Characterization of Molecular Tweezer Binding on α-Synuclein with Native Top-Down Mass Spectrometry and Ion Mobility-Mass Spectrometry Reveals a Mechanism for Aggregation Inhibition.

Parkinson's disease, a neurodegenerative disease that affects 15 million people worldwide, is characterized by deposition of α-synuclein into Lewy Bodies in brain neurons. Although this disease is prevalent worldwide, a therapy or cure has yet to be found. Several small compounds have been reported to disrupt fibril formation. Among these compounds is a molecular tweezer known as CLR01 that targets lysine and arginine residues. This study aims to characterize how CLR01 interacts with various proteoforms of α-synuclein and how the structure of α-synuclein is subsequently altered. Native mass spectrometry (nMS) measurements of α-synuclein/CLR01 complexes reveal that multiple CLR01 molecules can bind to α-synuclein proteoforms such as α-synuclein phosphorylated at Ser-129 and α-synuclein bound with copper and manganese ions. The binding of one CLR01 molecule shifts the ability for α-synuclein to bind other ligands. Electron capture dissociation (ECD) with Fourier transform-ion cyclotron resonance (FT-ICR) top-down (TD) mass spectrometry of α-synuclein/CLR01 complexes pinpoints the locations of the modifications on each proteoform and reveals that CLR01 binds to the N-terminal region of α-synuclein. CLR01 binding compacts the gas-phase structure of α-synuclein, as shown by ion mobility-mass spectrometry (IM-MS). These data suggest that when multiple CLR01 molecules bind, the N-terminus of α-synuclein shifts toward a more compact state. This compaction suggests a mechanism for CLR01 halting the formation of oligomers and fibrils involved in many neurodegenerative diseases.

Development of a Novel Electrochemiluminescence ELISA for Quantification of α-Synuclein Phosphorylated at Ser129 in Biological Samples.

Synucleinopathies are a group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). These diseases are characterized by the aggregation and deposition of α-synuclein (α-syn) in Lewy bodies (LBs) in PD and DLB or as glial cytoplasmic inclusions in MSA. In healthy brains, only ∼4% of α-syn is phosphorylated at Ser129 (pS129-α-syn), whereas >90% pS129-α-syn may be found in LBs, suggesting that pS129-α-syn could be a useful biomarker for synucleinopathies. However, a widely available, robust, sensitive, and reproducible method for measuring pS129-α-syn in biological fluids is currently missing. We used Meso Scale Discovery (MSD)'s electrochemiluminescence platform to create a new assay for sensitive detection of pS129-α-syn. We evaluated several combinations of capture and detection antibodies and used semisynthetic pS129-α-syn as a standard for the assay at a concentration range from 0.5 to 6.6 × 104 pg/mL. Using the antibody EP1536Y for capture and an anti-human α-syn antibody (MSD) for detection was the best combination in terms of assay sensitivity, specificity, and reproducibility. We tested the utility of the assay for the detection and quantification of pS129-α-syn in human cerebrospinal fluid, serum, plasma, saliva, and CNS-originating small extracellular vesicles, as well as in mouse brain lysates. Our data suggest that the assay can become a widely used method for detecting pS129-α-syn in biomedical studies including when only a limited volume of sample is available and high sensitivity is required, offering new opportunities for diagnostic biomarkers, monitoring disease progression, and quantifying outcome measures in clinical trials.

NLRP3 inflammasome in neurodegenerative disease.

Neurodegenerative diseases are characterized by a dysregulated neuro-glial microenvironment, culminating in functional deficits resulting from neuronal cell death. Inflammation is a hallmark of the neurodegenerative microenvironment and despite a critical role in tissue homeostasis, increasing evidence suggests that chronic inflammatory insult can contribute to progressive neuronal loss. Inflammation has been studied in the context of neurodegenerative disorders for decades but few anti-inflammatory treatments have advanced to clinical use. This is likely due to the related challenges of predicting and mitigating off-target effects impacting the normal immune response while detecting inflammatory signatures that are specific to the progression of neurological disorders. Inflammasomes are pro-inflammatory cytosolic pattern recognition receptors functioning in the innate immune system. Compelling pre-clinical data has prompted an intense interest in the role of the NLR family pyrin domain containing 3 (NLRP3) inflammasome in neurodegenerative disease. NLRP3 is typically inactive but can respond to sterile triggers commonly associated with neurodegenerative disorders including protein misfolding and aggregation, mitochondrial and oxidative stress, and exposure to disease-associated environmental toxicants. Clear evidence of enhanced NLRP3 inflammasome activity in common neurodegenerative diseases has coincided with rapid advancement of novel small molecule therapeutics making the NLRP3 inflammasome an attractive target for near-term interventional studies. In this review, we highlight evidence from model systems and patients indicating inflammasome activity in neurodegenerative disease associated with the NLRP3 inflammasome's ability to recognize pathologic forms of amyloid-ß, tau, and α-synuclein. We discuss inflammasome-driven pyroptotic processes highlighting the potential utility of evaluating extracellular inflammasome-related proteins in the context of biomarker discovery. We complete the report by pointing out gaps in our understanding of intracellular modifiers of inflammasome activity and mechanisms regulating the resolution of inflammasome activation. The literature review and perspectives provide a conceptual platform for continued analysis of inflammation in neurodegenerative diseases through the study of inflammasomes and pyroptosis, mechanisms of inflammation and cell death now recognized to function in multiple highly prevalent neurological disorders.

The molecular tweezer CLR01 reduces aggregated, pathologic, and seeding-competent α-synuclein in experimental multiple system atrophy.

Multiple system atrophy (MSA) is a fatal, adult-onset neurodegenerative disorder that has no cure and very limited treatment options. MSA is characterized by deposition of fibrillar α-synuclein (α-syn) in glial cytoplasmic inclusions in oligodendrocytes. Similar to other synucleinopathies, α-syn self-assembly is thought to be a key pathologic event and a prominent target for disease modification in MSA. Molecular tweezers are broad-spectrum nanochaperones that prevent formation of toxic protein assemblies and enhance their clearance. The current lead compound, CLR01, has been shown to inhibit α-syn aggregation but has not yet been tested in the context of MSA. To fill this gap, here, we conducted a proof-of-concept study to assess the efficacy of CLR01 in remodeling MSA-like α-syn pathology in the PLP-α-syn mouse model of MSA. Six-month-old mice received intracerebroventricular CLR01 (0.3 or 1 mg/kg per day) or vehicle for 32 days. Open-field test revealed a significant, dose-dependent amelioration of an anxiety-like phenotype. Subsequently, immunohistochemical and biochemical analyses showed dose-dependent reduction of pathological and seeding-competent forms of α-syn, which correlated with the behavioral phenotype. CLR01 treatment also promoted dopaminergic neuron survival in the substantia nigra. To our knowledge, this is the first demonstration of an agent that reduces formation of putative high-molecular-weight oligomers and seeding-competent α-syn in a mouse model of MSA, supporting the view that these species are key to the neurodegenerative process and its cell-to-cell progression in MSA. Our study suggests that CLR01 is an attractive therapeutic candidate for disease modification in MSA and related synucleinopathies, supporting further preclinical development.