Alpha-synuclein (aSyn) misfolding and aggregation are pathological features common to several neurodegenerative diseases, including Parkinson's disease (PD). Mounting evidence suggests that aSyn can be secreted and transferred from cell to cell, participating in the propagation and spreading of pathological events. Rab11, a small GTPase, is an important regulator in both endocytic and secretory pathways. Here, we show that Rab11 is involved in regulating aSyn secretion. Rab11 knockdown or overexpression of either Rab11a wild-type (Rab11a WT) or Rab11a GDP-bound mutant (Rab11a S25N) increased secretion of aSyn. Furthermore, we demonstrate that Rab11 interacts with aSyn and is present in intracellular inclusions together with aSyn. Moreover, Rab11 reduces aSyn aggregation and toxicity. Our results suggest that Rab11 is involved in modulating the processes of aSyn secretion and aggregation, both of which are important mechanisms in the progression of aSyn pathology in PD and other synucleinopathies.
Alpha-synuclein (ASYN) is central in Parkinson's disease (PD) pathogenesis. Converging pieces of evidence suggest that the levels of ASYN expression play a critical role in both familial and sporadic Parkinson's disease. ASYN fibrils are the main component of inclusions called Lewy Bodies (LBs) which are found mainly in the surviving neurons of the substantia nigra. Despite the accumulated knowledge regarding the involvement of ASYN in molecular mechanisms underlying the development of PD, there is much information missing which prevents understanding the causes of the disease and how to stop its progression.
?-Synuclein (AS) plays a crucial role in Parkinsons 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 Parkinsons disease.
The detection of ?-synuclein (?-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 ?-syn as a biological marker for Dementia with Lewy bodies (DLB) vs. normal subjects and patients with Alzheimers 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 ?-syn in CSF were measured using a novel enzyme-linked immunosorbent assay. There was a significant increase of CSF ?-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 ?-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 ?-syn as a surrogate biomarker for DLB.
Substantial genetic, neuropathological, and biochemical evidence implicates the presynaptic neuronal protein ?-synuclein in Parkinsons disease and related Lewy body disorders. How dysregulation of ?-synuclein leads to neurodegeneration is, however, unclear. Soluble oligomeric, but not fully fibrillar, ?-synuclein is thought to be toxic. The major neuronal target of aberrant ?-synuclein might be the synapse. The effects of aberrant ?-synuclein might include alteration of calcium homoeostasis or mitochondrial fragmentation and, in turn, mitochondrial dysfunction, which could link ?-synuclein dysfunction to recessive and toxin-induced parkinsonism. ?-Synuclein also seems to be linked to other genetic forms of Parkinsons disease, such as those linked to mutations in GBA or LRRK2, possibly through common effects on autophagy and lysosomal function. Finally, ?-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 ?-synuclein might be targets for therapy.
Genetic, biochemical, and animal model studies strongly suggest a central role for ?-synuclein in the pathogenesis of Parkinsons disease. ?-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 ?-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 ?-synuclein in brain interstitial fluid. We show for the first time that ?-synuclein is readily detected in the interstitial fluid of both ?-synuclein transgenic mice and human patients with traumatic brain injury. Our data suggest that ?-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 Parkinsons disease.
alpha-Synuclein is central in Parkinsons disease pathogenesis. Although initially alpha-synuclein was considered a purely intracellular protein, recent data suggest that it can be detected in the plasma and CSF of humans and in the culture media of neuronal cells. To address a role of secreted alpha-synuclein in neuronal homeostasis, we have generated wild-type alpha-synuclein and beta-galactosidase inducible SH-SY5Y cells. Soluble oligomeric and monomeric species of alpha-synuclein are readily detected in the conditioned media (CM) of these cells at concentrations similar to those observed in human CSF. We have found that, in this model, alpha-synuclein is secreted by externalized vesicles in a calcium-dependent manner. Electron microscopy and liquid chromatography-mass spectrometry proteomic analysis demonstrate that these vesicles have the characteristic hallmarks of exosomes, secreted intraluminar vesicles of multivesicular bodies. Application of CM containing secreted alpha-synuclein causes cell death of recipient neuronal cells, which can be reversed after alpha-synuclein immunodepletion from the CM. High- and low-molecular-weight alpha-synuclein species, isolated from this CM, significantly decrease cell viability. Importantly, treatment of the CM with oligomer-interfering compounds before application rescues the recipient neuronal cells from the observed toxicity. Our results show for the first time that cell-produced alpha-synuclein is secreted via an exosomal, calcium-dependent mechanism and suggest that alpha-synuclein secretion serves to amplify and propagate Parkinsons disease-related pathology.
Alpha-synuclein (ASYN) is central in Parkinsons disease pathogenesis. Converging pieces of evidence suggest that the levels of ASYN expression play a critical role in both familial and sporadic Parkinsons disease. To elucidate the mechanism underlying wild type (WT) ASYN-mediated neurotoxicity, we have generated a novel Tet-Off SHSY-5Y cell line, conditionally expressing WT ASYN. Induction of human WT ASYN in retinoic acid-differentiated SHSY-5Y cells leads to accumulation of soluble ASYN oligomers, in the absence of inclusions, and to gradual cellular degeneration. Morphologically, the death observed is non-apoptotic. Caspases other than caspase 3, including caspase 9, are activated and caspase inhibition diminishes death by acting at a point upstream of cytochrome c release. Application of Scyllo-inositol, an oligomer-stabilizing compound, prevents neuronal death in this model. These findings are consistent with a model in which oligomeric ASYN triggers the initial activation of the apoptotic pathway, which is however blocked downstream of the mitochondrial checkpoint, thus leading to a death combining in a unique fashion both apoptotic and non-apoptotic features. This novel inducible cell model system may prove valuable in the deciphering of WT ASYN-induced pathogenic effects and in the assessment and screening of potential therapeutic strategies.
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