Α-synuclein Preformed Fibrils Research Articles

Intercellular transmission of alpha-synuclein.

An emerging theme in Parkinson's disease (PD) is the propagation of α-synuclein pathology as the disease progresses. Research involving the injection of preformed α-synuclein fibrils (PFFs) in animal models has recapitulated the pathological spread observed in PD patients. At the cellular and molecular levels, this intercellular spread requires the translocation of α-synuclein across various membrane barriers. Recent studies have identified subcellular organelles and protein machineries that facilitate these processes. In this review, we discuss the proposed pathways for α-synuclein intercellular transmission, including unconventional secretion, receptor-mediated uptake, endosome escape and nanotube-mediated transfer. In addition, we advocate for a rigorous examination of the evidence for the localization of α-synuclein in extracellular vesicles.

Interspecies chimerism with human embryonic stem cells generates functional human dopamine neurons at low efficiency

Interspecies chimeras offer great potential for regenerative medicine and the creation of human disease models. Whether human pluripotent stem cell-derived neurons in an interspecies chimera can differentiate into functional neurons and integrate into host neural circuity is not known. Here, we show, using Engrailed 1 (En1) as a development niche, that human naive-like embryonic stem cells (ESCs) can incorporate into embryonic and adult mouse brains. Human-derived neurons including tyrosine hydroxylase (TH)+ neurons integrate into the mouse brain at low efficiency. These TH+ neurons have electrophysiologic properties consistent with their human origin. In addition, these human-derived neurons in the mouse brain accumulate pathologic phosphorylated α-synuclein in response to α-synuclein preformed fibrils. Optimization of human/mouse chimeras could be used to study human neuronal differentiation and human brain disorders.

6PPD-quinone exposure induces neuronal mitochondrial dysfunction to exacerbate Lewy neurites formation induced by α-synuclein preformed fibrils seeding

The emerging toxicant N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine quinone (6PPD-Q) is of wide concern due to its ubiquitous occurrence and high toxicity. Despite regular human exposure, limited evidence exists about its presence in the body and potential health risks. Herein, we analyzed cerebrospinal fluid (CSF) samples from Parkinson's disease (PD) patients and controls. The CSF levels of 6PPD-Q were twice as high in PD patients compared to controls. Immunostaining assays performed with primary dopaminergic neurons confirm that 6PPD-Q at environmentally relevant concentrations can exacerbate the formation of Lewy neurites induced by α-synuclein preformed fibrils (α-syn PFF). Assessment of cellular respiration reveals a considerable decrease in neuronal spare respiratory and ATP-linked respiration, potentially due to changes in mitochondrial membrane potential. Moreover, 6PPD-Q-induced mitochondrial impairment correlates with an upsurge in mitochondrial reactive oxygen species (mROS), and Mito-TEMPO-driven scavenging of mROS can lessen the amount of pathologic phospho-serine 129 α-synuclein. Untargeted metabolomics provides supporting evidence for the connection between 6PPD-Q exposure and changes in neuronal metabolite profiles. In-depth targeted metabolomics further unveils an overall reduction in glycolysis metabolite pool and fluctuations in the quantity of TCA cycle intermediates. Given its potentially harmful attributes, the presence of 6PPD-Q in human brain could potentially be a risk factor for PD.

MLKL deficiency alleviates neuroinflammation and motor deficits in the α-synuclein transgenic mouse model of Parkinson’s disease

Parkinson’s disease (PD), one of the most devastating neurodegenerative brain disorders, is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and deposits of α-synuclein aggregates. Currently, pharmacological interventions for PD remain inadequate. The cell necroptosis executor protein MLKL (Mixed-lineage kinase domain-like) is involved in various diseases, including inflammatory bowel disease and neurodegenerative diseases; however, its precise role in PD remains unclear. Here, we investigated the neuroprotective role of MLKL inhibition or ablation against primary neuronal cells and human iPSC-derived midbrain organoids induced by toxic α-Synuclein preformed fibrils (PFFs). Using a mouse model (Tg-Mlkl−/−) generated by crossbreeding the SNCA A53T synuclein transgenic mice with MLKL knockout (KO)mice, we assessed the impact of MLKL deficiency on the progression of Parkinsonian traits. Our findings demonstrate that Tg-Mlkl−/− mice exhibited a significant improvement in motor symptoms and reduced phosphorylated α-synuclein expression compared to the classic A53T transgenic mice. Furthermore, MLKL deficiency alleviated tyrosine hydroxylase (TH)-positive neuron loss and attenuated neuroinflammation by inhibiting the activation of microglia and astrocytes. Single-cell RNA-seq (scRNA-seq) analysis of the SN of Tg-Mlkl−/− mice revealed a unique cell type-specific transcriptome profile, including downregulated prostaglandin D synthase (PTGDS) expression, indicating reduced microglial cells and dampened neuron death. Thus, MLKL represents a critical therapeutic target for reducing neuroinflammation and preventing motor deficits in PD.

Enhanced mTORC1 signaling and protein synthesis in pathologic α-synuclein cellular and animal models of Parkinson's disease.

Pathologic α-synuclein plays an important role in the pathogenesis of α-synucleinopathies such as Parkinson's disease (PD). Disruption of proteostasis is thought to be central to pathologic α-synuclein toxicity; however, the molecular mechanism of this deregulation is poorly understood. Complementary proteomic approaches in cellular and animal models of PD were used to identify and characterize the pathologic α-synuclein interactome. We report that the highest biological processes that interacted with pathologic α-synuclein in mice included RNA processing and translation initiation. Regulation of catabolic processes that include autophagy were also identified. Pathologic α-synuclein was found to bind with the tuberous sclerosis protein 2 (TSC2) and to trigger the activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which augmented mRNA translation and protein synthesis, leading to neurodegeneration. Genetic and pharmacologic inhibition of mTOR and protein synthesis rescued the dopamine neuron loss, behavioral deficits, and aberrant biochemical signaling in the α-synuclein preformed fibril mouse model and Drosophila transgenic models of pathologic α-synuclein-induced degeneration. Pathologic α-synuclein furthermore led to a destabilization of the TSC1-TSC2 complex, which plays an important role in mTORC1 activity. Constitutive overexpression of TSC2 rescued motor deficits and neuropathology in α-synuclein flies. Biochemical examination of PD postmortem brain tissues also suggested deregulated mTORC1 signaling. These findings establish a connection between mRNA translation deregulation and mTORC1 pathway activation that is induced by pathologic α-synuclein in cellular and animal models of PD.

α-Synuclein pathology from the body to the brain: so many seeds so close to the central soil.

α-Synuclein is a protein that mainly exists in the presynaptic terminals. Abnormal folding and accumulation of α-synuclein are found in several neurodegenerative diseases, including Parkinson's disease. Aggregated and highly phosphorylated α-synuclein constitutes the main component of Lewy bodies in the brain, the pathological hallmark of Parkinson's disease. For decades, much attention has been focused on the accumulation of α-synuclein in the brain parenchyma rather than considering Parkinson's disease as a systemic disease. Recent evidence demonstrates that, at least in some patients, the initial α-synuclein pathology originates in the peripheral organs and spreads to the brain. Injection of α-synuclein preformed fibrils into the gastrointestinal tract triggers the gut-to-brain propagation of α-synuclein pathology. However, whether α-synuclein pathology can occur spontaneously in peripheral organs independent of exogenous α-synuclein preformed fibrils or pathological α-synuclein leakage from the central nervous system remains under investigation. In this review, we aimed to summarize the role of peripheral α-synuclein pathology in the pathogenesis of Parkinson's disease. We also discuss the pathways by which α-synuclein pathology spreads from the body to the brain.

Synaptotagmin-11 regulates immune functions of microglia invivo.

Membrane trafficking pathways mediate key microglial activities such as cell migration, cytokine secretion, and phagocytosis. However, the underlying molecular mechanism remains poorly understood. Previously, we found that synaptotagmin-11 (Syt11), a non-Ca2+ -binding Syt associated with Parkinson's disease (PD) and schizophrenia, inhibits cytokine release and phagocytosis in primary microglia. Here we reported the invivo function of Syt11 in microglial immune responses using an inducible microglia-specific Syt11-conditional-knockout (cKO) mouse strain. Syt11-cKO resulted in activation of microglia and elevated mRNA levels of IL-6, TNF-α, IL-1β, and iNOS in various brain regions under both resting state and LPS-induced acute inflammation state in adult mice. In a PD mouse model generated by microinjection of preformed α-synuclein fibrils into the striatum, a reduced number of microglia migrated toward the injection sites and an enhanced phagocytosis of α-synuclein fibrils by microglia were found in Syt11-cKO mice. To understand the molecular mechanism of Syt11 function, we identified its direct binding proteins vps10p-tail-interactor-1a (vti1a) and vti1b. The linker domain of Syt11 interacted with both proteins and a peptide derived from it competitively inhibited the interaction of Syt11 with vti1a/vti1b invitro and in cells. Importantly, application of this peptide induced more cytokine secretion in wild-type microglia upon LPS treatment, phenocopying defects in Syt11 knockdown cells. Altogether, we propose that Syt11 inhibits microglial activation invivo and regulates cytokine secretion through interactions with vti1a and vti1b.

Formation of templated inclusions in a forebrain α-synuclein mouse model is independent of LRRK2

Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein share enigmatic roles in the pathobiology of Parkinson's disease (PD). LRRK2 mutations are a common genetic cause of PD which, in addition to neurodegeneration, often present with abnormal deposits of α-synuclein in the form of Lewy-related pathology. As Lewy-related pathology is a prominent neuropathologic finding in sporadic PD, the relationship between LRRK2 and α-synuclein has garnered considerable interest. However, whether and how LRRK2 might influence the accumulation of Lewy-related pathology remains poorly understood. Through stereotactic injection of mouse α-synuclein pre-formed fibrils (PFF), we modeled the spread of Lewy-related pathology within forebrain regions where LRRK2 is most highly expressed. The impact of LRRK2 genotype on the formation of α-synuclein inclusions was evaluated at 1-month post-injection. Neither deletion of LRRK2 nor G2019S LRRK2 knockin appreciably altered the burden of α-synuclein pathology at this early timepoint. These observations fail to provide support for a robust pathophysiologic interaction between LRRK2 and α-synuclein in the forebrain in vivo. There was, however, a modest reduction in microglial activation induced by PFF delivery in the hippocampus of LRRK2 knockout mice, suggesting that LRRK2 may contribute to α-synuclein-induced neuroinflammation. Collectively, our data indicate that the pathological accumulation of α-synuclein in the mouse forebrain is largely independent of LRRK2.

Modelling α-synuclein processing in primary patient cells for pharmacological intervention

Aim: Parkinson’s disease (PD) is a complex, chronic neurodegenerative disorder with predominately sporadic etiology. Intricate genetic-environmental interactions lead to the hallmarks of the disease: degeneration of dopaminergic neurons and the deposition of α-synuclein aggregates. The aim of this study was to establish a novel primary patient cell model as an in vitro screen to study α-synuclein processing for drug screening. Methods: Primary patient olfactory neuroepithelial-derived cells (ONS) were exposed to α-synuclein and examined for cytotoxicity, processing, and solubility over 48 h. Epigallocatechin gallate (EGCG), which is known to destabilise α-synuclein fibrils, was used to investigate the solubilisation of α-synuclein in the model system. Results: Exposure to 0.1 μmol/L α-synuclein preformed fibrils was not toxic to ONS over 48 h. ONS processing of α-synuclein was observed to be different in PD cells by their increased accumulation in the cytoplasm. Processing deficits in the PD ONS were confirmed by immunoblotting with an increase in sodium dodecyl sulfate (SDS)-insoluble α-synuclein after 48 h. Conclusions: The data has illustrated the utility of primary patient ONS as a model system to understand the processing of α-synuclein. Considerable differences in α-synuclein processing were identified in PD ONS. Furthermore, the data suggests that primary patient ONS are a viable in vitro drug screening platform for α-synuclein pathology in PD.

Dopaminergic neurodegeneration in the substantia nigra is associated with olfactory dysfunction in mice models of Parkinson’s disease

Olfactory dysfunction represents a prodromal stage in Parkinson’s disease (PD). However, the mechanisms underlying hyposmia are not specified yet. In this study, we first observed an early olfactory dysfunction in mice with intragastric rotenone administration, consistent with dopaminergic neurons loss and α-synuclein pathology in the olfactory bulb. However, a much severer olfactory dysfunction was observed without severer pathology in olfactory bulb when the loss of dopaminergic neurons in the substantia nigra occurred. Then, we established the mice models by intrastriatal α-synuclein preformed fibrils injection and demonstrated the performance in the olfactory discrimination test was correlated to the loss of dopaminergic neurons in the substantia nigra, without any changes in the olfactory bulb analyzed by RNA-sequence. In mice with intranasal ferric ammonium citrate administration, we observed olfactory dysfunction when dopaminergic neurodegeneration in substantia nigra occurred and was restored when dopaminergic neurons were rescued. Finally we demonstrated that chemogenetic inhibition of dopaminergic neurons in the substantia nigra was sufficient to cause hyposmia and motor incoordination. Taken together, this study shows a direct relationship between nigral dopaminergic neurodegeneration and olfactory dysfunction in PD models and put forward the understandings that olfactory dysfunction represents the early stage of neurodegeneration in PD progression.

α-Synuclein propagation leads to synaptic abnormalities in the cortex through microglial synapse phagocytosis

The major neuropathologic feature of Parkinson’s disease is the presence of widespread intracellular inclusions of α-synuclein known as Lewy bodies. Evidence suggests that these misfolded protein inclusions spread through the brain with disease progression. Changes in synaptic function precede neurodegeneration, and this extracellular α-synuclein can affect synaptic transmission. However, whether and how the spreading of α-synuclein aggregates modulates synaptic function before neuronal loss remains unknown. In the present study, we investigated the effect of intrastriatal injection of α-synuclein preformed fibrils (PFFs) on synaptic activity in the somatosensory cortex using a combination of whole-cell patch-clamp electrophysiology, histology, and Golgi-Cox staining. Intrastriatal PFF injection was followed by formation of phosphorylated α-synuclein inclusions in layer 5 of the somatosensory cortex, leading to a decrease in synapse density, dendritic spines, and spontaneous excitatory post-synaptic currents, without apparent neuronal loss. Additionally, three-dimensional reconstruction of microglia using confocal imaging showed an increase in the engulfment of synapses. Collectively, our data indicate that propagation of α-synuclein through neural networks causes abnormalities in synaptic structure and dynamics prior to neuronal loss.

Developmental exposure to the Parkinson's disease-associated organochlorine pesticide dieldrin alters dopamine neurotransmission in α-synuclein pre-formed fibril (PFF)-injected mice.

Parkinson's disease (PD) is the fastest-growing neurological disease worldwide, with increases outpacing aging and occurring most rapidly in recently industrialized areas, suggesting a role of environmental factors. Epidemiological, post-mortem, and mechanistic studies suggest that persistent organic pollutants, including the organochlorine pesticide dieldrin, increase PD risk. In mice, developmental dieldrin exposure causes male-specific exacerbation of neuronal susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and synucleinopathy. Specifically, in the α-synuclein (α-syn) pre-formed fibril (PFF) model, exposure leads to increased deficits in striatal dopamine (DA) turnover and motor deficits on the challenging beam. Here, we hypothesized that alterations in DA handling contribute to the observed changes and assessed vesicular monoamine transporter 2 (VMAT2) function and DA release in this dieldrin/PFF 2-hit model. Female C57BL/6 mice were exposed to 0.3 mg/kg dieldrin or vehicle every 3 days by feeding, starting at 8 weeks of age and continuing throughout breeding, gestation, and lactation. Male offspring from independent litters underwent unilateral, intrastriatal injections of α-syn PFFs at 12 weeks of age, and vesicular 3H-DA uptake assays and fast-scan cyclic voltammetry were performed 4 months post-PFF injection. Dieldrin-induced an increase in DA release in striatal slices in PFF-injected animals, but no change in VMAT2 activity. These results suggest that developmental dieldrin exposure increases a compensatory response to synucleinopathy-triggered striatal DA loss. These findings are consistent with silent neurotoxicity, where developmental exposure to dieldrin primes the nigrostriatal striatal system to have an exacerbated response to synucleinopathy in the absence of observable changes in typical markers of nigrostriatal dysfunction and degeneration.

The Poly-Arginine Peptide R18D Interferes with the Internalisation of α-Synuclein Pre-Formed Fibrils in STC-1 Enteroendocrine Cells.

In Parkinson's disease (PD), gut inflammation is hypothesised to contribute to α-synuclein aggregation, but gastrointestinal α-synuclein expression is poorly characterised. Cationic arginine-rich peptides (CARPs) are an emerging therapeutic option that exerts various neuroprotective effects and may target the transmission of protein aggregates. This study aimed to investigate endogenous α-synuclein expression in enteroendocrine STC-1 cells and the potential of the CARP, R18D (18-mer of D-arginine), to prevent internalisation of pre-formed α-synuclein fibrils (PFFs) in enteroendocrine cells in vitro. Through confocal microscopy, the immunoreactivity of full-length α-synuclein and the serine-129 phosphorylated form (pS129) was investigated in STC-1 (mouse enteroendocrine) cells. Thereafter, STC-1 cells were exposed to PFFs tagged with Alexa-Fluor 488 (PFF-488) for 2 and 24 h and R18D-FITC for 10 min. After confirming the uptake of both PFFs and R18D-FITC through fluorescent microscopy, STC-1 cells were pre-treated with R18D (5 or 10 μM) for 10 min prior to 2 h of PFF-488 exposure. Immunoreactivity for endogenous α-synuclein and pS129 was evident in STC-1 cells, with prominent pS129 staining along cytoplasmic processes and in perinuclear areas. STC-1 cells internalised PFFs, confirmed through co-localisation of PFF-488 and human-specific α-synuclein immunoreactivity. R18D-FITC entered STC-1 cells within 10 min and pre-treatment of STC-1 cells with R18D interfered with PFF uptake. The endogenous presence of α-synuclein in enteroendocrine cells, coupled with their rapid uptake of PFFs, demonstrates a potential for pathogenic spread of α-synuclein aggregates in the gut. R18D is a novel therapeutic approach to reduce the intercellular transmission of α-synuclein pathology.

Myo1d promotes alpha-synuclein transfer from brain microvascular endothelial cells to pericytes through tunneling nanotubes

α-Synuclein preformed fibrils (α-syn PFF) in the blood can cross the blood-brain barrier and invade the central nervous system. Our previous study proved that α-syn PFF can be taken up by brain microvascular endothelial cells (BMVECs). Here, we found that α-syn PFF spread from BMVECs to pericytes with the highest transmission efficiency. We observed abundant tunneling nanotubes (TNTs) connecting BMVECs and pericytes, and α-syn PFF transmitted through these TNTs. Furthermore, α-syn PFF accumulation in BMVECs did not promote TNT formation, but activated the molecular motor Myo1d. Inhibition of Myo1d prevented α-syn PFF transfer from BMVECs to pericytes and decreased the colocalization of Myo1d and F-actin in BMVECs. In summary, we are the first to demonstrate that α-syn PFF spread from BMVECs to pericytes through a mechanism involving TNTs and myosin. Targeting Myo1d may be a promising approach to prevent α-syn spreading from the blood to the brain.

Monitoring α-synuclein Aggregation Induced by Preformed α-synuclein Fibrils in an In Vitro Model System.

Parkinson's disease (PD) is characterized by the presence of α-synuclein (α-syn) inclusions in the brain and the degeneration of dopamine-producing neurons. There is evidence to suggest that the progression of PD may be due to the prion-like spread of α-syn aggregates, so understanding and limiting α-syn propagation is a key area of research for developing PD treatments. Several cellular and animal model systems have been established to monitor α-syn aggregation and propagation. In this study, we developed an in vitro model using A53T α-syn-EGFP overexpressing SH-SY5Y cells and validated its usefulness for high-throughput screening of potential therapeutic targets. Treatment with preformed recombinant α-syn fibrils induced the formation of aggregation puncta of A53T α-syn-EGFP in these cells, which were analyzed using four indices: number of dots per cell, size of dots, intensity of dots, and percentage of cells containing aggregation puncta. Four indices are reliable indicators of the effectiveness of interventions against α-syn propagation in a one-day treatment model to minimize the screening time. This simple and efficient in vitro model system can be used for high-throughput screening to discover new targets for inhibiting α-syn propagation.

Retina-to-brain spreading of α-synuclein after intravitreal injection of preformed fibrils

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the aggregation of misfolded α-synuclein and progressive spreading of the aggregates from a few discrete regions to wider brain regions. Although PD has been classically considered a movement disorder, a large body of clinical evidence has revealed the progressive occurrence of non-motor symptoms. Patients present visual symptoms in the initial stages of the disease, and accumulation of phospho-α-synuclein, dopaminergic neuronal loss, and retinal thinning has been observed in the retinas of PD patients. Based on such human data, we hypothesized that α-synuclein aggregation can initiate in the retina and spread to the brain through the visual pathway. Here, we demonstrate accumulation of α-synuclein in the retinas and brains of naive mice after intravitreal injection of α-synuclein preformed fibrils (PFFs). Histological analyses showed deposition of phospho-α-synuclein inclusions within the retina 2 months after injection, with increased oxidative stress leading to loss of retinal ganglion cells and dopaminergic dysfunction. In addition, we found accumulation of phospho-α-synuclein in cortical areas with accompanying neuroinflammation after 5 months. Collectively, our findings suggest that retinal synucleinopathy lesions initiated by intravitreal injection of α-synuclein PFFs spread to various brain regions through the visual pathway in mice.

Ca2+ -Calmodulin-Calcineurin Signaling Modulates α-Synuclein Transmission.

The intercellular transmission of pathogenic proteins plays a crucial role in the progression of neurodegenerative diseases. Previous research has shown that the neuronal uptake of such proteins is activity-dependent; however, the detailed mechanisms underlying activity-dependent α-synuclein transmission in Parkinson's disease remain unclear. To examine whether α-synuclein transmission is affected by Ca2+ -calmodulin-calcineurin signaling in cultured cells and mouse models of Parkinson's disease. Mouse primary hippocampal neurons were used to examine the effects of the modulation of Ca2+ -calmodulin-calcineurin signaling on the neuronal uptake of α-synuclein preformed fibrils. The effects of modulating Ca2+ -calmodulin-calcineurin signaling on the development of α-synuclein pathology were examined using a mouse model injected with α-synuclein preformed fibrils. Modulation of Ca2+ -calmodulin-calcineurin signaling by inhibiting voltage-gated Ca2+ channels, calmodulin, and calcineurin blocked the neuronal uptake of α-synuclein preformed fibrils via macropinocytosis. Different subtypes of voltage-gated Ca2+ channel differentially contributed to the neuronal uptake of α-synuclein preformed fibrils. In wild-type mice inoculated with α-synuclein preformed fibrils, we found that inhibiting calcineurin ameliorated the development of α-synuclein pathology. Our data suggest that Ca2+ -calmodulin-calcineurin signaling modulates α-synuclein transmission and has potential as a therapeutic target for Parkinson's disease. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Modulating SQSTM1/p62-dependent selective autophagy of neurons by activating Nrf2 with multifunctional nanoparticles to eliminate α-synuclein aggregates and boost therapy of Parkinson’s disease

The misfolding and aggregation of α-synuclein is one of the key pathogeneses in Parkinson's disease (PD). Development of innovative and effective approaches to eliminate α-synuclein aggregates is crucial for PD treatment. Herein, we report that quercetin-modified ultrasmall Cu2-xSe antioxidative nanoparticles (abbreviated as CSPQ) can promote neurons to efficiently scavenge α-synuclein aggregates and boost PD therapy by activating their nuclear factor-erythroid 2-related factor 2 (Nrf2) to enhance the SQSTM1/p62-dependent selective autophagy. We reveal that CSPQ nanoparticles can promote the degradation of Kelch-like ECH-associated protein 1 (Keap1) and thus activate Nrf2/p62 pathway of neurons by stimulating their nuclear translocation of transcription factor EB (TFEB). Moreover, these nanoparticles can successfully reduce α-synuclein associated pathology to alleviate neuronal damage in vivo and improve motor disorder and exploration ability of α-synuclein preformed fibrils (PFFs)-induced PD mice. Our work demonstrates the importance and novelty of modulating the selective autophagy of neurons by nanoparticles in clearance of α-synuclein aggregates for PD therapy, and highlights the great potential of CSPQ nanoparticles in PD therapy.

ASC specks exacerbate α‑synuclein pathology via amplifying NLRP3 inflammasome activities

BackgroundInflammasome activation has a pathogenic role in Parkinson’s disease (PD). Up-regulated expressions of inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) and assembly of ASC specks have been observed in postmortems of human PD brains and experimental PD models. Extracellular ASC specks behave like danger signals and sustain prolonged inflammasome activation. However, the contribution of ASC specks in propagation of inflammasome activation and pathological progression in PD has not been fully established.MethodsHerein, we used human A53T mutant α-synuclein preformed fibrils (PFFs)-stimulated microglia in vitro and unilateral striatal stereotaxic injection of PFFs-induced mice model of PD in vivo, to investigate the significance of ASC specks in PD pathological progression. Rotarod and open-field tests were performed to measure motor behaviors of indicated mice. Changes in the molecular expression were evaluated by immunofluorescence and immunoblotting (IB). Intracellular knockdown of the ASC in BV2 cells was performed using si-RNA. Microglial and neuronal cells were co-cultured in a trans-well system to determine the effects of ASC knockdown on cytoprotection.ResultsWe observed a direct relationship between levels of ASC protein and misfolded α‑synuclein aggregates in PD mice brains. ASC specks amplified NLRP3 inflammasome activation driven by α-synuclein PFFs stimulation, which aggravated reactive microgliosis and accelerated α‑synuclein pathology, dopaminergic neurodegeneration and motor deficits. Endogenous ASC knockdown suppressed microglial inflammasome activation and neuronal α‑synuclein aggregation.ConclusionsIn conclusion, our study elucidated that ASC specks contribute to the propagation of inflammasome activation-associated α‑synuclein pathology in PD, which forms the basis for targeting ASC as a potential therapy for PD.

The purely thermodynamic anti-prionic mode of action for the treatment of neurodegenerative diseases - realized by target protein specific all-D-peptides.

Neurodegenerative protein-misfolding diseases, like Alzheimer's (AD) and Parkinson's disease (PD), are driven by prion-like self-replicating and propagating protein assemblies of Aβ, α-synuclein, and many more. Therefore, we have developed the so-called anti-prionic mode of action (MoA). It is realized by all-D-enantiomeric peptide ligands that bind the monomeric protein of interest with high affinity, thereby stabilizing the physiological intrinsically disordered monomer structure. We call this purely thermodynamic driven mode of action “anti-prionic”, because it is eliminating already existing prion-like aggregates by disassembling them into non-toxic and functional monomers. I will summarize the current, and yet unpublished, progress in realizing the anti-prionic MoA for the target proteins α-synuclein and Aβ. Briefly, the most promising all-D-enantiomeric ligand for α-synuclein, SVD-1a, disassembles preformed α-synuclein fibrils (PFF) as shown by AFM, DLS and SEC analysis. SPR and NMR demonstrate picomolar affinity of SVD-1a to α-synuclein monomers, while keeping them in their physiological intrinsically disordered conformation. The all-D-enantiomeric ligand for Aβ, RD2, disassembles Aβ oligomers obtained from brain tissue of former AD patients. A clinical phase Ib, double-bind, placebo-controlled study with 20 ATN positive patients treated once daily orally with RD2 or placebo for 4 weeks with an additional 4 weeks follow up period demonstrates good safety and tolerability, and yields interesting results on efficacy. This is valuable information for the scheduled phase II study. I will also acknowledge the many contributors of both developments that are too many to be included here in the abstract. Conclusion: The unique anti-prionic mode of action for the treatment of AD, PD and other protein misfolding diseases is promising.