Protein Α-synuclein Research Articles

Parkinson’s Disease: Biomarkers for Diagnosis and Disease Progression

Parkinson’s disease (PD) is a progressive neurological disease that causes both motor and nonmotor symptoms. While our understanding of putative mechanisms has advanced significantly, it remains challenging to verify biomarkers with sufficient evidence for regular clinical use. Clinical symptoms are the primary basis for diagnosing the disease, which can be mild in the early stages and overlap with other neurological disorders. As a result, clinical testing and medical records are mostly relied upon for diagnosis, posing substantial challenges during both the initial diagnosis and the continuous disease monitoring. Recent biochemical, neuroimaging, and genetic biomarkers have helped us understand the pathophysiology of Parkinson’s disease. This comprehensive study focuses on these biomarkers, which were chosen based on their relevance, methodological excellence, and contribution to the field. Biochemical biomarkers, including α-synuclein and glial fibrillary acidic protein (GFAP), can predict disease severity and progression. The dopaminergic system is widely used as a neuroimaging biomarker to diagnose PD. Numerous genes and genome wide association study (GWAS) sites have been related to the development of PD. Recent research on the SNCA gene and leucine-rich repeat protein kinase 2 (LRRK2) has shown promising results. By evaluating current studies, this review intends to uncover gaps in biomarker validation and use, while also highlighting promising improvements. It emphasizes the need for dependable and reproducible indicators in improving PD diagnosis and prognosis. These biomarkers may open up new avenues for early diagnosis, disease progression tracking, and the development of personalized treatment programs.

Targeting Glucose Metabolism: A Novel Therapeutic Approach for Parkinson’s Disease

Glucose metabolism is essential for the maintenance and function of the central nervous system. Although the brain constitutes only 2% of the body weight, it consumes approximately 20% of the body’s total energy, predominantly derived from glucose. This high energy demand of the brain underscores its reliance on glucose to fuel various functions, including neuronal activity, synaptic transmission, and the maintenance of ion gradients necessary for nerve impulse transmission. Increasing evidence shows that many neurodegenerative diseases, including Parkinson’s disease (PD), are associated with abnormalities in glucose metabolism. PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra, accompanied by the accumulation of α-synuclein protein aggregates. These pathological features are exacerbated by mitochondrial dysfunction, oxidative stress, and neuroinflammation, all of which are influenced by glucose metabolism disruptions. Emerging evidence suggests that targeting glucose metabolism could offer therapeutic benefits for PD. Several antidiabetic drugs have shown promise in animal models and clinical trials for mitigating the symptoms and progression of PD. This review explores the current understanding of the association between PD and glucose metabolism, emphasizing the potential of antidiabetic medications as a novel therapeutic approach. By improving glucose uptake and utilization, enhancing mitochondrial function, and reducing neuroinflammation, these drugs could address key pathophysiological mechanisms in PD, offering hope for more effective management of this debilitating disease.

Novel tools to quantify total, phospho-Ser129 and aggregated alpha-synuclein in the mouse brain

Assays for quantifying aggregated and phosphorylated (S129) human α-synuclein protein are widely used to evaluate pathological burden in patients suffering from synucleinopathy disorders. Many of these assays, however, do not cross-react with mouse α-synuclein or exhibit poor sensitivity for this target, which is problematic considering the preponderance of mouse models at the forefront of pre-clinical α-synuclein research. In this project, we addressed this unmet need by reformulating two existing AlphaLISA® SureFire® Ultra™ total and pS129 α-synuclein assay kits to yield robust and ultrasensitive (LLoQ ≤ 0.5 pg/mL) quantification of mouse and human wild-type and pS129 α-synuclein protein. We then employed these assays, together with the BioLegend α-synuclein aggregate ELISA, to assess α-synuclein S129 phosphorylation and aggregation in different mouse brain tissue preparations. Overall, we highlight the compatibility of these new immunoassays with rodent models and demonstrate their potential to advance knowledge surrounding α-synuclein phosphorylation and aggregation in synucleinopathies.

Retraction Note: 27-Hydroxycholesterol increases α-synuclein protein levels through proteasomal inhibition in human dopaminergic neurons

Retraction Note: 27-Hydroxycholesterol increases α-synuclein protein levels through proteasomal inhibition in human dopaminergic neurons

Interaction between intrinsically disordered proteins Praja1 and α-synuclein on gold electrodes

Abstract The behavior of Praja1 (PJA1) interacting with alpha-synuclein (α-Syn) on gold electrode was investigated using electrochemical methods, where both PJA1 and α-Syn are intrinsically disordered proteins. From the electrode reaction of ferricyanide ions added as a probe, it was suggested that PJA1 interacts with α-Syn even in its adsorbed state on gold substrate and that the state of PJA1 on gold electrode can be classified into four categories depending on the α-Syn concentration.

TMEM16F regulates pathologic α-synuclein secretion and spread in cellular and mouse models of Parkinson's disease.

One of the main hallmarks of Parkinson's disease (PD) pathology is the spread of the aggregate-prone protein α-synuclein (α-syn), which can be detected in the plasma and cerebrospinal fluid of patients as well as in the extracellular environment of neuronal cells. The secreted α-syn can exhibit "prion-like" behavior and transmission to naïve cells can promote conformational changes and pathology. The precise role of plasma membrane proteins in the pathologic process of α-syn is yet to be fully resolved. The TMEM16 family of lipid scramblases and ion channels has been recently associated with cancer and infectious diseases but is less known for its role in aging-related diseases. To elucidate the role of TMEM16F in α-syn spread, we transduced neurons derived from TMEM16F knockout mice with a reporter system that enables the distinction between donor and recipient neurons of pathologic α-synA53T. We found that the spread of α-synA53T was reduced in neurons derived from TMEM16F-knockout mice. These findings were recapitulated invivo in a mouse model of PD, where attenuated α-synA53T spread was observed when TMEM16F was ablated. Moreover, we identified a single nucleotide polymorphism in TMEM16F of Ashkenazi Jewish PD patients resulting in a missense Ala703Ser mutation with enhanced lipid scramblase activity. This mutation is associated with altered regulation of α-synA53T extracellular secretion in cellular models of PD. Our study highlights TMEM16F as a novel regulator of α-syn spread and as a potential therapeutic target in synucleinopathies.

Distinct Effects of SARS-CoV-2 Protein Segments on Structural Stability, Amyloidogenic Potential, and α-Synuclein Aggregation.

Amyloidosis is characterized by the abnormal accumulation of misfolded proteins, called amyloid fibrils, leading to diverse clinical manifestations. Recent studies on the amyloidogenesis of SARS-CoV‑2 protein segments have raised concerns on their potential link to post-infection neurodegeneration, however, the mechanisms remain unclear. Herein, we investigated the structure, stability, and amyloidogenic propensity of a nine-residue segment (SK9) of the SARS-CoV-2 envelope protein and their impact on neuronal protein α-synuclein (αSyn) aggregation. Specifically, the amino acid sequence of the SK9 wildtype has been modified from a basic and positively charged peptide (SFYVYSRVK), to a nearly neutral and more hydrophobic peptide (SAAVASAVK, labelled as SK9 var1), and to an acidic and positively charged peptide (SFYVYSRVK, labelled as SK9 var2). Our findings reveal that the SK9 wildtype exhibited a pronounced amyloidogenic propensity due to its disordered and unstable nature, while the SK9 variants possessed more ordered and stable structures preventing the amyloid formation. Significantly, the SK9 wildtype demonstrated distinct effect on αSyn aggregation kinetics and aggregate morphology to facilitate the formation of αSyn aggregates with enhanced resistance against enzymatic degradation. This study highlights the potential of modifying short peptide sequences to fine-tune their properties, providing insights into understanding and regulating viral-induced amyloid aggregations.

CK and LRRK2 Involvement in Neurodegenerative Diseases.

Neurodegenerative diseases (NDDs) are currently the most widespread neuronal pathologies in the world. Among these, the most widespread are Alzheimer's disease (AD), dementia, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD)-all characterized by a progressive loss of neurons in specific regions of the brain leading to varied clinical symptoms. At the basis of neurodegenerative diseases, an emerging role is played by genetic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene that cause increased LRRK2 activity with consequent alteration of neuronal autophagy pathways. LRRK2 kinase activity requires GTPase activity which functions independently of kinase activity and is required for neurotoxicity and to potentiate neuronal death. Important in the neurodegeneration process is the upregulation of casein kinase (CK), which causes the alteration of the AMPK pathway by enhancing the phosphorylation of α-synuclein and huntingtin proteins, known to be involved in PD and HD, and increasing the accumulation of the amyloid-β protein (Aβ) for AD. Recent research has identified CK of the kinases upstream of LRRK2 as a regulator of the stability of the LRRK2 protein. Based on this evidence, this review aims to understand the direct involvement of individual kinases in NDDs and how their crosstalk may impact the pathogenesis and early onset of neurodegenerative diseases.

Lactiplantibacillus plantarum SG5 inhibits neuroinflammation in MPTP-induced PD mice through GLP-1/PGC-1α pathway

Mounting evidence suggests that alterations in gut microbial composition play an active role in the pathogenesis of Parkinson's disease (PD). Probiotics are believed to modulate gut microbiota, potentially influencing PD development through the microbiota-gut-brain axis. However, the potential beneficial effects of Lactiplantibacillus plantarum SG5 (formerly known as Lactobacillus plantarum, abbreviated as L. plantarum) on PD and its underlying mechanisms remain unclear. In this study, we employed immunofluorescence, Western blotting, ELISA, and 16S rRNA gene sequencing to investigate the neuroprotective effects of L. plantarum SG5 against neuroinflammation in an MPTP-induced PD model and to explore the underlying mechanisms. Our results demonstrated that L. plantarum SG5 ameliorated MPTP-induced motor deficits, dopaminergic neuron loss, and elevated α-synuclein protein levels. Furthermore, SG5 inhibited MPTP-triggered overactivation of microglia and astrocytes in the substantia nigra (SN), attenuated disruption of both blood-brain and intestinal barriers, and suppressed the release of inflammatory factors in the colon and SN. Notably, SG5 modulated the composition and structure of the gut microbiota in mice. The MPTP-induced decrease in colonic GLP-1 secretion was reversed by SG5 treatment, accompanied by increased expression of GLP-1R and PGC-1α in the SN. Importantly, the GLP-1R antagonist Exendin 9–39 and PGC-1α inhibitor SR18292 attenuated the protective effects of SG5 in PD mice. In conclusion, we demonstrate a neuroprotective role of L. plantarum SG5 in the MPTP-induced PD mouse model, which likely involves modulation of the gut microbiota and, significantly, the GLP-1/PGC-1α signaling pathway.

The use of CRISPR-Cas9 technology for targeted gene therapy in Parkinson's disease focusing on LRRK2 and SNCA genes

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, leading to the start of motor and non-motor symptoms. These include bradykinesia, resting tremor, rigidity, and postural instability, alongside cognitive impairments, mood disorders, and autonomic dysfunctions. The hallmark of PD is the accumulation of α-synuclein protein aggregates, forming Lewy bodies within the neurons. Given the numerous causes of Parkinson’s, involving genetic, epigenetic, and environmental factors, therapeutic treatments are hard to come by. Traditional pharmacological treatments, such as levodopa and dopamine agonists, primarily offer symptomatic relief and are associated with diminishing efficacy over time. Therefore, there is a need for new therapeutic approaches that target the disease at its roots. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, with the RNA-guided Cas9 endonuclease enables precise, targeted modifications at the genomic level. This technology offers potential for terminating the genetic factors causing parkinsons and aid in developing gene-based therapies. By attempting to regulate gene expression, CRISPR holds promise for addressing the fundamental causes of neurodegeneration in Parkinsons. One of the key genetic targets in PD is the leucine-rich repeat kinase 2 (LRRK2) gene, where specific gain-of- function mutations, such as G2019S, lead to hyperactivation of its kinase activity, contributing to neurotoxicity. CRISPR-Cas9 can be employed to introduce precise double-strand breaks at the mutation site, followed by homology-directed repair (HDR) to restore the wild-type sequence, thereby normalizing LRRK2 function and preventing neuronal death. Similarly, CRISPR technology can target the SNCA gene, which encodes α-synuclein. Pathogenic duplicates or triplicates of SNCA result in overproduction of α-synuclein and subsequent aggregation. Utilizing CRISPR to disrupt these extra copies can reduce α-synuclein levels, alleviating its toxic effects.CRISPR activation (CRISPRa) and interference (CRISPRi) systems enable fine-tuned upregulation or downregulation of gene expression, offering strategies to increase the expression of neuroprotective factors or suppress the expression of deleterious genes.

Blood Glucocerebrosidase Activity and α-Synuclein Levels in Patients with GBA1-Associated Parkinson's Disease and Asymptomatic <i>GBA1</i> Mutation Carriers

Introduction. Mutations in a GBA1 gene, which encodes a lysosomal enzyme called glucocerebrosidase (GCase), are the most common genetic risk factor for Parkinson's disease (PD). The pathogenesis of PD results from the death of dopaminergic neurons in the substantia nigra of the brain, which is associated with the aggregation of α-synuclein protein. However, not all GBA1 mutation carriers develop PD during their lifetime. The aim of this study was to evaluate GCase activity and α-synuclein levels in CD45+ blood cells of patients with PD associated with GBA1 mutations (GBА1-PD), asymptomatic carriers of GBA1 mutations (GBА1-carriers), and patients with sporadic PD (sPD), as well as correlation between the study parameters in the study groups. Materials and methods. The study included patients with GBА1-PD (n = 25) and sPD (n = 147), and GBА1-carriers (n = 16). A control group included healthy volunteers (n = 154). The level of α-synuclein in CD45+ cells was measured by enzyme-linked immunosorbent assay, and GCase activity in dried blood spots was detected by high-performance liquid chromatography with tandem mass spectrometry. Results. Increased level of α-synuclein protein was detected in CD45+ blood cells of patients with GBA1-PD, sPD, and GBA1-carriers compared to controls (p = 0.0043; p = 0.0002; p = 0.032, respectively). Decreased GCase activity was reported in GBA1-PD patients and GBA1-carriers compared to sPD patients (p = 0.0003; p = 0.003, respectively) and controls (p 0.0001; p 0.0001, respectively). However, negative correlation between α-synuclein levels and GCase activity was observed only in GBA1-PD patients, but not in GBA1-carriers. Conclusion. Our data suggest a possible functional relationship between the activity of GCase and the metabolism of α-synuclein in PD associated with GBA1 mutations.

Imaging flow cytometry reveals LPS-induced changes to intracellular intensity and distribution of α-synuclein in a TLR4-dependent manner in STC-1 cells

BackgroundParkinson’s disease is a chronic neurodegenerative disorder, where pathological protein aggregates largely composed of phosphorylated α-synuclein are implicated in disease pathogenesis and progression. Emerging evidence suggests that the interaction between pro-inflammatory microbial factors and the gut epithelium contributes to α-synuclein aggregation in the enteric nervous system. However, the cellular sources and mechanisms for α-synuclein pathology in the gut are still unclear. MethodsThe STC-1 cell line, which models an enteroendocrine population capable of communicating with the microbiota, immune and nervous systems, was treated with a TLR4 inhibitor (TAK-242) prior to microbial lipopolysaccharide (LPS) exposure to investigate the role of TLR4 signalling in α-synuclein alterations. Antibodies targeting the full-length protein (α-synuclein) and the Serine-129 phosphorylated form (pS129) were used. Complex, multi-parametric image analysis was conducted through confocal microscopy (with Zen 3.8 analysis) and imaging flow cytometry (with IDEAS® analysis). ResultsConfocal microscopy revealed heterogenous distribution of α-synuclein and pS129 in STC-1 cells, with prominent pS129 staining along cytoplasmic processes. Imaging flow cytometry further quantified the relationship between various α-synuclein morphometric features. Thereafter, imaging flow cytometry demonstrated a dose-specific effect of LPS, where the low (8 μg/mL), but not high dose (32 μg/mL), significantly altered measures related to α-synuclein intensity, distribution, and localisation. Pre-treatment with a TLR4 inhibitor TAK-242 alleviated some of these significant alterations. ConclusionThis study demonstrates that LPS-TLR4 signalling alters the intracellular localisation of α-synuclein in enteroendocrine cells in vitro and showcases the utility of combining imaging flow cytometry to investigate subtle protein changes that may not be apparent through confocal microscopy alone. Further investigation is required to understand the apparent dose-dependent effects of LPS on α-synuclein in the gut epithelium in healthy states as well as conditions such as Parkinson’s disease.

Redox Modulation of Meniere Disease by Coriolus versicolor Treatment, a Nutritional Mushroom Approach with Neuroprotective Potential.

Meniere's disease (MD) is a cochlear neurodegenerative disease. Hearing loss appears to be triggered by oxidative stress in the ganglion neurons of the inner ear. Here, we confirm the variation of markers of oxidative stress and inflammation in patients with Meniere and hypothesize that chronic treatment with Coriolus mushroom helps in the response to oxidative stress and acts on α-synuclein and on NF-kB-mediated inflammatory processes Methods: Markers of oxidative stress and inflammation were evaluated in MD patients with or without Coriolus treatment for 3 or 6 months. MD patients had a small increase in Nrf2, HO-1, γ-GC, Hsp70, Trx and sirtuin-1, which were further increased by Coriolus treatment, especially after 6 months. Increased markers of oxidative damage, such as protein carbonyls, HNE, and ultraweak chemiluminescence, associated with a decrease in plasma GSH/GSSG ratio, were also observed in lymphocytes from MD patients. These parameters were restored to values similar to the baseline in patients treated with Coriolus for both 3 and 6 months. Furthermore, treated MD subjects showed decreased expression of α-synuclein, GFAP and Iba-1 proteins and modulation of the NF-kB pathway, which were impaired in MD patients. These changes were greatest in subjects taking supplements for 6 months. Our study suggests MD as a model of cochlear neurodegenerative disease for the identification of potent inducers of the Nrf2-vitagene pathway, able to reduce the deleterious consequences associated with neurodegenerative damage, probably by indirectly acting on a-synuclein expression and on inflammatory processes NF-kB-mediated.

Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins

Ubiquitin carboxyl-terminal hydrolase 19 (USP19) is a unique deubiquitinase (DUB), characterized by multiple variants generated by alternative splicing. Several variants bear a C-terminal transmembrane domain that anchors them to the endoplasmic reticulum (ER). Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from ER-associated protein degradation (ERAD) in a catalytic-independent manner, promote autophagy and address proteins to lysosomal degradation via endosomal microautophagy. USP19 has recently emerged as the protein responsible for the unconventional secretion of misfolded proteins including Parkinson's disease-associated protein α-synuclein. Despite mounting evidence that USP19 plays crucial roles in several biological processes, the underlying mechanisms are unclear due to lack of information on the physiological substrates of USP19. Herein, we used high-resolution quantitative proteomics to analyze changes in the secretome and cell proteome induced by loss of USP19 to identify proteins whose secretion or turnover is regulated by USP19. We found that ablation of USP19 induced significant proteomic alterations both in and out of the cell. Loss of USP19 impaired the release of several lysosomal proteins, including legumain (LGMN) and several cathepsins. In order to understand the underlaying mechanism, we dissected the USP19-regulated secretion of LGMN in several cell types. We found that LGMN was not a DUB substrate of USP19 and that its USP19-dependent release did not require their direct interaction. LGMN secretion occurred by a mechanism that involved the Golgi apparatus, autophagosome formation and lysosome function. This mechanism resembled the recently described "lysosomal exocytosis", by which lysosomal hydrolases are secreted, when ubiquitination of p62 is increased in cells lacking deubiquitinases such as USP15 and USP17. In conclusion, our proteomic characterization of USP19 has identified a collection of proteins in the secretome and within the cell that are regulated by USP19, which link USP19 to secretion of lysosomal proteins, including LGMN.

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.

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.

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.

Fluorescence-Based Monitoring of Early-Stage Aggregation of Amyloid-β, Amylin Peptide, Tau, and α-Synuclein Proteins.

Early-stage aggregates of amyloid-forming proteins, specifically soluble oligomers, are implicated in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Protein aggregation is typically monitored by fluorescence using the amyloid-binding fluorophore thioflavin T (ThT). Thioflavin T interacts, however, preferentially with fibrillar amyloid structures rather than with soluble, early-stage aggregates. In contrast, the two fluorophores, aminonaphthalene 2-cyanoacrylate-spiropyran (AN-SP) and triazole-containing boron-dipyrromethene (taBODIPY), were reported to bind preferentially to early-stage aggregates of amyloidogenic proteins. The present study compares ThT with AN-SP and taBODIPY with regard to their ability to monitor early stages of aggregation of four different amyloid-forming proteins, including amyloid-β (Aβ), tau protein, amylin, and α-synuclein. The results show that the three fluorophores vary in their suitability to monitor the early aggregation of different amyloid-forming proteins. For instance, in the presence of Aβ and amylin, the fluorescence intensity of AN-SP increased at an earlier stage of aggregation than the fluorescence of ThT, albeit with only a small fluorescence increase in the case of AN-SP. In contrast, in the presence of tau and amylin, the fluorescence intensity of taBODIPY increased at an earlier stage of aggregation than the fluorescence of ThT. Finally, α-synuclein aggregation could only be monitored by ThT fluorescence; neither AN-SP nor taBODIPY showed a significant increase in fluorescence over the course of aggregation of α-synuclein. These results demonstrate the ability of AN-SP and taBODIPY to monitor the formation of early-stage aggregates from specific amyloid-forming proteins at an early stage of aggregation, although moderate increases in fluorescence intensity, relatively large uncertainties in fluorescence values, and limited solubility of both fluorophores limit their usefulness for some amyloid proteins. The capability to monitor early aggregation of some amyloid proteins, such as amylin, might accelerate the discovery of aggregation inhibitors to minimize the formation of toxic oligomeric species for potential therapeutic use.

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.

Lauric acid with or without levodopa ameliorates Parkinsonism in genetically modified model of Drosophila melanogaster via the oxidative-inflammatory-apoptotic pathway.

Parkinson's disease (PD), the most prevalent type of Parkinsonism, is a progressive neurological condition characterized by a range of motor and non-motor symptoms. The complicated etiology of PD is thought to involve a summation of aging, genetic predisposition, and environmental variables. However, the α-synuclein protein plays a significant role in the disease's pathophysiology. The UAS-α-Syn and Ddc-Gal4 strains were crossed to produce offspring referred to as PD flies. The entire population of flies was divided into five groups, each having about 100 flies and five replicates. The control group (w1118) and the PD group not receiving treatment were exposed to lauric acid (LA)/levodopa (LD)-free diet, while the PD groups that received treatments were fed with either a 250mg/kg LA diet, a 250mg/kg LD diet, or a combination of the two for 21 days. Longevity, geotaxis, and olfactory assays were performed in addition to other biochemical tests. As a result of the overexpression of α-synuclein, the locomotive capacity, lifespan, and antioxidant status were all significantly (p<.05)reduced, and the apoptotic and neuroinflammatory activities were increased. Nevertheless, the majority of the treated flies improved significantly (p<.05). LA, whether combined with LD or not, elicited a significant response in α-synuclein/dopa decarboxylase genetically modified Drosophila melanogaster Parkinsonism models.