Evangelia Emmanouilidou

Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival.

α-Synuclein is central in Parkinsons disease pathogenesis. Although initially α-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 α-synuclein in neuronal homeostasis, we have generated wild-type α-synuclein and β-galactosidase inducible SH-SY5Y cells. Soluble oligomeric and monomeric species of α-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, α-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 α-synuclein causes cell death of recipient neuronal cells, which can be reversed after α-synuclein immunodepletion from the CM. High- and low-molecular-weight α-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 α-synuclein is secreted via an exosomal, calcium-dependent mechanism and suggest that α-synuclein secretion serves to amplify and propagate Parkinsons disease-related pathology.

Pathological roles of α-synuclein in neurological disorders

Substantial genetic, neuropathological, and biochemical evidence implicates the presynaptic neuronal protein α-synuclein in Parkinsons disease and related Lewy body disorders. How dysregulation of α-synuclein leads to neurodegeneration is, however, unclear. Soluble oligomeric, but not fully fibrillar, α-synuclein is thought to be toxic. The major neuronal target of aberrant α-synuclein might be the synapse. The effects of aberrant α-synuclein might include alteration of calcium homoeostasis or mitochondrial fragmentation and, in turn, mitochondrial dysfunction, which could link α-synuclein dysfunction to recessive and toxin-induced parkinsonism. α-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, α-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 α-synuclein might be targets for therapy.

Cell-produced α-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 Parkinsons Disease, may lead to proteasomal dysfunction. Using PC12 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.

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 Parkinsons 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 Parkinsons disease.

Inducible over‐expression of wild type α‐synuclein in human neuronal cells leads to caspase‐dependent non‐apoptotic death

Alpha‐synuclein (ASYN) is central in Parkinson’s disease pathogenesis. Converging pieces of evidence suggest that the levels of ASYN expression play a critical role in both familial and sporadic Parkinson’s 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.

The small GTPase Rab11 co-localizes with α-synuclein in intracellular inclusions and modulates its aggregation, secretion and toxicity

Alpha-synuclein (aSyn) misfolding and aggregation are pathological features common to several neurodegenerative diseases, including Parkinsons 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.

Deregulation of calcium homeostasis mediates secreted α–synuclein-induced neurotoxicity

α-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 diagnostic value of CSF α-synuclein in the differential diagnosis of dementia with Lewy bodies vs. normal subjects and patients with Alzheimer's disease.

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.

Exocytosis and Spreading of Normal and Aberrant α‐Synuclein

It is now established that α‐synuclein can be physiologically secreted to the extracellular space. In this sense, mechanisms that govern the secretion of the protein may be of importance in the initiation and progress of synucleinopathies. It is possible that increased secretion may aid the formation of toxic seeds extracellularly. Alternatively, reduced presence of extracellular α‐synuclein due to impaired secretion may increase the intracellular load and trigger intracellular seeding. Once outside, α‐synuclein can exert various paracrine actions on neighboring cells again by mechanisms that have not been fully elucidated. It has been demonstrated that, when applied extracellularly, α‐synuclein species can induce multiple neurotoxic and inflammatory responses, and aid the transmission of pathology between neurons. Still, the exact mechanism(s) by which secreted α‐synuclein affects the homeostasis of other neurons is still not well understood. A portion of α‐synuclein has been shown to be associated with the surface and lumen of exosomes which can transfer it to the surrounding cells, and potentially trigger seeding. Interestingly, increased exosome release has been linked to pathological situations of lysosomal dysfunction as observed in Parkinsons disease (PD). However, the possibility that the observed α‐synuclein pathology spread is attributable to the passive diffusion of the initial injected α‐synuclein strains cannot be excluded. Importantly, most of the studies that have so far addressed the role of extracellular α‐synuclein have not employed naturally secreted forms of the protein. It is plausible that deregulation in the normal processing of secreted α‐synuclein may aid the formation of “toxic” species and as such it may also be a causative risk factor for PD. In this capacity, elucidation of the underlying mechanisms that regulate the protein‐levels of extracellular α‐synuclein becomes essential. Such mechanisms could involve its proteolytic clearance from the extracellular milieu.

GABA transmission via ATP-dependent K+ channels regulates α-synuclein secretion in mouse striatum

α-Synuclein is readily released in human and mouse brain parenchyma, even though the normal function of the secreted protein has not been yet elucidated. Under pathological conditions, such as in Parkinsons disease, pathologically relevant species of α-synuclein have been shown to propagate between neurons in a prion-like manner, although the mechanism by which α-synuclein transfer induces degeneration remains to be identified. Due to this evidence extracellular α-synuclein is now considered a critical target to hinder disease progression in Parkinsons disease. Given the importance of extracellular α-synuclein levels, we have now investigated the molecular pathway of α-synuclein secretion in mouse brain. To this end, we have identified a novel synaptic network that regulates α-synuclein release in mouse striatum. In this brain area, the majority of α-synuclein is localized in corticostriatal glutamatergic terminals. Absence of α-synuclein from the lumen of brain-isolated synaptic vesicles suggested that they are unlikely to mediate its release. To dissect the mechanism of α-synuclein release, we have used reverse microdialysis to locally administer reagents that locally target specific cellular pathways. Using this approach, we show that α-synuclein secretion in vivo is a calcium-regulated process that depends on the activation of sulfonylurea receptor 1-sensitive ATP-regulated potassium channels. Sulfonylurea receptor 1 is distributed in the cytoplasm of GABAergic neurons from where the ATP-dependent channel regulates GABA release. Using a combination of specific agonists and antagonists, we were able to show that, in the striatum, modulation of GABA release through the sulfonylurea receptor 1-regulated ATP-dependent potassium channels located on GABAergic neurons controls α-synuclein release from the glutamatergic terminals through activation of the presynaptic GABAB receptors. Considering that sulfonylurea receptors can be selectively targeted, our study highlights the potential use of the key molecules in the α-synuclein secretory pathway to aid the discovery of novel therapeutic interventions for Parkinsons disease.