Deregulation of calcium homeostasis mediates secreted aesynuclein - induced neurotoxicity

α-Synuclein (AS) plays a crucial role in Parkinson's disease pathogenesis. AS is normally secreted from neuronal cells and can thus exert paracrine effects. We have previously demonstrated that naturally secreted AS species, derived from SH-SY5Y cells inducibly overexpressing human wild type AS, can be toxic to recipient neuronal cells. In the current study, we show that application of secreted AS alters membrane fluidity and increases calcium (Ca2+) entry. This influx is reduced on pharmacological inhibition of voltage-operated Ca2+ channels. Although no change in free cytosolic Ca2+ levels is observed, a significantly increased mitochondrial Ca2+ sequestration is found in recipient cells. Application of voltage-operated Ca2+ channel blockers or Ca2+ chelators abolishes AS-mediated toxicity. AS-treated cells exhibit increased calpain activation, and calpain inhibition greatly alleviates the observed toxicity. Collectively, our data suggest that secreted AS exerts toxicity through engagement, at least in part, of the Ca2+ homeostatic machinery. Therefore, manipulating Ca2+ signaling pathways might represent a potential therapeutic strategy for Parkinson's disease

Assessment of α-Synuclein Secretion in Mouse and Human Brain Parenchyma

Genetic, biochemical, and animal model studies strongly suggest a central role for α-synuclein in the pathogenesis of Parkinson's disease. α-synuclein lacks a signal peptide sequence and has thus been considered a cytosolic protein. Recent data has suggested that the protein may be released from cells via a non-classical secretory pathway and may therefore exert paracrine effects in the extracellular environment. However, proof that α-synuclein is actually secreted into the brain extracellular space in vivo has not been obtained. We developed a novel highly sensitive ELISA in conjugation with an in vivo microdialysis technique to measure α-synuclein in brain interstitial fluid. We show for the first time that α-synuclein is readily detected in the interstitial fluid of both α-synuclein transgenic mice and human patients with traumatic brain injury. Our data suggest that α-synuclein is physiologically secreted by neurons in vivo. This interstitial fluid pool of the protein may have a role in the propagation of synuclein pathology and progression of Parkinson's disease

Determination of prostate specific cancer markers in peripheral blood using polymerase chain reaction and chemiluminometric assays

Prostate cancer is the most commonly diagnosed neoplasm in men in the Western world. The sensitivity of current staging modalities is inadequate and more than one third of men with clinically localized prostate cancer are found to be understaged at the time of surgery. Recent studies have suggested that both detection and quantification of circulating prostate cancer cells strongly indicate the presence of metastasis.In the present study, the development of two quantitative methods for the mRNA determination of two specific prostate cancer molecular markers, PSA and PSMA, is described. The simultaneous qualitative detection of both PSA and PSMA mRNA messages in peripheral blood is also reported. The aforementioned tests utilize RT-PCR and a chemiluminometric hybridization assay in a microtiter well-based format.For the PSA mRNA quantification assay, the method includes the construction of a recombinant RNA IS that has the same sequence and primer binding sites as PSA mRNA but differs only in a 24-bp segment. A constant amount of RNA IS is added to the cell lysate prior to RNA isolation. Total RNA from a 5-ml venous blood sample is coextracted with PSA-IS RNA and subjected to RT-PCR. Amplified sequences are labeled with biotin through PCR by using a biotinylated upstream primer. Following heat-denaturation, the biotinylated products (PSA and PSA-IS) are hybridized with oligonucleotide-specific probes that are immobilized on separate microtiter wells. Immobilization of probes is achieved by absorption of their conjugates with BSA. The hybrids are measured using AP- labeled streptavidin and a dioxetane chemiluminogenic substrate. The ratio of the luminescence values obtained for the PSA mRNA and the PSA-IS RNA is a linear function of the initial amount of PSA mRNA present in the sample prior to RT-PCR amplification.The same technique for immobilization of probes is used for the development of the PSA/PSMA hybridization assay. Again total RNA from peripheral blood is subjected to RT-PCR, the biotinylated amplified products (PSA and PSMA) are heat denatured and captured on separate microtiter wells coated with probes specific for each PSA and PSMA target DNA. Addition of strepiavidin-conjugated alkaline phosphatase allows the detection of the hybridization products.As with PSA, the quantitation of PSMA mRNA involves the use of a recombinant RNA IS with similar characteristics. A constant amount of PSMA-IS RNA is added in the reverse transcription mixture. Following RT-PCR, the biotinylated amplified DNA products (PSMA and PSMA-IS) are captured on microtiter wells coated with streptavidin. Immobilized products are then allowed to hybridize to specific alkaline phosphatase- conjugated probes. The hybrids are finally detected by the addition of a dioxetane chemiluminogenic AP substate. The ratio of the luminescence values obtained for PSMA mRNA and PSMA-IS mRNA is linearly related to the initial amount of PSMA mRNA in the sample prior to RT-PCR amplification.In addition, the conjugation chemistry for the covalent coupling of oligonucleotides to proteins is described in detail. Since this chemistry involves more than one reaction steps, optimization conditions for each step are also discussed.As implied by their analytical performance, the proposed methods combine high sensitivity and reproducibility with a wide linear range. Validation of the assays using total RNA extracted from LNCaP cells, a prostate cancer-derived cell line, indicated that as low as 0.5 cancer cell equivalents can be detected from a 5 ml blood sample.From the three methods described above, the first two were also applied to clinical samples from both prostate cancer patients and healthy volunteers. Using the PSA mRNA quantification assay, 50% of the samples from patients with prostate cancer gave results above the detection limit. No PSA mRNA was detected in healthy individuals. On the other hand, the simultaneous detection of PSA and PSMA expression in peripheral blood confirmed the notion that the combined PSA/PSMA assay provides greater sensitivity than PSA or PSMA assay alone in detecting circulating prostatic cells.Ο καρκίνος του προστάτη είναι μια από τις συχνότερα εμφανιζόμενες μορφές καρκίνου στον ανδρικό πληθυσμό των ανεπτυγμένων χωρών. Η ευαισθησία των σύγχρονων μεθόδων σταδιοποίησης του καρκίνου του προστάτη είναι ανεπαρκής με αποτέλεσμα μεγάλο ποσοστό ασθενών (-40%) που αρχικά διαγνώσθηκαν με εντοπισμένο καρκίνο να βρίσκονται εκ των υστέρων ότι είχαν μετάσταση. Τα τελευταία χρόνια έχει επικρατήσει η άποψη ότι η ανίχνευση καθώς και ο ποσοτικός προσδιορισμός προστατικών καρκινικών κυττάρων στο περιφερικό αίμα αποτελεί ισχυρή ένδειξη ύπαρξης μεταστάσεων.Στην παρούσα εργασία περιγράφεται η ανάπτυξη δύο μεθόδων για τον ποσοτικό προσδιορισμό του mRNA δύο ειδικών προστατικών καρκινικών δεικτών, του PSA και PSMA, σε περιφερικό αίμα. Επιπρόσθετα, παρουσιάζεται μεθοδολογία για την ταυτόχρονη ανίχνευση του mRNA των μορίων αυτών σε περιφερικό αίμα. Όλες οι μέθοδοι που αναπτύχθηκαν βασίζονται στην τεχνική της RT-PCR και σε χημειοφωταυγή ανίχνευση των προϊόντων μέσω δοκιμασίας υβριδισμού σε πλακίδια μικροτιτλοδότησης.Για τον ποσοτικό προσδιορισμό του mRNA του PSA, η μέθοδος περιλαμβάνει τη χρήση εσωτερικού προτύπου (IS) που διαθέτει τις ίδιες περιοχές σύνδεσης εκκινητών με το mRNA του γονιδίου-στόχου. Τα προϊόντα της RT-PCR (PSA και PSA-IS) έχουν το ίδιο μέγεθος και διαφέρουν μόνο ως προς μια κεντρική αλληλουχία 24 b η οποία και επιτρέπει τελικά τη διάκρισή τους. Από δείγμα ολικού αίματος (5 ml) γίνεται απομόνωση ολικού RNA παρουσία σταθερής ποσότητας PSA-IS RNA και τμήμα του ολικού RNA υποβάλλεται σε RT-PCR. Τα προϊόντα της RT-PCR είναι επισημασμένα με βιοτίνη στο 5' τελικό άκρο τους και δεσμεύονται σε δύο σειρές φρεατίων μέσω υβριδισμού σε συμπληρωματικά ολιγονουκλεοτίδια (ανιχνευτές). Ακολουθεί προσθήκη στρεπταβιδίνης με ALP και η ανίχνευση των προϊόντων υβριδοποίησης γίνεται με προσθήκη χημειοφωταυγούς υποστρώματος της ALP. Η ακινητοποίηση των ανιχνευτών επιτυγχάνεται μέσω σύζευξής τους με BSA και προσρόφηση της πρωτεΐνης στη στερεή επιφάνεια. Ο λόγος των αναλυτικών σημάτων για το PSA mRNA και το PSA-IS RNA είναι ανάλογος της αρχικής ποσότητας του PSA mRNA στο αρχικό δείγμα.Η ταυτόχρονη ανίχνευση του PSA mRNA και του PSMA mRNA πραγματοποιείται με το ίδιο σύστημα ακινητοποίησης των παραγόμενων DNA τμημάτων μέσω σύζευξης των αντίστοιχων ανιχνευτών με BSA. Ολικό RNA απομονώνεται από το δείγμα, τμήμα αυτού υποβάλλεται σε RT-PCR και τα βιοτινυλιωμένα PSA και PSMA προϊόντα ανιχνεύονται με διαφορετικές δοκιμασίες υβριδισμού με μέτρηση της χημειοφωταύγειας.Όπως και στην περίπτωση του PSA, ο ποσοτικός προσδιορισμός του mRNA του PSMA βασίζεται στην κατασκευή εσωτερικού προτύπου (PSMA-IS RNA) με αντίστοιχα χαρακτηριστικά. Όμως, το PSMA-IS RNA προστίθεται τώρα στο στάδιο της αντίστροφης μεταγραφής κι όχι της απομόνωσης του ολικού RNA. Μετά από RT-PCR, τα βιοτινυλιωμένα προϊόντα (PSMA και PSMA-IS) ακινητοποιούνται στην επιφάνεια δύο σειρών φρεατίων επικαλυμένων με στρεπταβιδίνη. Ακολουθεί προσθήκη του ανιχνευτή ο οποίος είναι ιχνηθετημένος με ALP και η ανίχνευση των προϊόντων υβριδοποίησης γίνεται με προσθήκη χημειοφωταυγούς υποστρώματος της ALP. Ο λόγος των αναλυτικών σημάτων για το PSMA mRNA και το PSMA-IS RNA συσχετίζεται γραμμικά με την αρχική ποσότητα του PSMA mRNA στο δείγμα.Οι αντιδράσεις σύνθεσης των προϊόντων σύζευξης μεταξύ ολιγονουκλεοτιδίων και πρωτεϊνών που χρησιμοποιήθηκαν περιγράφονται διεξοδικά. Δεδομένου ότι πρόκειται για πορείες που περιλαμβάνουν περισσότερα του ενός στάδια, αναλύονται οι παράμετροι που βελτιστοποιήθηκαν ώστε το προϊόν σύζευξης να είναι σταθερό και να παρέχει την καλύτερη δυνατή ευαισθησία.Τα αναλυτικά χαρακτηριστικά των προτεινόμενων μεθόδων συνηγορούν στο ότι πρόκειται για μεθόδους με εξαιρετική ευαισθησία, ευρεία γραμμική περιοχή και πολύ καλή επαναληψιμότητα. Η εγκυρότητα κάθε μεθοδολογίας ελέγχθηκε με προσδιορισμό των mRNA των γονιδίων-στόχων σε ολικό RNA από την προστατική καρκινική σειρά, LNCaP. Μέχρι και 1 ισοδύναμο καρκινικό κύτταρο σε 10 mL δείγματος ολικού αίματος ήταν δυνατό να προσδιοριστεί με αξιοπιστία.Η μέθοδος ποσοτικού προσδιορισμού του PSA mRNA και η μέθοδος ταυτόχρονης ανίχνευσης των PSA mRNA και PSMA mRNA εφαρμόστηκαν σε δείγματα περιφερικού αίματος ασθενών με καρκίνο του προστάτη και υγιών εθελοντών. Στην πρώτη περίπτωση 50% των δειγμάτων των ασθενών έδωσαν αποτελέσματα πάνω από το όριο ανίχνευσης της μεθόδου. Κανένα από τα δείγματα υγιών ατόμων δεν έδωσε ανιχνεύσιμο αριθμό καρκινικών κυττάρων. Στη δεύτερη περίπτωση η ανάλυση των κλινικών δειγμάτων επιβεβαίωσε την άποψη ότι σε επίπεδο ανίχνευσης mRNA, η ταυτόχρονη ανίχνευση PSA/PSMA παρέχει υψηλότερη ευαισθησία σε σύγκριση με τη μεμονωμένη ανίχνευση του PSA ή του PSMA.

Cell-produced alpha-synuclein oligomers are targeted to, and impair, the 26S proteasome

Proteasomal dysfunction may play a role in neurodegenerative conditions and protein aggregation. Overexpression in neuronal cells of alpha-synuclein, a molecule linked to Parkinson’s Disease, may lead to proteasomal dysfunction. Using PCl2 cells stably expressing wild-type or mutant alpha-synuclein and gel filtration, we demonstrate that soluble, intermediate size oligomers of alpha-synuclein co-elute with the 26S proteasome. These soluble oligomers associate with the 26S proteasome and are significantly increased following treatment with proteasomal, but not lysosomal, inhibitors, indicating specific degradation of these particular species by the 26S proteasome. Importantly, expression of alpha-synuclein resulted in a significant inhibition of all proteasomal activities without affecting the levels or assembly of the 26S proteasome. Pharmacological dissociation of alpha-synuclein oligomers restored proteasomal function and reduced polyubiquitinated protein load in intact cells. Our findings suggest a model where only a subset of specific soluble cell-derived alpha-synuclein oligomers is targeted to the 26S proteasome for degradation, and simultaneously inhibit its function, likely by impeding access of other proteasomal substrates. (C) 2008 Elsevier Inc. All rights reserved

Assessment of cerebrospinal fluid α-synuclein as a potential biomarker in Parkinson’s disease and synucleinopathies

The discovery of diagnostic and prognostic biomarkers for neurodegenerative diseases represents an unmet clinical challenge. For example, the diagnosis of Parkinson’s disease (PD) relies mainly on the presence of clinical symptoms. Therefore, the identification and use of novel PD biomarkers would allow the application of disease-modifying treatments at the very early stages of neurodegeneration. The presynaptic protein, α-synuclein, has been genetically and biochemically linked with PD pathogenesis and has been considered as a potential biomarker for the diagnosis of PD and the related synucleinopathies. The vast majority of studies have assessed the measurement of α-synuclein, alone or in combination with other biomarkers in the cerebrospinal fluid (CSF), since it is the biofluid that most closely reflects the pathophysiology of the brain. The diagnostic value of the monomeric α-synuclein but also the oligomeric, the phosphorylated and the aggregated forms of the protein has been evaluated using a variety of immunoassays. The results have so far been reproducible but the assays used are still lacking the required diagnostic accuracy. Recent reports have shown that Protein misfolding cyclic amplification is a technique that has the potential to detect α-synuclein seeds in samples of CSF with high sensitivity and across different synucleinopathies. In an effort to increase the source of biomarker for PD and related synucleinopathies, α-synuclein has also been measured in neuronal exosomes, small vesicles of endosomal origin that are secreted from neurons into the CSF or the periphery. The potential diagnostic value of exosomes stems from the notion that exosomes carry a disease-specific repertoire of marker proteins. Therefore, the assessment of exosome-associated α-synuclein species may also open up new avenues for disease diagnosis in different synucleinopathies

Pathological roles of alpha-synuclein in neurological disorders

Substantial genetic, neuropathological, and biochemical evidence implicates the presynaptic neuronal protein alpha-synuclein in Parkinson’s disease and related Lewy body disorders. How dysregulation of alpha-synuclein leads to neurodegeneration is, however, unclear. Soluble oligomeric, but not fully fibrillar, alpha-synuclein is thought to be toxic. The major neuronal target of aberrant alpha-synuclein might be the synapse. The effects of aberrant alpha-synuclein might include alteration of calcium homoeostasis or mitochondrial fragmentation and, in turn, mitochondrial dysfunction, which could link alpha-synuclein dysfunction to recessive and toxin-induced parkinsonism. alpha-Synuclein also seems to be linked to other genetic forms of Parkinson’s disease, such as those linked to mutations in GBA or LRRK2, possibly through common effects on autophagy and lysosomal function. Finally, alpha-synuclein is physiologically secreted, and this extracellular form could lead to the spread of pathological accumulations and disease progression. Consequently, factors that regulate the levels, post-translational modifications, specific aberrant cellular effects, or secretion of alpha-synuclein might be targets for therapy

The Diagnostic Value of CSF α-Synuclein in the Differential Diagnosis of Dementia with Lewy Bodies vs. Normal Subjects and Patients with Alzheimer’s Disease

<div><p>The detection of α-synuclein (α-syn) in the cerebrospinal fluid (CSF) of patients with synucleinopathy has yielded promising but inconclusive results. The aim of the present study was to determine the diagnostic value of α-syn as a biological marker for Dementia with Lewy bodies (DLB) vs. normal subjects and patients with Alzheimer’s disease (AD), after strict control of several recognized confounders. Sixteen patients with DLB, 18 patients with AD and 22 age- and sex-matched normal controls (CTRL) were recruited. The levels of total α-syn in CSF were measured using a novel enzyme-linked immunosorbent assay. There was a significant increase of CSF α-syn levels in DLB patients as compared to the CTRL and AD groups (P= 0.049 and 0.01 respectively). ROC analysis revealed that increased α-syn was 81.8% specific for the discrimination of DLB vs. CTRL and 90% vs. AD. However, sensitivity was lower (56.2 % and 50% respectively). These findings provide evidence for a possible diagnostic role of α-syn as a surrogate biomarker for DLB.</p> </div

How is alpha-synuclein cleared from the cell?

The levels and conformers of alpha-synuclein are critical in the pathogenesis of Parkinson's Disease and related synucleinopathies. Homeostatic mechanisms in protein degradation and secretion have been identified as regulators of alpha-synuclein at different stages of its intracellular trafficking and transcellular propagation. Here we review pathways involved in the removal of various forms of alpha-synuclein from both the intracellular and extracellular environment. Proteasomes and lysosomes are likely to play complementary roles in the removal of intracellular alpha-synuclein species, in a manner that depends on alpha-synuclein post-translational modifications. Extracellular alpha-synuclein is cleared by extracellular proteolytic enzymes, or taken up by neighboring cells, especially microglia and astrocytes, and degraded within lysosomes. Exosomes, on the other hand, represent a vehicle for egress of excess burden of the intracellular protein, potentially contributing to the transfer of alpha-synuclein between cells. Dysfunction in any one of these clearance mechanisms, or a combination thereof, may be involved in the initiation or progression of Parkinson's disease, whereas targeting these pathways may offer an opportunity for therapeutic intervention. (Figure presented.)

Analytical characteristics of α-Syn ELISA.

<p>(A) Generation of a standard curve of the α-Syn ELISA in the low concentration range (0.1-2.7 ng/ml) showing the assay sensitivity (5 pg/ml). (B) Assessment of assay specificity by measuring different concentrations of recombinant α-Syn and β-Syn (0.1-0.9 ng/ml). (C) ELISA specificity was tested by measuring brain homogenates from KO, WT and α-Syn Tg mice. No α-Syn was detected in the KO animals. α-Syn levels in Tg brain extracts were 3.7-fold higher than in WT (***: p<0.001, n=4, 1-way ANOVA test followed by Tukey’s test) (D) Calibration graphs for the assessment of the day-to-day reproducibility of the assay during a period of 6 months (n=7 curves). The average of the S/B ratios for each concentration were plotted vs. the input of recombinant α-Syn. Error bars correspond to the reproducibility of the assay.</p