Activation of parkin by a molecular glue.

Mutations in parkin and PINK1 cause early-onset Parkinson's disease (EOPD). The ubiquitin ligase parkin is recruited to damaged mitochondria and activated by PINK1, a kinase that phosphorylates ubiquitin and the ubiquitin-like domain of parkin. Activated phospho-parkin then ubiquitinates mitochondrial proteins to target the damaged organelle for degradation. Here, we present the mechanism of activation of a new class of small molecule allosteric modulators that enhance parkin activity. The compounds act as molecular glues to enhance the ability of phospho-ubiquitin (pUb) to activate parkin. Ubiquitination assays and isothermal titration calorimetry with the most active compound (BIO-2007817) identify the mechanism of action. We present the crystal structure of a closely related compound (BIO-1975900) bound to a complex of parkin and two pUb molecules. The compound binds next to pUb on RING0 and contacts both proteins. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments confirm that activation occurs through release of the catalytic Rcat domain. In organello and mitophagy assays demonstrate that BIO-2007817 partially rescues the activity of parkin EOPD mutants, R42P and V56E, offering a basis for the design of activators as therapeutics for Parkinson's disease.

Individual-specific metabolic network based on 18F-FDG PET revealing multi-level aberrant metabolisms in Parkinson's disease.

Metabolic network analysis in Parkinson's disease (PD) based on 18F-FDG PET has revealed PD-related metabolic patterns. However, alterations at the systemic metabolic network level and at the connection level between different brain regions still remain unknown. This study aimed to explore metabolic network alterations at multiple network levels among PD patients using an individual-specific metabolic network (ISMN) approach. 18F-FDG-PET images of patients with PD (n = 34) and healthy subjects (n = 47) were collected. Healthy subjects were further separated into reference group (n = 28) and control group (n = 19) randomly. Standardized uptake value normalized by lean body mass ratio (SULr) maps was calculated from the PET images. ISMNs were constructed based on SULr maps for PD patients and controls with reference to the reference group. Comparisons of nodal and edge features were performed between PD and control groups. Correlation analysis was conducted between multilevel network properties and clinical scales in PD group. A linear classifier was trained based on nodal or edge features to distinguish PD from controls. The distance from each patient's ISMN to the group-level difference network showed a negative correlation with Hoehn and Yahr stage (r = -0.390, p = .023). Eight nodes from ISMN were identified which exhibited significantly increased nodal degree in PD patients compared to controls (p < .05). Eleven edges were observed which demonstrated significant distinctions in Z-score values in comparisons to the control group (p < .05). Furthermore, the nodal and edge features showed comparable performances in PD diagnosis compared to the traditional SULr values, with area under the receiver operating characteristic curve larger than 0.91. The proposed ISMN approach revealed systemic metabolic deviations, as well as nodal and edge distinctions in PD, which might be supplementary to the existing findings on PD-related metabolic patterns.

Multiomic Profiling and Neuroprotective Bioactivity of Salvia Hairy Root-Derived Extracellular Vesicles in a Cellular Model of Parkinson's Disease.

Purpose: Extracellular vesicles (EVs) are promising tools for nanomedicine and nanobiotechnology. The purification of mammalian-derived EVs involves intensive processes, and their therapeutic application raises multiple safety and regulatory issues. Plants have the potential to serve as nonconventional sources of therapeutically relevant EVs. In this context, we recently identified hairy roots (HRs) of medicinal plants as a novel biotechnological platform to produce EVs for human health. Methods: Herein, we report the purification, omics profiling, and bioactivity of EVs isolated from HRs of the medicinal plants S. sclarea and S. dominica. EVs were isolated from conditioned media of HR cultures using differential ultracentrifugation (dUC) and size exclusion chromatography (SEC). The isolated EVs were characterized by nanoparticle tracking analysis (NTA) and electron microscopy. The proteomic and metabolomic profiles of the EVs were determined using mass spectrometry. Uptake studies and bioactivity assays, including confocal microscopy, MTT, flow cytometry, ROS quantification, and untargeted metabolomics analyses, were conducted in SH-SY5Y cells treated with the neurotoxin 6-hydroxydopamine (6-OHDA) to evaluate the therapeutic potential of EVs in an in vitro model of Parkinson's disease. Results: S. sclarea HRs released nanosized round-shaped EVs with a distinctive molecular signature. HR EVs from S. sclarea and S. dominica revealed conserved cargo of secondary metabolites, predominantly triterpenoids, which are known for their antioxidant properties. We showed that HR EVs are safe, enter the cells, and strongly inhibit apoptosis in a cellular model of Parkinson's disease. Cellular metabolomics revealed that EVs preserved metabolic homeostasis and mitigated cellular oxidative stress when co-administered with 6-OHDA. Mechanistically, HR EVs inhibited 6-OHDA autoxidation and substantially reduced the accumulation of its oxidative products, which are responsible for 6-OHDA-induced toxicity. Conclusion: Collectively, our findings provide compelling evidence that EVs isolated from the hairy roots of Salvia species are promising, non-mammalian alternative for the design of novel therapies targeting neurological disorders.

Unveiling Lobophytum sp. the neuroprotective potential of Parkinson's disease through multifaceted mechanisms, supported by metabolomic analysis and network pharmacology.

A main feature of neurodegenerative diseases is the loss of neurons. One of the most prevalent neurodegenerative illnesses is Parkinson disease (PD). Although several medications are already approved to treat neurodegenerative disorders, most of them only address associated symptoms. The main aim of the current study was to examine the neuroprotective efficacy and underlying mechanism of Lobophytum sp. crude extract in a rotenone-induced rat model of neurodegeneration mimicking PD in humans. The influence of the treatment on antioxidant, inflammatory, and apoptotic markers was assessed in addition to the investigation of TH (tyrosine hydroxylase) immunochemistry, histopathological changes, and α-synuclein. Metabolomic profiling of Lobophytum sp. crude extract was done by using High-Resolution Liquid Chromatography coupled with Mass Spectrometry (HR-LC-ESI-MS), which revealed the presence of 20 compounds (1-20) belonging to several classes of secondary metabolites including diterpenoids, sesquiterpenoids, steroids, and steroid glycosides. From our experimental results, we report that Lobophytum sp. extract conferred neuroprotection against rotenone-induced PD by inhibiting ROS formation, apoptosis, and inflammatory mediators including IL-6, IL-1ß, and TNF-α, NF-кB, and subsequent neurodegeneration as evidenced by decreased α-synuclein deposition and enhanced tyrosine hydroxylase immunoreactivity. Moreover, a computational network pharmacology study was performed for the dereplicated compounds from Lobophytum sp. using PubChem, SwissTarget Prediction, STRING, DisGeNET, and ShinyGO databases. Among the studied genes, CYP19A1 was the top gene related to Parkinson's disease. Dendrinolide compounds annotated a high number of parkinsonism genes. The vascular endothelial growth factor (VEGF) pathway was the top signaling pathway related to the studied genes. Therefore, we speculate that Lobophytum sp. extract, owing to its pleiotropic mechanisms, could be further developed as a possible therapeutic drug for treating Parkinson's disease.

Transcriptional profiling in microglia across physiological and pathological states identifies a transcriptional module associated with neurodegeneration.

Microglia are the resident immune cells of the central nervous system and are involved in brain development, homeostasis, and disease. New imaging and genomics technologies are revealing microglial complexity across developmental and functional states, brain regions, and diseases. We curated a set of publicly available gene expression datasets from human microglia spanning disease and health to identify sets of genes reflecting physiological and pathological microglial states. We also integrated multiple human microglial single-cell RNA-seq datasets in Alzheimer's disease (AD), multiple sclerosis (MS), and Parkinson's disease, and identified a distinct microglial transcriptional signature shared across diseases. Analysis of germ-line DNA identified genes with variants associated with AD and MS that are overrepresented in microglial gene sets, including the disease-associated transcriptional signature. This work points to genes that are dysregulated in disease states and provides a resource for the analysis of diseases in which microglia are implicated by genetic evidence.

Multivalency drives interactions of alpha-synuclein fibrils with tau.

Age-related neurodegenerative disorders like Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by deposits of protein aggregates, or amyloid, in various regions of the brain. Historically, aggregation of a single protein was observed to be correlated with these different pathologies: tau in AD and α-synuclein (αS) in PD. However, there is increasing evidence that the pathologies of these two diseases overlap, and the individual proteins may even promote each other's aggregation. Both tau and αS are intrinsically disordered proteins (IDPs), lacking stable secondary and tertiary structure under physiological conditions. In this study we used a combination of biochemical and biophysical techniques to interrogate the interaction of tau with both soluble and fibrillar αS. Fluorescence correlation spectroscopy (FCS) was used to assess the interactions of specific domains of fluorescently labeled tau with full length and C-terminally truncated αS in both monomer and fibrillar forms. We found that full-length tau as well as individual tau domains interact with monomer αS weakly, but this interaction is much more pronounced with αS aggregates. αS aggregates also mildly slow the rate of tau aggregation, although not the final degree of aggregation. Our findings suggest that co-occurrence of tau and αS in disease are more likely to occur through monomer-fiber binding interactions, rather than monomer-monomer or co-aggregation.

Direct evidence for ultrastructures of the α-synuclein-associated synaptic vesicle pool in presynaptic terminals.

SNCA/PARK1 encodes α-synuclein, which is associated with familial Parkinson's disease. Despite its abundance in presynaptic terminals, the aggregation mechanism of α-synuclein and its relationship with Parkinson's disease have not yet been elucidated. Moreover, the ultrastructures of α-synuclein localization sites in neuronal presynaptic terminals remain unclear. Therefore, we herein generated transgenic mice expressing human α-synuclein tagged with mKate2 (hSNCA-mKate2 mice). These mice exhibited normal growth and fertility and had no motor dysfunction relative to their wild-type littermates, even at one year old. α-Synuclein-mKate2 accumulated in presynaptic terminals, particularly between Purkinje cells in the cerebellum and neurons in cerebellar nuclei. α-Synuclein-mKate2 was associated with the presynaptic marker, synaptophysin. In-resin CLEM and immunoelectron or electron microscopy revealed that α-synuclein-mKate2 localized on the surface of synaptic vesicles that were tightly arranged and assembled to form large synaptic pools in the cerebellum with negligible effects on the active zone. These results suggest that α-synuclein-associated ultrastructures in the presynaptic terminals of hSNCA-mKate2 mice reflect the structures of α-synuclein-assembled synaptic vesicle pools, and the size of vesicle pools increased. This transgenic mouse model will be a valuable tool for studying α-synuclein-associated synaptic vesicle pools.

Unveiling MRI markers for Parkinson's Disease: GABAergic dysfunction and cortical changes.

OBJECTIVE: The study aimed to investigate changes in basal levels of the inhibitory γ-aminobutyric acid (GABA) neurotransmitter in the sensorimotor cortex (SMC) and cortical gyrification in patients with Parkinson's disease (PD), which could further identify potential imaging biomarkers for PD, particularly in patients with early-onset Parkinson's disease (EOPD). METHOD: Fifty patients with PD (EOPD: 10, late-onset Parkinson's disease [LOPD]: 40) and fifty-two age- and gender-matched healthy controls (HC) underwent GABA-edited 1H MRS of the SMC and high-resolution 3D T1-weighted brain imaging. GABA levels and local gyrification index (LGI) were calculated to assess GABAergic and cortical gyrification deficits in PD. RESULT: The Pearson correlation coefficients revealed significant negative associations between eight indicators, including GABA/Cr level and local gyrification index (LGI) of specific cortical regions (precentral, postcentral, entorhinal, superiortemporal, posteriorcingulate, cuneus, and transversetemporal cortex), and the likelihood of Parkinson's disease (r < -0.4, p < 0.001). Additionally, GABA levels were significantly lower in the SMC region of both EOPD and LOPD patients compared to healthy controls (mean ± SD [u.i.]: EOPD=0.081 ± 0.022 vs. Young-HC=0.112 ± 0.021, p = 0.003; LOPD=0.054 ± 0.024 vs. Old-HC=0.099 ± 0.021, p < 0.001). The logistic regression model was established by using multivariate analysis, identifying two statistically significant indicators: GABA/Cr and LGI of the transversetemporal. The combined model exhibited the highest AUC values in both younger and older populations. CONCLUSION: GABAergic dysfunction may play an important role in the pathogenesis of PD patients. Changes in neurotransmitter and morphological may serve as potential markers for the preclinical diagnosis and progression of PD, including EOPD.

Targeting ferroptosis with the lipoxygenase inhibitor PTC-041 as a therapeutic strategy for the treatment of Parkinson's disease.

Parkinson's disease is the second most common neurodegenerative disorder, affecting nearly 10 million people worldwide. Ferroptosis, a recently identified form of regulated cell death characterized by 15-lipoxygenase-mediated hydroperoxidation of membrane lipids, has been implicated in neurodegenerative disorders including amyotrophic lateral sclerosis and Parkinson's disease. Pharmacological inhibition of 15 -lipoxygenase to prevent iron- and lipid peroxidation-associated ferroptotic cell death is a rational strategy for the treatment of Parkinson's disease. We report here the characterization of PTC-041 as an anti-ferroptotic reductive lipoxygenase inhibitor developed for the treatment of Parkinson's disease. In these studies, PTC-041 potently protects primary human Parkinson's disease patient-derived fibroblasts from lipid peroxidation and subsequent ferroptotic cell death and prevents ferroptosis-related neuronal loss and astrogliosis in primary rat neuronal cultures. Additionally, PTC-041 prevents ferroptotic-mediated α-synuclein protein aggregation and nitrosylation in vitro, suggesting a potential role for anti-ferroptotic lipoxygenase inhibitors in mitigating pathogenic aspects of synucleinopathies such as Parkinson's disease. We further found that PTC-041 protects against synucleinopathy in vivo, demonstrating that PTC-041 treatment of Line 61 transgenic mice protects against α-synuclein aggregation and phosphorylation as well as prevents associated neuronal and non-neuronal cell death. Finally, we show that. PTC-041 protects against 6-hydroxydopamine-induced motor deficits in a hemiparkinsonian rat model, further validating the potential therapeutic benefits of lipoxygenase inhibitors in the treatment of Parkinson's disease.

The p53 target DRAM1 modulates calcium homeostasis and ER stress by promoting contact between lysosomes and the ER through STIM1.

It is well established that DNA Damage Regulated Autophagy Modulator 1 (DRAM1), a lysosomal protein and a target of p53, participates in autophagy. The cellular functions of DRAM1 beyond autophagy remain elusive. Here, we show p53-dependent upregulation of DRAM1 in mitochondrial damage-induced Parkinson's disease (PD) models and exacerbation of disease phenotypes by DRAM1. We find that the lysosomal location of DRAM1 relies on its intact structure including the cytosol-facing C-terminal domain. Excess DRAM1 disrupts endoplasmic reticulum (ER) structure, triggers ER stress, and induces protective ER-phagy. Mechanistically, DRAM1 interacts with stromal interacting molecule 1 (STIM1) to tether lysosomes to the ER and perturb STIM1 function in maintaining intracellular calcium homeostasis. STIM1 overexpression promotes cellular health by restoring calcium homeostasis, ER stress response, ER-phagy, and AMP-activated protein kinase (AMPK)-Unc-51 like autophagy activating kinase 1 (ULK1) signaling in cells with excess DRAM1. Thus, by promoting organelle contact between lysosomes and the ER, DRAM1 modulates ER structure and function and cell survival under stress. Our results suggest that DRAM1 as a lysosomal protein performs diverse roles in cellular homeostasis and stress response. These findings may have significant implications for our understanding of the role of the p53/DRAM1 axis in human diseases, from cancer to neurodegenerative diseases.

The regulation of NFKB1 on CD200R1 expression and their potential roles in Parkinson's disease.

BACKGROUND: Overactivated microglia are a key contributor to Parkinson's disease (PD) by inducing neuroinflammation. CD200R1, a membrane glycoprotein mainly found on microglia, is crucial for maintaining quiescence with its dysregulation linked to microglia's abnormal activation. We and other groups have reported a decline in CD200R1 levels in several neurological disorders including PD. However, the mechanism regulating CD200R1 expression and the specific reasons for its reduction in PD remain largely unexplored. Given the pivotal role of transcription factors in gene expression, this study aimed to elucidate the transcriptional regulation of CD200R1 and its implications in PD. METHODS: The CD200R1 promoter core region was identified via luciferase assays. Potential transcription factors were predicted using the UCSC ChIP-seq database and JASPAR. NFKB1 binding to the CD200R1 core promoter was substantiated through electrophoretic mobility shift and chromatin immunoprecipitation assays. Knocking-down or overexpressing NFKB1 validated its regulatory effect on CD200R1. Correlation between decreased CD200R1 and deficient NFKB1 was studied using Genotype-Tissue Expression database. The clinical samples of the peripheral blood mononuclear cells were acquired from 44 PD patients (mean age 64.13 ± 9.78, 43.2% male, median Hoehn-Yahr stage 1.77) and 45 controls (mean age 64.70 ± 9.41, 52.1% male). NFKB1 knockout mice were utilized to study the impact of NFKB1 on CD200R1 expression and to assess their roles in PD pathophysiology. RESULTS: The study identified the CD200R1 core promoter region, located 482 to 146 bp upstream of its translation initiation site, was directly regulated by NFKB1. Significant correlation between NFKB1 and CD200R1 expression was observed in human PMBCs. Both NFKB1 and CD200R1 were significantly decreased in PD patient samples. Furthermore, NFKB1-/- mice exhibited exacerbated microglia activation and dopaminergic neuron loss after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. CONCLUSION: Our study identified that NFKB1 served as a direct regulator of CD200R1. Reduced NFKB1 played a critical role in CD200R1 dysregulation and subsequent microglia overactivation in PD. These findings provide evidence that targeting the NFKB1-CD200R1 axis would be a novel therapeutic strategy for PD.

Acetyl-DL-leucine in two individuals with REM sleep behavior disorder improves symptoms, reverses loss of striatal dopamine-transporter binding and stabilizes pathological metabolic brain pattern-case reports.

Isolated REM Sleep Behavior Disorder (iRBD) is considered a prodrome of Parkinson's disease (PD). We investigate whether the potentially disease-modifying compound acetyl-DL-leucine (ADLL; 5 g/d) has an effect on prodromal PD progression in 2 iRBD-patients. Outcome parameters are RBD-severity sum-score (RBD-SS-3), dopamine-transporter single-photon emission computerized tomography (DAT-SPECT) and metabolic "Parkinson-Disease-related-Pattern (PDRP)"-z-score in 18F-fluorodeoxyglucose positron emission tomography (FDG-PET). After 3 weeks ADLL-treatment, the RBD-SS-3 drops markedly in both patients and remains reduced for >18 months of ADLL-treatment. In patient 1 (female), the DAT-SPECT putaminal binding ratio (PBR) decreases in the 5 years pretreatment from normal (1.88) to pathological (1.22) and the patient's FDG-PET-PDRP-z-score rises from 1.72 to 3.28 (pathological). After 22 months of ADLL-treatment, the DAT-SPECT-PBR increases to 1.67 and the FDG-PET-PDRP-z-score stabilizes at 3.18. Similar results are seen in patient 2 (male): his DAT-SPECT-PBR rises from a pretreatment value of 1.42 to 1.72 (close to normal) and the FDG-PET-PDRP-z-score decreases from 1.02 to 0.30 after 18 months of ADLL-treatment. These results support exploration of whether ADLL may have disease-modifying properties in prodromal PD.

Lysosomal dysfunction in α-synuclein pathology: molecular mechanisms and therapeutic strategies.

In orchestrating cell signaling, facilitating plasma membrane repair, supervising protein secretion, managing waste elimination, and regulating energy consumption, lysosomes are indispensable guardians that play a crucial role in preserving intracellular homeostasis. Neurons are terminally differentiated post-mitotic cells. Neuronal function and waste elimination depend on normal lysosomal function. Converging data suggest that lysosomal dysfunction is a critical event in the etiology of Parkinson's disease (PD). Mutations in Glucosylceramidase Beta 1 (GBA1) and leucine-rich repeat kinase 2 (LRRK2) confer an increased risk for the development of parkinsonism. Furthermore, lysosomal dysfunction has been observed in the affected neurons of sporadic PD (sPD) patients. Given that lysosomal hydrolases actively contribute to the breakdown of impaired organelles and misfolded proteins, any compromise in lysosomal integrity could incite abnormal accumulation of proteins, including α-synuclein, the major component of Lewy bodies in PD. Clinical observations have shown that lysosomal protein levels in cerebrospinal fluid may serve as potential biomarkers for PD diagnosis and as signs of lysosomal dysfunction. In this review, we summarize the current evidence regarding lysosomal dysfunction in PD and discuss the intimate relationship between lysosomal dysfunction and pathological α-synuclein. In addition, we discuss therapeutic strategies that target lysosomes to treat PD.

Tilorone mitigates the propagation of α-synucleinopathy in a midbrain-like organoid model.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative condition characterized by the loss of dopaminergic neurons and the accumulation of Lewy-body protein aggregates containing misfolded α-synuclein (α-syn) in a phosphorylated form. The lack of effective models for drug screens has hindered drug development studies for PD. However, the recent development of in vitro brain-like organoids provides a new opportunity for evaluating therapeutic agents to slow the progression of this chronic disease. METHODS: In this study, we used a 3D brain-like organoid model to investigate the potential of repurposing Tilorone, an anti-viral drug, for impeding the propagation of α-synucleinopathy. We assessed the effect of Tilorone on the uptake of fluorescently labeled α-syn preformed fibrils (sPFF) and sPFF-induced apoptosis using confocal microscopy. We also examined Tilorone's impact on the phosphorylation of endogenous α-syn induced by pathogenic sPFF by immunoblotting midbrain-like organoid extracts. Additionally, quantitative RT-PCR and proteomic profiling of sPFF-treated organoids were conducted to evaluate the global impact of Tilorone treatment on tissue homeostasis in the 3D organoid model. RESULTS: Tilorone inhibits the uptake of sPFF in both mouse primary neurons and human midbrain-like organoids. Tilorone also reduces the phosphorylation of endogenous α-syn induced by pathogenic α-syn fibrils and mitigates α-syn fibril-induced apoptosis in midbrain-like organoids. Proteomic profiling of fibril-treated organoids reveals substantial alterations in lipid homeostasis by α-syn fibrils, which are reversed by Tilorone treatment. Given its safety profile in clinics, Tilorone may be further developed as a therapeutic intervention to alleviate the propagation of synucleinopathy in PD patients.

ADVANCEMENT IN ALPHA-SYNUCLEIN PROTEOMICS: EXPLORING ANALYTICAL TECHNIQUES AND THEIR CLINICAL IMPLICATIONS IN PARKINSON'S DISEASE.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, affecting around 500,000 to 1 million Americans, with a significant portion diagnosed before age 50. Despite advances in treatments such as dopamine replacement therapy and deep brain stimulation, no therapies currently exist to halt or slow disease progression in advanced stages. Research is increasingly focused on identifying early biomarkers for PD to enable earlier intervention. Alpha-synuclein (α-Syn), a key protein implicated in PD pathology, is studied using various proteomics techniques like mass spectrometry, gel electrophoresis, and chromatography, to understand its role and alterations in PD. These techniques help in extracting, analyzing, and characterizing α-Syn from brain samples, providing insights into disease mechanisms and potential diagnostic and therapeutic applications.

Olfactory deficit and gastrointestinal dysfunction precede motor abnormalities in alpha-Synuclein G51D knock-in mice.

Parkinson's disease (PD) is typically a sporadic late-onset disorder, which has made it difficult to model in mice. Several transgenic mouse models bearing mutations in SNCA, which encodes alpha-Synuclein (α-Syn), have been made, but these lines do not express SNCA in a physiologically accurate spatiotemporal pattern, which limits the ability of the mice to recapitulate the features of human PD. Here, we generated knock-in mice bearing the G51D SNCA mutation. After establishing that their motor symptoms begin at 9 mo of age, we then sought earlier pathologies. We assessed the phosphorylation at Serine 129 of α-Syn in different tissues and detected phospho-α-Syn in the olfactory bulb and enteric nervous system at 3 mo of age. Olfactory deficit and impaired gut transit followed at 6 mo, preceding motor symptoms. The SncaG51D mice thus parallel the progression of human PD and will enable us to study PD pathogenesis and test future therapies.

Neuronal tissue collection from intra-cranial instruments used in deep brain stimulation surgery for Parkinson's disease with implications for study of alpha-synuclein.

Alpha-synuclein (αSyn) forms pathologic aggregates in Parkinson's disease (PD) and is implicated in mechanisms underlying neurodegeneration. While pathologic αSyn has been extensively studied, there is currently no method to evaluate αSyn within the brains of living patients. Patients with PD are often treated with deep brain stimulation (DBS) surgery in which surgical instruments are in direct contact with neuronal tissue; herein, we describe a method by which tissue is collected from DBS surgical instruments in PD and essential tremor (ET) patients and demonstrate that αSyn is detected. 24 patients undergoing DBS surgery for PD (17 patients) or ET (7 patients) were enrolled; from patient samples, 81.2 ± 44.8 µg of protein (n = 15), on average, was collected from surgical instruments. Light microscopy revealed axons, capillaries, and blood cells as the primary components of purified tissue (n = 3). ELISA assay further confirmed the presence of neuronal and glial tissue in DBS samples (n = 4). Further analysis was conducted using western blot, demonstrating that multiple αSyn antibodies are reactive in PD (n = 5) and ET (n = 3) samples; truncated αSyn (1-125 αSyn) was significantly increased in PD (n = 5) compared to ET (n = 3), in which αSyn misfolding is not expected (0.64 ± 0.25 vs. 0.25 ± 0.12, P = 0.046), thus showing that multiple forms of αSyn can be detected from living PD patients with this method.

Tet1-mediated activation of the Ampk signaling by Trpv1 DNA hydroxymethylation exerts neuroprotective effects in a rat model of Parkinson's disease.

Epigenetic regulation plays a role in Parkinson's disease (PD), and ten-eleven translocation methylcytosine dioxygenase 1 (TET1) catalyzes the first step in DNA demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine. We investigated whether TET1 binds to the promoter of the transient receptor potential cation channel subfamily V member 1 (TRPV1) and regulates its expression, thereby controlling oxidative stress in PD. TRPV1 was identified as an oxidative stress-associated gene in the GSE20186 dataset including substantia nigra from 14 patients with PD and 14 healthy controls and the Genecards database. Lentiviral vectors were used to manipulate Trpv1 expression in rats, followed by 6-hydroxydopamine hydrochloride (6-OHDA) injection for modeling. Behavioral tests, immunofluorescence, Nissl staining, western blot assays, DHE fluorescent probe, biochemical analysis, and ELISA were conducted to assess oxidative stress and neurotoxicity. Trpv1 expression was significantly reduced in the brain tissues of 6-OHDA-treated Parkinsonian rats. Trpv1 alleviated behavioral dysfunction, oxidative stress, and dopamine neuron loss in rats. TET1 mediated TRPV1 hydroxymethylation to promote its expression, and Trpv1 inhibition reversed the mitigating effect of Tet1 on oxidative stress and behavioral dysfunction in PD. TRPV1 activated the AMPK signaling by promoting AMPK phosphorylation to alleviate neurotoxicity and oxidative stress in SH-SY5Y cells. Tet1-mediated Trpv1 hydroxymethylation modification promotes the Ampk signaling activation, thereby eliciting neuroprotection in 6-OHDA-treated Parkinsonian rats. These findings provide experimental evidence that targeting the TET1/TRPV1 axis may be neuroprotective for PD by acting on the AMPK signaling.

The Bifunctional Dimer Caffeine-Indan Attenuates α-Synuclein Misfolding, Neurodegeneration and Behavioral Deficits after Chronic Stimulation of Adenosine A1 Receptors.

We previously found that chronic adenosine A1 receptor stimulation with N6-Cyclopentyladenosine increased α-synuclein misfolding and neurodegeneration in a novel α-synucleinopathy model, a hallmark of Parkinson's disease. Here, we aimed to synthesize a dimer caffeine-indan linked by a 6-carbon chain to cross the blood-brain barrier and tested its ability to bind α-synuclein, reducing misfolding, behavioral abnormalities, and neurodegeneration in our rodent model. Behavioral tests and histological stains assessed neuroprotective effects of the dimer compound. A rapid synthesis of the 18F-labeled analogue enabled Positron Emission Tomography and Computed Tomography imaging for biodistribution measurement. Molecular docking analysis showed that the dimer binds to α-synuclein N- and C-termini and the non-amyloid-ß-component (NAC) domain, similar to 1-aminoindan, and this binding promotes a neuroprotective α-synuclein "loop" conformation. The dimer also binds to the orthosteric binding site for adenosine within the adenosine A1 receptor. Immunohistochemistry and confocal imaging showed the dimer abolished α-synuclein upregulation and aggregation in the substantia nigra and hippocampus, and the dimer mitigated cognitive deficits, anxiety, despair, and motor abnormalities. The 18F-labeled dimer remained stable post-injection and distributed in various organs, notably in the brain, suggesting its potential as a Positron Emission Tomography tracer for α-synuclein and adenosine A1 receptor in Parkinson's disease therapy.