[Mechanisms Underlying the Propagation of α-Synuclein Seeds Derived from the Blood of Patients with α-Synucleinopathies].

The aggregation of α-synuclein (α-syn), associated with Parkinson's disease (PD) extends from the peripheral autonomic nervous system to the cerebral cortex, indicating a neural circuit-based mechanism of spread. However, recent studies, have proposed alternative propagation routes beyond neural pathways, including transmission via bodily fluids, such as the blood. This notion expands our understanding of PD progression, underscoring the complexity of α-syn spread and its implications in disease management and therapeutic strategies.

Alpha-synuclein aggregates are phosphatase resistant.

Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.

A multiverse of α-synuclein: investigation of prion strain properties with carboxyl-terminal truncation specific antibodies in animal models.

Synucleinopathies are a group of neurodegenerative disorders characterized by the presence of misfolded α-Synuclein (αSyn) in the brain. These conditions manifest with diverse clinical and pathophysiological characteristics. This disease diversity is hypothesized to be driven by αSyn strains with differing biophysical properties, potentially influencing prion-type propagation and consequentially the progression of illness. Previously, we investigated this hypothesis by injecting brain lysate (seeds) from deceased individuals with various synucleinopathies or human recombinant αSyn preformed fibrils (PFFs) into transgenic mice overexpressing either wild type or A53T human αSyn. In the studies herein, we expanded on these experiments, utilizing a panel of antibodies specific for the major carboxyl-terminally truncated forms of αSyn (αSynΔC). These modified forms of αSyn are found enriched in human disease brains to inform on potential strain-specific proteolytic patterns. With monoclonal antibodies specific for human αSyn cleaved at residues 103, 114, 122, 125, and 129, we demonstrate that multiple system atrophy (MSA) seeds and PFFs induce differing neuroanatomical spread of αSyn pathology associated with host specific profiles. Overall, αSyn cleaved at residue 103 was most widely present in the induced pathological inclusions. Furthermore, αSynΔC-positive inclusions were present in astrocytes, but more frequently in activated microglia, with patterns dependent on host and inoculum. These findings support the hypothesis that synucleinopathy heterogeneity might stem from αSyn strains with unique biochemical properties that include proteolytic processing, which could result in dominant strain properties.

Parkinson's Disease and Mitotherapy-Based Approaches towards α-Synucleinopathies.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta region of the midbrain and the formation of intracellular protein aggregates known as Lewy bodies, of which a major component is the protein α-synuclein. Several studies have suggested that mitochondria play a central role in the pathogenesis of PD, encompassing both familial and sporadic forms of the disease. Mitochondrial dysfunction is attributed to bioenergetic impairment, increased oxidative stress, damage to mitochondrial DNA, and alteration in mitochondrial morphology. These alterations may contribute to improper functioning of the central nervous system and ultimately lead to neurodegeneration. The perturbation of mitochondrial function makes it a potential target, worthy of exploration for neuroprotective therapies and to improve mitochondrial health in PD. Thus, in the current review, we provide an update on mitochondria-based therapeutic approaches toward α-synucleinopathies in PD.

Positron Emission Tomography with [18F]ROStrace Reveals Progressive Elevations in Oxidative Stress in a Mouse Model of Alpha-Synucleinopathy.

The synucleinopathies are a diverse group of neurodegenerative disorders characterized by the accumulation of aggregated alpha-synuclein (aSyn) in vulnerable populations of brain cells. Oxidative stress is both a cause and a consequence of aSyn aggregation in the synucleinopathies; however, noninvasive methods for detecting oxidative stress in living animals have proven elusive. In this study, we used the reactive oxygen species (ROS)-sensitive positron emission tomography (PET) radiotracer [18F]ROStrace to detect increases in oxidative stress in the widely-used A53T mouse model of synucleinopathy. A53T-specific elevations in [18F]ROStrace signal emerged at a relatively early age (6-8 months) and became more widespread within the brain over time, a pattern which paralleled the progressive development of aSyn pathology and oxidative damage in A53T brain tissue. Systemic administration of lipopolysaccharide (LPS) also caused rapid and long-lasting elevations in [18F]ROStrace signal in A53T mice, suggesting that chronic, aSyn-associated oxidative stress may render these animals more vulnerable to further inflammatory insult. Collectively, these results provide novel evidence that oxidative stress is an early and chronic process during the development of synucleinopathy and suggest that PET imaging with [18F]ROStrace holds promise as a means of detecting aSyn-associated oxidative stress noninvasively.

Single-domain antibody-based protein degrader for synucleinopathies.

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.

The αSynuclein half-life conundrum.

αSynuclein (αSyn) misfolding and aggregation frequently precedes neuronal loss associated with Parkinson's Disease (PD) and other Synucleinopathies. The progressive buildup of pathological αSyn species results from alterations on αSyn gene and protein sequence, increased local concentrations, variations in αSyn interactome and protein network. Therefore, under physiological conditions, it is mandatory to regulate αSyn proteostasis as an equilibrium among synthesis, trafficking, degradation and extracellular release. In this frame, a crucial parameter is protein half-life. It provides indications of the turnover of a specific protein and depends on mRNA synthesis and translation regulation, subcellular localization, function and clearance by the designated degradative pathways. For αSyn, the molecular mechanisms regulating its proteostasis in neurons have been extensively investigated in various cellular models, either using biochemical or imaging approaches. Nevertheless, a converging estimate of αSyn half-life has not emerged yet. Here, we discuss the challenges in studying αSyn proteostasis under physiological and pathological conditions, the advantages and disadvantages of the experimental strategies proposed so far, and the relevance of determining αSyn half-life from a translational perspective.

Gut microbiota produces biofilm-associated amyloids with potential for neurodegeneration.

Age-related neurodegenerative diseases involving amyloid aggregation remain one of the biggest challenges of modern medicine. Alterations in the gastrointestinal microbiome play an active role in the aetiology of neurological disorders. Here, we dissect the amyloidogenic properties of biofilm-associated proteins (BAPs) of the gut microbiota and their implications for synucleinopathies. We demonstrate that BAPs are naturally assembled as amyloid-like fibrils in insoluble fractions isolated from the human gut microbiota. We show that BAP genes are part of the accessory genomes, revealing microbiome variability. Remarkably, the abundance of certain BAP genes in the gut microbiome is correlated with Parkinson's disease (PD) incidence. Using cultured dopaminergic neurons and Caenorhabditis elegans models, we report that BAP-derived amyloids induce α-synuclein aggregation. Our results show that the chaperone-mediated autophagy is compromised by BAP amyloids. Indeed, inoculation of BAP fibrils into the brains of wild-type mice promote key pathological features of PD. Therefore, our findings establish the use of BAP amyloids as potential targets and biomarkers of α-synucleinopathies.

Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy.

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of α-Synuclein (α-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the α-Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress α-Syn replicate several features of PD. Decreasing total α-Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of α-Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of α-Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the α-Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical α-Syn pathology, indicated by reduced numbers of pS129 α-Syn-containing cells. In primary neurons, we found that Cdk14 depletion protects against the propagation of toxic α-Syn species. We further validated these findings on pS129 α-Syn levels in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated α-Syn in human neurons, in PFF-challenged rat neurons and in the brains of α-Syn-humanized mice. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

The mechanistic interaction, aggregation and neurotoxicity of α-synuclein after interaction with glycyrrhizic acid: Modulation of synucleinopathies.

This article reveals the binding mechanism between glycyrrhizic acid (GA) and α-synuclein to may provide further information for the modulation of synucleinopathies using bioactive compounds. Therefore, the inhibitory activities of GA against α-synuclein aggregation and induced neurotoxicity were evaluated using different assays. Results showed that α-synuclein-GA binding was mediated by intermolecular hydrogen bonds leading to the formation of a slightly folded complex. Theoretical studies revealed that GA binds to the N-terminal domain of α-synuclein and triggers a compact structure around a major part of the N-terminal and the NAC regions along with fluctuations in the C-terminal domain, which are prerequisites for the inhibition of α-synuclein aggregation. Then, the cellular assays showed that GA as a potential small molecule can inhibit the oligomerization of α-synuclein and relevant neurotoxicity through modulation of neural viability, membrane leakage, and ROS formation in a concentration-dependent manner. As a result, the primary mechanism of GA's anti-aggregation and neuroprotective activities is the reorganized α-synuclein structure and fluctuating C-terminal domain, which promotes long-range transient intramolecular contacts between the N-terminal and the C-terminal domain.

Role of the Gut Microbiome and Bacterial Amyloids in the Development of Synucleinopathies.

Less than ten years ago, evidence began to accumulate about association between the changes in the composition of gut microbiota and development of human synucleinopathies, in particular sporadic form of Parkinson's disease. We collected data from more than one hundred and thirty experimental studies that reported similar results and summarized the frequencies of detection of different groups of bacteria in these studies. It is important to note that it is extremely rare that a unidirectional change in the population of one or another group of microorganisms (only an elevation or only a reduction) was detected in the patients with Parkinson's disease. However, we were able to identify several groups of bacteria that were overrepresented in the patients with Parkinson's disease in the analyzed studies. There are various hypotheses about the molecular mechanisms that explain such relationships. Usually, α-synuclein aggregation is associated with the development of inflammatory processes that occur in response to the changes in the microbiome. However, experimental evidence is accumulating on the influence of bacterial proteins, including amyloids (curli), as well as various metabolites, on the α-synuclein aggregation. In the review, we provided up-to-date information about such examples.

Loss of monomeric alpha-synuclein (synucleinopenia) and the origin of Parkinson's disease.

These facts argue against the gain-of-function synucleinopathy hypothesis, which proposes that Lewy pathology causes Parkinson's disease: (1) most brains from people without neurological symptoms have multiple pathologies; (2) neither pathology type nor distribution correlate with disease severity or progression in Parkinson's disease; (3) aggregated α-synuclein in the form of Lewy bodies is not a space-occupying lesion but the insoluble fraction of its precursor, soluble monomeric α-synuclein; (4) pathology spread is passive, occurring by irreversible nucleation, not active replication; and (5) low cerebrospinal fluid α-synuclein levels predict brain atrophy and clinical disease progression. The transformation of α-synuclein into Lewy pathology may occur as a response to biological, toxic, or infectious stressors whose persistence perpetuates the nucleation process, depleting normal α-synuclein and eventually leading to Parkinson's symptoms from neuronal death. We propose testing the loss-of-function synucleinopenia hypothesis by evaluating the clinical and neurodegenerative rescue effect of replenishing the levels of monomeric α-synuclein.

Systemic inflammation activates coagulation and immune cell infiltration pathways in brains with propagating α-synuclein fibril aggregates.

Synucleinopathies are a group of diseases characterized by brain aggregates of α-synuclein (α-syn). The gradual accumulation of α-syn and the role of inflammation in early-stage pathogenesis remain poorly understood. We explored this interaction by inducing chronic inflammation in a common pre-clinical synucleinopathy mouse model. Three weeks post unilateral intra-striatal injections of human α-syn pre-formed fibrils (PFF), mice underwent repeated intraperitoneal injections of 1 mg/ml lipopolysaccharide (LPS) for 3 weeks. Histological examinations of the ipsilateral site showed phospho-α-syn regional spread and LPS-induced neutrophil recruitment to the brain vasculature. Biochemical assessment of the contralateral site confirmed spreading of α-syn aggregation to frontal cortex and a rise in intracerebral TNF-α, IL-1ß, IL-10 and KC/GRO cytokines levels due to LPS. No LPS-induced exacerbation of α-syn pathology load was observed at this stage. Proteomic analysis was performed contralateral to the PFF injection site using LC-MS/MS. Subsequent downstream Reactome Gene-Set Analysis indicated that α-syn pathology alters mitochondrial metabolism and synaptic signaling. Chronic LPS-induced inflammation further lead to an overrepresentation of pathways related to fibrin clotting as well as integrin and B cell receptor signaling. Western blotting confirmed a PFF-induced increase in fibrinogen brain levels and a PFF + LPS increase in Iba1 levels, indicating activated microglia. Splenocyte profiling revealed changes in T and B cells, monocytes, and neutrophils populations due to LPS treatment in PFF injected animals. In summary, early α-syn pathology impacts energy homeostasis pathways, synaptic signaling and brain fibrinogen levels. Concurrent mild systemic inflammation may prime brain immune pathways in interaction with peripheral immunity.

CRISPR-based identification of N-terminal acetylation in synucleinopathies.

A recent study by Kumar et al. identified several biological pathways that regulate the levels of endogenous alpha-synuclein (α-synuclein). They specifically highlighted the N-terminal acetylation (NTA) pathway as an important factor in maintaining the stability of endogenous α-synuclein, suggesting targeting the NTA pathway as a potential therapeutic approach.

FABP2 is Involved in Intestinal α-Synuclein Pathologies.

BACKGROUND: Recently, the hypothesis that pathological α-Synuclein propagates from the gut to the brain has gained attention. Although results from animal studies support this hypothesis, the specific mechanism remains unclear. This study focused on the intestinal fatty acid-binding protein (FABP2), which is one of the subtypes of fatty acid binding proteins localizing in the gut, with the hypothesis that FABP2 is involved in the gut-to-brain propagation of α-synuclein. The aim of this study was to clarify the pathological significance of FABP2 in the pathogenesis and progression of synucleinopathy. METHODS: We examined the relationship between FABP2 and α-Synuclein in the uptake of α-Synuclein into enteric neurons using primary cultured neurons derived from mouse small intestinal myenteric plexus. We also quantified disease-related protein concentrations in the plasma of patients with synucleinopathy and related diseases, and analyzed the relationship between plasma FABP2 level and progression of the disease. RESULTS: Experiments on α-Synuclein uptake in primary cultured enteric neurons showed that following uptake, α-Synuclein was concentrated in areas where FABP2 was localized. Moreover, analysis of the plasma protein levels of patients with Parkinson's disease revealed that the plasma FABP2 and α-Synuclein levels fluctuate with disease duration. The FABP2/α-Synuclein ratio fluctuated more markedly than either FABP2 or α-Synuclein alone, depending on the duration of disease, indicating a higher discriminant ability of early Parkinson's disease patients from healthy patients. CONCLUSIONS: These results suggest that FABP2 potentially contributes to the pathogenesis and progression of α-synucleinopathies. Thus, FABP2 is an important molecule that has the potential to elucidate the consistent mechanisms that lead from the prodromal phase to the onset and subsequent progression of synucleinopathies.

Aquaporin-4 and Parkinson's Disease.

The water-selective channel aquaporin-4 (AQP4) is implicated in water homeostasis and the functioning of the glymphatic system, which eliminates various metabolites from the brain tissue, including amyloidogenic proteins. Misfolding of the α-synuclein protein and its post-translational modifications play a crucial role in the development of Parkinson's disease (PD) and other synucleopathies, leading to the formation of cytotoxic oligomers and aggregates that cause neurodegeneration. Human and animal studies have shown an interconnection between AQP4 dysfunction and α-synuclein accumulation; however, the specific role of AQP4 in these mechanisms remains unclear. This review summarizes the current knowledge on the role of AQP4 dysfunction in the progression of α-synuclein pathology, considering the possible effects of AQP4 dysregulation on brain molecular mechanisms that can impact α-synuclein modification, accumulation and aggregation. It also highlights future directions that can help study the role of AQP4 in the functioning of the protective mechanisms of the brain during the development of PD and other neurodegenerative diseases.

α-Synuclein seed amplification technology for Parkinson's disease and related synucleinopathies.

Synucleinopathies are a group of neurodegenerative diseases (NDs) associated with cerebral accumulation of α-synuclein (αSyn) misfolded aggregates. At this time, there is no effective treatment to stop or slow down disease progression, which in part is due to the lack of an early and objective biochemical diagnosis. In the past 5 years, the seed amplification technology has emerged for highly sensitive identification of these diseases, even at the preclinical stage of the illness. Much research has been done in multiple laboratories to validate the efficacy and reproducibility of this assay. This article provides a comprehensive review of this technology, including its conceptual basis and its multiple applications for disease diagnosis, as well for understanding of the disease biology and therapeutic development.

Defining a Lewy Body: Running Up the Hill of Shifting Definitions and Evolving Concepts.

Lewy bodies (LBs) are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies, characterized by the accumulation of α-synuclein (αSyn) protein in the brain. While LBs were first described a century ago, their formation and morphogenesis mechanisms remain incompletely understood. Here, we present a historical overview of LB definitions and highlight the importance of semantic clarity and precise definitions when describing brain inclusions. Recent breakthroughs in imaging revealed shared features within LB subsets and the enrichment of membrane-bound organelles in these structures, challenging the conventional LB formation model. We discuss the involvement of emerging concepts of liquid-liquid phase separation, where biomolecules demix from a solution to form dense condensates, as a potential LB formation mechanism. Finally, we emphasize the need for the operational definitions of LBs based on morphological characteristics and detection protocols, particularly in studies investigating LB formation mechanisms. A better understanding of LB organization and ultrastructure can contribute to the development of targeted therapeutic strategies for synucleinopathies.

The involvement of α-synucleinopathy in the disruption of microglial homeostasis contributes to the pathogenesis of Parkinson's disease.

The intracellular deposition and intercellular transmission of α-synuclein (α-syn) are shared pathological characteristics among neurodegenerative disorders collectively known as α-synucleinopathies, including Parkinson's disease (PD). Although the precise triggers of α-synucleinopathies remain unclear, recent findings indicate that disruption of microglial homeostasis contributes to the pathogenesis of PD. Microglia play a crucial role in maintaining optimal neuronal function by ensuring a homeostatic environment, but this function is disrupted during the progression of α-syn pathology. The involvement of microglia in the accumulation, uptake, and clearance of aggregated proteins is critical for managing disease spread and progression caused by α-syn pathology. This review summarizes current knowledge on the interrelationships between microglia and α-synucleinopathies, focusing on the remarkable ability of microglia to recognize and internalize extracellular α-syn through diverse pathways. Microglia process α-syn intracellularly and intercellularly to facilitate the α-syn neuronal aggregation and cell-to-cell propagation. The conformational state of α-synuclein distinctly influences microglial inflammation, which can affect peripheral immune cells such as macrophages and lymphocytes and may regulate the pathogenesis of α-synucleinopathies. We also discuss ongoing research efforts to identify potential therapeutic approaches targeting both α-syn accumulation and inflammation in PD. Video Abstract.

Endogenous Alpha-Synuclein is Essential for the Transfer of Pathology by Exosome-Enriched Extracellular Vesicles, Following Inoculation with Preformed Fibrils in vivo.

The main pathological hallmark of Parkinson's disease (PD) and related synucleinopathies is the presence of intracellular proteinaceous aggregates, enriched in the presynaptic protein alpha-Synuclein (α-Syn). α-Syn association with exosomes has been previously documented both as a physiological process of secretion and as a pathological process of disease transmission, however, critical information about the mechanisms governing this interplay is still lacking. To address this, we utilized the α-Syn preformed fibril (PFF) mouse model of PD, as a source of brain-derived exosome-enriched extracellular vesicles (ExE-EVs) and assessed their pathogenic capacity following intrastriatal injections in host wild type (WT) mouse brain. We further investigated the impact of the fibrillar α-Syn on the exosomal cargo independent of the endogenous α-Syn, by isolating ExE-EVs from PFF-injected α-Syn knockout mice. Although PFF inoculation does not alter the morphology, size distribution, and quantity of brain-derived ExE-EVs, it triggers changes in the exosomal proteome related to synaptic and mitochondrial function, as well as metabolic processes. Importantly, we showed that the presence of the endogenous α-Syn is essential for the ExE-EVs to acquire a pathogenic capacity, allowing them to mediate disease transmission by inducing phosphorylated-α-Syn pathology. Notably, misfolded α-Syn containing ExE-EVs when injected in WT mice were able to induce astrogliosis and synaptic alterations in the host brain, at very early stages of α-Syn pathology, preceding the formation of the insoluble α-Syn accumulations. Collectively, our data suggest that exosomal cargo defines their ability to spread α-Syn pathology.