Structural Polymorphism of α-Synuclein Amyloid Fibrils is Strongly Influenced by pH and Buffer Conditions

Keskeiset käsitteet

The aggregation of α-synuclein protein into amyloid fibrils is closely associated with neurodegenerative disorders. The structural polymorphism of these fibrils is highly dependent on the pH and buffer conditions, with distinct polymorphs favored at different pH levels. Even in the presence of seeds, the polymorph selection during aggregation is largely determined by the environmental factors rather than the seed structure.

Tiivistelmä

The study investigates the structural polymorphism of α-synuclein amyloid fibrils and how it is influenced by the pH and buffer conditions. Key findings:

1. In the physiological pH range of 5.8-7.4, a pH-dependent selection between Type 1, 2 and 3 polymorphs of α-synuclein fibrils was observed.

2. At pH 5.8, only Type 3 polymorphs (3B and 3C) were observed. At pH 6.5, a mixture of Type 2 and 3 polymorphs was found.

3. At pH 7.0, a new Type 5 polymorph and a monofilament Type 1 polymorph (similar to the juvenile-onset synucleinopathy polymorph) were discovered.

4. Seeding experiments showed that even in the presence of preformed seeds, the polymorph selection was largely determined by the pH and buffer conditions rather than the seed structure. This suggests that secondary nucleation processes, which are non-polymorph-specific, play a major role in the in vitro aggregation of α-synuclein.

5. The results highlight the shallow energy landscape of α-synuclein amyloid formation, where subtle changes in the environment can lead to the selection of different polymorphic structures.

6. The ability to produce disease-relevant polymorphs, like the juvenile-onset synucleinopathy polymorph, in vitro opens up new possibilities for studying the structural basis of synucleinopathies.

Tilastot

"The speed and abundance of polymorphs being discovered have led to inconsistent naming conventions."
"In the five independent amyloid samples that we prepared at pH 5.8, none of them contained a detectable level of Type 2 fibrils."
"In the sample prepared at pH 6.5, we did indeed see a mixture of Type 2 and 3 (2A:2B:3B:3C at 25%:18%:31%:26%)."
"In several pH 7.0 aggregation samples, we also observed a Type 1 monofilament fibril polymorph (termed 1M), similar to those from the MSA and Parkinsons-CSF seeding experiments."
"In yet another of the pH 7.0 aggregation samples we found a completely new polymorph which we have termed Type 5."

Lainaukset

"The speed and abundance of polymorphs being discovered have led to inconsistent naming conventions."
"Seeding experiments have also been performed with cerebrospinal fluid (CSF) of Parkinson's disease patients at pH 6.5, this time with N-terminally acetylated α-Syn, yielding once again the 3B and 3C polymorphs."
"Considering the strong temperature dependence of the Tris pKa, it is worth noting that our Tris buffer was prepared for pH 7.0 at the fibrillization temperature of 37 °C."

Tärkeimmät oivallukset

On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seed-based amyloid propagation

by Frey,L., Gho... klo www.biorxiv.org 06-26-2023

https://www.biorxiv.org/content/10.1101/2023.06.25.546428v6

Syvällisempiä Kysymyksiä

What other environmental factors beyond pH and buffer composition could influence the selection of α-synuclein amyloid polymorphs

In addition to pH and buffer composition, several other environmental factors could influence the selection of α-synuclein amyloid polymorphs. One crucial factor is the presence of co-factors or co-aggregates that may interact with α-synuclein and influence the folding and aggregation process. These co-factors could include lipids, metal ions, small molecules, or other proteins that modulate the conformational landscape of α-synuclein. Temperature is another critical environmental factor that can impact the kinetics and thermodynamics of amyloid formation. Changes in temperature can alter the stability of different polymorphs and shift the equilibrium towards the formation of specific structures. The presence of crowding agents, such as molecular chaperones or other cellular components, can also affect the aggregation pathway and polymorph selection by altering the local concentration and molecular interactions of α-synuclein. Furthermore, variations in ionic strength, redox conditions, and the presence of post-translational modifications can all play a role in determining the structural diversity of α-synuclein amyloid aggregates.

How can the in vitro production of disease-relevant polymorphs, like the juvenile-onset synucleinopathy polymorph, be further optimized and scaled up to enable more detailed structural and functional studies

To optimize and scale up the in vitro production of disease-relevant polymorphs, such as the juvenile-onset synucleinopathy polymorph, several strategies can be employed. Firstly, a systematic screening of a wide range of environmental conditions beyond pH and buffer composition should be conducted to identify the key factors that promote the formation of the desired polymorph. This may involve testing different salts, additives, temperatures, and co-factors to mimic the physiological conditions associated with the disease state. Additionally, the use of advanced structural biology techniques, such as cryo-electron microscopy and solid-state NMR, can provide detailed insights into the structural features of disease-relevant polymorphs and guide the optimization process. Collaborations with experts in the field of neurodegenerative diseases and structural biology can also help in interpreting the structural and functional implications of the disease-specific polymorphs. Furthermore, the development of high-throughput screening methods and automation of the aggregation assays can facilitate the rapid screening of a large number of conditions to identify the optimal conditions for producing disease-relevant polymorphs. Finally, the establishment of standardized protocols and quality control measures can ensure reproducibility and reliability in the production of disease-relevant α-synuclein polymorphs for detailed structural and functional studies.

Could the insights from this study on the role of secondary nucleation in polymorph selection be applied to develop new strategies for polymorph-specific amplification of α-synuclein aggregates from patient samples for diagnostic purposes

The insights from this study on the role of secondary nucleation in polymorph selection can be leveraged to develop new strategies for polymorph-specific amplification of α-synuclein aggregates from patient samples for diagnostic purposes. By understanding the influence of secondary nucleation on polymorph selection, researchers can design seeding assays that minimize secondary nucleation processes and focus on fibril elongation, which is more likely to preserve the structural characteristics of the seed polymorph. This could involve optimizing the seed concentration, incubation conditions, and buffer composition to favor fibril elongation over secondary nucleation. Additionally, the identification of specific inhibitors of secondary nucleation, such as small molecules or peptides that target the C-terminal region of α-synuclein, could be explored to selectively amplify disease-relevant polymorphs from patient samples. By combining the knowledge of polymorph-specific amplification with advanced structural characterization techniques, such as cryo-EM and mass spectrometry, researchers can develop reliable and sensitive diagnostic assays for differentiating between α-synuclein polymorphs associated with distinct synucleinopathies.