Environmental pollutants and alpha-synuclein toxicity in Parkinson's disease.

Parkinson's Disease (PD) is a chronic and progressive neurodebilitating disorder that affects both motor and non-motor functions. PD is the second most commonly occurring brain disorder after Alzheimer's disease. The incidence rate of PD was found to be 17 per 100000 per year. The prevalence of the disease is at its peak at age 70 and older. One of the major reasons for the failure to devise a complete therapeutic cure for PD is an inability to identify the exact pathological cause. Recent research has also stated that PD originates in the gut way before the symptoms begin to manifest in an affected person. This might be due to the transmission of pathological alpha-synuclein (α-syn) from the gut to the brain via the vagus nerve. The involvement of toxic environmental exposure in the generation of major disorders like cancer, brain disorders etc, is not an entirely new notion. Our genes are affected directly by the environment. Simultaneously, a number of environmental pollutants may contribute significantly to the trigger of alpha-synuclein misfolding in the brain during PD. In the present review, we will mainly focus on understanding the pathological cascade of PD and how it is triggered by environmental pollutants.

α-Synuclein pathology disrupts mitochondrial function in dopaminergic and cholinergic neurons at-risk in Parkinson's disease.

BACKGROUND: Pathological accumulation of aggregated α-synuclein (aSYN) is a common feature of Parkinson's disease (PD). However, the mechanisms by which intracellular aSYN pathology contributes to dysfunction and degeneration of neurons in the brain are still unclear. A potentially relevant target of aSYN is the mitochondrion. To test this hypothesis, genetic and physiological methods were used to monitor mitochondrial function in substantia nigra pars compacta (SNc) dopaminergic and pedunculopontine nucleus (PPN) cholinergic neurons after stereotaxic injection of aSYN pre-formed fibrils (PFFs) into the mouse brain. METHODS: aSYN PFFs were stereotaxically injected into the SNc or PPN of mice. Twelve weeks later, mice were studied using a combination of approaches, including immunocytochemical analysis, cell-type specific transcriptomic profiling, electron microscopy, electrophysiology and two-photon-laser-scanning microscopy of genetically encoded sensors for bioenergetic and redox status. RESULTS: In addition to inducing a significant neuronal loss, SNc injection of PFFs induced the formation of intracellular, phosphorylated aSYN aggregates selectively in dopaminergic neurons. In these neurons, PFF-exposure decreased mitochondrial gene expression, reduced the number of mitochondria, increased oxidant stress, and profoundly disrupted mitochondrial adenosine triphosphate production. Consistent with an aSYN-induced bioenergetic deficit, the autonomous spiking of dopaminergic neurons slowed or stopped. PFFs also up-regulated lysosomal gene expression and increased lysosomal abundance, leading to the formation of Lewy-like inclusions. Similar changes were observed in PPN cholinergic neurons following aSYN PFF exposure. CONCLUSIONS: Taken together, our findings suggest that disruption of mitochondrial function, and the subsequent bioenergetic deficit, is a proximal step in the cascade of events induced by aSYN pathology leading to dysfunction and degeneration of neurons at-risk in PD.

Spiral volumetric optoacoustic tomography of reduced oxygen saturation in the spinal cord of M83 mouse model of Parkinson's disease.

PURPOSE: Metabolism and bioenergetics in the central nervous system play important roles in the pathophysiology of Parkinson's disease (PD). Here, we employed a multimodal imaging approach to assess oxygenation changes in the spinal cord of the transgenic M83 murine model of PD overexpressing the mutated A53T alpha-synuclein form in comparison with non-transgenic littermates. METHODS: In vivo spiral volumetric optoacoustic tomography (SVOT) was performed to assess oxygen saturation (sO2) in the spinal cords of M83 mice and non-transgenic littermates. Ex vivo high-field T1-weighted (T1w) magnetic resonance imaging (MRI) at 9.4T was used to assess volumetric alterations in the spinal cord. 3D SVOT analysis and deep learning-based automatic segmentation of T1w MRI data for the mouse spinal cord were developed for quantification. Immunostaining for phosphorylated alpha-synuclein (pS129 α-syn), as well as vascular organization (CD31 and GLUT1), was performed after MRI scan. RESULTS: In vivo SVOT imaging revealed a lower sO2SVOT in the spinal cord of M83 mice compared to non-transgenic littermates at sub-100 µm spatial resolution. Ex vivo MRI-assisted by in-house developed deep learning-based automatic segmentation (validated by manual analysis) revealed no volumetric atrophy in the spinal cord of M83 mice compared to non-transgenic littermates at 50 µm spatial resolution. The vascular network was not impaired in the spinal cord of M83 mice in the presence of pS129 α-syn accumulation. CONCLUSION: We developed tools for deep-learning-based analysis for the segmentation of mouse spinal cord structural MRI data, and volumetric analysis of sO2SVOT data. We demonstrated non-invasive high-resolution imaging of reduced sO2SVOT in the absence of volumetric structural changes in the spinal cord of PD M83 mouse model.

Interplay of α-Synuclein Oligomers and Endoplasmic Reticulum Stress in Parkinson'S Disease: Insights into Cellular Dysfunctions.

Oligomeric forms of α-synuclein (α-syn) are critical in the formation of α-synuclein fibrils, exhibiting neurotoxic properties that are pivotal in the pathogenesis of Parkinson's disease (PD). A salient feature of this pathology is the disruption of the protein folding capacity of the endoplasmic reticulum (ER), leading to a perturbation in the ER's protein quality control mechanisms. The accumulation of unfolded or misfolded proteins instigates ER stress. However, the onset of ER stress and the consequent activation of the Unfolded Protein Response (UPR) and Endoplasmic Reticulum-Associated Degradation (ERAD) pathways do not merely culminate in apoptosis when they fail to restore cellular homeostasis. More critically, this condition initiates a cascade of reactions involving ER-related structures and organelles, resulting in multifaceted cellular damage and, potentially, a feedback loop that precipitates neuroinflammation. In this review, we elucidate the interplay between UPR and ERAD, as well as the intricate crosstalk among the ER and other organelles such as mitochondria, lysosomes, and the Golgi apparatus, underscoring their roles in the neurodegenerative process.

Possible regulation of the immune modulator tetraspanin CD81 by alpha-synuclein in melanoma.

We probed the mechanism by which the Parkinson's disease-associated protein α-synuclein (α-syn)/SNCA promotes the pathogenesis and progression of melanoma. We found that the human melanoma cell line SK-MEL-28 in which SNCA is knocked out (SNCA-KO) has low levels of tetraspanin CD81, which is a cell-surface protein that promotes invasion, migration, and immune suppression. Analyzing data from the Cancer Genome Atlas, we show that SNCA and CD81 mRNA levels are positively correlated in melanoma; melanoma survival is inversely related to the levels of SNCA and CD81; and SNCA/CD81 are inversely related to the expression of key cytokine genes (IL12A, IL12B, IFN, IFNG, PRF1 and GZMB) for immune activation and immune cell-mediated killing of melanoma cells. We propose that high levels of α-syn and CD81 in melanoma and in immune cells drive invasion and migration and in parallel cause an immunosuppressive microenvironment; these contributing factors lead to aggressive melanomas.

Chitosan-enhanced sensitivity of mercaptoundecanoic acid (MUA)- capped gold nanorod based localized surface plasmon resonance (LSPR) biosensor for detection of alpha-synuclein oligomer biomarker in parkinson's disease.

Alpha-synuclein oligomers play a crucial role in the early diagnosis of Parkinson's disease (PD). In this study, a mercaptoundecanoic acid (MUA)-capped gold nanorod (GNR)-coated and chitosan (CH)-immobilized fiber optic probe has shown considerable sensitivity of its detection. The proposed U-shaped fiber optic biosensor based on localized surface plasmon resonance (LSPR) was applied to detect α-syn oligomer (OA) biomarker. By analyzing OA concentrations, the biosensor achieved a limit of detection of (LOD) 11 pM within the concentration range of 10-100 pM and the sensitivity value was found as 502.69 Δλ/RIU. Upon analysis of the CV% (coefficient of variation) and accuracy/recovery values, it is revealed that the sensor successfully fulfilled the criteria for success, displaying accuracy/recovery values within the range of 80%-120% and CV% values below 20%. This sensor presents significant advantages, including high sensitivity, specificity, and ability to detect very low concentrations of OA. In conclusion, the suggested U-shaped fiber optic biosensor has the potential to be valuable in the early detection of PD from a clinical perspective.

Serum Alpha-synuclein in Restless Legs Syndrome.

STUDY OBJECTIVES: To determine any correlation between serum alpha-synuclein (α-syn) concentrations and restless legs syndrome (RLS), and to explore the impact of intravenous iron supplementation on serum α-syn levels. METHODS: We collected clinical data on 113 RLS patients in whom serum α-syn levels were quantified using an ELISA kit and compared to a group of 45 age matched controls. A subset of 9 RLS patients who received intravenous (IV) iron underwent pre- and post-treatment blood sampling to assess α-syn and ferritin response. RESULTS: Family history of RLS was reported by 62.8% of patients, and current dopaminergic augmentation was observed in 31.0%. Low serum ferritin levels below 75 µg/L were seen in 39.8%. Serum α-syn levels were found to be significantly decreased in RLS patients (mean: 7.7 ng/ml) compared to controls (mean: 10.7 ng/ml,), p<0.05. Stratification based on sex, age and age of onset, did not reveal significant differences in α-syn levels. In 9 RLS patients who received IV iron treatment, a linear correlation between fold change in α-syn and ferritin was observed (R: 0.7, p <0.05). The temporal relation between serum α-syn and IV iron treatment showed a gradual decline of α-syn and ferritin by time correlation (p = 0.023, R: -0.739). CONCLUSION: In our study of 113 RLS subjects, serum α-syn levels were decreased in RLS patients compared to healthy controls and increased in the 9 patients who received IV iron treatment in correlation with ferritin. This correlation could suggest a mechanism for reduced dopamine transmission in RLS.

Clinical, neuropathological, and molecular characteristics of rapidly progressive dementia with Lewy bodies: a distinct clinicopathological entity?

BACKGROUND: The term rapidly progressive dementia (RPD) with Lewy bodies (rpDLB) is used for DLB patients who develop a rapidly progressive neurological syndrome and have reduced survival. Here, we characterise the clinical, neuropathological, and molecular characteristics of a large rpDLB neuropathological series. METHODS: We included all RPD patients with a disease duration < 4 years submitted to our prion disease referral centre between 2003 and 2022 who showed Lewy body pathology (LBP) in limbic or neocortical stages as primary neuropathological diagnosis, had no systemic condition justifying the rapid deterioration and were previously neurologically unimpaired. Clinical features were retrieved and compared with Creutzfeldt-Jakob disease (CJD) and rapidly progressive Alzheimer's disease (rpAD) cohorts. Neuropathological and genetic (whole exome sequencing, APOE genotyping, and C9orf72 repeat expansion analysis) characteristics of rpDLB patients were systematically investigated. We scored semi-quantitatively the LBP load and performed a α-synuclein (αSyn) RT-QuIC seeding amplification assay (SAA) on cerebrospinal fluid (CSF) and tenfold serially diluted brain homogenates from different brain areas in rpDLB patients and typical long-lasting Lewy body disease (LBD) with dementia patients as control group. RESULTS: RpDLB patients were older (p = 0.047) and presented more cognitive fluctuations (p = 0.005), visual hallucinations (p = 0.020), neuropsychiatric symptoms (p = 0.006) and seizures (p = 0.032), and fewer cerebellar (p < 0.001) and visual (p = 0.004) signs than CJD ones. Delirium onset was more common than in both CJD (p < 0.001) and rpAD (p = 0.008). Atypical LBD signs (pyramidal, myoclonus, akinetic mutism) were common. All tested patients were positive by CSF αSyn SAA. Concomitant pathologies were common, with only four cases showing relatively "pure" LBP. LBP load and αSyn seeding activity measured through αSyn RT-QuIC SAA were not significantly different between rpDLB patients and typical LBD. We found a likely pathogenic variant in GBA in one patient. CONCLUSIONS: Our results indicate that: 1) rpDLB exhibits a distinct clinical signature (2) CSF αSyn SAA is a reliable diagnostic test; 3) rpDLB is a heterogeneous neuropathological entity that can be underlain by both widespread pure LBP, or multiple copathologies 4) rpDLB is likely not sustained by distinct αSyn conformational strains; 5) genetic defects may, at least occasionally, contribute to the poor prognosis in these patients.

Understanding alpha-synuclein aggregation propensity in animals and humans.

Alpha-synuclein (α-syn) aggregation plays a critical role in the pathogenicity of Parkinson's Disease (PD). This study aims to evaluate the aggregation propensity of α-syn fragment peptides designed using the variability found in humans and animals. Thioflavin T (ThT) and transmission electron microscopy (TEM) were used to validate the formation of fibrils to identify important amino acid residues. Human α-syn fragments 51-75, 37-61, 62-86, 76-100, and 116-140 demonstrate a significantly higher tendency to aggregate compared to fragments 1-25, 26-50, and 91-115. All species analyzed of the α-syn 37-61 and 62-86 regions were shown to form fibrils on both ThT and TEM. The α-syn 37-61 and 62-86 fragment regions exhibited a high susceptibility to aggregation, with fibril formation observed in all species. The A53T mutation in several α-syn 37-61 fragments may enhance their propensity for aggregation, suggesting a correlation between this mutation and the capacity for fibril formation. Furthermore, the presence of the non-amyloid-ß component (NAC) region, specifically in α-syn 62-86, was consistently observed in several fragments that displayed fibril formation, indicating a potential correlation between the NAC region and the process of fibril formation in α-syn. Finally, the combination of a high quantity of valine and a low quantity of acidic amino acids in these fragments may serve as indicators of α-syn fibril formation.

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.

Simulating the anti-aggregative effect of fasudil in early dimerisation process of α-synuclein.

The aggregation of the protein α-synuclein into amyloid deposits is associated with multiple neurological disorders, including Parkinson's disease. Soluble amyloid oligomers are reported to exhibit higher toxicity than insoluble amyloid fibrils, with dimers being the smallest toxic oligomer. Small molecule drugs, such as fasudil, have shown potential in targeting α-synuclein aggregation and reducing its toxicity. In this study, we use atomistic molecular dynamics simulations to demonstrate how fasudil affects the earliest stage of aggregation, namely dimerization. Our results show that the presence of fasudil reduces the propensity for intermolecular contact formation between protein chains. Consistent with previous reports, our analysis confirms that fasudil predominantly interacts with the negatively charged C-terminal region of α-synuclein. However, we also observe transient interactions with residues in the charged N-terminal and hydrophobic NAC regions. Our simulations indicate that while fasudil prominently interacts with the C-terminal region, it is the transient interactions with residues in the N-terminal and NAC regions that effectively block the formation of intermolecular contacts between protein chains and prevent early dimerization of this disordered protein.

Modulation of cannabinoid receptor 2 alters neuroinflammation and reduces formation of alpha-synuclein aggregates in a rat model of nigral synucleinopathy.

Research into the disequilibrium of microglial phenotypes has become an area of intense focus in neurodegenerative disease as a potential mechanism that contributes to chronic neuroinflammation and neuronal loss in Parkinson's disease (PD). There is growing evidence that neuroinflammation accompanies and may promote progression of alpha-synuclein (Asyn)-induced nigral dopaminergic (DA) degeneration. From a therapeutic perspective, development of immunomodulatory strategies that dampen overproduction of pro-inflammatory cytokines from chronically activated immune cells and induce a pro-phagocytic phenotype is expected to promote Asyn removal and protect vulnerable neurons. Cannabinoid receptor-2 (CB2) is highly expressed on activated microglia and peripheral immune cells, is upregulated in the substantia nigra of individuals with PD and in mouse models of nigral degeneration. Furthermore, modulation of CB2 protects against rotenone-induced nigral degeneration; however, CB2 has not been pharmacologically and selectively targeted in an Asyn model of PD. Here, we report that 7 weeks of peripheral administration of CB2 inverse agonist SMM-189 reduced phosphorylated (pSer129) Asyn in the substantia nigra compared to vehicle treatment. Additionally, SMM-189 delayed Asyn-induced immune cell infiltration into the brain as determined by flow cytometry, increased CD68 protein expression, and elevated wound-healing-immune-mediator gene expression. Additionally, peripheral immune cells increased wound-healing non-classical monocytes and decreased pro-inflammatory classical monocytes. In vitro analysis of RAW264.7 macrophages treated with lipopolysaccharide (LPS) and SMM-189 revealed increased phagocytosis as measured by the uptake of fluorescence of pHrodo E. coli bioparticles. Together, results suggest that targeting CB2 with SMM-189 skews immune cell function toward a phagocytic phenotype and reduces toxic aggregated species of Asyn. Our novel findings demonstrate that CB2 may be a target to modulate inflammatory and immune responses in proteinopathies.

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.

Viral and nonviral approaches.

Neurodegenerative diseases pose a substantial unmet medical need, and no disease-modifying treatments exist. Neurotrophic factors have been studied for decades as a therapy to slow down or stop the progression of these diseases. In this chapter, we focus on Parkinson disease, the second most common neurodegenerative disorder, and on studies carried out with neurotrophic factors. We explore the routes of administration, how the invasive intracranial administration is the challenge, and different ways to deliver the therapeutic proteins, for example, gene therapy and protein therapy. This therapy concept has been developed to mostly work on the restoration of the lost nigrostriatal dopaminergic neuronal connectivity in the brain. However, in recent years, the center of attention of neurotrophic factors has been on maintaining proteostasis and dissolving and preventing protein inclusions called Lewy bodies. We describe the most studied neurotrophic factor families and compare different preclinical experiments that have been carried out. We also analyze several clinical trials and describe their challenges and breakthroughs and discuss the prospects and challenges of neurotrophic support as a therapy for neurodegenerative diseases. In this chapter, we discuss why they still do and why it is essential to continue to work with this area of neurorestorative research around neurotrophic factors.

Bee Venom Reduces Early Inflammation and Oxidative Stress Associated with Lipopolysaccharide-induced Alpha-synuclein in the Substantia Nigra-striatum Axis.

Neuroinflammation and oxidative stress are important features in the pathogenesis and development of synucleinopathies, the glial activation and upregulation of pro-inflammatory and oxidative mediators induce alpha-synuclein (α-syn) accumulation. Recent studies have shown that bee venom (BV) has beneficial effects on symptoms of these neurodegenerative diseases. BV is known to exert anti-inflammatory and anti-oxidative effects. Here, we investigated the effects of BV over the different inflammatory and oxidative markers, and in the expression of α-syn and tyrosine hydroxylase (TH) in a lipopolysaccharide (LPS)-induced rat model of synucleinopathies. We examined whether BV (1.5 mg/kg by acupoint injection ST36 six times every 48 h) could change the α-syn and TH expression measured by western blotting, also, observed the activation of microglia and astrocytes by immunofluorescence, quantified the proinflammatory cytokines levels of tumoral necrosis factor-α (TNF-α) and Interleukin-1ß (IL-1ß) by enzyme-linked immunosorbent assay (ELISA), and estimated the lipid peroxidation and the activity of superoxide dismutase (SOD) and catalase (CAT) by colorimetric kits in LPS-treated rats (2.5 µg by a single dose intranigral injection) in substantia nigra (SN) and striatum (STR) brain areas. In the LPS-injected rat brain, BV treatment reduced α-syn levels and increased the TH levels. In addition, we observed lower microglia and astrocyte activation in SN and STR. Furthermore, BV decreases IL-1ß and lipid peroxidation and increases the CAT activity in the STR. These results indicate that BV can restore the α-syn and TH levels possibly by the inhibition of LPS-induced neuroinflammation and oxidation, also, these results suggest that BV could be a promising treatment option for synucleinopathies.

Viral overexpression of human alpha-synuclein in mouse substantia nigra dopamine neurons results in hyperdopaminergia but no neurodegeneration.

Loss of select neuronal populations such as midbrain dopamine (DA) neurons is a pathological hallmark of Parkinson's disease (PD). The small neuronal protein α-synuclein has been related both genetically and neuropathologically to PD, yet how and if it contributes to selective vulnerability remains elusive. Here, we describe the generation of a novel adeno-associated viral vector (AAV) for Cre-dependent overexpression of wild-type human α-synuclein. Our strategy allows us to restrict α-synuclein to select neuronal populations and hence investigate the cell-autonomous effects of elevated α-synuclein in genetically-defined cell types. Since DA neurons in the substantia nigra pars compacta (SNc) are particularly vulnerable in PD, we investigated in more detail the effects of increased α-synuclein in these cells. AAV-mediated overexpression of wildtype human α-synuclein in SNc DA neurons increased the levels of α-synuclein within these cells and augmented phosphorylation of α-synuclein at serine-129, which is considered a pathological feature of PD and other synucleinopathies. However, despite abundant α-synuclein overexpression and hyperphosphorylation we did not observe any dopaminergic neurodegeneration up to 90 days post virus infusion. In contrast, we noticed that overexpression of α-synuclein resulted in increased locomotor activity and elevated striatal DA levels suggesting that α-synuclein enhanced dopaminergic activity. We therefore conclude that cell-autonomous effects of elevated α-synuclein are not sufficient to trigger acute dopaminergic neurodegeneration.

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.

Dose-Dependent Alterations of Lysosomal Activity and Alpha-Synuclein in Peripheral Blood Monocyte-Derived Macrophages and SH-SY5Y Neuroblastoma Cell Line by upon Inhibition of MTOR Protein Kinase - Assessment of the Prospects of Parkinson's Disease Therapy.

To date, the molecular mechanisms of the common neurodegenerative disorder Parkinson's disease (PD) are unknown and, as a result, there is no neuroprotective therapy that may stop or slow down the process of neuronal cell death. The aim of the current study was to evaluate the prospects of using the mTOR molecule as a potential target for PD therapy due to the dose-dependent effect of mTOR kinase activity inhibition on cellular parameters associated with, PD pathogenesis. The study used peripheral blood monocyte-derived macrophages and SH-SY5Y neuroblastoma cell line. As a result, we have for the first time showed that inhibition of mTOR by Torin1 only at a concentration of 100 nM affects the level of the lysosomal enzyme glucocerebrosidase (GCase), encoded by the GBA1 gene. Mutations in GBA1 are considered a high-risk factor for PD development. This concentration led a decrease in pathological phosphorylated alpha-synuclein (Ser129), an increase in its stable tetrameric form with no changes in the lysosomal enzyme activities and concentrations of lysosphingolipids. Our findings suggest that inhibition of the mTOR protein kinase could be a promising approach for developing therapies for PD, particularly for GBA1-associated PD.

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 ß-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.

Neuronal alpha-Synuclein Disease Integrated Staging System performance in PPMI, PASADENA, and SPARK baseline cohorts.

The Neuronal alpha-Synuclein Disease (NSD) biological definition and Integrated Staging System (NSD-ISS) provide a research framework to identify individuals with Lewy body pathology and stage them based on underlying biology and increasing degree of functional impairment. Utilizing data from the PPMI, PASADENA and SPARK studies, we developed and applied biologic and clinical data-informed definitions for the NSD-ISS across the disease continuum. Individuals enrolled as Parkinson's disease, Prodromal, or Healthy Controls were defined and staged based on biological, clinical, and functional anchors at baseline. Across the three studies 1,741 participants had SAA data and of these 1,030 (59%) were S+ consistent with NSD. Among sporadic PD, 683/736 (93%) were NSD, and the distribution for Stages 2B, 3, and 4 was 25%, 63%, and 9%, respectively. Median (95% CI) time to developing a clinically meaningful outcome was 8.3 (6.2, 10.1), 5.9 (4.1, 6.0), and 2.4 (1.0, 4.0) years for baseline stage 2B, 3, and 4, respectively. We propose pilot biologic and clinical anchors for NSD-ISS. Our results highlight the baseline heterogeneity of individuals currently defined as early PD. Baseline stage predicts time to progression to clinically meaningful milestones. Further research on validation of the anchors in longitudinal cohorts is necessary.