Small molecules for modulating protein driven liquid-liquid phase separation in treating neurodegenerative disease

Background:

The relatively nascent concept of biomolecular condensates has stirred up increasing interest in recent years, emerging as a fundamental organizational principle in cells¹. Multiple groups have described how these compartments – also described as ‘membraneless organelles’ – are formed by liquid-liquid phase separation and are functionally important for various biological processes². Interestingly, phase separation or demixing also underlies the formation of stress granules: ribonucleoprotein assemblies that sequester mRNAs from the translation machinery under conditions of stress³. These assemblies enable cells to prioritize synthesis of proteins that are required to deal with the emergency, by temporarily halting the translation of non-essential genes³. However, there also appears to be a direct link between stress granules and disease. For example, amyotrophic lateral sclerosis (ALS) is associated with mutations in stress granule proteins⁴. Further, ALS-linked mutations have been shown to alter the material states of condensates to less liquid-like⁵. One might speculate that a productive intervention would target the material properties of stress granules, without a) affecting other phase-separated compartments and b) relying on highly specific interactions with granule components ^6,7.

In this exciting new collaborative study, the authors monitor the localization and phase behavior of FUS to screen for compounds capable of modulating these properties. FUS is a protein that normally resides in the nucleus but localizes to stress granules under conditions of stress. Mutations in FUS, many of which shift its localization towards the cytoplasm, are associated with familial forms of ALS. The candidates obtained in the screen are then used to probe the alteration in properties of the phase separated condensates, and to test for reversal of ALS-linked detrimental phenotypes in patient-derived iPSC neurons and in organisms such as worms and flies.

 

Key findings:

• Candidate identification and verification: A cell-based screen identified lipoamide and the closely related compound lipoic acid as having a strong effect on numbers of FUS condensates and intracellular FUS localization in stressed cells. Lipoic acid is a cofactor required for aerobic metabolism and has been previously proposed to be an antioxidant. Although an effect of redox chemistry cannot be ruled out completely, the authors demonstrate that the compounds have additional effects on the phase behavior of stress granules. Further they find that both candidates were able to reduce stress granules induced by diverse stresses, and that the modulatory effect is not restricted to FUS – other stress granule-resident proteins are also similarly affected.
• Specificity towards stress granules: Lipoamide did not affect a range of other functional condensates indicating that it preferentially modulates stress granules. This is a leap forward from known stress granule modulators like 1,6-hexanediol which is required in very high concentrations to elicit any effect and alters other membraneless organelles like the nucleolus. Further, lipoamide did not negatively impact the beneficial recruitment of FUS at sites of DNA damage within the nucleus. It is noteworthy that both candidates are non-toxic and lipoic acid also has well-characterized pharmacokinetics.
• Proposed mechanism of action: Lipoamide treatment affected key physical properties of the condensates resulting in a) improved liquidity in vitro, and b) delayed pathological ‘aging’ of condensates containing G156E FUS into fibers characteristic of ALS. NMR-based experiments further indicated that the most likely mechanism by which lipoamide acts is the alteration of condensate properties, rather than high-affinity binding with stress granule components.
• Demonstration of beneficial effects, in vivo: The candidates were able to reverse protein aggregation and deleterious phenotypes in multiple models. Treatment with lipoic acid reduced the aggregation of PAB-1, a stress granule-related protein in elegans. Further, both lipoic acid and lipoamide treatment reduced aggregation in patient-derived P525L FUS iPSC lines. The compounds also recovered axonal transport and prevented axon die-back in iPSC motor neurons expressing the same ALS-associated FUS mutation. Finally, severe motor neuron defects caused by expressing human FUS mutants in Drosophila were also successfully reversed by dietary lipoamide/lipoic acid supplementation, as estimated by restoration of their ability to climb.

 

 

Why I chose this preprint:

Currently approved treatments available for ALS either target specific symptoms or serve only to slow down functional deterioration and reduce discomfort. Mutations in core stress granule proteins like TIA-1 have been associated not just with ALS but also in ALS patients with accompanying Frontotemporal Dementia⁸. Further, TIA-1-positive stress granules have been suggested to stimulate tau pathophysiology in Alzheimer’s Disease⁹. Therefore, understanding stress granule biology and identifying interventions to modulate their formation, physico-chemical properties and stability, is key to designing any potential therapeutic approach. This study accomplishes a mammoth task of identifying small molecules from a cell-based screen, demonstrating their effects on FUS condensates in vitro and in cells, and further in reversing FUS-associated defects at the level of individual neurons and organisms. Taken together, it is the first study indicating that a direct link may exist between modulation of phase behavior of subcellular condensates and the outcome on a disease-linked state.

Further, the authors show that lipoamide does not interact via high-affinity binding to FUS. Instead, the proposed mechanism of action is through modulation of physical properties of the condensates, resulting in altered phase behavior. There are likely other compounds/small molecules that can also beneficially modulate biomolecular condensates linked with ALS or with other neurodegenerative diseases.

Finally, this study provides evidence in favor of the argument that modulation of stress granules leading to recovery of normal localization of proteins like FUS, is indeed beneficial.

 

 Questions for authors:

• It appears that the dietary supplements in flies leading to reversal of motor defects were supplied chronically throughout life. If so, do you have a sense for whether the effect is preventive and if the timing of introduction of lipoamide/lipoic acid matters? Can an acute intervention in flies that already demonstrate the climbing defects still produce the beneficial effect? In other words, how late is too late?
• Lipoamide treatment did not dissolve multiple nuclear compartments either containing FUS or otherwise. In the preprint it is speculated that the interactions and physical chemistry driving different types of condensates could account for this specificity. However, assuming the nucleus and cytoplasm to be already two separate assemblies, could the nuclear envelope somehow represent a stronger permeability barrier to these compounds, thereby protecting the nuclear condensates? How universal is this protection of diverse non-stress granule condensates in the cytoplasm (beyond the example of non-disruption of P-bodies containing DCP1A)?
• The formation of stress granules represents a protective cellular mechanism to cope with adverse environmental conditions. Would you expect undesirable side effects when treating patients with any compound that inhibits stress granule formation? In order to be of therapeutic value, would the dose need to be fine-tuned to still allow a sufficient level of protection against cellular stress?

 

Acknowledgements:

I would like to thank my fellow preLights team members Tessa Sinnige and Monika Magon for very helpful feedback and discussion, preprint author Richard Wheeler for comments on technical accuracy, and Suvrajit Saha for critical comments.

 

References:

1. Alberti S. Phase separation in biology (2017). Phase separation in biology. Curr. Biol. 27
2. Banani SF, Lee HO, Hyman AA and Rosen MK (2017). Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18
3. Decker CJ & Parker R (2012). P‑Bodies and stress granules: possible roles in the control of translation and mRNA degradation. Cold Spring Harb. Perspect. Biol. 4
4. Bosco DA, Lemay N, Ko HK, Zhou H, Burke C, et. al. (2010). Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules. Hum. Mol. Genet. 19
5. Patel A, Lee HO, Jawerth L, Maharana S, Jahnel M, al. (2015). A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162
6. Kroschwald S, Maharana S, Mateju D, Malinovska L, Nuske E, et. al. (2015). Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules. eLife 4
7. Wheeler RJ & Hyman AA (2018). Controlling compartmentalization by non-membrane-bound organelles. Philos. Trans. R. Soc. B Biol. Sci. 373
8. Mackenzie IR, Nicholson AM, Sarkar M, Messing J, Purice MD, et. al. (2017). TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics. Neuron 95
9. Vanderweyde T, Yu H, Varnum M, Liu-Yesucevitz L, Citro A, et. al. (2012). Contrasting pathology of the stress granule proteins TIA-1 and G3BP in tauopathies. J. Neurosci. 32

 

Further reading:

1. This blog post about the same preprint, by Derek Lowe.
2. Alberti S. & Dormann D. (2019) Liquid-liquid phase separation in disease Annu. Rev. Genet. 53

Tags: amyotrophic lateral sclerosis, fus, liquid-liquid phase separation, small molecules, stress granules

Posted on: 30 August 2019 , updated on: 31 August 2019

doi: https://doi.org/10.1242/prelights.13626

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