that non-fibrillar oligomeric species are likely to play a critical role in the pathogenesis of FBD and suggest that a similar process may also operate in other neurodegenerative diseases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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