January's ALS Research Roundup 2023

Sustained therapeutic benefits by transient reduction of TDP-43 using ENA-modified antisense oligonucleotides in ALS/FTD mice

(Takeuchi et al., 2023)

Antisense oligonucleotides (ASOs) are small pieces of DNA or RNA that can bind to specific RNAs, blocking their ability to function. They have been investigated with regards to many neurodegenerative disorders with complex RNA and protein interactions including Huntington’s, Parkinson’s and Alzheimer’s diseases. ALS genes have likewise been targeted with ASOs, including SOD1, FUS and C9orf72. However, ASO therapies targeting TDP-43, the most ubiquitous aggregating protein in ALS, have not yet been explored. Previous research has shown that shutting down TDP-43 expression reduces pathology in similar disease models, particularly in early stages, so ASO-based inhibition is expected to produce positive changes in disease course. A major reason for the lack of TDP-43-targeted ASOs is the crucial cellular function of TDP-43, which has led previous researchers to investigate reducing the toxicity of TDP-43 without directly reducing its levels.

The ASO used in this study used 2’-O,4’-C-ethylene nucleic acids (ENAs), modified nucleic acids with high stability. These were administered to a TDP-43 mouse model of ALS. This treatment showed impressive results, with a single injection leading to long-lasting improvement on both a molecular and clinical level. Pathologically aggregated TDP-43 was significantly reduced in the brain and spinal cord, leading to suppression of disease phenotypes and recovery of locomotor function. which lasted 4-6 months post-injection. Mice injected at 6 weeks of age showed reduced TDP-43 aggregation on cortical neurons at 18 months of age, indicating that it may possess an even stronger preventative function. This is well beyond the expected duration of even the highly stable ENA-modified ASOs, suggesting that it has a beneficial interaction outside of its direct activity. One possible explanation could be that suppressing early stages of aggregation prevented the initiation of gradual development of pathology, preventing or at least slowing an escalating pathological process.

The ASO therapy demonstrated here has a great deal of potential as a therapeutic avenue. It showed success in reducing disease-driving aggregates, clinical symptoms and may have even stronger preventative function. However, the study did lack any data on potentially toxic side effects. Considering this was a major reason why TDP-43 ASOs had not been previously tested, this is potentially a major limitation. However, with the effectiveness demonstrated, the treatment definitely merits further study to determine this. If it is possible to prevent the first stages of pathological buildup, well before the self-reinforcing cycles observed in ALS emerge, it could be a key avenue of addressing the disease as a whole.

TAGS: therapeutics, ASO, TDP-43, mouse

Stress granule assembly?in vivo?is deficient in the CNS of mutant TDP-43 ALS mice

(Dubinski et al., 2023)

Stress granules are dense, membraneless aggregates which form in the cytoplasms of cells, particularly in response to cellular stress. They are primarily comprised of RNA-binding proteins and stalled mRNAs, preventing them from carrying out certain cellular functions. Stress granules have been linked to ALS by several possible interactions. Many of these proteins (but not mRNAs) are also captured by aggregates formed in ALS, possibly increasing the vulnerability of cells to ALS by accelerating the loss of function. Stress granules may also act as a scaffold on which ALS-associated TDP-43 aggregates could form, accelerating the onset of pathology. However, others have theorised a possible protective effect, isolating proteins and RNAs to prevent pathological effects. This study investigated the effects of heat-induced stress granule formation in the spinal cords of transgenic TDP-43-ALS mice, testing the hypothesis that defective stress granules increase the vulnerability of motor neurons to ALS pathology.

Surprisingly, expression of mutant TDP-43 was associated with defects in the assembly of stress granules, despite TDP-43 not accumulating near them. TDP-43 mice suffered stress granule disruption at 4 months equivalent to wild-type mice at 18 months, well before the onset of symptoms. This may be linked to depletion of several proteins key to stress granule formation in TDP-43 mice. Previous research found that a SOD1 mouse model of ALS had neither stress granule dysfunction or depletion of RNA-binding proteins, supporting this theory. The general appearance of the stress granules in heat-treated animals was quite different compared to those found in in vitro experiments. It may be that this heat treatment model alters the stress granules formed in an unexpected manner. The concept of different stress granule structures based on cell type and stressor has been theorised, but not conclusively demonstrated before. It is also of note that the heat stress used was acute, lasting only 20 minutes, and so may differ from chronic stressors. ALS-associated movement of TDP-43 from the nucleus to the cytoplasm was still observed despite stress granule dysfunction, providing evidence that these are independent processes.

Given the large role that protein aggregation is believed to play in the pathology of ALS, understanding the factors which drive and inhibit this aggregation may be key to controlling disease progression. This study provided evidence that rather than contributing to the disease, stress granules are themselves negatively affected by ALS-associated TDP-43 aggregation. This does not necessarily mean that stress granules don’t provide a beneficial environment for TDP-43, but does suggest that this is not one of the self-propagating cycles which are seen in several aspects of ALS pathology. It would be interesting to assess changes in disease course if stress granule formation is bolstered. If it does prove protective, this could emerge as a novel therapeutic avenue for certain forms of ALS.

TAGS: mechanistic, stress_granules, TDP-43, mouse

Metamorphism in TDP-43 prion-like domain determines chaperone recognition

(Carrasco et al., 2023)

Molecular chaperones are proteins which help other proteins to take on functionally active forms, typically contributing to the folding and unfolding of protein complexes. Mutations in several of these proteins have been shown to contribute to ALS, resulting in misfolded proteins which are prone to pathological aggregation. However, while it is known that molecular chaperones are protective against this aggregation, exactly how they do so is often unclear. A key element of this is phase-separated liquid compartments within the liquid cytoplasm. These are created by molecular chaperones to isolate misfolded proteins in healthy conditions, but often act as crucibles for pathological aggregation in ALS. This study investigated the factors which distinguish functional from disease-associated aggregates, using nuclear magnetic resonance imaging (NMR) to investigate the structures involved in harmful phase separation, and its interactions with molecular chaperones.

The analysis performed here showed that molecular chaperones primarily recognize the structured elements in TDP-43′s prion-like domain (PLD). Increased PLD misfolding within phase-separated regions can overpower elements such as chaperones intended to remove these, which is exacerbated by lower levels of molecular chaperones in ALS patients. While specific chaperones including HSP70 and HSP90 chaperones promote TDP-43 phase separation, co-chaperones from the three classes of the large human HSP40 family (namely DNAJA2, DNAJB1, DNAJB4 and DNAJC7) were found to interact with different regions of the protein and presumably have more varied effects. Given the importance of TDP-43’s PLD, it is not surprising that disruption of part of the PLD using methionine sulfoxidation was able to impact phase separation and aberrant protein aggregation. This supports the possibility of structural modification of key regions of TDP-43 as a therapeutic avenue, preventing key molecular interactions which drive pathological phase separation and protein aggregation.

Despite extensive study, we still know relatively little about the protein aggregation processes involved in ALS pathology. They appear to vary between variants, proteins and even time points, with multiple stages of aggregation obscuring clear images of structural forms. However, understanding how the misfolded proteins interact with each other and avoid regulatory mechanisms such as molecular chaperones can help us to counteract these pathology-driving processes and develop more effective molecular therapies. While enacting structural modifications like was done here is naturally difficult in a living organism, understanding the processes involved may lead to valuable means of preventing both the formation and propagation of aggregates, slowing or even stopping the spread of ALS pathology.

TAGS: mechanistic, PLD, stress_granules, TDP-43, protein

Cervical spinal cord atrophy in amyotrophic lateral sclerosis across disease stages

(Nigri et al., 2023)

One method of assessing the progression of ALS outside of clinical symptoms is measuring the decay of cells, particularly in certain regions of the brain and spinal cord. This study assessed gray and white matter atrophy in the cervical region of the spinal cord, the section located just below the brain, in 44 early-stage ALS patients. While spinal cord atrophy has been assessed before, this has predominantly studied it as a whole, rather than distinguishing tissue types. This distinction was made using phase sensitive inversion recovery (PSIR), an imaging method commonly used in cardiac MRI to enhance contrast between tissue types. The aim was to identify a spinal cord imaging marker for disease progression that can be easily employed in a clinical context without any invasive tissue extraction.

The first thing assessed was patterns of gray and white matter atrophy. Overall, both gray and white matter were reduced in ALS patients compared to controls, in all cervical segments. Cervical atrophy was then compared with King’s stages, an anatomical staging system based on the number of anatomical regions involved. They found that in stage 1, where only a single region is involved, there was only significant atrophy in the gray matter, which is composed of neuronal cell bodies. White matter, which is predominantly nerve fibres, only became involved in later stages when multiple CNS regions were involved. Total atrophy, as well as atrophy of gray and white matter was correlated with clinical loss of function in all segments of the cervical spinal cord. However, the earliest involvement was the C3-C4 segments, which started to atrophy 7-20 months before symptom onset according to a mathematical model developed from the observed data. This seemed independent of the site of onset of ALS (upper or motor neurons), as well as the clinical phenotypes.

While the different rates of atrophy in gray and white matter were interesting, the finding that stood out was the presymptomatic atrophy in the C3-C4 segments. These may emerge as a way of diagnosing, or at least warning of ALS long before symptom onset, and without any invasive procedures. It should be noted that this was based on a mathematical model rather than direct observation, due to issues with identifying ALS cases before symptom onset. As such, this would benefit from validation in animal models. The lack of association with clinical phenotypes is surprising, as it indicates that this atrophy does not directly contribute to ALS pathology. It would be interesting to see whether this early atrophy is associated with atrophy of any brain regions, or whether it emerges independently.

TAGS: diagnosis, anatomy, presymptomatic, human

SOMAscan proteomics identifies novel plasma proteins in amyotrophic lateral sclerosis patients

(Berrone et al., 2023)

Directly assessing various biological factors in ALS is made difficult by both complex interactions between relevant genes and clinical outcomes, and the most relevant effects taking place inside the sealed environment of the central nervous system. This study attempted to address both by employing a novel technique to assess protein profiles in the blood, which is far more accessible than the CNS but can carry valuable disease markers. Proteins are more directly causative of disease phenotypes than genes are, so can provide valuable evidence of the biological pathways involved. Several techniques exist to assess proteins levels, but large-scale studies are difficult due to technical limitations. The specific technology used here, SOMAscan, determines levels of circulating proteins using aptamers, artificial strands of DNA or RNA which bind specific target molecules.

Around 1300 proteins were assessed in 47 ALS patients and 32 controls, with the aim of finding differentially expressed proteins between the two groups. 42 proteins were identified to have altered levels in circulation in ALS patients, from which four were selected for greater statistical and biological significance. These were validated in a partially independent cohort and all found to be significantly increased in the blood plasma of ALS patients. Interestingly, they also varied significantly between sporadic and familial ALS patients. The first, TARC, is a chemokine associated with neuroinflammation and microglial activity. The second, TIMP-3, is a tissue inhibitor linked to the extracellular matrix which surrounds cells and which likely plays a role in cell growth, death and tissue repair. Overexpression of this protein may contribute to apoptotic cell death, particularly in early stages of ALS. The last two, NID-1 and NID-2, are secreted glycoproteins which help to separate tissues including blood vessels, nerves and muscles. Their exact function is unknown, but loss of both caused death in transgenic mice.?

The analysis performed here was able to distinguish controls, sporadic and familial ALS based on protein levels in peripheral blood. They are not the first to do so, but their ability to specifically assess a large range of proteins using aptamer-based technologies is potentially extremely valuable. However, the technology does still have some limitations. Aptamers can have multiple targets, and alterations in protein structure can prevent proper binding, but could still be a valuable tool for assessment of specific protein biomarkers in the blood. The differentially expressed proteins identified here were either only recently, or not yet linked to ALS, but all have plausible mechanisms of action. This adds to a growing body of possible ALS biomarkers, which can be assessed without the invasive procedures needed to access the CNS. It would be interesting to see more in-depth exploration of how levels of these proteins change alongside clinical development of ALS, and whether they could be used as prognostic as well as diagnostic biomarkers.

TAGS: biomarkers, aptamers, proteomics, human

Integrative genetic analysis illuminates ALS heritability and identifies risk genes

(Megat et al., 2023)

Understanding the inheritance of ALS is a complex undertaking, with a broad range of gene and non-gene factors appearing to contribute. An area which has been achieving much greater awareness in recent years is that of variants in splicing, the process by which RNA is fragmented and recombined to create multiple different proteins. Splicing variants are typically the result of mutations in the boundaries between these fragmented regions (introns and exons), resulting in changes in the location of these boundaries and so different proteins.

This study underwent genetic analysis of a cohort of several thousand ALS patients and controls, finding significant differences in splicing variants and RNA binding sites in six key genes. These included the well-established ALS genes TDP-43 and FUS, as well as several unusual ones. Splicing-associated mutation sites were found to be significantly associated with ALS risk, particularly in genes expressed in neurons. This was theorised to be the result of increased vulnerability to the loss of TDP-43 and FUS in ALS, both being RNA-binding proteins which interact with the splicing process. Of the six genes identified, NUP50 was further explored as it was the only one to be identified from two separate ALS cohorts. NUP50 is a nuclear pore protein, which is significant since nuclear pore dysfunction is often an early event in sporadic ALS, and is exacerbated by aggregation of both TDP-43 and FUS. In addition, NUP50 has been found to be less expressed in the cortexes of ALS patients, as well as several experimental models of the disease. The key mutations explored here were those connected to haploinsufficiency, deactivation of one copy of a gene in a situation where the remaining copy is not capable of maintaining healthy function. Loss of NUP50 was assessed in three different ALS models (cultured neurons, fruit flies and zebrafish), with ALS-like outcomes in either loss of motor function or cell death.

The main outcome of this study is identifying that splicing-associated genetic variants, particularly in genes expressed in neurons, have a disproportionately strong connection to ALS. As such, researchers may benefit from these being more highly prioritised when assessing disease-driving genes. It may also help to explain the massive role of TDP-43 in ALS, disrupting splicing variants whose loss can contribute to the pathology. NUP50 has been only recently identified as an ALS risk factor, and this study helped to explore its functional role and cement its place as a gene of note.

TAGS: mechanistic, RNA, NUP50, human

Symptoms timeline and outcomes in amyotrophic lateral sclerosis using artificial intelligence

(Segura et al., 2023)

The patterns of symptom progression and clinical outcomes of ALS vary wildly from patient to patient, making accurate prediction difficult. Various measures have been used to predict the course of the disease, from levels of protein expression to biomarkers in the blood to rates of degeneration across different regions of the brain. This study used a an artificial intelligence model to explore the health records of 250 ALS patients. The most significant machine learning tool used was Natural Language Processing (NLP), which helps computers to parse human language into usable data. They aimed to use this to characterise features of the ALS population including demographics, delays between symptom onset and diagnosis (diagnostic delay) and the timing of certain manifestations of the disease.

The demographic data found here was largely in line with prior studies, with some interesting exceptions. They noted two major diseases which co-occurred with ALS: hypertension, in 44% of patients, and dyslipidaemia in 21%. There is an existing debate as to the role of these in ALS, with hypertension possibly playing a protective role and lipid dysfunction possibly contributing to ALS pathology. The diagnostic delay was found to be an average of 11 months, towards the lower end of the range identified previously. Early referral to a neurologist reduced this, although under 40% of patients visited one before diagnosis. Survival time was also in line with prior findings, as was rates of treatment with riluzole and tracheostomy, which increased survival times.

While none of the outcomes found here were particularly groundbreaking, the findings validated and were validated by prior clinical data. This supports the accuracy of the machine learning model used, which could be applied to less well-explored research data in the future. Being able to investigate less quantitative data may provide access to much more diverse datasets, particularly with regards to retrospective data.

TAGS: model, machine_learning, human_clinical

References

Berrone, E., Chiorino, G., Guana, F., Benedetti, V., Palmitessa, C., Gallo, M., Calvo, A., Casale, F., Manera, U., Favole, A., Crociara, P., Testori, C., Carta, V., Tessarolo, C., D’angelo, A., Marco, G.D., Caramelli, M., Chiò, A., Casalone, C., and Corona, C. 2023. SOMAscan Proteomics Identifies Novel Plasma Proteins in Amyotrophic Lateral Sclerosis Patients. International Journal of Molecular Sciences [Online], 24.

Carrasco, J., Antón, R., Valbuena, A., Pantoja-Uceda, D., Mukhi, M., Hervás, R., Laurents, D.V., Gasset, M., and Oroz, J. (2023). Metamorphism in TDP-43 prion-like domain determines chaperone recognition. Nature Communications 14, 466.

Dubinski, A., Gagné, M., Peyrard, S., Gordon, D., Talbot, K., and Vande Velde, C. (2023). Stress granule assembly in vivo is deficient in the CNS of mutant TDP-43 ALS mice. Human Molecular Genetics 32, 319-332.

Megat, S., Mora, N., Sanogo, J., Roman, O., Catanese, A., Alami, N.O., Freischmidt, A., Mingaj, X., De Calbiac, H., Muratet, F., Dirrig-Grosch, S., Dieterle, S., Van Bakel, N., Müller, K., Sieverding, K., Weishaupt, J., Andersen, P.M., Weber, M., Neuwirth, C., Margelisch, M., Sommacal, A., Van Eijk, K.R., Veldink, J.H., Lautrette, G., Couratier, P., Camuzat, A., Le Ber, I., Grassano, M., Chio, A., Boeckers, T., Ludolph, A.C., Roselli, F., Yilmazer-Hanke, D., Millecamps, S., Kabashi, E., Storkebaum, E., Sellier, C., Dupuis, L., and Project Mine Als Sequencing, C. (2023). Integrative genetic analysis illuminates ALS heritability and identifies risk genes. Nature Communications 14, 342.

Nigri, A., Dalla Bella, E., Ferraro, S., Medina Carrion, J.P., Demichelis, G., Bersano, E., Consonni, M., Bischof, A., Stanziano, M., Palermo, S., Lauria, G., Bruzzone, M.G., and Papinutto, N. (2023). Cervical spinal cord atrophy in amyotrophic lateral sclerosis across disease stages. Ann Clin Transl Neurol.

Segura, T., Medrano, I.H., Collazo, S., Maté, C., Sguera, C., Del Rio-Bermudez, C., Casero, H., Salcedo, I., García-García, J., Alcahut-Rodríguez, C., Aquino, J., Casadevall, D., Donaire, D., Marin-Corral, J., Menke, S., Polo, N., Taberna, M., and Savana Research, G. (2023). Symptoms timeline and outcomes in amyotrophic lateral sclerosis using artificial intelligence. Scientific Reports 13, 702.

Takeuchi, T., Maeta, K., Xin, D., Oe, Y., Takeda, A., Inoue, M., Nagano, S., Fujihara, T., Matsuda, S., Ishigaki, S., Sahashi, K., Minakawa, E.N., Mochizuki, H., Neya, M., Sobue, G., and Nagai, Y. (2023). Sustained therapeutic benefits by transient reduction of TDP-43 using ENA-modified antisense oligonucleotides in ALS/FTD mice. Molecular Therapy - Nucleic Acids.