The Influence of Mutations of FUS on Amyotrophic Lateral Sclerosis Brooke Gushen Biology 470: Neuroscience II Byron Jones The

Problem This is critical analysis of three different scientific research articles discussing the role of the FUS gene in Amyotrophic Lateral Sclerosis (ALS). ALS is an adult-onset, rapidly progressive and ultimately fatal neurodegenerative disorder characterised by degeneration of upper and lower motor neurons. This leads to weakness, muscular atrophy and spasticity, which relentlessly progress to complete paralysis with a low survival rate beyond 5 years from symptom onset. [2] ALS can be either familial (FALS), which is ~10% of cases, or sporatic (SALS), which is ~90% of cases. Although few discoveries have been made for SALS, several genes including SOD1, TDP-43, and FUS have been correlated with FALS. [1] The FUS gene, in particular, is a hot topic in the current world of neuroscience. Mutations of the FUS protein have been linked to about 4.4% of familial ALS. [2] Many experimental research labs have taken on the project of narrowing down which part of the genome is being affected, and others are working on discovering the actual mechanism from start to finish. The labs working on the genome have found that specific exons of the FUS gene are responsible for shuttling RNA from the nucleus to the cytoplasm. When mutations occur, the FUS protein, which is supposed to be housed in the nucleus, ends up in the cytoplasm, forming aggregations of the protein. [1] Further research was done proposing a mechanism explaining why this leads to neurodegeneration. They are hypothesizing that when the FUS protein is redistributed to the cytoplasm, an overexpression of mutant FUS is detected by the cell. This triggers a stress response, leading to the formation of stress granules (SGs). It is possible that this disturbs mRNA transport, thus further disturbing spine maintenance of the neuron. This could possibly lead to motor neuron degeneration. [3] The Significance This is significant because it can be seen that neural atrophy quickly leads to muscular atrophy. ALS is a disease that progresses rapidly, and usually by the time the symptoms begin to surface, the damage to the nervous and muscular system is already irreversible. Research being done in this field is important because the causes are completely unknown. It is like putting together the pieces of a puzzle without knowing what the big picture looks like. A few links are being made with hereditary genes, and researchers hope that they can use that

knowledge to work to find the physiological causes of the sporadic ALS. There is still many more directions to go in the future, but success is being found currently in many neuroscience labs. Discussion of the Three Positions Analysis of FUS gene mutation in familial amyotrophic lateral sclerosis within an Italian cohort In this 2009 study, an analysis was conducted on 94 Italian patients with FALS, whom did not show mutations in SOD1 and TDP-43. They found that four specific missense mutations were found in five patients out of the 94 on exons 5, 6, 14, and 15. Two of the mutations (G156E, R234L) caused amino acid alterations that could not be connected to any specific function but were explained to be highly evolutionarily conserved across several different animal classes. This means that these genes are found in many different types of animals that evolved apart many years ago, therefore the genes have been around for a while and probably for a good reason, even if science does not know what that reason is. They are thought to affect the function of the FUS protein somehow. The other two mutations (R521C, R521G) are found in the C-terminus region of the FUS gene, both leading to substitutions of an arginine. This is thought to disrupt the nuclear localization signal (NLS), causing FUS protein to be redistributed to the cytoplasm and aggregating. All of these alterations in amino acid sequences were checked against a control population of 376 healthy Italian individuals, to make sure that these were uncommon mutations. With the study finding five patients testing positively for FUS mutation out of 94 FALS patients, it was determined that FUS is responsible for ~4% of the FALS cases, making it the third most significant cause of ALS in Italy behind mutations of SOD1 and TDP-43. One interesting note brought up was the difference in clinical symptoms of patients suffering from FALS and expressing mutant FUS. Instead of weakness beginning in the extremities of a single limb, the patients showed symmetric, proximal, and axial weakness at onset. [1] Mutations of FUS gene in sporadic amyotrophic lateral sclerosis In this 2010 study, the researchers decided to look not only at familial ALS but also sporadic ALS, while taking a much larger sample size. Here they analyzed the FUS gene of 1802 participants: 45 with FALS, 964 with SALS, and 793 control subjects. These patients also did not have mutations of the genes SOD1 and TDP-43, similar to the previous study. Here they found that 16 unrelated patients showed mutations in the C-Terminus (two at specifically R521C). There were also six other mutations, each only affecting one or two patients. The R521C mutation was found in both a FALS patient and a SALS patient. This was one of the specific mutations identified in the previous study. In this

study they suggested that the amino acid alteration also affects the nuclear transport in a similar fashion. What is significant about this finding is the effect on both forms of ALS. This raises the question whether there is an environmental factor that can cause gene mutation or was the SALS patient really sporadic at all. It is possible that the older generations had ALS but died of another cause before a diagnosis was made. The lack of family history makes it difficult to really distinguish between FALS and SALS accurately. Looking at the other mutations found on exon 6, this region of the FUS gene is characteristically glycine-rich. The mutations come in both missense and deletion of amino acids. This leads to deletion of glycine tracts of varying length from two to four in length. Most cases of SALS were found to be involved in this glycine-rich area rather than the C-terminus, suggesting that perhaps these changes show low penetrance FUS variations, hence why it is seen as sporadic. Although there does seem to be a connection to glycine here, these changes are not formally attributed to ALS because there is not enough evidence. There was also one mutation found to benign. In this study, there also were unusual symptoms in the patients showing a mutation at R521C. This leads researchers to wonder if a type of selective degeneration is occurring in the forms of ALS involving FUS mutations. Another association found with FUS mutations in ALS patients was that those testing positively for mutant FUS tend to have an earlier onset of ALS. [2] Nuclear Transport Impairment of Amyotrophic Lateral Sclerosis-Linked Mutations in FUS/TLS In this 2011 study, researchers attempted to recreate the proposed mechanism that mutant FUS triggers to better understand the influence on ALS. They were able to confirm some hypotheses and disprove others, and still some questions were left unanswered on the biochemistry of this gene. FUS mutants were created and transfected into host cells. They were then subjected to various immunofluorescent tests. In the first tests, they found that 5-13% of cells with FUS mutants showed inclusion bodies (IBs) in their cytoplasm. From these tests they were able to discover that FUS mutations were unlikely to affect post-translational fragments, which is unlike the mutations of SOD1, TDP-43, and seipin. They also compared the formation of FUS IBs to the formation of aggresomes, which are characteristic of Huntingtons Disease and Parkinsons Disease, and found no correlation in the mechanisms. There were also questions of ubiquitination being a cause in the pathology of ALS; however, ubiquitin was not detected in the FUS IBs. This lab did put together a proposed mechanism that works step-by-step from the mutation to the degeneration. Nuclear import is regulated by an unknown nuclear transport factor and Ran GTPase. A disturbance of the nuclear transport is triggered by the ALS-linked mutations of FUS. The FUS mutants are redistributed and accumulate in the cytoplasm. Under stress or pathological

conditions stress granules (SGs) form, interfering with RNA quality control systems. This is theoretically what leads to the motor neuron degeneration. Comparing the Different Positions

[3]

Reading through these studies chronologically shows how this field of research has been developing over the past three years. In the first study, the focus was solely familial ALS in Italy because it was a smaller population that could be tracked back in history, in an attempt to get rid of as many external factors as possible, even if it was just a few. The mechanism began as recognizing the aggregates present in the cytoplasm. It is not until later in the third study that the chemistry behind the aggregates is looked into at great length. The first study also brings up this idea of genes that are evolutionarily highly conserved, an idea that is revisited in the second study but with little change in understanding. With similar software and means of DNA analysis, the second study takes the first a step farther and includes sporadic ALS patients in an attempt to make any connection between the factors that cause either form of the disease. They were able to find more mutations with the increased sample size, however, it was still the R521C mutation from the first study that maintained the only strong correlation, and it did establish a connection between FALS and SALS, even if it was not significant. This study also opened research up to the involvement of glycine in the mechanism leading to ALS. The influence of glycine is not focused on in this study. The third study did not even mention the influence of glycine on the chemistry of the mechanism. There was a statement that FUS is involved in multiple pathways of neurodegeneration, so in this particular study, glycine is not touched on. This study differs from the other two in that it does not deal with human patients at all. Most of the research is performed in petri dishes. They are, however, very effective in narrowing down the functions of the mechanism of ALS-linked FUS mutation. They try to describe how the SGs can lead to the observed change in morphology of the dendritic spines and the structure of the synapse, ultimately causing the motor neuron degeneration. Critical Evaluations of the Authors Ticozzi, N., Silani, V., LeClerc, A.L., et al The authors of this first article are very successful in conveying their research in a clear, intellectual way. I felt that I understood the writing through most of the text, other than the machinery involved in the methods I was not personally familiar with. The method of research was effective in finding the inconsistencies in the FUS gene from the ALS patients to the controls. I also thought that since this study focused on the familial form, the use of the family pedigrees helped to show where the genes were coming from. However, the abundance of deceased

relatives made it impossible to even attempt to compare blood samples. Also, the tables with the patient information had spaces reading N/A creating a lot of inconclusive interpretation. The conclusion that 4.4% of FALS patients are afflicted with FUS mutations is true for this population, however, an increase in population size would strength the conclusive argument. [1] Corrado, L., Del Bo, R., Castellotti, B., et al. This second article and the previous article had a few overlapping authors, so the format and background information is very similar. So, I also found this study to be very clear and did not have any trouble following or understanding the bulk of the text. The method of research for this study was very similar to the previous study, but they did improve, in that, they increased the sample size dramatically. They ended up finding about the same percentage, further proving the numbers of the first study. Even though there was a good amount of overlap, the researchers made an effort to create separation in the studies by not reusing the data of the FALS patients from the first study. They did, however, merge some data and numbers from previous studies in one part of the discussion to give an even larger sample size. One confusing figure had a column labeled Disease duration, and in the text it stated that no further survival data follow-up was available. That leads the reader to wonder if they died or what. Patient follow-up is crucial to a complete study. The conclusion of this study was that the FUS gene was not only involved in familial ALS but also sporadic ALS. This was supported by the findings that two patients with no family history of ALS showed mutations of the FUS gene. There is some questioning, however, of whether the family history is really accurate and reliable without actual blood samples to compare against. [2] Ito, D., Seki, M., Tsunoda, Y., et al. Although the methods and language of this last article was pretty complex, the general results and discussion was very effectively communicated. I was able to follow the proposed mechanism without too much trouble. I cannot say that I completely understand every detail of the methods, so Im not sure if they went about their experimenting in the most effective way. But following the figures with the immunofluorescent stains, I was able to see what they had done and understand their conclusions made. They underwent a lot of different procedures to prove or disprove many hypotheses that had been made about this particular mechanism, and they supported their conclusions with their findings in their figures. The figures were very helpful in making the text make sense. They did mention at the end of the text that there is a possibility that the transfection of the FUS gene was the cause of the cytoplasmic IBs, rather than the dysfunction of the nuclear transport. This can be addressed in a future study.

The conclusion of this study was the mutation of FUS linked to ALS causes a disruption in the nuclear transport, leading to aggregations, and neurodegeneration. They support this by showing how there is an increase in SGs in the cytoplasm, and they discuss the relationship between the nuclear localization signal and the C-terminal region. [3] Future Research Directions I think that a longitudinal study of both FALS and SALS patients would be a great improvement on the first two studies. It would help to clear up whether the sporadic form is sometimes misdiagnosed. Also, more in-depth research on the glycine tract lengths that are characteristic of exons 5 and 6 could increase the understanding of one of the other pathways FUS influences. The third study discusses using a Knock-In study to solve the problem of the transfection in the original study. This method would be less traumatic on the cell. Also, it seemed like all the research done looking at the cytoplasm aggregates was done in vitro. Im wondering whether this aspect has been investigated through autopsies on deceased patients in physical human cells or if it is only seen in a lab environment. Or could it be possible to take some cell biopsy in a living patient to see if these aggregates actually exist in vivo. References [1] Ticozzi, N., Silani, V., LeClerc, A.L., et al. Analysis of FUS gene mutation in familial amyotrophic lateral sclerosis within an Italian cohort. Neurology 73: 1180-1185, 2009. [2] Corrado, L., Del Bo, R., Castellotti, B., et al. Mutations of FUS gene in sporadic amyotrophic lateral sclerosis. J. Med. Genet 47:190-194, 2010. [3] Ito, D., Seki, M., Tsunoda, Y., et al. Nuclear Transport Impairment of Amyotrophic Lateral Sclerosis-Linked Mutations in FUS/TLS. Ann. Neurology 69: 152-162, 2011.