When good proteins go bad: my quest to characterise TDP-43 and its role in ALS
Every movement we make, such as taking a walk, sending a text or hugging someone we love, depends on specialised nerve cells called motor neurones. These neurones send signals that tell muscles to contract, allowing the body to move. In people with Amyotrophic Lateral Sclerosis (ALS), these cells gradually stop working. As they die, muscles weaken, and everyday tasks become harder.
Scientists are still trying to understand how this disease develops. One protein that repeatedly appears in ALS research is TDP-43. Found in all our cells, TDP-43 helps control several aspects of RNA behaviour, including processing, stability and transport. Most of the time it stays in the nucleus, the cell’s control centre. Occasionally, it ventures out into the cytoplasm, the surrounding cellular environment, when the cell is under stress.
When stress is prolonged, TDP-43 accumulates in the cytoplasm, where it forms insoluble clumps known as aggregates. When this happens, the protein can no longer properly regulate RNA. As a result, neurones may struggle to produce the proteins they need to function properly. Read more here.
This raises an important question: how can a protein that is present in all of us contribute to disease in only some individuals? Previous research suggests that genetic and environmental stressors may increase TDP-43 pathology. Over 40 different genes have been linked to ALS, including TARDBP- the gene responsible for expressing TDP-43. Importantly, each mutation presents different physical symptoms and cellular patterns of pathology. This makes designing therapeutics and biomarkers difficult, as scientists don’t have a list of common symptoms to target.
This challenge forms the focus of my project: understanding how different patient mutations change the behaviour of TDP-43 inside cells. By identifying which mutations promote aggregation, we may be able to better understand why the disease develops and how future therapies could target these processes.
Using an ALS database and published literature, I identified a panel of common ALS patient mutations to study further. Based on what is known so far, mutations commonly associated with ALS should cause TDP-43 to leave the nucleus and aggregate more readily in the cytoplasm.
To investigate this, I designed several ALS-associated mutations in the laboratory, using a technique called site-directed mutagenesis, which is used to edit DNA sequences. The mutated gene is present in a plasmid, a small circular piece of DNA that acts as a delivery vehicle for introducing genes into cells. The plasmid contains a toolkit that helps the gene function inside cells and make the protein easier to study in the lab. One of these is a reporter enzyme called NanoLuc Luciferase, originally discovered in deep-sea shrimp. When the substrate furimazine is added, it glows. Cleverly, by attaching NanoLuc to TDP-43, the protein can theoretically be visualised under a specialised bioluminescence microscope.
Once the mutant plasmids were ready, I introduced them into human cell lines, including HEK293 and SH-SY5Y cells. Within 24–48 hours, the cells began producing the mutated TDP-43 protein, allowing me to observe how it behaves inside a living cell. Personally, this is the most exciting part of this project, looking down the microscope, not knowing whether the next image will show a perfectly normal cell or signs of protein aggregation.
In theory, this sounds like a straightforward process. However, science rarely works exactly as planned. In many ways, the beauty of research lies in adapting to unexpected challenges. So that is what I did.
When I tried to visualise TDP-43 using a bioluminescence microscope, spotting aggregates proved surprisingly difficult. The microscope's limited resolution meant that images appeared too blurry to confirm their presence. I was stuck wondering: are there truly no aggregates in the cell, or is the microscope not strong enough to visualise them?
To overcome this, I explored two troubleshooting strategies. Both involved shifting from bioluminescence to fluorescence, which required using a different type of microscope.
Firstly, I took advantage of the plasmid’s toolkit, specifically a small molecular label called a HA tag, which acts like a flag attached to the protein. Using a technique called immunohistochemistry, I added fluorescent antibodies that recognise this flag, allowing the protein to be visualised under a high-resolution confocal microscope. These microscopes scan fluorescent molecules with a focused laser, producing sharper images and clearer results.
Alongside this, I recreated the mutants in a different plasmid, which uses Green Fluorescent Protein (GFP) instead. Here, GFP is directly attached to TDP-43, and fluoresces green when exposed to specific wavelengths of light. This allows me to visualise the protein, under a confocal, without the need for antibody staining. By reducing the number of experimental steps and increasing imaging resolution, this approach improves the chances of identifying aggregates.
My early observations suggest that three out of twelve mutations may lead to the formation of small aggregates within the nucleus. This was unexpected, as we initially predicted that aggregates would form in the cytoplasm instead. However, this simply reflects the unpredictable nature of ALS pathology. Also, the remaining nine mutations still need to be analysed before we can rule out the presence of cytoplasmic aggregates entirely.
Overall, understanding this process is only one piece of the puzzle, but it may help provide a more rounded understanding of TDP-43 behaviour. Thus, helping scientists develop treatments that protect the neurones that allow us to move and communicate.
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