Targeting the Biophysical Roots of Amyotrophic lateral sclerosis (ALS)

Veneta Salyahetdinova

18 hours ago

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder which destroys motor neurons in the brain and central nervous system, and eventually causes the devastating prognosis of complete paralysis ¹. ALS can be caused by genetic mutations. One of the genes that can be affected is C9orf72, which encodes a protein that shares the same name and is involved in autophagy — clearance of cellular waste in the cytoplasm. Of particular interest is the hexanucleotide expansion mutation in this gene, which in essence is a six-letter DNA sequence GGGGCC, repeated many times ².

The cellular manifestation of this mutation begins once the repeat-containing RNA is translated by the ribosome. The process of translation encapsulates the synthesis of a final product (the protein) by the cellular protein factories (the ribosomes). In this important biological event, mRNA serves as the recipe by which the ribosome builds the correct protein. This leads to the production of highly basic arginine-rich proteins, poly-GR (glycine-arginine) and poly-PR (proline-arginine) ³. It is only then that the genetic mutation takes form in the cell, and this form is a polypeptide (protein) that contains highly basic residues such as arginine (Arg). Due to the charged nature of these polypeptides at bodily pH, it is not surprising that they interfere with normal cellular activity, especially because many components in the cell are charged.

This is where the physical manifestation of ALS becomes directly relevant to physical chemistry and intermolecular interactions in the cell. The toxicity is not only genetic in origin, but also expressed through electrostatic interactions involving the ribosome and other charged species. Kriachkov et al. provide one of the most insightful studies on repeats resulting from the C9orf72 mutation ⁴. They made several observations. Firstly, stalling during translation was more likely with longer mutated protein products, with some forms of mutation causing near-complete stalling at around 75 repeats. Dipeptide repeats containing charged residues such as Arg caused the largest decrease in translation. Kriachkov et al. identified Arg-rich dipeptide repeat (DPR) stalling as a characteristic inducer of translational stress, while also acknowledging that the mechanism remains unclear. In this article, we will explore how charged interactions between the newly-formed polypeptide and the ribosome could influence ALS development.

Independent studies, outside the direct scope of ALS but strongly relevant to it, have explored the effect of charged repeats on the interaction between the newly synthesised protein and the ribosome, and more specifically the ribosome exit tunnel — the structural compartment through which the protein chain is ejected. An important cellular event is co-translational folding, which refers to the process of the protein adopting its three-dimensional structure as it is emerging from the ribosome. One study by Nissley et al. explored how the dynamics of this event are influenced by the ribosome, again highlighting the importance of specific electrostatic interactions ⁵. To understand the exact mode of toxicity in ALS, it is worth considering these studies which had a different purpose in mind, but whose findings are highly relevant.

Nissley et al. simulated the ejection of 122 E. coli proteins from the ribosome and compiled their ejection times. This study gives results that are highly coherent with the biophysical manifestation of ALS. One key finding directly relevant to ALS is that newly-formed proteins containing negatively charged residues near one of their ends eject the fastest, while those enriched with positive charge eject the slowest. A direct parallel can be drawn with the arginine-rich nature of DPR repeats in ALS. A highly basic protein in their study, 4DCM, exhibited complete ejection from the exit tunnel in only 23 out of 50 simulation trajectories. This is striking evidence that basic residues, through their charged nature, can and do participate in favourable interactions with the ribosomal surface that interfere with healthy translational dynamics.

The study took this one step further by exploring the direct implications of the length of time that newly-formed protein spends in the exit tunnel. One implication is delayed ribosome recycling. Ribosome recycling is the process by which molecular factors bind to the ribosome and aid the dissociation of the large and small subunits ⁶. This is a crucial process, because it allows for the ribosomal machinery to be reused in new translational events. Their key finding was that slow ejectors have, on average, 3.3-times higher ribosome density at a particular point in the protein compared with fast ejecting proteins. If arginine-rich DPRs in ALS behave as extremely slow-ejecting newly-formed proteins, their prolonged residence in the ribosomal exit tunnel could increase ribosome density, delay recycling, and create conditions for stalling, subsequent collisions and hence disease progression.

A direct parallel to ALS can be drawn here too. Stress granules are often discussed in relation to ALS and other neurodegenerative diseases, and they can be connected to translational inhibition and ribosome-associated stress ⁷. Stress granules are RNA-protein condensates found in the cell that form when translation initiation is suppressed, which allows cells to adapt to stress. However, when stress is chronic or unresolved, persistent stress granules become part of the pathological landscape of neurodegeneration in ALS.

This is where ribosome recycling becomes relevant. Ribosome-associated quality control (RQC) in healthy cells detects stalled or collided ribosomes, and in turn promotes splitting of ribosomal subunits, and tags incomplete newly-formed proteins for degradation. If ribosomes however remain stalled, collided, or fail to recycle efficiently, broader stress signalling pathways are activated, including the integrated stress response (ISR) ⁸.

In a mouse model, some patterns of ribosome stalling were linked to integrated stress response hallmarks, such as phosphorylated eIF2α ⁹. This is highly relevant here because it suggests that stalled ribosomes interacting with arginine-rich newly-formed proteins cause wider cellular stress. Since ISR activation suppresses translational initiation, and stress granules form under conditions of translational inhibition, unresolved ribosome stalling may indirectly feed into stress granule formation. Hence, repeat mutations may contribute to toxicity in more than one way.

This is another reason to think that charged interactions involving mutated repeats may drive cellular toxicity through mechanisms similar to those explained by Nissley and colleagues. Unresolved ribosome collisions and unrecycled ribosomes have the potential to be even more detrimental if the standard RQC and ribosome rescue machinery does not work efficiently. There is strong indication that this may be the case in ALS, as reported by Kriachkov and his team. They pointed out that poly-K, a well-known RQC-related stalling substrate, induces a different pattern of RQC gene expression compared to 102xPR. These differential and poorly managed ribosome collisions are worth exploring further, as finding ways to support the natural RQC support system of the cell might be one way to reduce ALS-associated cellular stress.

Switching gears now, because the centre of the mini-review comprises interactions between the basic nature of the protein and the ribosome, thermodynamics should also be taken into account. Streit et al. have emphasised how the thermodynamics of protein folding are affected by the interaction between the ribosome and the mutated protein ¹⁰. They show, through experimental and computational approaches, that the energetics of folding (a key component of forming a functional protein) differ depending on whether a newly-formed protein folds in isolation or folds while emerging from the ribosome. In a nutshell, the entropic penalty (aka the energy tax) that a protein needs to pay in order to fold into its native form is lower once it is ejected from the ribosome. This is mainly because while still bound to the exit vestibule of the ribosome in the unfolded state, the protein gets more heavily solvated, which means that a high number of water molecules are in a highly ordered state around it. Upon ejection, the protein is freed from the ribosomal constraints, allowing it to fully collapse and release these trapped water molecules back into the bulk. This massive release of ordered water is very energetically favourable and provides a powerful entropic drive for post-ejection folding of the protein. If we take a look at protein folding in the absence of the ribosome, the protein is less heavily solvated. Hence, the retention of the protein on the ribosome for a certain time serves an entropically important function. These studies might not seem directly related to ALS at first sight, but the energetics of folding dictate whether a polypeptide chain will potentially misfold, which is also a direct route of toxicity in ALS. The ejection times of highly basic residues is abnormally long, which may alter the thermodynamics of folding even more. While still bound to the ribosome, the protein may form intermediate conformations which are not its true native functional state. Streit et al. found that interactions between the ribosome and the nascent chain stabilise the unfolded state entropically. Essentially, the long-range electrostatic interactions between the negatively charged ribosomal surface, largely caused by rRNA, and the protein side residues are responsible for the retention of the nascent chain on the ribosome. In the case of ALS, these basic repeats render those times longer compared to normal proteins, due to the enhanced nature of these electrostatic interactions. The longer the chain stays on the ribosome, the higher the likelihood of either partially folded inactive or toxic conformations forming, or translational stalling of the ribosome and subsequent ribotoxicity through collisions or faulty recycling.

Another insightful study relevant to ALS biophysical manifestation is done by Cassaignau et al., who found that the folding of proteins as they are being made can be interrupted by interactions between the newly-formed protein and the ribosome ¹¹. To investigate these interactions they studied a specific part (domain) of a protein called FLN5 while it was still tethered to the ribosome.

This study again proves the significance of long-range electrostatic interactions. Interestingly, even though 31 residues is the minimum required linker length for the entire FLN5 domain to span outside the exit tunnel of the ribosome, the protein does not fully fold into its native form at this length and remains partially unfolded. This indicates that interactions with the ribosome stay in the way of folding. It is only at a linker length of 41 residues that complete FLN5 folding can be observed.

This study added additional evidence that basic residues as in ALS are of critical importance in these interactions. To prove this, the researchers used an advanced technique called Nuclear Magnetic Resonance (NMR) spectroscopy. NMR essentially tracks how a molecule’s local environment changes, producing a signature spectrum of signals. When parts of the emerging FLN5 protein interact with the ribosome, their specific signals change — a phenomenon known as line broadening. This in turn confirms that the basic parts of FLN5 protein were actively sticking to the ribosome. Interestingly, the team discovered that these changes can be reversed by increasing ionic strength (salt concentration) of the surrounding fluid. Because salt disrupts electrical charges, this observation confirms the electrostatic nature of the interaction between the ribosome and the FLN5 protein. Most excitingly, this proves that these cellular interactions are highly dynamic and could potentially be modified by future medical interventions.

Further clarification of what makes these electrostatic interactions favourable in ALS would allow for new approaches to tilt the equilibrium away from stabilisation of the basic chain on the ribosome and hence away from stalling and collisions. A small-molecule approach could be one way through which these interactions can be shielded. On the other hand, modulating stress granule formation dynamics and supporting the natural RQC of the cell may also present viable approaches

Overall, this article proposes that ALS toxicity, at least in the context of C9orf72 arginine-rich DPRs can be viewed not only as a biochemical problem, but also as a physical manifestation inside the cell. The mutation creates a product whose charge, repetition, and supramolecular behaviour alter how it interacts with the ribosome and the wider intracellular environment. If the pathological consequences are a result partly from these interactions, then targeting the biophysical manifestation of the disease may become an additional way of future intervention.

Edited by: Cameron McKeddie

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Veneta Salyahetdinova

Veneta is a second year Biochemistry student at the University of Glasgow who has always been deeply passionate about science, vividly remembering her sisters buying her science journals for kids and being completely consumed by how intriguing the human body is. In high school, she took part in many initiatives related to science such as "The Young Scientists of Bulgaria", organised by the Bulgarian Academy of Sciences. She aims to stay socially active, which is why she was part of "The Youth Parliament" of her hometown, Blagoevgrad. She organised initiatives, in collaboration with the Regional Healthcare Institution, the Municipality and the Regional museum. Through all of these activities she aimed to improve Blagoevgrad's culture. Her desire is to transform her love and knowledge for science into intriguing and informative scientific articles.

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