Erythrocytic α-Synuclein and the Gut Microbiome: Kindling of the Gut-Brain Axis in Parkinson's Disease.

BACKGROUND: Progressive spreading of α-synuclein via gut-brain axis has been hypothesized in the pathogenesis of Parkinson's disease (PD). However, the source of seeding-capable α-synuclein in the gastrointestinal tract (GIT) has not been fully investigated. Additionally, the mechanism by which the GIT microbiome contributes to PD pathogenesis remains to be characterized. OBJECTIVES: We aimed to investigate whether blood-derived α-synuclein might contribute to PD pathology via a gut-driven pathway and involve GIT microbiota. METHODS: The GIT expression of α-synuclein and the transmission of extracellular vesicles (EVs) derived from erythrocytes/red blood cells (RBCs), with their cargo α-synuclein, to the GIT were explored with various methods, including radioactive labeling of RBC-EVs and direct analysis of the transfer of α-synuclein protein. The potential role of microbiota on the EVs transmission was further investigated by administering butyrate, the short-chain fatty acids produced by gut microbiota and studying mice with different α-synuclein genotypes. RESULTS: This study demonstrated that RBC-EVs can effectively transport α-synuclein to the GIT in a region-dependent manner, along with variations closely associated with regional differences in the expression of gut-vascular barrier markers. The investigation further revealed that the infiltration of α-synuclein into the GIT was influenced significantly by butyrate and α-synuclein genotypes, which may also affect the GIT microbiome directly. CONCLUSION: By demonstrating the transportation of α-synuclein through RBC-EVs to the GIT, and its potential association with gut-vascular barrier markers and gut microbiome, this work highlights a potential mechanism by which RBC α-synuclein may impact PD initiation and/or progression. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Blood extracellular vesicles carrying synaptic function- and brain-related proteins as potential biomarkers for Alzheimer's disease.

INTRODUCTION: Objective and accessible markers for Alzheimer's disease (AD) and other dementias are critically needed. METHODS: We identified NMDAR2A, a protein related to synaptic function, as a novel marker of central nervous system (CNS)-derived plasma extracellular vesicles (EVs) and developed a flow cytometry-based technology for detecting such plasma EVs readily. The assay was initially tested in our local cross-sectional study to distinguish AD patients from healthy controls (HCs) or from Parkinson's disease (PD) patients, followed by a validation study using an independent cohort collected from multiple medical centers (the Alzheimer's Disease Neuroimaging Initiative). Cerebrospinal fluid AD molecular signature was used to confirm diagnoses of all AD participants. RESULTS: Likely CNS-derived EVs in plasma were significantly reduced in AD compared to HCs in both cohorts. Integrative models including CNS-derived EV markers and AD markers present on EVs reached area under the curve of 0.915 in discovery cohort and 0.810 in validation cohort. DISCUSSION: This study demonstrated that robust and rapid analysis of individual neuron-derived synaptic function-related EVs in peripheral blood may serve as a helpful marker of synaptic dysfunction in AD and dementia.

Reduced oligodendrocyte exosome secretion in multiple system atrophy involves SNARE dysfunction.

Transportation of key proteins via extracellular vesicles has been recently implicated in various neurodegenerative disorders, including Parkinson's disease, as a new mechanism of disease spreading and a new source of biomarkers. Extracellular vesicles likely to be derived from the brain can be isolated from peripheral blood and have been reported to contain higher levels of α-synuclein (α-syn) in Parkinson's disease patients. However, very little is known about extracellular vesicles in multiple system atrophy, a disease that, like Parkinson's disease, involves pathological α-syn aggregation, though the process is centred around oligodendrocytes in multiple system atrophy. In this study, a novel immunocapture technology was developed to isolate blood CNPase-positive, oligodendrocyte-derived enriched microvesicles (OEMVs), followed by fluorescent nanoparticle tracking analysis and assessment of α-syn levels contained within the OEMVs. The results demonstrated that the concentrations of OEMVs were significantly lower in multiple system atrophy patients, compared to Parkinson's disease patients and healthy control subjects. It is also noted that the population of OEMVs involved was mainly in the size range closer to that of exosomes, and that the average α-syn concentrations (per vesicle) contained in these OEMVs were not significantly different among the three groups. The phenomenon of reduced OEMVs was again observed in a transgenic mouse model of multiple system atrophy and in primary oligodendrocyte cultures, and the mechanism involved was likely related, at least in part, to an α-syn-mediated interference in the interaction between syntaxin 4 and VAMP2, leading to the dysfunction of the SNARE complex. These results suggest that reduced OEMVs could be an important mechanism related to pathological α-syn aggregation in oligodendrocytes, and the OEMVs found in peripheral blood could be further explored for their potential as multiple system atrophy biomarkers.

Immunoregulation of microglial polarization: an unrecognized physiological function of α-synuclein.

BACKGROUND: Microglial function is vital for maintaining the health of the brain, and their activation is an essential component of neurodegeneration. There is significant research on factors that provoke "reactive" or "inflammatory" phenotypes in conditions of injury or disease. One such factor, exposure to the aggregated or oligomeric forms of α-synuclein, an abundant brain protein, plays an essential role in driving microglial activation; including chemotactic migration and production of inflammatory mediators in Lewy body (LB) diseases such as Parkinson's disease. On the other hand, it is increasingly recognized that microglia also undergo changes, dependent on the cellular environment, that promote mainly reconstructive and anti-inflammatory functions, i.e., mostly desirable functions of microglia in a physiological state. What maintains microglia in this physiological state is essentially unknown. METHODS: In this study, using in vitro and in vivo models, we challenged primary microglia or BV2 microglia with LPS + IFN-γ, IL-4 + IL-13, α-synuclein monomer, and α-synuclein oligomer, and examined microglia phenotype and the underlying mechanism by RT-PCR, Western blot, ELISA, IF, IHC, Co-IP. RESULTS: We described a novel physiological function of α-synuclein, in which it modulates microglia toward an anti-inflammatory phenotype by interaction with extracellular signal-regulated kinase (ERK) and recruitment of the ERK, nuclear factor kappa B (NF-κB), and peroxisome proliferator-activated receptor γ (PPARγ) pathways. CONCLUSIONS: These findings suggest a previously unrecognized function of monomeric α-synuclein that likely gives new insights into the pathogenesis and potential therapies for Lewy body-related diseases and beyond, given the abundance and multiple functions of α-synuclein in brain tissue.

Mass spectrometry: A platform for biomarker discovery and validation for Alzheimer's and Parkinson's diseases.

Accurate, reliable, and objective biomarkers for Alzheimer's disease (AD), Parkinson's disease (PD), and related age-associated neurodegenerative disorders are urgently needed to assist in both diagnosis, particularly at early stages, and monitoring of disease progression. Technological advancements in protein detection platforms over the last few decades have resulted in a plethora of reported molecular biomarker candidates for both AD and PD; however, very few of these candidates are developed beyond the discovery phase of the biomarker development pipeline, a reflection of the current bottleneck within the field. In this review, the expanded use of selected reaction monitoring (SRM) targeted mass spectrometry will be discussed in detail as a platform for systematic verification of large panels of protein biomarker candidates prior to costly validation testing. We also advocate for the coupling of discovery-based proteomics with modern targeted MS-based approaches (e.g., SRM) within a single study in future workflows to expedite biomarker development and validation for AD and PD. It is our hope that improving the efficiency within the biomarker development process by use of an SRM pipeline may ultimately hasten the development of biomarkers that both decrease misdiagnosis of AD and PD and ultimately lead to detection at early stages of disease and objective assessment of disease progression. This article is part of the special issue "Proteomics".

Plasma α-synuclein and cognitive impairment in the Parkinson's Associated Risk Syndrome: A pilot study.

Plasma total and nervous system derived exosomal (NDE) α-synuclein have been determined as potential biomarkers of Parkinson's disease (PD). To explore the utility of plasma α-synuclein in the prodromal phase of PD, plasma total and NDE α-synuclein were evaluated in baseline and 2-year follow-up samples from 256 individuals recruited as part of the Parkinson's Associated Risk Syndrome (PARS) study. The results demonstrated that baseline and longitudinal increases in total α-synuclein predicted progression of cognitive decline in hyposmic individuals with dopamine transporter (DAT) binding reduction. On the other hand, a longitudinal decrease in NDE α-synuclein predicted worsening cognitive scores in hyposmic individuals with DAT binding reduction. Finally, in individuals with faster DAT progression, decreasing NDE/total α-synuclein ratio was associated with a larger reduction in DAT from baseline to follow-up. These results suggest that, though underlying mechanisms remain to be defined, alterations in plasma total and NDE α-synuclein concentrations are likely associated with PD progression, especially in the aspect of cognitive impairment, at early stages of the disease.

α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation.

Malformed α-Synuclein (α-syn) aggregates in neurons are released into the extracellular space, activating microglia to induce chronic neuroinflammation that further enhances neuronal damage in α-synucleinopathies, such as Parkinson's disease. The mechanisms by which α-syn aggregates activate and recruit microglia remain unclear, however. Here we show that α-syn aggregates act as chemoattractants to direct microglia toward damaged neurons. In addition, we describe a mechanism underlying this directional migration of microglia. Specifically, chemotaxis occurs when α-syn binds to integrin CD11b, leading to H2O2 production by NADPH oxidase. H2O2 directly attracts microglia via a process in which extracellularly generated H2O2 diffuses into the cytoplasm and tyrosine protein kinase Lyn, phosphorylates the F-actin-associated protein cortactin after sensing changes in the microglial intracellular concentration of H2O2. Finally, phosphorylated cortactin mediates actin cytoskeleton rearrangement and facilitates directional cell migration. These findings have significant implications, given that α-syn-mediated microglial migration reaches beyond Parkinson's disease.

Mass-Spectrometry-Based Method To Quantify in Parallel Tau and Amyloid ß 1-42 in CSF for the Diagnosis of Alzheimer's Disease.

Alzheimer's disease (AD), the most common form of dementia, afflicts about 50 million people worldwide. Currently, AD diagnosis is primarily based on psychological evaluation and can only be confirmed post-mortem. Reliable and objective biomarkers for prognosis and diagnosis have been sought for years. Together, tau and amyloid ß 1-42 (Aß42) in cerebrospinal fluid (CSF) have been shown to provide good diagnostic sensitivity and specificity. Additionally, phosphorylated forms of tau, such as tau pS181, have also shown promising results. However, the measurement of such markers currently relies on antibody-based immunoassays that have shown variability, leading to discrepant results across laboratories. To date, mass spectrometry methods developed to evaluate CSF tau and Aß42 are not compatible. We present in this article the development of a mass-spectrometry-based method of quantification for CSF tau and Aß42 in parallel. The absolute concentrations of tau and Aß42 we measured are on average 50 ng/mL (7-130 ng/mL) and 7.1 ng/mL (3-13 ng/mL), respectively. Analyses of CSF tau and Aß42, in a cohort of patients with AD, mild cognitive impairment, and healthy controls (30 subjects), provide significant group differences evaluated with ROC curves (AUC(control-AD) and AUC(control-MCI) = 1, AUC(MCI-AD) = 0.76), with at least equivalent diagnostic utility to immunoassay measurements in the same sample set. Finally, a significant and negative correlation was found between the tau and Aß peptides ratio and the disease severity.

Cerebrospinal fluid peptides as potential Parkinson disease biomarkers: a staged pipeline for discovery and validation.

Finding robust biomarkers for Parkinson disease (PD) is currently hampered by inherent technical limitations associated with imaging or antibody-based protein assays. To circumvent the challenges, we adapted a staged pipeline, starting from our previous proteomic profiling followed by high-throughput targeted mass spectrometry (MS), to identify peptides in human cerebrospinal fluid (CSF) for PD diagnosis and disease severity correlation. In this multicenter study consisting of training and validation sets, a total of 178 subjects were randomly selected from a retrospective cohort, matching age and sex between PD patients, healthy controls, and neurological controls with Alzheimer disease (AD). From ∼14,000 unique peptides displaying differences between PD and healthy control in proteomic investigations, 126 peptides were selected based on relevance and observability in CSF using bioinformatic analysis and MS screening, and then quantified by highly accurate and sensitive selected reaction monitoring (SRM) in the CSF of 30 PD patients versus 30 healthy controls (training set), followed by diagnostic (receiver operating characteristics) and disease severity correlation analyses. The most promising candidates were further tested in an independent cohort of 40 PD patients, 38 AD patients, and 40 healthy controls (validation set). A panel of five peptides (derived from SPP1, LRP1, CSF1R, EPHA4, and TIMP1) was identified to provide an area under curve (AUC) of 0.873 (sensitivity = 76.7%, specificity = 80.0%) for PD versus healthy controls in the training set. The performance was essentially confirmed in the validation set (AUC = 0.853, sensitivity = 82.5%, specificity = 82.5%). Additionally, this panel could also differentiate the PD and AD groups (AUC = 0.990, sensitivity = 95.0%, specificity = 97.4%). Furthermore, a combination of two peptides belonging to proteins TIMP1 and APLP1 significantly correlated with disease severity as determined by the Unified Parkinson's Disease Rating Scale motor scores in both the training (r = 0.381, p = 0.038)j and the validation (r = 0.339, p = 0.032) sets. The novel panel of CSF peptides, if validated in independent cohorts, could be used to assist in clinical diagnosis of PD and has the potential to help monitoring or predicting disease progression.

Identification of a specific α-synuclein peptide (α-Syn 29-40) capable of eliciting microglial superoxide production to damage dopaminergic neurons.

BACKGROUND: Misfolded α-synuclein (α-Syn) aggregates participate in the pathogenesis of synucleinopathies, such as Parkinson's disease. Whereas much is known about how the various domains within full-length α-Syn (FL-α-Syn) contribute to the formation of α-Syn aggregates and therefore to their neurotoxicity, little is known about whether the individual peptides that can be generated from α-syn, possibly as intermediate metabolites during degradation of misfolded α-Syn aggregates, are neurotoxic themselves. METHODS: A series of synthesized α-Syn peptides, corresponding to the locus in FL-α-Syn containing alanine 30, substitution of which with a proline causes a familial form of Parkinson's disease, were examined for their capacity of inducing release of microglial superoxide. The neurotoxicity of these peptides was measured according to their influence on the ability of neuroglial cultures deficient in gp91 (phox) , the catalytic unit of NADPH oxidase (Nox2), or wild-type cultures to take up (3)H-labeled dopamine and on the number of tyrosine hydroxylase-staining-positive neurons. Western blots and confocal images were utilized to analyze membrane translocation of p47 (phox) and p67 (phox) , phosphorylation of p47 (phox) and Erk1/2 kinase, and binding of α-Syn peptides to gp91 (phox) . Activation of brain microglia in mice injected with α-Syn peptides was demonstrated by immunostaining for major histocompatibility complex (MHC)-II along with qPCR for Iba-1 and MHC-II. RESULTS: We report α-Syn (29-40) as a specific peptide capable of activating microglial Nox2 to produce superoxide and cause dopaminergic neuronal damage. Administered to mice, this peptide also activated brain microglia to increase expression of MHC-II and Iba-1 and stimulated oxidation reaction. Exploring the underlying mechanisms showed that α-Syn (29-40) peptide triggered Nox2 to generate extracellular superoxide and its metabolite H2O2 by binding to the catalytic unit gp91 (phox) of Nox2; diffusing into cytosol, H2O2 activated Erk1/2 kinase to phosphorylate p47 (phox) and p67 (phox) and further activated Nox2, establishing a positive feedback loop to amplify the Nox2-mediated response. CONCLUSIONS: Collectively, our study suggests novel information regarding how α-Syn causes neuronal injury, possibly including mechanisms involving abnormal metabolites of α-Syn aggregates.

Astrocytic dynamin-like protein 1 regulates neuronal protection against excitotoxicity in Parkinson disease.

Mitochondrial dynamics has recently become an area of piqued interest in neurodegenerative disorders, including Parkinson disease (PD); however, the contribution of astrocytes to these disorders remains unclear. Here, we show that the level of dynamin-like protein 1 (Dlp1; official name DNM1L), which promotes mitochondrial fission, is lower in astrocytes from the brains of PD patients, and that decreased astrocytic Dlp1 likely represents a relatively early event in PD pathogenesis. In support of this conclusion, we show that Dlp1 knockdown dramatically affects mitochondrial morphological characteristics and localization in astrocytes, impairs the ability of astrocytes to adequately protect neurons from the excitotoxic effects of glutamate, and increases intracellular Ca(2+) in response to extracellular glutamate, resulting from compromised intracellular Ca(2+) buffering. Taken together, our results suggest that astrocytic mitochondrial Dlp1 is a key protein in mitochondrial dynamics and decreased Dlp1 may interfere with neuron survival in PD by disrupting Ca(2+)-coupled glutamate uptake.

CNS tau efflux via exosomes is likely increased in Parkinson's disease but not in Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) and Parkinson's disease (PD) involve tau pathology. Tau is detectable in blood, but its clearance from neuronal cells and the brain is poorly understood. METHODS: Tau efflux from the brain to the blood was evaluated by administering radioactively labeled and unlabeled tau intracerebroventricularly in wild-type and tau knock-out mice, respectively. Central nervous system (CNS)-derived tau in L1CAM-containing exosomes was further characterized extensively in human plasma, including by single molecule array technology with 303 subjects. RESULTS: The efflux of Tau, including a fraction via CNS-derived L1CAM exosomes, was observed in mice. In human plasma, tau was explicitly identified within L1CAM exosomes. In contrast to AD patients, L1CAM exosomal tau was significantly higher in PD patients than controls and correlated with cerebrospinal fluid tau. CONCLUSIONS: Tau is readily transported from the brain to the blood. The mechanisms of CNS tau efflux are likely different between AD and PD.

Cerebrospinal fluid α-synuclein predicts cognitive decline in Parkinson disease progression in the DATATOP cohort.

Most patients with Parkinson disease (PD) develop both cognitive and motor impairment, and biomarkers for progression are urgently needed. Although α-synuclein is altered in cerebrospinal fluid of patients with PD, it is not known whether it predicts motor or cognitive deterioration. We examined clinical data and α-synuclein in >300 unmedicated patients with PD who participated in the deprenyl and tocopherol antioxidative therapy of parkinsonism (DATATOP) study, with up to 8 years of follow-up. Longitudinal measures of motor and cognitive function were studied before (phase 1) and during (phase 2) levodopa therapy; cerebrospinal fluid was collected at the beginning of each phase. Correlations and linear mixed models were used to assess α-synuclein association with disease severity and prediction of progression in the subsequent follow-up period. Despite decreasing α-synuclein (phase 1 to phase 2 change of -0.05 ± 0.21 log-transformed values, P < 0.001), no correlations were observed between α-synuclein and motor symptoms. Longitudinally, lower α-synuclein predicted better preservation of cognitive function by several measures [Selective Reminding Test total recall α-synuclein × time interaction effect coefficient, -0.12 (P = 0.037); delayed recall, -0.05 (P = 0.002); New Dot Test, -0.03 (P = 0.002)]. Thus, α-synuclein, although not clinically useful for motor progression, might predict cognitive decline, and future longitudinal studies should include this outcome for further validation.

Targeted discovery and validation of plasma biomarkers of Parkinson's disease.

Despite extensive research, an unmet need remains for protein biomarkers of Parkinson's disease (PD) in peripheral body fluids, especially blood, which is easily accessible clinically. The discovery of such biomarkers is challenging, however, due to the enormous complexity and huge dynamic range of human blood proteins, which are derived from nearly all organ systems, with those originating specifically from the central nervous system (CNS) being exceptionally low in abundance. In this investigation of a relatively large cohort (∼300 subjects), selected reaction monitoring (SRM) assays (a targeted approach) were used to probe plasma peptides derived from glycoproteins previously found to be altered in the CNS based on PD diagnosis or severity. Next, the detected peptides were interrogated for their diagnostic sensitivity and specificity as well as the correlation with PD severity, as determined by the Unified Parkinson's Disease Rating Scale (UPDRS). The results revealed that 12 of the 50 candidate glycopeptides were reliably and consistently identified in plasma samples, with three of them displaying significant differences among diagnostic groups. A combination of four peptides (derived from PRNP, HSPG2, MEGF8, and NCAM1) provided an overall area under curve (AUC) of 0.753 (sensitivity: 90.4%; specificity: 50.0%). Additionally, combining two peptides (derived from MEGF8 and ICAM1) yielded significant correlation with PD severity, that is, UPDRS (r = 0.293, p = 0.004). The significance of these results is at least two-fold: (1) it is possible to use a targeted approach to identify otherwise very difficult to detect CNS related biomarkers in peripheral blood and (2) the novel biomarkers, if validated in independent cohorts, can be employed to assist with clinical diagnosis of PD as well as monitoring disease progression.

GV-971 attenuates α-Synuclein aggregation and related pathology.

RATIONALE: Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), share a distinct pathological feature, that is, a widespread accumulation of α-synuclein (α-syn) in the brain. There is a significant clinical unmet need for disease-modifying treatments for synucleinopathies. Recently, a seaweed-derived mixture of oligosaccharides sodium oligomannate, GV-971, was approved for Phase 2 clinical trials for PD. This study aimed to further evaluate the therapeutic effects of GV-971 on synucleinopathies using cellular and animal models and explore its associated molecular mechanisms. METHODS: α-Syn aggregation was assessed, in vitro and ex vivo, by ThT assay. A dopaminergic neuron cell line, Prnp-SNCAA53T mice, and brain slices from PD and DLB patients were used to determine the efficacy of GV-971 in ameliorating α-syn pathology. Measurements of motor functions, including pole, cylinder, and rotarod tests, were conducted on Prnp-SNCAA53T mice 4 weeks after intragastric administration of GV-971 (200 mg day-1 kg-1 ). RESULTS: GV-971 effectively prevented α-syn aggregation and even disassembled pre-aggregated α-syn fibrils, in vitro and ex vivo. In addition, GV-971 was able to rescue α-syn-induced neuronal damage and reduced release of extracellular vesicles (EVs), likely via modulating Alix expression. In the Prnp-SNCAA53T mouse model, when treated at the age of 5 months, GV-971 significantly decreased α-syn deposition in the cortex, midbrain, and cerebellum regions, along with ameliorating the motor dysfunctions. CONCLUSIONS: Our results indicate that GV-971, when administered at a relatively early stage of the disease process, significantly reduced α-syn accumulation and aggregation in Prnp-SNCAA53T mice. Furthermore, GV-971 corrected α-syn-induced inhibition of EVs release in neurons, contributing to neuronal protection. Future studies are needed to further assess GV-971 as a promising disease-modifying therapy for PD and other synucleinopathies.

Cerebrospinal fluid efflux through dynamic paracellular pores on venules as a missing piece of the brain drainage system.

The glymphatic system plays a key role in the clearance of waste from the parenchyma, and its dysfunction has been associated with the pathogenesis of Alzheimer's disease (AD). However, questions remain regarding its complete mechanisms. Here, we report that efflux of cerebrospinal fluid (CSF)/interstitial fluid (ISF) solutes occurs through a triphasic process that cannot be explained by the current model, but rather hints at the possibility of other, previously undiscovered routes from paravenous spaces to the blood. Using real-time, in vivo observation of efflux, a novel drainage pathway was discovered, in which CSF molecules enter the bloodstream directly through dynamically assembled, trumpet-shaped pores (basolateral ϕ<8 µm; apical ϕ < 2 µm) on the walls of brain venules. As Zn2+ could facilitate the brain clearance of macromolecular ISF solutes, Zn2+-induced reconstruction of the tight junctions (TJs) in vascular endothelial cells may participate in pore formation. Thus, an updated model for glymphatic clearance of brain metabolites and potential regulation is postulated. In addition, deficient clearance of Aß through these asymmetric venule pores was observed in AD model mice, supporting the notion that impaired brain drainage function contributes to Aß accumulation and pathogenic dilation of the perivascular space in AD.

Longitudinal assessment of tau and amyloid beta in cerebrospinal fluid of Parkinson disease.

Tau gene has been consistently associated with the risk of Parkinson disease in recent genome wide association studies. In addition, alterations of the levels of total tau, phosphorylated tau [181P], and amyloid beta 1-42 in cerebrospinal fluid have been reported in patients with sporadic Parkinson disease and asymptomatic carriers of leucine-rich repeat kinase 2 mutations, in patterns that clearly differ from those typically described for patients with Alzheimer disease. To further determine the potential roles of these molecules in Parkinson disease pathogenesis and/or in tracking the disease progression, especially at early stages, the current study assessed all three proteins in 403 Parkinson disease patients enrolled in the DATATOP (Deprenyl and tocopherol antioxidative therapy of parkinsonism) placebo-controlled clinical trial, the largest cohort to date with cerebrospinal fluid samples collected longitudinally. These initially drug-naive patients at early disease stages were clinically evaluated, and cerebrospinal fluid was collected at baseline and then at endpoint, defined as the time at which symptomatic anti-Parkinson disease medications were determined to be required. General linear models were used to test for associations between baseline cerebrospinal fluid biomarker levels or their rates of change and changes in the Unified Parkinson Disease Rating Scale (total or part III motor score) over time. Robust associations among candidate markers are readily noted. Baseline levels of amyloid beta were weakly but negatively correlated with baseline Unified Parkinson Disease Rating Scale total scores. Baseline phosphorylated tau/total tau and phosphorylated tau/amyloid beta were significantly and negatively correlated with the rates of the Unified Parkinson Disease Rating Scale change. While medications (deprenyl and/or tocopherol) did not appear to alter biomarkers appreciably, a weak but significant positive correlation between the rate of change in total tau or total tau/amyloid beta levels and the change of the Unified Parkinson Disease Rating Scale was observed. Notably, these correlations did not appear to be influenced by APOE genotype. These results are one of the very first pieces of evidence suggesting that tau and amyloid beta are critically involved in early Parkinson disease progression, potentially by a different mechanism than that in Alzheimer disease, although their applications as Parkinson disease progression markers will likely require the addition of other proteins.

Calcium influx: An essential process by which α-Synuclein regulates morphology of erythrocytes.

INTRODUCTION: Morphological abnormalities of erythrocytes/red blood cells (RBCs), e.g., increased acanthocytes, in Parkinson's disease (PD) have been reported previously, although the underlying mechanisms remain to be characterized. In this study, the potential roles of α-synuclein (α-syn), a protein critically involved in PD and highly abundant in RBCs, were studied in PD patients as well as in a PD mouse model. METHODS: Transgenic [PAC-Tg (SNCAA53T), A53T] mice overexpressing A53T mutant α-syn and SNCA knockout mice were employed to characterize the effect of α-syn on RBC morphology. In addition to A53T and SNCA knockout mice, the morphology of RBCs of PD patients was also examined using scanning electron microscopy. The potential roles of α-syn were further investigated in cultured RBCs and mice. RESULTS: Morphological abnormalities of RBCs and increased accumulation of aggregated α-syn on the RBC membrane were observed in PD patients. A similar phenomenon was also observed in A53T mice. Furthermore, while mice lacking α-syn expression showed a lower proportion of acanthocytes, treating RBCs derived from SNCA knockout mice with aggregated α-syn resulted in a higher percentage of acanthocytes. In a follow-up proteomic investigation, several major classes of proteins were identified as α-syn-associated proteins on the RBC membrane, seven of which were calcium-binding proteins. Applying aggregated α-syn to the RBC membrane directly induced extracellular calcium influx along with morphological changes; both observations were adequately reversed by blocking calcium influx. CONCLUSIONS: This study demonstrated that α-syn plays a critical role in PD-associated morphological abnormalities of RBCs, at least partially via a process mediated by extracellular calcium influx.

Applying bioinformatics to proteomics: is machine learning the answer to biomarker discovery for PD and MSA?

Bioinformatics tools are increasingly being applied to proteomic data to facilitate the identification of biomarkers and classification of patients. In the June, 2012 issue, Ishigami et al. used principal component analysis (PCA) to extract features and support vector machine (SVM) to differentiate and classify cerebrospinal fluid (CSF) samples from two small cohorts of patients diagnosed with either Parkinson's disease (PD) or multiple system atrophy (MSA) based on differences in the patterns of peaks generated with matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). PCA accurately segregated patients with PD and MSA from controls when the cohorts were combined, but did not perform well when segregating PD from MSA. On the other hand, SVM, a machine learning classification model, correctly classified the samples from patients with early PD or MSA, and the peak at m/z 6250 was identified as a strong contributor to the ability of SVM to distinguish the proteomic profiles of either cohort when trained on one cohort. This study, while preliminary, provides promising results for the application of bioinformatics tools to proteomic data, an approach that may eventually facilitate the ability of clinicians to differentiate and diagnose closely related parkinsonian disorders.

Antiepileptic and antiepileptogenic performance of carisbamate after head injury in the rat: blind and randomized studies.

Carisbamate (CRS) exhibits broad acute anticonvulsant activity in conventional anticonvulsant screens, genetic models of absence epilepsy and audiogenic seizures, and chronic spontaneous motor seizures arising after chemoconvulsant-induced status epilepticus. In add-on phase III trials with pharmacoresistant patients CRS induced < 30% average decreases in partial-onset seizure frequency. We assessed the antiepileptogenic and antiepileptic performance of subchronic CRS administration on posttraumatic epilepsy (PTE) induced by rostral parasaggital fluid percussion injury (rpFPI), which closely replicates human contusive closed head injury. Studies were blind and randomized, and treatment effects were assessed on the basis of sensitive electrocorticography (ECoG) recordings. Antiepileptogenic effects were assessed in independent groups of control and CRS-treated rats, at 1 and 3 months postinjury, after completion of a 2-week prophylactic treatment initiated 15 min after injury. The antiepileptic effects of 1-week CRS treatments were assessed in repeated measures experiments at 1 and 4 months postinjury. The studies were powered to detect ~50 and ~40% decreases in epilepsy incidence and frequency of seizures, respectively. Drug/vehicle treatment, ECoG analysis, and [CRS](plasma) determination all were performed blind. We detected no antiepileptogenic and an equivocal transient antiepileptic effects of CRS despite [CRS](plasma) comparable with or higher than levels attained in previous preclinical and clinical studies. These findings contrast with previous preclinical data demonstrating large efficacy of CRS, but agree with the average effect of CRS seen in clinical trials. The data support the use of rpFPI-induced PTE in the adolescent rat as a model of pharmacoresistant epilepsy for preclinical development.