Quaternary structure of patient-homogenate amplified α-synuclein fibrils modulates seeding of endogenous α-synuclein.

Parkinson's disease (PD) and Multiple System Atrophy (MSA) are progressive and unremitting neurological diseases that are neuropathologically characterized by α-synuclein inclusions. Increasing evidence supports the aggregation of α-synuclein in specific brain areas early in the disease course, followed by the spreading of α-synuclein pathology to multiple brain regions. However, little is known about how the structure of α-synuclein fibrils influence its ability to seed endogenous α-synuclein in recipient cells. Here, we aggregated α-synuclein by seeding with homogenates of PD- and MSA-confirmed brain tissue, determined the resulting α-synuclein fibril structures by cryo-electron microscopy, and characterized their seeding potential in mouse primary oligodendroglial cultures. The combined analysis shows that the two patient material-amplified α-synuclein fibrils share a similar protofilament fold but differ in their inter-protofilament interface and their ability to recruit endogenous α-synuclein. Our study indicates that the quaternary structure of α-synuclein fibrils modulates the seeding of α-synuclein pathology inside recipient cells. It thus provides an important advance in the quest to understand the connection between the structure of α-synuclein fibrils, cellular seeding/spreading, and ultimately the clinical manifestations of different synucleinopathies.

Exosomes in Alpha-Synucleinopathies: Propagators of Pathology or Potential Candidates for Nanotherapeutics?

The pathological accumulation of alpha-synuclein governs the pathogenesis of neurodegenerative disorders, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, collectively termed alpha-synucleinopathies. Alpha-synuclein can be released in the extracellular space, partly via exosomes, and this extracellular protein pool may contribute to disease progression by facilitating the spread of pathological alpha-synuclein or activating immune cells. The content of exosomes depends on their origin and includes specific proteins, lipids, functional mRNAs and various non-coding RNAs. Given their ability to mediate intercellular communication via the transport of multilevel information, exosomes are considered to be transporters of toxic agents. Beyond neurons, glial cells also release exosomes, which may contain inflammatory molecules and this glia-to-neuron or neuron-to-glia transmission of exosomal alpha-synuclein may contribute to the propagation of pathology and neuroinflammation throughout the brain. In addition, as their content varies as per their originating and recipient cells, these vesicles can be utilized as a diagnostic biomarker for early disease detection, whereas targeted exosomes may be used as scaffolds to deliver therapeutic agents into the brain. This review summarizes the current knowledge regarding the role of exosomes in the progression of alpha-synuclein-related pathology and their potential use as biomarkers and nanotherapeutics in alpha-synucleinopathies.

Autophagy mediates the clearance of oligodendroglial SNCA/alpha-synuclein and TPPP/p25A in multiple system atrophy models.

Accumulation of the neuronal protein SNCA/alpha-synuclein and of the oligodendroglial phosphoprotein TPPP/p25A within the glial cytoplasmic inclusions (GCIs) represents the key histophathological hallmark of multiple system atrophy (MSA). Even though the levels/distribution of both oligodendroglial SNCA and TPPP/p25A proteins are critical for disease pathogenesis, the proteolytic mechanisms involved in their turnover in health and disease remain poorly understood. Herein, by pharmacological and molecular modulation of the autophagy-lysosome pathway (ALP) and the proteasome we demonstrate that the endogenous oligodendroglial SNCA and TPPP/p25A are degraded mainly by the ALP in murine primary oligodendrocytes and oligodendroglial cell lines under basal conditions. We also identify a KFERQ-like motif in the TPPP/p25A sequence that enables its effective degradation via chaperone-mediated autophagy (CMA) in an in vitro system of rat brain lysosomes. Furthermore, in a MSA-like setting established by addition of human recombinant SNCA pre-formed fibrils (PFFs) as seeds of pathological SNCA, we thoroughly characterize the contribution of CMA and macroautophagy in particular, in the removal of the exogenously added and the seeded oligodendroglial SNCA pathological assemblies. We also show that PFF treatment impairs autophagic flux and that TPPP/p25A exerts an inhibitory effect on macroautophagy, while at the same time CMA is upregulated to remove the pathological SNCA species formed within oligodendrocytes. Finally, augmentation of CMA or macroautophagy accelerates the removal of the engendered pathological SNCA conformations further suggesting that autophagy targeting may represent a successful approach for the clearance of pathological SNCA and/or TPPP/p25A in the context of MSA.Abbreviations: 3MA: 3-methyladenine; ACTB: actin, beta; ALP: autophagy-lysosome pathway; ATG5: autophagy related 5; AR7: atypical retinoid 7; CMA: chaperone-mediated autophagy; CMV: cytomegalovirus; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; Epox: epoxomicin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCIs: glial cytoplasmic inclusions; GFP: green fluorescent protein; HMW: high molecular weight; h: hours; HSPA8/HSC70: heat shock protein 8; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mcherry: monomeric cherry; MFI: mean fluorescence intensity; mRFP: monomeric red fluorescent protein; MSA: multiple system atrophy; OLN: oligodendrocytes; OPCs: oligodendroglial progenitor cells; PBS: phosphate-buffered saline; PC12: pheochromocytoma cell line; PD: Parkinson disease; PFFs: pre-formed fibrils; PIs: protease inhibitors; PSMB5: proteasome (prosome, macropain) subunit, beta type 5; Rap: rapamycin; RFP: red fluorescent protein; Scr: scrambled; SDS: sodium dodecyl sulfate; SE: standard error; siRNAs: small interfering RNAs; SNCA: synuclein, alpha; SQSTM1: sequestosome 1; TPPP: tubulin polymerization promoting protein; TUBA: tubulin, alpha; UPS: ubiquitin-proteasome system; WT: wild type.

Neurons and Glia Interplay in α-Synucleinopathies.

Accumulation of the neuronal presynaptic protein alpha-synuclein within proteinaceous inclusions represents the key histophathological hallmark of a spectrum of neurodegenerative disorders, referred to by the umbrella term a-synucleinopathies. Even though alpha-synuclein is expressed predominantly in neurons, pathological aggregates of the protein are also found in the glial cells of the brain. In Parkinson's disease and dementia with Lewy bodies, alpha-synuclein accumulates mainly in neurons forming the Lewy bodies and Lewy neurites, whereas in multiple system atrophy, the protein aggregates mostly in the glial cytoplasmic inclusions within oligodendrocytes. In addition, astrogliosis and microgliosis are found in the synucleinopathy brains, whereas both astrocytes and microglia internalize alpha-synuclein and contribute to the spread of pathology. The mechanisms underlying the pathological accumulation of alpha-synuclein in glial cells that under physiological conditions express low to non-detectable levels of the protein are an area of intense research. Undoubtedly, the presence of aggregated alpha-synuclein can disrupt glial function in general and can contribute to neurodegeneration through numerous pathways. Herein, we summarize the current knowledge on the role of alpha-synuclein in both neurons and glia, highlighting the contribution of the neuron-glia connectome in the disease initiation and progression, which may represent potential therapeutic target for a-synucleinopathies.

In Search of Effective Treatments Targeting α-Synuclein Toxicity in Synucleinopathies: Pros and Cons.

Parkinson's disease (PD), multiple system atrophy (MSA) and Dementia with Lewy bodies (DLB) represent pathologically similar, progressive neurodegenerative disorders characterized by the pathological aggregation of the neuronal protein α-synuclein. PD and DLB are characterized by the abnormal accumulation and aggregation of α-synuclein in proteinaceous inclusions within neurons named Lewy bodies (LBs) and Lewy neurites (LNs), whereas in MSA α-synuclein inclusions are mainly detected within oligodendrocytes named glial cytoplasmic inclusions (GCIs). The presence of pathologically aggregated α-synuclein along with components of the protein degradation machinery, such as ubiquitin and p62, in LBs and GCIs is considered to underlie the pathogenic cascade that eventually leads to the severe neurodegeneration and neuroinflammation that characterizes these diseases. Importantly, α-synuclein is proposed to undergo pathogenic misfolding and oligomerization into higher-order structures, revealing self-templating conformations, and to exert the ability of "prion-like" spreading between cells. Therefore, the manner in which the protein is produced, is modified within neural cells and is degraded, represents a major focus of current research efforts in the field. Given that α-synuclein protein load is critical to disease pathogenesis, the identification of means to limit intracellular protein burden and halt α-synuclein propagation represents an obvious therapeutic approach in synucleinopathies. However, up to date the development of effective therapeutic strategies to prevent degeneration in synucleinopathies is limited, due to the lack of knowledge regarding the precise mechanisms underlying the observed pathology. This review critically summarizes the recent developed strategies to counteract α-synuclein toxicity, including those aimed to increase protein degradation, to prevent protein aggregation and cell-to-cell propagation, or to engage antibodies against α-synuclein and discuss open questions and unknowns for future therapeutic approaches.

Correction to: Endogenous oligodendroglial alpha-synuclein and TPPP/p25α orchestrate alpha-synuclein pathology in experimental multiple system atrophy models.

he original version of this article.

Endogenous oligodendroglial alpha-synuclein and TPPP/p25α orchestrate alpha-synuclein pathology in experimental multiple system atrophy models.

Multiple system atrophy (MSA) is characterized by the presence of distinctive glial cytoplasmic inclusions (GCIs) within oligodendrocytes that contain the neuronal protein alpha-synuclein (aSyn) and the oligodendroglia-specific phosphoprotein TPPP/p25α. However, the role of oligodendroglial aSyn and p25α in the formation of aSyn-rich GCIs remains unclear. To address this conundrum, we have applied human aSyn (haSyn) pre-formed fibrils (PFFs) to rat wild-type (WT)-, haSyn-, or p25α-overexpressing oligodendroglial cells and to primary differentiated oligodendrocytes derived from WT, knockout (KO)-aSyn, and PLP-haSyn-transgenic mice. HaSyn PFFs are readily taken up by oligodendroglial cells and can recruit minute amounts of endogenous aSyn into the formation of insoluble, highly aggregated, pathological assemblies. The overexpression of haSyn or p25α accelerates the recruitment of endogenous protein and the generation of such aberrant species. In haSyn PFF-treated primary oligodendrocytes, the microtubule and myelin networks are disrupted, thus recapitulating a pathological hallmark of MSA, in a manner totally dependent upon the seeding of endogenous aSyn. Furthermore, using oligodendroglial and primary cortical cultures, we demonstrated that pathology-related S129 aSyn phosphorylation depends on aSyn and p25α protein load and may involve different aSyn "strains" present in oligodendroglial and neuronal synucleinopathies. Importantly, this hypothesis was further supported by data obtained from human post-mortem brain material derived from patients with MSA and dementia with Lewy bodies. Finally, delivery of haSyn PFFs into the mouse brain led to the formation of aberrant aSyn forms, including the endogenous protein, within oligodendroglia and evoked myelin decompaction in WT mice, but not in KO-aSyn mice. This line of research highlights the role of endogenous aSyn and p25α in the formation of pathological aSyn assemblies in oligodendrocytes and provides in vivo evidence of the contribution of oligodendroglial aSyn in the establishment of aSyn pathology in MSA.

Impairment of chaperone-mediated autophagy affects neuronal homeostasis through altered expression of DJ-1 and CRMP-2 proteins.

Chaperone-mediated autophagy (CMA) is a substrate-specific mode of lysosomal proteolysis, with multiple lines of evidence connecting its dysfunction to both ageing and disease. We have recently shown that CMA impairment through knock-down of the lysosomal receptor LAMP2A is detrimental to neuronal viability in vivo; however, it is not clear which subset of proteins regulated by the CMA pathway mediate such changes. In this study, we have manipulated CMA function through alterations of LAMP2A abundance in primary rat cortical neurons, to identify potential changes to the neuronal proteome occurring prior to neurotoxic effects. We have identified a list of proteins with significant, >2-fold change in abundance following our manipulations, of which PARK7/DJ-1 - an anti-oxidant implicated in hereditary forms of Parkinson's Disease (PD), and DPYSL2/CRMP-2 - a microtubule-binding phosphoprotein involved in schizophrenia pathogenesis - were both found to have measurable effects on neuronal homeostasis and phenotype. Taken together, this study describes alterations in the abundance of neuronal proteins involved in neuropsychiatric disorders upon CMA manipulation, and suggests that such alterations may in part be responsible for the neurodegeneration observed upon CMA impairment in vivo.

Oxygen and Glucose Deprivation Alter Synaptic Distribution of Tau Protein: The Role of Phosphorylation.

Alterations in tau synaptic distribution are considered to underlie synaptic dysfunction observed in Alzheimer's disease (AD). In the present study, brain blood hypoperfusion was simulated in mouse brain slices, and tau levels and phosphorylation were investigated in total extracts, as well as in postsynaptic density fractions (PSDs) and non-PSDs obtained through differential extraction and centrifugation. Oxygen deprivation (OD) resulted in tau dephosphorylation at several AD-related residues and activation of GSK3ß and phosphatase PP2A. On the contrary, glucose deprivation (GD) did not affect total levels of cellular tau or its phosphorylation despite inactivation of GSK3ß. However, tau distribution in PSD and non-PSD fractions and the pattern of tau phosphorylation in these compartments is highly complex. In PSDs, tau was increased under GD conditions and decreased under OD conditions. GD resulted in tau dephosphorylation at Ser199, Ser262, and Ser396 while OD resulted in tau hyperphosphorylation at Ser199 and Ser404. In the non-PSD fraction, GD or OD resulted in lower levels of tau, but the phosphorylation status of tau was differentially affected. In GD conditions, tau was found dephosphorylated at Ser199, Thr205, and Ser404 and hyperphosphorylated at Ser262. However, in OD conditions tau was found hyperphosphorylated at Thr205, SerSer356, Ser396, and Ser404. Combined OD and GD resulted in degradation of cellular tau and dephosphorylation of PSD tau at Ser396 and Ser404. These results indicate that oxygen deprivation causes dephosphorylation of tau, while GD and OD differentially affect distribution of total tau and tau phosphorylation variants in neuronal compartments by activating different mechanisms.

Differential effects of calcium on PI3K-Akt and HIF-1α survival pathways.

Calcium signaling participates in the regulation of numberless cellular functions including cell cycle progression and cellular migration, important processes for cancer expansion. Cancer cell growth, migration, and invasion are typically supported by PI3K/Akt activation, while a hypoxic environment is critical in cancer development. Accordingly, in the present study, we aimed at investigating whether perturbations in calcium homeostasis induce alterations of HIF-1α and activate Akt levels in epithelial A549 and A431 cells. Survival was drastically reduced in the presence of calcium chelator BAPTA-AM and thapsigargin, a SERCA inhibitor inducing store-operated calcium entry, to a lesser extent. Calcium chelation provoked a transient but strong upregulation of HIF-1α protein levels and accumulation in the nucleus, whereas in the presence of thapsigargin, HIF-1α levels were rapidly abolished before reaching and exceeding control levels. Despite cell death, calcium chelation merely inhibited Akt, which was significantly activated in the presence of thapsigargin. Moreover, when store-operated calcium entry was simulated by reintroducing calcium ions in cell suspensions, Akt was rapidly activated in the absence of any growth factor. These data further underscore the growing importance of calcium entry and directly link this elementary event of calcium homeostasis to the Akt pathway, which is commonly deregulated in cancer.

Comparative assessment of HIF-1α and Akt responses in human lung and skin cells exposed to benzo[α]pyrene: Effect of conditioned medium from pre-exposed primary fibroblasts.

Exposure to atmospheric pollutants has been accused for many adverse health effects. Benzo[α]pyrene (Β[α]Ρ) in particular, the most extensively studied member of pollutants, is implicated in both cancer initiation and promotion. In the present study, we compared the effects of noncytotoxic doses of Β[α]Ρ, between human skin and lung epithelial cells A431 and A549, respectively, focusing on Akt kinase and HIF-1α, as it is well known that these proteins are upregulated in various human cancers promoting survival, angiogenesis and metastasis of tumor cells. Also, taking into consideration that fibroblasts are involved in cancer progression, we tested the possible modulation of epithelial cell response by paracrine factors secreted by Β[α]Ρ-treated fibroblasts. Low doses of Β[α]Ρ were found to enhance epithelial cell proliferation and upregulate both Akt kinase and HIF-1α, with A549 cells exhibiting a more sustained profile of upregulation. It is to notice that, the response of HIF-1α was remarkably early, acting as a sensitive marker in response to airborne pollutants. Also, HIF-1α was induced by Β[α]Ρ in both lung and skin fibroblasts indicating that this effect may be conserved throughout different cell types and tissues. Interestingly however, the response of both proteins was differentially modified upon treatment with conditioned medium from Β[α]Ρ-exposed fibroblasts. This is particularly evident in A459 cells and confirms the critical role of intercellular and paracrine factors in the modulation of the final response to an extracellular signal. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1103-1112, 2016.