Seeking a cure for ALS


A Fortuitous Discovery

Back in 1995, in the midst of the HIV epidemic, research from Dr Sai-Hong Ou at the University of Texas Southwestern discovered a protein called ‘TDP-43’ that could bind and repress the expression of the HIV-1 virus gene. At the time the discovery of this protein was paid very little attention to by scientists outside of the HIV field, and it wasn’t until 11 years later that it was found to play a crucial role in a devastating condition called Motor neuron disease (MND).

Since then, TDP-43 has been found to be abnormally present in a staggering 90-95% of patients with MND, and evidence demonstrating its role in the disease is increasing exponentially. To further investigate the role of TDP-43 in MND, I have recently published some work examining similarities that the TDP-43 protein and MND have in common with another devastating and fatal disease called ‘Prion disease’.

ALS – A Condition With No Cure

My publication reports some of the findings from my PhD based on a condition called Amyotrophic Lateral Sclerosis (ALS). ALS is the most common form of MND, and is a fatal incurable neurodegenerative disease which kills patients’ motor neurons leading to muscle weakness and paralysis and eventually death of the patient afflicted.

A small percentage of patients with ALS have a genetic cause of the disease; however, most of these cases have no known cause, which are known as sporadic. Therefore we focused our efforts to investigate what could be one of the causes of these sporadic cases.

This hypothesis was inspired by a landmark discovery from Manuela Neumann and colleagues in 2006, who discovered that this particular protein was found abnormally clumped in the affected brain and spinal cord of 90-95% of patients with ALS.

This discovery and later findings that mutations in the TDP-43 gene can cause ALS, profoundly changed the face of ALS research for good. Many researchers are now investigating the wide ranging functions of this protein and how it may be involved with ALS.

Inspiration From Further Afield

Hypotheses in science are more often than not inspired by different areas within the field and making new connections from these different areas based on previous observations. Indeed, our hypothesis – that a misfolded protein may cause a cell to die – has a firm basis in another devastating brain disease called ‘prion disease’. This is an infectious disease caused by a misfolded prion protein that affects mammals such as sheep, goats, elk, cows and also humans. Prion disease may not be a household name but it’s a condition that is commonly recognised from the ‘mad cow’ disease epidemic in the UK between 1986 and 1998.

The idea that a misfolded protein can act as an infectious entity (such as a virus or bacteria) is unusual, as misfolded proteins do not possess the usual machinery (DNA and RNA) to replicate themselves. However, in prion disease misfolded proteins do act in this way, corrupting healthy proteins and causing them to misfold.

The method prions use to replicate themselves is by acting as a template and converting the normal form of the protein into an abnormal misfolded form, which then results in the deposition of clumps of misfolded prion protein. This can then spread in the brain tissue causing cell death by as yet unknown mechanisms.

ALS and Prion Disease – How are they linked?

ALS and Prion disease are clinically very different diseases but they do share a few remarkable similarities. The main one being the unique spread and progression of cell death from cell to cell, which can be seen when observing the spread of symptoms in ALS patients. In addition to this both the abnormal TDP-43 and prion protein are found as abnormal misfolded clumps in neurons. Finally, the TDP-43 protein has also been demonstrated to have similarities in the protein sequence that allow it to behave in a similar manner to a prion protein. Therefore, although there is no evidence that abnormal TDP-43 is infective, the deposition of misfolded clumps of TDP-43 suggests the idea that this protein may actually be working in a similar fashion to misfolded prion protein.

Investigating The Hypothesis

To test this idea we decided to use the abnormal TDP-43 from some donated ALS patient brain and spinal cord tissue, and then reproduce this clumping of abnormal TDP-43 in a cell model we could study.

This was by no means an easy process and it took a whole year of tinkering and adjusting protocols until finally we came upon the correct solution. The technique we devised was to extract and purify the TDP-43 from these ALS samples and add it to cells producing large amounts of normal TDP-43 protein.

Once we had perfected this method, we found that this abnormal form of the TDP-43 protein could convert the normal form into an abnormal form and produce clumps of abnormal TDP-43 in a similar manner to a prion protein. Remarkably, we also observed that these clumps of misfolded TDP-43 protein formed identical shapes to the clumps of TDP-43 protein observed in patients with ALS.

It is known that prion protein replication can adapt and speed up; producing larger amounts of prion protein clumps, so our next step was to demonstrate this behaviour with abnormal TDP-43. We did this by adding extracts of cells containing abnormal TDP-43 into new cells producing large amounts of normal TDP-43, and observed that the introduction of abnormal TDP-43 did indeed result in significantly larger amounts of clumped TDP-43 protein than the first time round.

Another key characteristic of prion disease that we wanted to investigate is that the abnormal prion protein can spread from one cell to another. By mixing normal cells containing an identifiable fluorescent marker with cells containing abnormal TDP-43, we were able to observe that over time some of the normal cells eventually contained abnormal clumps of TDP-43, indicating that some of these clumps of TDP-43 had indeed spread to another cell.

Our findings so far had been incredibly illuminating. The only downside was that the cell line we were using was a human kidney cell line, which does not have much physiological relevance to motor neuron disease.

To add weight to our discoveries we wanted to demonstrate this clumping of TDP-43 in a physiologically relevant cell line to see if this process caused cell damage. We did this by using a cell line that displays many properties of motor neurons called NSC-34, and replicating the above process. As a result of this we observed that this TDP-43 clumping process causes significant damage to the cells.

Even though these clumps of misfolded protein are appearing in cells, many researchers have evidence from other misfolded proteins in other neurodegenerative diseases demonstrating that it is not the presence of these clumps themselves that kill the cells, but some smaller clumps of protein that form during the clumping process. We therefore successfully demonstrated that these smaller clumps of abnormal TDP-43 are indeed forming in our cells, and therefore may be responsible for damaging or killing the cells.

Publishing these findings – The tricky part

We were thrilled with the success of our investigation but we couldn’t celebrate just yet. For science researchers, making discoveries is only the beginning. Before your findings are accepted by the scientific community they must be rigorously tested by other scientists from your specialist field.

The process, known as ‘peer review’, involves collecting, analysing and writing up all this data and sending it to an appropriate journal where other scientists review and scrutinise your findings. This process is essential to ensure the validity and quality of the data, determining whether it can prove or disprove the original hypothesis. If you can pass this very strict, thorough and gruelling test of your work, then your research can be published.

The collection of the results for this paper was a very long and arduous process, which took many long evenings and weekends in the lab collecting and analysing results. As is often the case in science, I was also in competition with various other groups working on similar topics in a race to publish these findings. So, after a few amendments and resubmissions of this data – and numerous days and hours waiting with baited breath – it was finally accepted in the journal ‘Neurobiology of Disease’. The subsequent feeling of elation and relief upon the acceptance of the work was extraordinary, and resulted in a few celebrations on my behalf.

The future

Publishing is essential in science in order to build up your scientific CV and future career. However, more importantly, publishing is about communicating your discovery to the world and hopefully inspiring the next line of enquiry. In this case, any further lines of enquiry will hopefully build on ideas for developing therapies and treatments for this devastating disorder. This paper was my small piece in the large and complicated puzzle that is ALS, and by using these smaller pieces we can potentially build a more complete picture of this disease in the future.

Phillip Smethurst is a Postdoctoral researcher at the Patani Lab, UCL.

You can watch a video about Phil’s research and findings over on the Patani Stem Cell Lab YouTube channel: WATCH THE VIDEO