Α-syn Secretion Research Articles

Human induced pluripotent stem cell-derived dopaminergic neurons release alpha-synuclein through neuronal activity

Lewy body diseases, including Parkinson's disease (PD), are characterized by the spread of alpha-synuclein (αSyn) between neurons across synapses, a process crucial for understanding their pathophysiology and developing effective treatments. In this study, we aimed to investigate the role of neuronal activity in releasing αSyn from human induced pluripotent stem cell-derived dopaminergic neurons. We examined human induced pluripotent stem cell-derived dopaminergic neurons, both healthy and those with the αSyn gene mutation associated with PD. We employed pharmacological agents and optogenetic techniques and demonstrated that increased neuronal activity, induced by bicuculline or optogenetic stimulation, significantly enhances αSyn release. However, suppression of neuronal activity with cyanquixaline reduces αSyn secretion. These findings underscore the pivotal role of neuronal activity in αSyn transmission between neurons, showing its potential impact on the spread of Lewy pathology in patients with neurodegenerative diseases like PD. Therefore, this study advances our understanding of PD and opens new avenues for therapeutic strategies to mitigate Lewy body disease progression.

Neuronal activity promotes secretory autophagy for the extracellular release of α-synuclein

Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it was reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca2+ chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1+/− mice). Glutamate-induced α-syn and p62 secretion was suppressed by knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion was also reduced by knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca2+-dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers.

Mono-UFMylation promotes misfolding-associated secretion of α-synuclein.

Stressed cells secret misfolded proteins lacking signaling sequence via an unconventional protein secretion (UcPS) pathway, but how misfolded proteins are targeted selectively in UcPS is unclear. Here, we report that misfolded UcPS clients are subject to modification by a ubiquitin-like protein named ubiquitin-fold modifier 1 (UFM1). Using α-synuclein (α-Syn) as a UcPS model, we show that mutating the UFMylation sites in α-Syn or genetic inhibition of the UFMylation system mitigates α-Syn secretion, whereas overexpression of UFBP1, a component of the endoplasmic reticulum-associated UFMylation ligase complex, augments α-Syn secretion in mammalian cells and in model organisms. UFM1 itself is cosecreted with α-Syn, and the serum UFM1 level correlates with that of α-Syn. Because UFM1 can be directly recognized by ubiquitin specific peptidase 19 (USP19), a previously established UcPS stimulator known to associate with several chaperoning activities, UFMylation might facilitate substrate engagement by USP19, allowing stringent and regulated selection of misfolded proteins for secretion and proteotoxic stress alleviation.

Cell-to-cell transmitted alpha-synuclein recapitulates experimental Parkinson’s disease

Parkinson’s disease is characterized by a progressive accumulation of alpha-Synuclein (αSyn) neuronal inclusions called Lewy bodies in the nervous system. Lewy bodies can arise from the cell-to-cell propagation of αSyn, which can occur via sequential steps of secretion and uptake. Here, by fusing a removable short signal peptide to the N-terminus of αSyn, we developed a novel mouse model with enhanced αSyn secretion and cell-to-cell transmission. Expression of the secreted αSyn in the mouse brain was under the control of a novel hybrid promoter in combination with adeno-associated virus serotype 9 (AAV9). This combination of promoter and viral vector induced a robust expression in neurons but not in the glia of injected mice. Biochemical characterization of the secreted αSyn revealed that, in cultured cells, this protein is released to the extracellular milieu via conventional secretion. The released αSyn is then internalized and processed by acceptor cells via the endosome–lysosome pathway indicating that the secreted αSyn is cell-to-cell transmitted. The secreted αSyn is aggregation-prone and amyloidogenic, and when expressed in the brain of wild-type non-transgenic mice, it induces a Parkinson’s disease-like phenotype that includes a robust αSyn pathology in the substantia nigra, neuronal loss, neuroinflammation, and motor deficits, all the key features of experimental animal models of Parkinson’s disease. In summary, a novel animal model of Parkinson’s disease based on enhanced cell-to-cell transmission of αSyn was developed. The neuron-produced cell-to-cell transmitted αSyn triggers all phenotypic features of experimental Parkinson’s disease in mice.

IGF2 prevents dopaminergic neuronal loss and decreases intracellular alpha-synuclein accumulation in Parkinson’s disease models

Parkinson’s disease (PD) is the second most common late-onset neurodegenerative disease and the predominant cause of movement problems. PD is characterized by motor control impairment by extensive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). This selective dopaminergic neuronal loss is in part triggered by intracellular protein inclusions called Lewy bodies, which are composed mainly of misfolded alpha-synuclein (α-syn) protein. We previously reported insulin-like growth factor 2 (IGF2) as a key protein downregulated in PD patients. Here we demonstrated that IGF2 treatment or IGF2 overexpression reduced the α-syn aggregates and their toxicity by IGF2 receptor (IGF2R) activation in cellular PD models. Also, we observed IGF2 and its interaction with IGF2R enhance the α-syn secretion. To determine the possible IGF2 neuroprotective effect in vivo we used a gene therapy approach in an idiopathic PD model based on α-syn preformed fibrils intracerebral injection. IGF2 gene therapy revealed a significantly preventing of motor impairment in idiopathic PD model. Moreover, IGF2 expression prevents dopaminergic neuronal loss in the SN together with a decrease in α-syn accumulation (phospho-α-syn levels) in the striatum and SN brain region. Furthermore, the IGF2 neuroprotective effect was associated with the prevention of synaptic spines loss in dopaminergic neurons in vivo. The possible mechanism of IGF2 in cell survival effect could be associated with the decrease of the intracellular accumulation of α-syn and the improvement of dopaminergic synaptic function. Our results identify to IGF2 as a relevant factor for the prevention of α-syn toxicity in both in vitro and preclinical PD models.

Effect of Melatonin on Alpha Synuclein and Autophagy in Dopaminergic Neuronal Differentiation of Adipose Mesenchymal Stem Cells.

The current work investigated the effect of melatonin on differentiation of adipose mesenchymal stem cells (AD-MSCs) into dopamine producing cells and its effect on autophagy process and alpha-Synuclein (α-Syn) secretion. AD-MSCs were characterized by flow cytometry and divided into 4 groups; i) control group (AD-MSCs without any treatment), ii) M+MSCs group (MSCs treated with 1 µM melatonin for 12 days), iii) DN group (MSCs cultured in neurobasal A medium and essential neuronal growth factors for 12 days) and iv) DN+M group (MSCs cultured in neurobasal A medium and 1µM melatonin for 12 days. By the end of experiments, the dopamine and α-Syn levels using ELISA, the expression of MAP-2, m-TOR and α-Syn genes at the level of mRNA and detection of autophagosomes formation using transmission electron microscope were performed. We found that the isolated cells were MSCs due to their positivity expression for CD105 and CD90 and negativity expression for CD34 and CD45. The concentration of dopamine was significantly higher and α-Syn concentration was significantly lower in DN+M group when compared to other groups (P< 0.005). Also, this group showed the highly expression for MAP-2 gene and less expression for m-TOR and α-Syn genes (P< 0.005). Moreover, there was significantly increase in autophagosomes formation in this group than another group (P< 0.005). It is concluded that the melatonin promotes the differentiation of rat AD-MSCs into dopaminergic cells via induction of autophagy process and reduction of α-Syn secretion.

Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers.

Alpha-synuclein (α-syn), a major component of Lewy bodies found in Parkinson's disease (PD) patients, has been found exported outside of cells and may mediate its toxicity via cell-to-cell transmission. Here, we reconstituted soluble, monomeric α-syn secretion by the expression of DnaJ homolog subfamily C member 5 (DNAJC5) in HEK293T cells. DNAJC5 undergoes palmitoylation and anchors on the membrane. Palmitoylation is essential for DNAJC5-induced α-syn secretion, and the secretion is not limited by substrate size or unfolding. Cytosolic α-syn is actively translocated and sequestered in an endosomal membrane compartment in a DNAJC5-dependent manner. Reduction of α-syn secretion caused by a palmitoylation-deficient mutation in DNAJC5 can be reversed by a membrane-targeting peptide fusion-induced oligomerization of DNAJC5. The secretion of endogenous α-syn mediated by DNAJC5 is also found in a human neuroblastoma cell line, SH-SY5Y, differentiated into neurons in the presence of retinoic acid, and in human-induced pluripotent stem cell-derived midbrain dopamine neurons. We propose that DNAJC5 forms a palmitoylated oligomer to accommodate and export α-syn.

α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α

α-synucleinopathy is driven by an imbalance of synthesis and degradation of α-synuclein (αSyn), causing a build up of αSyn aggregates and post-translationally modified species, which not only interfere with normal cellular metabolism but also by their secretion propagates the disease. Therefore, a better understanding of αSyn degradation pathways is needed to address α-synucleinopathy. Here, we used the nerve growth factor-differentiated catecholaminergic PC12 neuronal cell line, which was conferred α-synucleinopathy by inducible expression of αSyn and tubulin polymerization-promoting protein p25α. p25α aggregates αSyn, and imposes a partial autophagosome-lysosome block to mimic aspects of lysosomal deficiency common in neurodegenerative disease. Under basal conditions, αSyn was degraded by multiple pathways but most prominently by macroautophagy and Nedd4/Ndfip1-mediated degradation. We found that expression of p25α induced strong p38MAPK activity. Remarkably, when opposed by inhibitor SB203580 or p38MAPK shRNA knockdown, endolysosomal localization and degradation of αSyn increased, and αSyn secretion and cytotoxicity decreased. This effect was specifically dependent on Hsc70 and the endosomal sorting complex required for transport machinery, but different from classical microautophagy, as the αSyn Hsc70 binding motif was unnecessary. Furthermore, in a primary neuronal (h)-αSyn seeding model, p38MAPK inhibition decreased pathological accumulation of phosphorylated serine-129-αSyn and cytotoxicity. In conclusion, p38MAPK inhibition shifts αSyn degradation from various forms of autophagy to an endosomal sorting complex required for transport-dependent uptake mechanism, resulting in increased αSyn turnover and cell viability in p25α-expressing cells. More generally, our results suggest that under conditions of autophagolysosomal malfunction, the uninterrupted endosomal pathway offers a possibility to achieve disease-associated protein degradation.

SNARE Proteins Mediate α-Synuclein Secretion via Multiple Vesicular Pathways.

The cell-to-cell transmission of pathological α-synuclein (α-syn) has been proposed to be a critical event in the development of synucleinopathies. Recent studies have begun to reveal the underlying molecular mechanism of α-syn propagation. As one of the central steps, α-syn secretion is reported to be Ca2+-dependent and mediated by unconventional exocytosis. However, the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) requirement and vesicle identity of α-syn secretion remain elusive. Here we found that α-syn secretion is SNARE-dependent by systematically knocking down Q-SNAREs and R-SNAREs in exocytosis pathways. α-Syn secretion was mainly mediated by syntaxin 4 (STX4) and synaptosomal-associated protein 23 (SNAP23), but did not require STX1 and SNAP25, in differentiated SH-SY5Y cells. On the other hand, vesicle-associated membrane protein 3 (VAMP3), VAMP7, and VAMP8 were all involved in α-syn secretion, most likely in overlapping pathways. Application of super-resolution microscopy revealed localization of both endogenous and overexpressed α-syn in endosomes, lysosomes, and autophagosomes in rat primary cortical neurons. α-Syn co-localized with microtubule-associated protein 1 light chain 3 (LC3) most extensively, suggesting its tight association with the autophagy pathway. Consistently, α-syn secretion was regulated by the autophagy-lysosome pathway. Collectively, our data suggest that α-syn secretion is SNARE-dependent and is mediated by multiple vesicular pathways including exocytosis of recycling endosomes, multivesicular bodies, autophagosomes, and lysosomes.

Monoamine Oxidase-B Inhibition Facilitates α-Synuclein Secretion In Vitro and Delays Its Aggregation in rAAV-Based Rat Models of Parkinson's Disease.

Cell-to-cell transmission of α-synuclein (α-syn) pathology is considered to underlie the spread of neurodegeneration in Parkinson's disease (PD). Previous studies have demonstrated that α-syn is secreted under physiological conditions in neuronal cell lines and primary neurons. However, the molecular mechanisms that regulate extracellular α-syn secretion remain unclear. In this study, we found that inhibition of monoamine oxidase-B (MAO-B) enzymatic activity facilitated α-syn secretion in human neuroblastoma SH-SY5Y cells. Both inhibition of MAO-B by selegiline or rasagiline and siRNA-mediated knock-down of MAO-B facilitated α-syn secretion. However, TVP-1022, the S-isomer of rasagiline that is 1000 times less active, failed to facilitate α-syn secretion. Additionally, the MAO-B inhibition-induced increase in α-syn secretion was unaffected by brefeldin A, which inhibits endoplasmic reticulum (ER)/Golgi transport, but was blocked by probenecid and glyburide, which inhibit ATP-binding cassette (ABC) transporter function. MAO-B inhibition preferentially facilitated the secretion of detergent-insoluble α-syn protein and decreased its intracellular accumulation under chloroquine-induced lysosomal dysfunction. Moreover, in a rat model (male Sprague Dawley rats) generated by injecting recombinant adeno-associated virus (rAAV)-A53T α-syn, subcutaneous administration of selegiline delayed the striatal formation of Ser129-phosphorylated α-syn aggregates, and mitigated loss of nigrostriatal dopaminergic neurons. Selegiline also delayed α-syn aggregation and dopaminergic neuronal loss in a cell-to-cell transmission rat model (male Sprague Dawley rats) generated by injecting rAAV-wild-type α-syn and externally inoculating α-syn fibrils into the striatum. These findings suggest that MAO-B inhibition modulates the intracellular clearance of detergent-insoluble α-syn via the ABC transporter-mediated non-classical secretion pathway, and temporarily suppresses the formation and transmission of α-syn aggregates.SIGNIFICANCE STATEMENT The identification of a neuroprotective agent that slows or stops the progression of motor impairments is required to treat Parkinson's disease (PD). The process of α-synuclein (α-syn) aggregation is thought to underlie neurodegeneration in PD. Here, we demonstrated that pharmacological inhibition or knock-down of monoamine oxidase-B (MAO-B) in SH-SY5Y cells facilitated α-syn secretion via a non-classical pathway involving an ATP-binding cassette (ABC) transporter. MAO-B inhibition preferentially facilitated secretion of detergent-insoluble α-syn protein and reduced its intracellular accumulation under chloroquine-induced lysosomal dysfunction. Additionally, MAO-B inhibition by selegiline protected A53T α-syn-induced nigrostriatal dopaminergic neuronal loss and suppressed the formation and cell-to-cell transmission of α-syn aggregates in rat models. We therefore propose a new function of MAO-B inhibition that modulates α-syn secretion and aggregation.

Analysis of α-synuclein levels related to LRRK2 kinase activity: from substantia nigra to urine of patients with Parkinson’s disease

ABSTRACT Research on Parkinson’s disease (PD) has been focused on the development of PD diagnostic tools as much as the development of PD therapeutics. Several genetic culprits of PD, including DJ-1, Leucine-rich repeat kinase 2 (LRRK2), and α-synuclein (α-syn), have been investigated as markers of PD in human biofluids. Unfortunately, the approaches to develop PD diagnostic tools are impractical, and there is a considerable demand for an appropriate marker of PD. The measurement of α-syn in biofluids has recently been made more accurate by examining monomers and aggregates separately using enzyme-linked immunosorbent assay (ELISA). Previously, we reported on the development of two types of sandwich ELISA for total α-syn and MJFR-14-6-4-2 antibody-specific α-syn fibrillar oligomers. The pathogenic LRRK2 G2019S mutation is related to increased α-syn secretion in the extracellular space. We tested our established ELISA using differentiated SH-SH5Y cells transfected with LRRK2 G2019S. The secretory levels of fibrillar oligomeric α-syn divided by total α-syn were significantly increased in LRRK2 G2019S-expressing cells. Additionally, substantia nigra lysates or concentrated urine from PD patients and non-PD subjects were analyzed. We observed ambiguous changes in the levels of total or fibrillar oligomeric α-syn and their ratio between PD and non-PD. Despite the insignificant increase in the relative levels of fibrillar oligomeric α-syn to total α-syn in PD, the duration of disease progression after diagnosis significantly corresponded to the relative levels of fibrillar oligomeric α-syn to total α-syn in the urine. These results might provide greater understanding for the next stage of development of α-syn ELISAs.

Prolyl oligopeptidase inhibition by KYP-2407 increases alpha-synuclein fibril degradation in neuron-like cells

Growing evidence emphasizes insufficient clearance of pathological alpha-synuclein (αSYN) aggregates in the progression of Parkinson’s disease (PD). Consequently, cellular degradation pathways represent a potential therapeutic target. Prolyl oligopeptidase (PREP) is highly expressed in the brain and has been suggested to increase αSYN aggregation and negatively regulate the autophagy pathway. Inhibition of PREP with a small molecule inhibitor, KYP-2407, stimulates autophagy and reduces the oligomeric species of αSYN aggregates in PD mouse models. However, whether PREP inhibition has any effects on intracellular αSYN fibrils has not been studied before. In this study, the effect of KYP2407 on αSYN preformed fibrils (PFFs) was tested in SH-SY5Y cells and human astrocytes. Immunostaining analysis revealed that both cell types accumulated αSYN PFFs intracellularly but KYP-2047 decreased intracellular αSYN deposits only in SH-SY5Y cells, as astrocytes did not show any PREP activity. Western blot analysis confirmed the reduction of high molecular weight αSYN species in SH-SY5Y cell lysates, and secretion of αSYN from SH-SY5Y cells also decreased in the presence of KYP-2407. Accumulation of αSYN inside the SH-SY5Y cells resulted in an increase of the auto-lysosomal proteins p62 and LC3BII, as well as calpain 1 and 2, which have been shown to be associated with PD pathology. Notably, treatment with KYP-2407 significantly reduced p62 and LC3BII levels, indicating an increased autophagic flux, and calpain 1 and 2 levels returned to normal in the presence of KYP-2407. Our findings indicate that PREP inhibition can potentially be used as therapy to reduce the insoluble intracellular αSYN aggregates.

Pathogenic Mutations Differentially Regulate Cell-to-Cell Transmission of α-Synuclein.

Recent studies suggest that the cell-to-cell spread of pathological α-synuclein (α-syn) plays important roles in the development of Parkinson’s disease (PD). PD patients who carry α-syn gene mutations often have an earlier onset and more severe clinical symptoms and pathology than sporadic PD cases who carry the wild-type (WT) α-syn gene. However, the molecular mechanism by which α-syn gene mutations promote PD remains unclear. Here, we hypothesized that pathogenic mutations facilitate the intercellular transfer and cytotoxicity of α-syn, favoring an early disease onset and faster progression. We investigated the effects of eight known pathogenic mutations in human α-syn (A18T, A29S, A30P, E46K, H50Q, G51D, A53E, and A53T) on its pathological transmission in terms of secretion, aggregation, intracellular level, cytotoxicity, seeding, and induction of neuroinflammation in SH-SY5Y neuroblastoma cells, cultured rat neurons, and microglia, and the rat substantia nigra pars compacta. We found that 2 of the 8 mutations (H50Q and A53T) significantly increased α-syn secretion while 6 mutations (A18T, A29S, A30P, G51D, A53E, and E46K) tended to enhance it. In vitroα-syn aggregation experiments showed that H50Q promoted while G51D delayed aggregation most strongly. Interestingly, 3 mutations (E46K, H50Q, and G51D) greatly increased the intracellular α-syn level when cultured cells were treated with preformed α-syn fibrils (PFFs) compared with the WT, while the other 5 had no effect. We also demonstrated that H50Q, G51D, and A53T PFFs, but not E46K PFFs, efficiently seeded in vivo and acutely induced neuroinflammation in rat substantia nigra pars compacta. Our data indicate that pathogenic mutations augment the prion-like spread of α-syn at different steps and blockade of this pathogenic propagation may serve as a promising therapeutic intervention for PD.

Voltage-Gated Calcium Channels and α-Synuclein: Implications in Parkinson's Disease.

Alpha-synuclein (α-syn) is biochemically and genetically linked to Parkinson’s disease (PD) and other synucleinopathies. It is now widely accepted that α-syn can be released in the extracellular space, even though the mechanism of its release is still unclear. In addition, pathology-related aggregated species of α-syn have been shown to propagate between neurons in synaptically connected areas of the brain thereby assisting the spreading of pathology in healthy neighboring neuronal cells. In neurons, calcium channels are key signaling elements that modulate the release of bioactive molecules (hormones, proteins, and neurotransmitters) through calcium sensing. Such calcium sensing activity is determined by the distinct biophysical and pharmacological properties and the ability of calcium channels to interact with other modulatory proteins. Although the function of extracellular α-syn is currently unknown, previous work suggested the presence of a calcium-dependent mechanism for α-syn secretion both in vitro, in neuronal cells in culture, and also in vivo, in the context of a trans-neuronal network in brain. Mechanisms regulating extracellular α-syn levels may be of particular importance as they could represent novel therapeutic targets. We discuss here how calcium channel activity may contribute to α-syn aggregation and secretion as a pathway to disease progression in synucleinopathies.

Increased Lysosomal Exocytosis Induced by Lysosomal Ca2+ Channel Agonists Protects Human Dopaminergic Neurons from α-Synuclein Toxicity.

The accumulation of misfolded proteins is a common pathological feature of many neurodegenerative disorders, including synucleinopathies such as Parkinson's disease (PD), which is characterized by the presence of α-synuclein (α-syn)-containing Lewy bodies. However, although recent studies have investigated α-syn accumulation and propagation in neurons, the molecular mechanisms underlying α-syn transmission have been largely unexplored. Here, we examined a monogenic form of synucleinopathy caused by loss-of-function mutations in lysosomal ATP13A2/PARK9. These studies revealed that lysosomal exocytosis regulates intracellular levels of α-syn in human neurons. Loss of PARK9 function in patient-derived dopaminergic neurons disrupted lysosomal Ca2+ homeostasis, reduced lysosomal Ca2+ storage, increased cytosolic Ca2+, and impaired lysosomal exocytosis. Importantly, this dysfunction in lysosomal exocytosis impaired α-syn secretion from both axons and soma, promoting α-syn accumulation. However, activation of the lysosomal Ca2+ channel transient receptor potential mucolipin 1 (TRPML1) was sufficient to upregulate lysosomal exocytosis, rescue defective α-syn secretion, and prevent α-syn accumulation. Together, these results suggest that intracellular α-syn levels are regulated by lysosomal exocytosis in human dopaminergic neurons and may represent a potential therapeutic target for PD and other synucleinopathies.SIGNIFICANCE STATEMENT Parkinson's disease (PD) is the second most common neurodegenerative disease linked to the accumulation of α-synuclein (α-syn) in patient neurons. However, it is unclear what the mechanism might be. Here, we demonstrate a novel role for lysosomal exocytosis in clearing intracellular α-syn and show that impairment of this pathway by mutations in the PD-linked gene ATP13A2/PARK9 contributes to α-syn accumulation in human dopaminergic neurons. Importantly, upregulating lysosomal exocytosis by increasing lysosomal Ca2+ levels was sufficient to rescue defective α-syn secretion and accumulation in patient neurons. These studies identify lysosomal exocytosis as a potential therapeutic target in diseases characterized by the accumulation of α-syn, including PD.

Manganese promotes the aggregation and prion-like cell-to-cell exosomal transmission of α-synuclein.

The aggregation of α-synuclein (αSyn) is considered a key pathophysiological feature of certain neurodegenerative disorders, collectively termed synucleinopathies. Given that a prion-like, cell-to-cell transfer of misfolded αSyn has been recognized in the spreading of αSyn pathology in synucleinopathies, we investigated the biological mechanisms underlying the propagation of the disease with respect to environmental neurotoxic stress. Considering the potential role of the divalent metal manganese (Mn2+) in protein aggregation, we characterized its effect on αSyn misfolding and transmission in experimental models of Parkinson's disease. In cultured dopaminergic neuronal cells stably expressing wild-type human αSyn, misfolded αSyn was secreted through exosomes into the extracellular medium upon Mn2+ exposure. These exosomes were endocytosed through caveolae into primary microglial cells, thereby mounting neuroinflammatory responses. Furthermore, Mn2+-elicited exosomes exerted a neurotoxic effect in a human dopaminergic neuronal model (LUHMES cells). Moreover, bimolecular fluorescence complementation (BiFC) analysis revealed that Mn2+ accelerated the cell-to-cell transmission of αSyn, resulting in dopaminergic neurotoxicity in a mouse model of Mn2+ exposure. Welders exposed to Mn2+ had increased misfolded αSyn content in their serum exosomes. Stereotaxically delivering αSyn-containing exosomes, isolated from Mn2+-treated αSyn-expressing cells, into the striatum initiated Parkinsonian-like pathological features in mice. Together, these results indicate that Mn2+ exposure promotes αSyn secretion in exosomal vesicles, which subsequently evokes proinflammatory and neurodegenerative responses in both cell culture and animal models.

Microglia as modulators of exosomal alpha-synuclein transmission

Recent researches regarding to exosomal involvement in alpha-synuclein (α-syn) transmission relating to the pathological process of Parkinson’s disease (PD) have attracted considerable attention. It is highly desirable to make clear the diffusion process and cellular uptake of α-syn-associated exosomes and the underlying mechanism of exosomes-involved communication in the synucleinopathy pathogenesis. To determine the contribution of α-syn-associated exosomes to the initiation and progression of PD, plasma exosomes derived from PD patients were stereotaxically injected into the striatum of mice brains. Exosomes extracted from plasma diagnosed with PD contained monomeric and oligomeric α-syn. Here, we found that microglia display a high potency for uptake of plasma exosomes derived from PD patients, and therefore could be activated by exogenous exosomes in vitro and in vivo. In addition, immunofluorescent double staining verified the transfer of exogenous human exosomal α-syn to neurons. The release of human exosomal α-syn from microglia may facilitate this propagation. Finally, we described a mechanism underlying this potential role of microglia in the transmission of exosomal α-syn. Specifically, exogenous exosomes were found to dysregulate autophagy of the BV2 mouse microglia cell line with presentation of increased accumulation of intracellular α-syn and accelerated secretion of α-syn into extracellular space. These results suggest that microglia play a crucial role in the transmission of α-syn via exosomal pathways, in additional to idea that the progression of PD may be altered by the modulation of exosome secretion and/or microglial states.

The interplay between parkin and alpha-synuclein; possible implications for the pathogenesis of Parkinson’s disease

Parkin and alpha-synuclein (α-syn) are two key proteins involved in the pathophysiology of Parkinson's disease (PD). Oligomerization/aggregation and excessive secretion of α-syn contributes to PD through free radical stress, mitochondrial impairment, and synaptic dysfunction. Parkin, an E3 ubiquitin ligase, is considered to be a pleiotropic, neuroprotective protein that modulates metabolic turnover and the accumulation of α-syn. This is in addition to parkin's role in counteracting the more distant effects of α-syn on cellular survival by altering proteasomal, autophagic, and calpain-mediated protein degradation pathways that can reduce α-syn levels. Moreover, parkin regulates mitochondrial turnover, cell survival, and immune phenomena - processes that are all known to be disturbed in PD. In addition, parkin might have an impact on the spreading and propagation of α-syn by controlling its post-translational modifications. On the other hand, recent research has shown that α-syn oligomers affect the expression, post-translational modification, and activity of parkin. This review focuses on the molecular mechanisms of cross-talk between parkin and α-syn in PD. The physical and functional interactions between α-syn and parkin, which have been incompletely characterized to-date, may present a new therapeutic avenue in PD and related synucleinopathies. The development of effective, clinically feasible modulators may offer great hopes for the the rapy of PD.

Secretion and Uptake of \u03b1-Synuclein Via Extracellular Vesicles in Cultured Cells

In Parkinson’s disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular α-synuclein (α-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different α-syn variants, including α-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted α-syn, 0.1–2% was associated with vesicles. The major part of EV α-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For α-syn expressed with N-terminal hemi-Venus, the relative levels associated with EVs were higher than for WT α-syn. Moreover, such EV-associated α-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T α-syn displayed an increased association with EVs. Taken together, our data suggest that α-syn species with presumably lost physiological functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful α-syn species and thereby contribute to the pathology in α-synucleinopathies.

The Golgi-localized, gamma ear-containing, ARF-binding (GGA) protein family alters alpha synuclein (α-syn) oligomerization and secretion.

Several age-related neurodegenerative disorders are associated with protein misfolding and aggregation of toxic peptides. α-synuclein (α-syn) aggregation and the resulting cytotoxicity is a hallmark of Parkinson's disease (PD) as well as dementia with Lewy bodies. Rising evidence points to oligomeric and pre-fibrillar forms as the pathogenic species, and oligomer secretion seems to be crucial for the spreading and progression of PD pathology. Recent studies implicate that dysfunctions in endolysosomal/autophagosomal pathways increase α-syn secretion. Mutation in the retromer-complex protein VPS35, which is involved in endosome to Golgi transport, was suggested to cause familial PD. GGA proteins regulate vesicular traffic between Golgi and endosomes and might work as antagonists for retromer complex mediated transport. To investigate the role of the GGAs in the α-syn oligomerization and/or secretion process we utilized protein-fragment complementation assays (PCA). We here demonstrate that GGAs alter α-syn oligomer secretion and α-syn oligomer-mediated toxicity. Specifically, we determined that GGA3 modifies extracellular α-syn species in an exosome-independent manner. Our data suggest that GGA3 drives α-syn oligomerization in endosomal compartments and thus facilitates α-syn oligomer secretion. Preventing the early events in α-syn oligomer release may be a novel approach to halt disease spreading in PD and other synucleinopathies.