Periodic Reporting for period 1 - SynDrops (Understanding the physiological and pathological relevance of liquid-liquid phase separation by synuclein family of proteins)
Reporting period: 2022-06-01 to 2024-05-31
Context and Overall Objectives of the Project:
Project SynDrops investigates the phase separation behavior of α-synuclein (α-Syn), a protein associated with Parkinson's disease (PD) and other neurodegenerative disorders. The primary focus is on how pathological mutations and terminal truncations affect the thermodynamics and physical properties of α-Syn phase condensates and their role in modulating amyloid aggregation.
Key Background and Motivation: Understanding the phase separation behavior of α-Syn, particularly how mutations and truncations influence this process, is crucial as it is believed to be a precursor to amyloid fibril formation—a hallmark of synucleinopathies. In addition, recent findings of nanoscale phase separation challenge classical theories, suggesting the presence of stable, small molecular clusters below the critical concentration for protein phase separation. We also discover and characterize these nanoscale assemblies for α-Syn and study the effects of pathological mutations on the nanoscale phase separation landscape.
Advanced Techniques Utilized:
1. Capflex and TDIPS: Techniques developed on the FIDA1 instrument to measure phase separation properties.
2. Mass Photometry: Employed to detect and characterize nanoscale phase separation of α-Syn.
Primary Objectives:
1. Impact of Pathological Mutations: Determine how familial mutations in α-Syn influence its phase separation properties, focusing on dilute phase concentrations and associated free energy changes (ΔG).
2. Examine Terminal Truncations: Investigate the effects of truncating the C-terminal region of α-Syn on its phase behavior and stability.
3. Characterize Nanocluster Formation: Explore the formation of α-Syn micelles and nanoclusters at varying ionic strengths and their transition into larger condensates.
4. Material Properties of Condensates: Analyze the material properties of α-Syn condensates, such as viscosity and resistance to dissolution.
5. Quantify Amyloid Fibril Formation: Develop methods to estimate amyloid fibril concentration within α-Syn condensates and correlate this with phase separation dynamics.
Project Pathway to Impact:
Scientific and Technical Impact:
• Understanding Disease Mechanisms: By elucidating how mutations and truncations influence α-Syn phase separation and aggregation, SynDrops aims to uncover the mechanisms driving neurodegenerative diseases.
• Novel Techniques: The validation of Capflex, TDIPS and mass photometry techniques provides valuable tools for broader applications in cellular and molecular biology in the context of phase separation.
Expected Contributions:
1. Enhanced Scientific Knowledge: Advancing our understanding of protein phase behavior and its implications for disease.
2. Therapeutic Development: Informing the design of interventions that can modulate protein phase behavior, offering new avenues for combating neurodegenerative disorders.
3. Diagnostic Tools: Detailed characterization of α-Syn nanoclusters and condensates could lead to new strategies for diagnosing and treating synucleinopathies by targeting early-stage aggregation events.
Project SynDrops investigates the phase separation behavior of α-synuclein (α-Syn), a protein associated with Parkinson's disease (PD) and other neurodegenerative disorders. The primary focus is on how pathological mutations and terminal truncations affect the thermodynamics and physical properties of α-Syn phase condensates and their role in modulating amyloid aggregation.
Key Background and Motivation: Understanding the phase separation behavior of α-Syn, particularly how mutations and truncations influence this process, is crucial as it is believed to be a precursor to amyloid fibril formation—a hallmark of synucleinopathies. In addition, recent findings of nanoscale phase separation challenge classical theories, suggesting the presence of stable, small molecular clusters below the critical concentration for protein phase separation. We also discover and characterize these nanoscale assemblies for α-Syn and study the effects of pathological mutations on the nanoscale phase separation landscape.
Advanced Techniques Utilized:
1. Capflex and TDIPS: Techniques developed on the FIDA1 instrument to measure phase separation properties.
2. Mass Photometry: Employed to detect and characterize nanoscale phase separation of α-Syn.
Primary Objectives:
1. Impact of Pathological Mutations: Determine how familial mutations in α-Syn influence its phase separation properties, focusing on dilute phase concentrations and associated free energy changes (ΔG).
2. Examine Terminal Truncations: Investigate the effects of truncating the C-terminal region of α-Syn on its phase behavior and stability.
3. Characterize Nanocluster Formation: Explore the formation of α-Syn micelles and nanoclusters at varying ionic strengths and their transition into larger condensates.
4. Material Properties of Condensates: Analyze the material properties of α-Syn condensates, such as viscosity and resistance to dissolution.
5. Quantify Amyloid Fibril Formation: Develop methods to estimate amyloid fibril concentration within α-Syn condensates and correlate this with phase separation dynamics.
Project Pathway to Impact:
Scientific and Technical Impact:
• Understanding Disease Mechanisms: By elucidating how mutations and truncations influence α-Syn phase separation and aggregation, SynDrops aims to uncover the mechanisms driving neurodegenerative diseases.
• Novel Techniques: The validation of Capflex, TDIPS and mass photometry techniques provides valuable tools for broader applications in cellular and molecular biology in the context of phase separation.
Expected Contributions:
1. Enhanced Scientific Knowledge: Advancing our understanding of protein phase behavior and its implications for disease.
2. Therapeutic Development: Informing the design of interventions that can modulate protein phase behavior, offering new avenues for combating neurodegenerative disorders.
3. Diagnostic Tools: Detailed characterization of α-Syn nanoclusters and condensates could lead to new strategies for diagnosing and treating synucleinopathies by targeting early-stage aggregation events.
Work Performed and Main Achievements:
1. Technique Development and Validation
• Capflex and TDIPS: Developed and validated techniques for measuring the thermodynamics of α-Syn phase separation.
• Mass Photometry: Used to discover and characterize nanoclusters, ensuring accuracy and reproducibility through rigorous testing and comparison with established methods.
2. Understanding Mutation Effects
• Quantitative studies on familial mutations in α-Syn, providing insights into how these mutations alter the stability and dynamics of phase-separated states.
3. Exploring Truncation and Electrostatics
• Investigated the effects of truncating the N and C-terminal region of α-Syn and the role of electrostatic interactions in phase separation.
4. Nanocluster Characterization
• Introduced mass photometry to study nanoscale phase separation, offering high-resolution detection of protein assemblies.
• Characterized α-Syn nanoclusters at different ionic strengths, understanding their transition to macroscopic condensates.
5. Analyzing the Material Properties of Condensates
• Measured viscosity and resistance to dissolution to determine the extent of aggregation within WT and mutant α-Syn condensates.
6. Quantifying Amyloid Fibril Formation
• Developed methods to quantify amyloid fibril formation within α-Syn condensates, linking phase separation behavior to amyloidogenesis.
1. Technique Development and Validation
• Capflex and TDIPS: Developed and validated techniques for measuring the thermodynamics of α-Syn phase separation.
• Mass Photometry: Used to discover and characterize nanoclusters, ensuring accuracy and reproducibility through rigorous testing and comparison with established methods.
2. Understanding Mutation Effects
• Quantitative studies on familial mutations in α-Syn, providing insights into how these mutations alter the stability and dynamics of phase-separated states.
3. Exploring Truncation and Electrostatics
• Investigated the effects of truncating the N and C-terminal region of α-Syn and the role of electrostatic interactions in phase separation.
4. Nanocluster Characterization
• Introduced mass photometry to study nanoscale phase separation, offering high-resolution detection of protein assemblies.
• Characterized α-Syn nanoclusters at different ionic strengths, understanding their transition to macroscopic condensates.
5. Analyzing the Material Properties of Condensates
• Measured viscosity and resistance to dissolution to determine the extent of aggregation within WT and mutant α-Syn condensates.
6. Quantifying Amyloid Fibril Formation
• Developed methods to quantify amyloid fibril formation within α-Syn condensates, linking phase separation behavior to amyloidogenesis.
Results and Potential Impacts:
Thermodynamics of α-Syn Phase Separation
• Quantified dilute phase concentrations (Cdil) and demonstrated how familial mutations and truncations affect phase separation free energy (ΔG).
• Revealed how NaCl concentration influences Cdil for WT and mutant α-Syn.
• Showed the importance of electrostatic interactions in phase separation through the study of recombinant α-Syn variants.
Micelle and Nanocluster Formation
• Identified stable nanoclusters at lower NaCl concentrations, transitioning to larger assemblies with increasing ionic strength, ultimately resulting in macroscopic condensates.
• Characterized micellar assemblies and nanoclusters, providing detailed insights into early-stage LLPS.
Material Properties of Condensates
• Demonstrated differences in translational dynamics and material properties between WT and mutant α-Syn condensates.
• Highlighted distinct viscosity differences, with truncated variants forming more viscous, gel-like condensates.
Amyloid Fibril Formation
• Showed rapid amyloid aggregation for truncated mutants under phase-separating conditions.
• Compared the concentration of amyloid fibrils within condensates, revealing familial mutants with higher fibril concentrations than WT.
Key Needs for Further Uptake and Success:
1. Further Research
• Mechanistic Studies: Investigate molecular mechanisms driving phase separation and aggregation.
• Extended Mutational Analysis: Examine additional α-Syn mutations.
2. Demonstration
• In Vivo Validation: Confirm in vitro findings in cellular and animal models.
• Functional Assays: Assess the functional consequences of altered phase separation and aggregation.
3. Access to Markets and Finance
• Funding: Secure financial support for large-scale and translational research.
• Partnerships: Collaborate with pharmaceutical companies.
4. Commercialization
• Therapeutic Development: Develop compounds that modulate α-Syn phase behavior.
• Biomarker Discovery: Use phase separation characteristics as biomarkers for disease detection and monitoring.
5. IPR Support
• Patent Protection: Secure intellectual property rights.
• Licensing: Establish licensing agreements for commercial exploitation.
6. Internationalization
• Global Collaborations: Engage with international research communities.
7. Supportive Regulatory and Standardization Framework
• Regulatory Guidance: Develop guidelines for evaluating phase separation-modulating therapies.
• Standardization: Establish protocols for studying protein phase separation and aggregation.
Taken together. the research findings from SynDrops provide significant insights into the phase separation behavior and aggregation kinetics of α-synuclein (α-Syn), a key protein implicated in neurodegenerative diseases such as Parkinson's. Utilizing innovative techniques like Capflex, TDIPS, and mass photometry, the study reveals how pathological mutations and terminal truncations influence the thermodynamics and physical properties of α-Syn phase condensates. The results highlight the formation of stable molecular clusters and nanoclusters, as well as the transition to larger condensates and amyloid fibril formation. Moreover, the research identifies differences in material properties between wild-type and mutant α-Syn condensates, providing valuable insights into their potential role in disease progression. Overall, these findings significantly advance our understanding of protein phase behavior and offer promising avenues for therapeutic interventions in neurodegenerative disorders.
Thermodynamics of α-Syn Phase Separation
• Quantified dilute phase concentrations (Cdil) and demonstrated how familial mutations and truncations affect phase separation free energy (ΔG).
• Revealed how NaCl concentration influences Cdil for WT and mutant α-Syn.
• Showed the importance of electrostatic interactions in phase separation through the study of recombinant α-Syn variants.
Micelle and Nanocluster Formation
• Identified stable nanoclusters at lower NaCl concentrations, transitioning to larger assemblies with increasing ionic strength, ultimately resulting in macroscopic condensates.
• Characterized micellar assemblies and nanoclusters, providing detailed insights into early-stage LLPS.
Material Properties of Condensates
• Demonstrated differences in translational dynamics and material properties between WT and mutant α-Syn condensates.
• Highlighted distinct viscosity differences, with truncated variants forming more viscous, gel-like condensates.
Amyloid Fibril Formation
• Showed rapid amyloid aggregation for truncated mutants under phase-separating conditions.
• Compared the concentration of amyloid fibrils within condensates, revealing familial mutants with higher fibril concentrations than WT.
Key Needs for Further Uptake and Success:
1. Further Research
• Mechanistic Studies: Investigate molecular mechanisms driving phase separation and aggregation.
• Extended Mutational Analysis: Examine additional α-Syn mutations.
2. Demonstration
• In Vivo Validation: Confirm in vitro findings in cellular and animal models.
• Functional Assays: Assess the functional consequences of altered phase separation and aggregation.
3. Access to Markets and Finance
• Funding: Secure financial support for large-scale and translational research.
• Partnerships: Collaborate with pharmaceutical companies.
4. Commercialization
• Therapeutic Development: Develop compounds that modulate α-Syn phase behavior.
• Biomarker Discovery: Use phase separation characteristics as biomarkers for disease detection and monitoring.
5. IPR Support
• Patent Protection: Secure intellectual property rights.
• Licensing: Establish licensing agreements for commercial exploitation.
6. Internationalization
• Global Collaborations: Engage with international research communities.
7. Supportive Regulatory and Standardization Framework
• Regulatory Guidance: Develop guidelines for evaluating phase separation-modulating therapies.
• Standardization: Establish protocols for studying protein phase separation and aggregation.
Taken together. the research findings from SynDrops provide significant insights into the phase separation behavior and aggregation kinetics of α-synuclein (α-Syn), a key protein implicated in neurodegenerative diseases such as Parkinson's. Utilizing innovative techniques like Capflex, TDIPS, and mass photometry, the study reveals how pathological mutations and terminal truncations influence the thermodynamics and physical properties of α-Syn phase condensates. The results highlight the formation of stable molecular clusters and nanoclusters, as well as the transition to larger condensates and amyloid fibril formation. Moreover, the research identifies differences in material properties between wild-type and mutant α-Syn condensates, providing valuable insights into their potential role in disease progression. Overall, these findings significantly advance our understanding of protein phase behavior and offer promising avenues for therapeutic interventions in neurodegenerative disorders.