Α-syn Protein Research Articles

Exploring the rhodanine universe: Design and synthesis of fluorescent rhodanine-based derivatives as anti-fibrillar and anti-oligomer agents against α-synuclein and 2N4R tau

Tau and α-synuclein (α-syn) are prone-to-aggregate proteins that coprecipitate in the brains of Alzheimer’s disease (AD) and Parkinson’s disease (PD) patients. The early-stage oligomers and protofibrils of tau are believed to be strongly linked to human cognitive impairment while the toxic α-syn oligomers are associated with behavioral motor deficits. Therefore, concurrent targeting of both proteinaceous aggregates and their lower dense oligomers are very challenging. Herein, rhodanine-based compounds were designed and synthesized to tackle the fibrils and oligomers of tau and α-syn proteins. In particular, the indole-containing rhodanines 5l and 5r displayed significantly high anti-aggregation activity towards α-syn fibrils by reduction of the thioflavin-T (ThT) fluorescence to lower than 5 %. Moreover, 5r showed a remarkable decrease in the fluorescence of thioflavin-S (ThS) when incubated with the non-phosphorylated tau 0N4R and 2N4R as well as the hyperphosphorylated tau isoform 1N4R, respectively. Transmission electron microscopy (TEM) analyses validated the powerful anti-fibrillar activity of 5l and 5r towards both protein aggregates. In addition, both 5l and 5r highly suppressed 0N4R tau and α-syn oligomer formation using the photo-induced cross-linking of unmodified Protein (PICUP) assay. The fluorescence emission intensity of 5l was quenched to almost half in the presence of both protein fibrils at 510 nm. 5r showed a similar fluorescence response upon binding to 2N4R fibrils while no quenching effect was observed with α-syn aggregates. Ex vivo disaggregation assay using extracted human Aβ plaques was employed to confirm the ability of 5l and 5r to disaggregate the dense fibrils. Both inhibitors showed no promising activity towards the cell-based α-syn inclusion formation. However, another indolyl derivative 5j prevented the α-syn inclusion at 5 µM. Collectively, the indolyl-rhodanine scaffold could act as a building block for further structural optimization to obtain dual targeting disease-modifying candidates for AD and PD.

Targeting protein aggregation using a cocoa-bean shell extract to reduce α-synuclein toxicity in models of Parkinson's disease

Neurodegenerative diseases are among the major challenges in modern medicine, due to the progressive aging of the world population. Among these, Parkinson's disease (PD) affects 10 million people worldwide and is associated with the aggregation of the presynaptic protein α-synuclein (α-syn). Here we use two different PD models, yeast cells and neuroblastoma cells overexpressing α-syn, to investigate the protective effect of an extract from the cocoa shell, which is a by-product of the roasting process of cocoa beans. The LC-ESI-qTOF-MS and NMR analyses allow the identification of amino acids (including the essential ones), organic acids, lactate and glycerol, confirming also the presence of the two methylxanthines, namely caffeine and theobromine. The present study demonstrates that the supplementation with the cocoa bean shell extract (CBSE) strongly improves the longevity of yeast cells expressing α-syn, reducing the level of reactive oxygen species, activating autophagy and reducing the intracellular protein aggresomes. These anti-aggregation properties are confirmed also in neuroblastoma cells, where CBSE treatment leads to activation of AMPK kinase and to a significant reduction of toxic α-syn oligomers. Results obtained by surface plasmon resonance (SPR) assay highlights that CBSE binds α-syn protein in a concentration-dependent manner, supporting its inhibitory role on the amyloid aggregation of α-syn. These findings suggest that the supplementation with CBSE in the form of nutraceuticals may represent a promising way to prevent neurodegenerative diseases associated with α-syn aggregation.

High-Resolution Cryo-EM Structure Determination of α-synuclein - A Prototypical Amyloid Fibril.

The physiological role of α-synuclein (α-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, α-syn will self-assemble into β-sheet rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient derived brain tissues have concluded that these inclusions are associated with Parkinson's disease, the second most common neurodegenerative disorder, and other synuclein related diseases called synucleinopathies. In addition, repetitions of and specific mutations to the SNCA gene, the gene that encodes α-syn, results in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of α-syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 Å. Here, we provide a protocol for α-syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step by step protocol for high resolution cryo-EM structure determination of α-syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high resolution cryo-EM map. This workflow resulted in a 2.04 Å structure of α-syn fibrils with excellent resolution of residues 36 to 97 and an additional island of density for residues 15 to 22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of α-synuclein and other amyloid fibrils.

Navigating the Neurobiology of Parkinson's: The Impact and Potential of α-Synuclein.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease worldwide; therefore, since its initial description, significant progress has been made, yet a mystery remains regarding its pathogenesis and elusive root cause. The widespread distribution of pathological α-synuclein (αSyn) aggregates throughout the body raises inquiries regarding the etiology, which has prompted several hypotheses, with the most prominent one being αSyn-associated proteinopathy. The identification of αSyn protein within Lewy bodies, coupled with genetic evidence linking αSyn locus duplication, triplication, as well as point mutations to familial Parkinson's disease, has underscored the significance of αSyn in initiating and propagating Lewy body pathology throughout the brain. In monogenic and sporadic PD, the presence of early inflammation and synaptic dysfunction leads to αSyn aggregation and neuronal death through mitochondrial, lysosomal, and endosomal functional impairment. However, much remains to be understood about αSyn pathogenesis, which is heavily grounded in biomarkers and treatment strategies. In this review, we provide emerging new evidence on the current knowledge about αSyn's pathophysiological impact on PD, and its presumable role as a specific disease biomarker or main target of disease-modifying therapies, highlighting that this understanding today offers the best potential of disease-modifying therapy in the near future.

Modeling Lewy body disease with SNCA triplication iPSC-derived cortical organoids and identifying therapeutic drugs.

Aggregated α-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we recapitulated α-SYN pathologies in human induced pluripotent stem cells (iPSCs)-derived cortical organoids generated from patients with LBD with SNCA gene triplication. Single-cell RNA sequencing, combined with functional and molecular validation, identified synaptic and mitochondrial dysfunction in excitatory neurons exhibiting high expression of the SNCA gene, aligning with observations in the cortex of autopsy-confirmed LBD human brains. Furthermore, we screened 1280 Food and Drug Administration-approved drugs and identified four candidates (entacapone, tolcapone, phenazopyridine hydrochloride, and zalcitabine) that inhibited α-SYN seeding activity in real-time quaking-induced conversion assays with human brains, reduced α-SYN aggregation, and alleviated mitochondrial dysfunction in SNCA triplication organoids and excitatory neurons. Our findings establish human cortical LBD models and suggest potential therapeutic drugs targeting α-SYN aggregation for LBD.

Immunotherapy for Parkinson's Disease and Alzheimer's Disease: A Promising Disease-Modifying Therapy.

Alzheimer's disease (AD) and Parkinson's disease (PD) are two neurodegenerative diseases posing a significant disease burden due to their increasing prevalence and socio-economic cost. Traditional therapeutic approaches for these diseases exist but provide limited symptomatic relief without addressing the underlying pathologies. This review examines the potential of immunotherapy, specifically monoclonal antibodies (mAbs), as disease-modifying treatments for AD and PD. We analyze the pathological mechanisms of AD and PD, focusing on the roles of amyloid-beta (Aβ), tau (τ), and alpha-synuclein (α-syn) proteins. We discuss the latest advancements in mAb therapies targeting these proteins, evaluating their efficacy in clinical trials and preclinical studies. We also explore the challenges faced in translating these therapies from bench to bedside, including issues related to safety, specificity, and clinical trial design. Additionally, we highlight future directions for research, emphasizing the need for combination therapies, improved biomarkers, and personalized treatment strategies. This review aims to provide insights into the current state and future potential of antibody-based immunotherapy in modifying the course of AD and PD, ultimately improving patient outcomes and quality of life.

A Versatile Method for Site-Specific Chemical Installation of Aromatic Posttranslational Modification Analogs into Proteins.

Posttranslational modifications (PTMs) of proteins play central roles in regulating the protein structure, interactome, and functions. A notable modification site is the aromatic side chain of Tyr, which undergoes modifications such as phosphorylation and nitration. Despite the biological and physiological importance of Tyr-PTMs, our current understanding of the mechanisms by which these modifications contribute to human health and disease remains incomplete. This knowledge gap arises from the absence of natural amino acids that can mimic these PTMs and the lack of synthetic tools for the site-specific introduction of aromatic PTMs into proteins. Herein, we describe a facile method for the site-specific chemical installation of aromatic PTMs into proteins through palladium-mediated S-C(sp2) bond formation under ambient conditions. We demonstrate the incorporation of novel PTMs such as Tyr-nitration and phosphorylation analogs to synthetic and recombinantly expressed Cys-containing peptides and proteins within minutes and in good yields. To demonstrate the versatility of our approach, we employed it to prepare 10 site-specifically modified proteins, including nitrated and phosphorylated analogs of Myc and Max proteins. Furthermore, we prepared a focused library of site-specifically nitrated and phosphorylated α-synuclein (α-Syn) protein, which enabled, for the first time, deciphering the role of these competing modifications in regulating α-Syn conformation aggregation in vitro. Our strategy offers advantages over synthetic or semisynthetic approaches, as it enables rapid and selective transfer of rarely explored aromatic PTMs into recombinant proteins, thus facilitating the generation of novel libraries of homogeneous posttranslationally modified proteins for biomarker discovery, mechanistic studies, and drug discovery.

Novel C. elegans models of Lewy body disease reveal pathological protein interactions and widespread miRNA dysregulation

Lewy body diseases (LBD) comprise a group of complex neurodegenerative conditions originating from accumulation of misfolded alpha-synuclein (α-syn) in the form of Lewy bodies. LBD pathologies are characterized by α-syn deposition in association with other proteins such as Amyloid β (Aβ), Tau, and TAR-DNA-binding protein. To investigate the complex interactions of these proteins, we constructed 2 novel transgenic overexpressing (OE) C. elegans strains (α-synA53T;Taupro-agg (OE) and α-synA53T;Aβ1-42;Taupro-agg (OE)) and compared them with previously established Parkinson’s, Alzheimer’s, and Lewy Body Dementia disease models. The LBD models presented here demonstrate impairments including uncoordinated movement, egg-laying deficits, altered serotonergic and cholinergic signaling, memory and posture deficits, as well as dopaminergic neuron damage and loss. Expression levels of total and prone to aggregation α-syn protein were increased in α-synA53T;Aβ1-42 but decreased in α-synA53T;Taupro-agg animals when compared to α-synA53T animals suggesting protein interactions. These alterations were also observed at the mRNA level suggesting a pre-transcriptional mechanism. miRNA-seq revealed that cel-miR-1018 was upregulated in LBD models α-synA53T, α-synA53T;Aβ1-42, and α-synA53T;Taupro-agg compared with WT. cel-miR-58c was upregulated in α-synA53T;Taupro-agg but downregulated in α-synA53T and α-synA53T;Aβ1-42 compared with WT. cel-miR-41-3p and cel-miR-355-5p were significantly downregulated in 3 LBD models. Our results obtained in a model organism provide evidence of interactions between different pathological proteins and alterations in specific miRNAs that may further exacerbate or ameliorate LBD pathology.

Zingiber officinale Roscoe extract improves nigrostriatal dopaminergic activity in rotenone-induced Parkinsonian mice: Implication of COX-2/TNF-α/IL-6 and antioxidant enzyme crosstalk in the immunoinflammatory responses

Ethnopharmacological relevanceParkinson's disease (PD) is a chronic neurodegenerative disease that occurs progressively with time. Current PD treatments are symptomatic with moderate effects on striatal dopamine release. Previously, the ethnomedicinal benefits of Zingiber officinale Roscoe have been demonstrated to possess a broad range of pharmacological benefits, but no single data on its effect against nigrostriatal degeneration in PD model has been reported. AimProactive neuroprotective and pharmacotherapeutic approaches with asymptomatic effects are needed to prevent the progression of Parkinson's disease (PD) and maintain the appropriate population of dopaminergic neurons. Current treatments are symptomatic and can only elevate the striatal dopamine levels. Hence, the objective of the study is to elucidate the neuroprotective and neurorestorative properties of Zingiber officinale methanol extract (MEZO) on nigrostriatal degeneration implicated by immunoinflammatory responses in rotenone-challenged PD mice model. MethodsMale Swiss mice were injected with rotenone (2.5 mg/kg) intraperitoneally to induce PD symptoms 30 minutes prior to MEZO (50 and 100 mg/kg) and LD-CD (10 mg/kg) oral treatment for 28 days. Motor/neuromuscular behavior, dopamine, acetylcholinesterase (AChE), tyrosine hydroxylase (TH), α-synuclein (α-syn), cyclooxygenase-2 (COX-2), oxidative stress markers, inflammatory cytokines, and histopathology were evaluated. ResultsOur data showed that MEZO treatment attenuated sensorimotor and neuromuscular incompetence by up-regulating the expression of TH protein and dopamine release and maintaining AChE enzyme in the symptomatic mice. We observed that MEZO treatment prevented the formation of Lewis bodies (LBs) by inhibiting the aggregation of nigrostriatal α-Syn proteins, decreasing the release of pro-oxidant and pro-inflammatory mediators and cyclooxygenase-2 (COX-2) protein expression, increasing glutathione enzymes, and moderately abated the loss dopaminergic neurons in a dose-dependent effect in the symptomatic mice. ConclusionThese findings demonstrated that MEZO exhibited the potential neuroprotective and neurorestorative effect in the treatment and management of PD.

Cheminformatics-Guided Exploration of Synthetic Marine Natural Product-Inspired Brominated Indole-3-Glyoxylamides and Their Potentials for Drug Discovery.

Marine natural products (MNPs) continue to be tested primarily in cellular toxicity assays, both mammalian and microbial, despite most being inactive at concentrations relevant to drug discovery. These MNPs become missed opportunities and represent a wasteful use of precious bioresources. The use of cheminformatics aligned with published bioactivity data can provide insights to direct the choice of bioassays for the evaluation of new MNPs. Cheminformatics analysis of MNPs found in MarinLit (n = 39,730) up to the end of 2023 highlighted indol-3-yl-glyoxylamides (IGAs, n = 24) as a group of MNPs with no reported bioactivities. However, a recent review of synthetic IGAs highlighted these scaffolds as privileged structures with several compounds under clinical evaluation. Herein, we report the synthesis of a library of 32 MNP-inspired brominated IGAs (25-56) using a simple one-pot, multistep method affording access to these diverse chemical scaffolds. Directed by a meta-analysis of the biological activities reported for marine indole alkaloids (MIAs) and synthetic IGAs, the brominated IGAs 25-56 were examined for their potential bioactivities against the Parkinson's Disease amyloid protein alpha synuclein (α-syn), antiplasmodial activities against chloroquine-resistant (3D7) and sensitive (Dd2) parasite strains of Plasmodium falciparum, and inhibition of mammalian (chymotrypsin and elastase) and viral (SARS-CoV-2 3CLpro) proteases. All of the synthetic IGAs tested exhibited binding affinity to the amyloid protein α-syn, while some showed inhibitory activities against P. falciparum, and the proteases, SARS-CoV-2 3CLpro, and chymotrypsin. The cellular safety of the IGAs was examined against cancerous and non-cancerous human cell lines, with all of the compounds tested inactive, thereby validating cheminformatics and meta-analyses results. The findings presented herein expand our knowledge of marine IGA bioactive chemical space and advocate expanding the scope of biological assays routinely used to investigate NP bioactivities, specifically those more suitable for non-toxic compounds. By integrating cheminformatics tools and functional assays into NP biological testing workflows, we can aim to enhance the potential of NPs and their scaffolds for future drug discovery and development.

The Role of Alpha-Synuclein and Tubulin-Associated Unit (Tau) Proteins in the Diagnosis, Prognosis, and Treatment of Parkinson's Disease: A Systematic Review.

Parkinson's disease (PD) is a degenerative neurological disorder resulting from the death of dopaminergic neurons, which, in turn, results in impaired motor and cognitive functions. Early diagnosis is important in achieving a good prognosis for PD. Currently, the only approved way to diagnose PD is through medical history, current symptoms, and neurological examination. This, however, can only happen after PD progresses far enough in patients. Biomarkers in cerebrospinal fluid (CSF) and blood plasma, however, may provide insight into the early progress of PD and potentially concurrent dementia, which can also aid in the development of novel treatments. Specifically, this systematic review explores alpha-synuclein (α-syn) and tubulin-associated unit (Tau) proteins and analyzes their potential roles as biomarkers while also touching on nilotinib and immunotherapy as potential treatment options. PubMed, PubMed Central (PMC), Medline, and Cochrane Library serve as the databases for relevant literature, upon which eligibility criteria and quality checks - Assessment of Multiple Systematic Review (AMSTAR) tool, Newcastle-Ottawa Quality Assessment Scale, Cochrane risk-of-bias assessment 2 (RoB2), and Scale for the Assessment of Narrative Review (SANRA) - were applied. The remaining literature examines the various aspects of PD and Parkinson's disease dementia (PDD) and associated biomarkers. From 10 studies, 2,361 participants, both PD patients and healthy controls (HCs), were assessed and compared. Various assessment scales, such as the Unified Parkinson's Disease Rating Scale part III (UPDRS III), were used to ascertain the severity or progression of PD in patients while also seeking a noticeable correlation with α-syn and total Tau (t-Tau). The lack of standardized clinical testing has led to conflicting reports. Thus, while the articles generally agree on the potential efficacy of α-syn and Tau protein analysis in the diagnosis, prognosis, and treatment of PD and PDD, they also argue for further testing and trials.

Elucidating the molecular mechanisms behind the therapeutic impact of median nerve stimulation on cognitive dysfunction post-traumatic brain injury

ObjectiveFerroptosis represents a form of regulated cellular death dependent upon iron and lipid peroxidation derivatives, holding considerable implications for cerebral and neurologic pathologies. In the present study, we endeavored to elucidate the molecular mechanisms governing ferroptosis and appraise the therapeutic value of electrical stimulation of median nerve in addressing cognitive impairments following traumatic brain injury (TBI), employing a rodent model. MethodsIn this study, we established a rat model to investigate the cognitive impairments resulting from TBI, followed by the application of median nerve stimulation (MNS). Initially, rats received an intraperitoneal injection of Erastin (2 mg/kg) prior to undergoing MNS. After 24 h of MNS treatment, the rats were subjected to an open field test to evaluate their cognitive and motor functions. Subsequently, we conducted biochemical assays to measure the serum levels of GSH, MDA and SOD. The structural integrity and cellular morphology of hippocampal tissue were examined through H&E staining, Nissl staining and transmission electron microscopy. Additionally, we assessed the expression levels of proteins crucial for neuronal health and function in the hippocampus, including VEGF, SLC7A11, GPX4, Nrf2, α-syn, NEUN and PSD95. ResultsCompared to the control group, rats in the stimulation group demonstrated enhanced mobility, reduced levels of tissue damage, a decrease in MDA concentration, and increased levels of GSH and SOD. Additionally, there was a significant upregulation in the expression of proteins critical for cellular defense and neuronal health, including GPX4, SLC7A11, Nrf2, VEGF, α-syn, NEUN, and PSD95 proteins. Conversely, rats in the Erastin group demonstrated decreased mobility, exacerbated pathological tissue damage, elevated MDA concentration, and decreased levels of GSH and SOD. There was also a notable decrease in the expression of GPX4, SLC7CA11, Nrf2, and VEGF proteins. The expression levels of α-syn, NEUN, and PSD95 were similarly diminished in the Erastin group. Each of these findings was statistically significant, indicating that MNS exerts neuroprotective effect in the hippocampal tissue of rats with TBI by inhibiting the ferroptosis pathway. Conclusion(1) MNS may enhance the cognitive and behavioral performance of rats after TBI; (2) MNS can play a neuroprotective role by promoting the expression of nerve injury-related proteins, alleviating oxidative stress and ferroptosis process; (3) MNS may inhibit ferroptosis of neuronal cells by activating Nrf2/ GPX4 signaling pathway, thereby improving cognitive impairment in TBI rats.

Selection of DNA aptamers that prevent the fibrillization of α-synuclein protein in cellular and mouse models

A neuropathological hallmark of Parkinson's Disease (PD) is the aggregation and spreading of misfolded α-synuclein (αSyn) protein. In this study, a selection method was developed to identify aptamers that showed affinity for monomeric αSyn and inhibition of αSyn aggregation. Aptamer a-syn-1 exhibited strong inhibition of αSyn aggregation in vitro by transmission electron microscopy and thioflavin T fluorescence. A-syn-1-treated SH-SY5Y cells incubated with pre-formed fibrils (PFFs) showed less intracellular aggregation of αSyn in comparison to a scrambled oligonucleotide control, as observed with fluorescent microscopy. Systemic delivery of a-syn-1 to the brain was achieved using a liposome vehicle and confirmed with fluorescence microscopy and qPCR. Transgenic mice overexpressing the human A53T variant of αSyn protein were injected with a-syn-1 loaded liposomes at 5 months of age both acutely (single i.p injection) and repeatedly (5 i.p. injections over 5 days). Western blot protein quantification revealed that both acute and repeated injections of a-syn-1 decreased levels of the aggregated form of αSyn in the transgenic mice in the prefrontal cortex, caudate, and substania nigra (SNc). These results provide in vitro and in vivo evidence that a-syn-1 can inhibit pathological αSyn aggregation and may have implications in treatment strategies to target dysregulation in PD.

High-Yield α-Synuclein Purification and Ionic Strength Modification Pivotal to Seed Amplification Assay Performance and Reproducibility.

Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 Escherichia coli, lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.

Effects of local reduction of endogenous α-synuclein using antisense oligonucleotides on the fibril-induced propagation of pathology through the neural network in wild-type mice

In Parkinson's disease and other synucleinopathies, fibrillar forms of α-synuclein (aSyn) are hypothesized to structurally convert and pathologize endogenous aSyn, which then propagates through the neural connections, forming Lewy pathologies and ultimately causing neurodegeneration. Inoculation of mouse-derived aSyn preformed fibrils (PFFs) into the unilateral striatum of wild-type mice causes widespread aSyn pathologies in the brain through the neural network. Here, we used the local injection of antisense oligonucleotides (ASOs) against Snca mRNA to confine the area of endogenous aSyn protein reduction and not to affect the PFFs properties in this model. We then varied the timing and location of ASOs injection to examine their impact on the initiation and propagation of aSyn pathologies in the whole brain and the therapeutic effect using abnormally-phosphorylated aSyn (pSyn) as an indicator. By injecting ASOs before or 0–14 days after the PFFs were inoculated into the same site in the left striatum, the reduction in endogenous aSyn in the striatum leads to the prevention and inhibition of the regional spread of pSyn pathologies to the whole brain including the contralateral right hemisphere. ASO post-injection inhibited extension from neuritic pathologies to somatic ones. Moreover, injection of ASOs into the right striatum prevented the remote regional spread of pSyn pathologies from the left striatum where PFFs were inoculated and no ASO treatment was conducted. This indicated that the reduction in endogenous aSyn protein levels at the propagation destination site can attenuate pSyn pathologies, even if those at the propagation initiation site are not inhibited, which is consistent with the original concept of prion-like propagation that endogenous aSyn is indispensable for this regional spread. Our results demonstrate the importance of recruiting endogenous aSyn in this neural network propagation model and indicate a possible potential for ASO treatment in synucleinopathies.

Exploring Structural Insights of Aβ42 and α-Synuclein Monomers and Heterodimer: A Comparative Study Using Implicit and Explicit Solvent Simulations.

Protein misfolding, aggregation, and fibril formation play a central role in the development of severe neurological disorders, including Alzheimer's and Parkinson's diseases. The structural stability of mature fibrils in these diseases is of great importance, as organisms struggle to effectively eliminate amyloid plaques. To address this issue, it is crucial to investigate the early stages of fibril formation when monomers aggregate into small, toxic, and soluble oligomers. However, these structures are inherently disordered, making them challenging to study through experimental approaches. Recently, it has been shown experimentally that amyloid-β 42 (Aβ42) and α-synuclein (α-Syn) can coassemble. This has motivated us to investigate the interaction between their monomers as a first step toward exploring the possibility of forming heterodimeric complexes. In particular, our study involves the utilization of various Amber and CHARMM force-fields, employing both implicit and explicit solvent models in replica exchange and conventional simulation modes. This comprehensive approach allowed us to assess the strengths and weaknesses of these solvent models and force fields in comparison to experimental and theoretical findings, ensuring the highest level of robustness. Our investigations revealed that Aβ42 and α-Syn monomers can indeed form stable heterodimers, and the resulting heterodimeric model exhibits stronger interactions compared to the Aβ42 dimer. The binding of α-Syn to Aβ42 reduces the propensity of Aβ42 to adopt fibril-prone conformations and induces significant changes in its conformational properties. Notably, in AMBER-FB15 and CHARMM36m force fields with the use of explicit solvent, the presence of Aβ42 significantly increases the β-content of α-Syn, consistent with the experiments showing that Aβ42 triggers α-Syn aggregation. Our analysis clearly shows that although the use of implicit solvent resulted in too large compactness of monomeric α-Syn, structural properties of monomeric Aβ42 and the heterodimer were preserved in explicit-solvent simulations. We anticipate that our study sheds light on the interaction between α-Syn and Aβ42 proteins, thus providing the atom-level model required to assess the initial stage of aggregation mechanisms related to Alzheimer's and Parkinson's diseases.

Teaghrelin protected dopaminergic neurons in MPTP-induced Parkinson's disease animal model by promoting PINK1/Parkin-mediated mitophagy and AMPK/SIRT1/PGC1-α-mediated mitochondrial biogenesis.

Mitochondrial dysfunction, a common cellular hallmark in both familial and sporadic forms of Parkinson's disease (PD), is assumed to play a significant role in pathologic development and progression of the disease. Teaghrelin, a unique bioactive compound in some oolong tea varieties, has been demonstrated to protect SH-SY5Y cells against 1-methyl-4-phenylpyridinium induced neurotoxicity by binding to the ghrelin receptor to activate the AMPK/SIRT1/PGC-1α pathway. In this study, an animal model was established using a neurotoxin, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP), a byproduct of a prohibited drug, to evaluate the oral efficacy of teaghrelin on PD by monitoring motor dysfunction of mice in open field, pole, and bean walking tests. The results showed that MPTP-induced motor dysfunction of mice was significantly attenuated by teaghrelin supplementation. Tyrosine hydroxylase and dopamine transporter protein were found reduced in the striatum and midbrain of MPTP-treated mice, and significantly mitigated by teaghrelin supplementation. Furthermore, teaghrelin administration enhanced mitophagy and mitochondria biogenesis, which maintained cell homeostasis and prevented the accumulation of αSyn and apoptosis-related proteins. It seemed that teaghrelin protected dopaminergic neurons in MPTP-treated mice by increasing PINK1/Parkin-mediated mitophagy and AMPK/SIRT1/PGC-1α-mediated mitochondria biogenesis, highlighting its potential therapeutic role in maintaining dopaminergic neurons function in PD. Mitochondrial dysfunction, a common cellular hallmark in both familial and sporadic forms of Parkinson's disease (PD), is assumed to play a significant role in pathologic development and progression of the disease. Teaghrelin, a unique bioactive compound in some oolong tea varieties, has been demonstrated to protect SH-SY5Y cells against 1-methyl-4-phenylpyridinium induced neurotoxicity by binding to the ghrelin receptor to activate the AMPK/SIRT1/PGC-1α pathway. In this study, an animal model was established using a neurotoxin, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP), a byproduct of a prohibited drug, to evaluate the oral efficacy of teaghrelin on PD by monitoring motor dysfunction of mice in open field, pole, and bean walking tests. The results showed that MPTP-induced motor dysfunction of mice was significantly attenuated by teaghrelin supplementation. Tyrosine hydroxylase and dopamine transporter protein were found reduced in the striatum and midbrain of MPTP-treated mice, and significantly mitigated by teaghrelin supplementation. Furthermore, teaghrelin administration enhanced mitophagy and mitochondria biogenesis, which maintained cell homeostasis and prevented the accumulation of αSyn and apoptosis-related proteins. It seemed that teaghrelin protected dopaminergic neurons in MPTP-treated mice by increasing PINK1/Parkin-mediated mitophagy and AMPK/SIRT1/PGC-1α-mediated mitochondria biogenesis, highlighting its potential therapeutic role in maintaining dopaminergic neurons function in PD.

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.

Discovery of a Gut Bacterial Metabolic Pathway that Drives α-Synuclein Aggregation.

Parkinson's disease (PD) etiology is associated with aggregation and accumulation of α-synuclein (α-syn) proteins in midbrain dopaminergic neurons. Emerging evidence suggests that in certain subtypes of PD, α-syn aggregates originate in the gut and subsequently spread to the brain. However, mechanisms that instigate α-syn aggregation in the gut have remained elusive. In the brain, the aggregation of α-syn is induced by oxidized dopamine. Such a mechanism has not been explored in the context of the gastrointestinal tract, a niche harboring 46% of the body's dopamine reservoirs. Here, we report that Enterobacteriaceae, a bacterial family prevalent in human gut microbiotas, induce α-syn aggregation. More specifically, our in vitro data indicate that respiration of nitrate by Escherichia coli K-12, which results in production of nitrite that mediates oxidation of Fe2+ to Fe3+, creates an oxidizing redox potential. These oxidizing conditions enabled the formation of dopamine-derived quinones and α-syn aggregates. Exposing nitrite, but not nitrate, to enteroendocrine STC-1 cells induced aggregation of α-syn that is natively expressed in these cells, which line the intestinal tract. Taken together, our findings indicate that bacterial nitrate reduction may be critical for initiating intestinal α-syn aggregation.

A topographical atlas of α-synuclein dosage and cell type-specific expression in adult mouse brain and peripheral organs

Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide and presents pathologically with Lewy pathology and dopaminergic neurodegeneration. Lewy pathology contains aggregated α-synuclein (αSyn), a protein encoded by the SNCA gene which is also mutated or duplicated in a subset of familial PD cases. Due to its predominant presynaptic localization, immunostaining for the protein results in a diffuse reactivity pattern, providing little insight into the types of cells expressing αSyn. As a result, insight into αSyn expression-driven cellular vulnerability has been difficult to ascertain. Using a combination of knock-in mice that target αSyn to the nucleus (SncaNLS) and in situ hybridization of Snca in wild-type mice, we systematically mapped the topography and cell types expressing αSyn in the mouse brain, spinal cord, retina, and gut. We find a high degree of correlation between αSyn protein and RNA levels and further identify cell types with low and high αSyn content. We also find high αSyn expression in neurons, particularly those involved in PD, and to a lower extent in non-neuronal cell types, notably those of oligodendrocyte lineage, which are relevant to multiple system atrophy pathogenesis. Surprisingly, we also found that αSyn is relatively absent from select neuron types, e.g., ChAT-positive motor neurons, whereas enteric neurons universally express some degree of αSyn. Together, this integrated atlas provides insight into the cellular topography of αSyn, and provides a quantitative map to test hypotheses about the role of αSyn in network vulnerability, and thus serves investigations into PD pathogenesis and other α-synucleinopathies.