FUS on ALS Paper

8/2/2019 FUS on ALS Paper

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knowledge to work to find the physiological causes of the sporadic ALS. There isstill many more directions to go in the future, but success is being found currentlyin many neuroscience labs.

Discussion of the Three Positions

Analysis of FUS gene mutation in familial amyotrophic lateral sclerosis within anItalian cohort

In this 2009 study, an analysis was conducted on 94 Italian patients withFALS, whom did not show mutations in SOD1 and TDP-43. They found that fourspecific 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 acidalterations that could not be connected to any specific function but wereexplained to be highly evolutionarily conserved across several different animalclasses. This means that these genes are found in many different types ofanimals 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 notknow what that reason is. They are thought to affect the function of the FUSprotein somehow.

The other two mutations (R521C, R521G) are found in the C-terminus regionof the FUS gene, both leading to substitutions of an arginine. This is thought todisrupt the nuclear localization signal (NLS), causing FUS protein to beredistributed to the cytoplasm and aggregating. All of these alterations in aminoacid sequences were checked against a control population of 376 healthy Italianindividuals, to make sure that these were uncommon mutations. With the studyfinding five patients testing positively for FUS mutation out of 94 FALS patients, itwas determined that FUS is responsible for ~4% of the FALS cases, making itthe third most significant cause of ALS in Italy behind mutations of SOD1 andTDP-43.

One interesting note brought up was the difference in clinical symptoms ofpatients suffering from FALS and expressing mutant FUS. Instead of weaknessbeginning 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 butalso sporadic ALS, while taking a much larger sample size. Here they analyzedthe FUS gene of 1802 participants: 45 with FALS, 964 with SALS, and 793control subjects. These patients also did not have mutations of the genes SOD1and TDP-43, similar to the previous study. Here they found that 16 unrelatedpatients showed mutations in the C-Terminus (two at specifically R521C). Therewere 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


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study they suggested that the amino acid alteration also affects the nucleartransport in a similar fashion. What is significant about this finding is the effect onboth forms of ALS. This raises the question whether there is an environmentalfactor that can cause gene mutation or was the SALS patient really sporadic atall. 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 reallydistinguish between FALS and SALS accurately.

Looking at the other mutations found on exon 6, this region of the FUS geneis characteristically glycine-rich. The mutations come in both missense anddeletion of amino acids. This leads to deletion of glycine tracts of varying lengthfrom two to four in length. Most cases of SALS were found to be involved in thisglycine-rich area rather than the C-terminus, suggesting that perhaps thesechanges 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 arenot formally attributed to ALS because there is not enough evidence. There wasalso one mutation found to benign.

In this study, there also were unusual symptoms in the patients showing amutation at R521C. This leads researchers to wonder if a type of selectivedegeneration is occurring in the forms of ALS involving FUS mutations. Anotherassociation found with FUS mutations in ALS patients was that those testingpositively for mutant FUS tend to have an earlier onset of ALS. [2]

Nuclear Transport Impairment of Amyotrophic LateralSclerosis-LinkedMutations in FUS/TLS

In this 2011 study, researchers attempted to recreate the proposedmechanism that mutant FUS triggers to better understand the influence on ALS.They were able to confirm some hypotheses and disprove others, and still somequestions were left unanswered on the biochemistry of this gene. FUS mutantswere created and transfected into host cells. They were then subjected to variousimmunofluorescent tests. In the first tests, they found that 5-13% of cells withFUS mutants showed inclusion bodies (IBs) in their cytoplasm.

From these tests they were able to discover that FUS mutations were unlikelyto affect post-translational fragments, which is unlike the mutations of SOD1,TDP-43, and seipin. They also compared the formation of FUS IBs to theformation of aggresomes, which are characteristic of Huntingtons Disease andParkinsons 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 fromthe mutation to the degeneration. Nuclear import is regulated by an unknownnuclear transport factor and Ran GTPase. A disturbance of the nuclear transportis triggered by the ALS-linked mutations of FUS. The FUS mutants areredistributed and accumulate in the cytoplasm. Under stress or pathological


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conditions stress granules (SGs) form, interfering with RNA quality controlsystems. This is theoretically what leads to the motor neuron degeneration. [3]

Comparing the Different Positions

Reading through these studies chronologically shows how this field of

research has been developing over the past three years. In the first study, thefocus was solely familial ALS in Italy because it was a smaller population thatcould be tracked back in history, in an attempt to get rid of as many externalfactors as possible, even if it was just a few. The mechanism began asrecognizing the aggregates present in the cytoplasm. It is not until later in thethird study that the chemistry behind the aggregates is looked into at greatlength. The first study also brings up this idea of genes that are evolutionarilyhighly conserved, an idea that is revisited in the second study but with littlechange 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 anyconnection between the factors that cause either form of the disease. They wereable to find more mutations with the increased sample size, however, it was stillthe R521C mutation from the first study that maintained the only strongcorrelation, and it did establish a connection between FALS and SALS, even if itwas not significant. This study also opened research up to the involvement ofglycine in the mechanism leading to ALS. The influence of glycine is not focusedon in this study.

The third study did not even mention the influence of glycine on the chemistryof the mechanism. There was a statement that FUS is involved in multiplepathways of neurodegeneration, so in this particular study, glycine is not touchedon. This study differs from the other two in that it does not deal with humanpatients at all. Most of the research is performed in petri dishes. They are,however, very effective in narrowing down the functions of the mechanism ofALS-linked FUS mutation. They try to describe how the SGs can lead to theobserved change in morphology of the dendritic spines and the structure of thesynapse, 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 researchin a clear, intellectual way. I felt that I understood the writing through most of thetext, other than the machinery involved in the methods I was not personallyfamiliar with.

The method of research was effective in finding the inconsistencies in theFUS gene from the ALS patients to the controls. I also thought that since thisstudy focused on the familial form, the use of the family pedigrees helped toshow where the genes were coming from. However, the abundance of deceased


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relatives made it impossible to even attempt to compare blood samples. Also, thetables with the patient information had spaces reading N/A creating a lot ofinconclusive interpretation.

The conclusion that 4.4% of FALS patients are afflicted with FUS mutations istrue for this population, however, an increase in population size would strengththe conclusive argument. [1]

Corrado, L., Del Bo, R., Castellotti, B., et al.

This second article and the previous article had a few overlapping authors, sothe format and background information is very similar. So, I also found this studyto be very clear and did not have any trouble following or understanding the bulkof 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. Theyended up finding about the same percentage, further proving the numbers of thefirst study. Even though there was a good amount of overlap, the researchersmade an effort to create separation in the studies by not reusing the data of theFALS patients from the first study. They did, however, merge some data andnumbers from previous studies in one part of the discussion to give an evenlarger sample size. One confusing figure had a column labeled Diseaseduration, and in the text it stated that no further survival data follow-up wasavailable. That leads the reader to wonder if they died or what. Patient follow -upis crucial to a complete study.

The conclusion of this study was that the FUS gene was not only involved infamilial 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. Thereis some questioning, however, of whether the family history is really accurate andreliable 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 ableto follow the proposed mechanism without too much trouble.

I cannot say that I completely understand every detail of the methods, so Imnot 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 whatthey had done and understand their conclusions made. They underwent a lot ofdifferent procedures to prove or disprove many hypotheses that had been madeabout this particular mechanism, and they supported their conclusions with theirfindings in their figures. The figures were very helpful in making the text makesense. They did mention at the end of the text that there is a possibility that thetransfection of the FUS gene was the cause of the cytoplasmic IBs, rather thanthe dysfunction of the nuclear transport. This can be addressed in a future study.


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The conclusion of this study was the mutation of FUS linked to ALS causes adisruption in the nuclear transport, leading to aggregations, andneurodegeneration. They support this by showing how there is an increase inSGs in the cytoplasm, and they discuss the relationship between the nuclearlocalization signal and the C-terminal region. [3]

Future Research Directions

I think that a longitudinal study of both FALS and SALS patients would be agreat improvement on the first two studies. It would help to clear up whether thesporadic form is sometimes misdiagnosed. Also, more in-depth research on theglycine tract lengths that are characteristic of exons 5 and 6 could increase theunderstanding of one of the other pathways FUS influences.

The third study discusses using a Knock-In study to solve the problem of thetransfection in the original study. This method would be less traumatic on the cell.

Also, it seemed like all the research done looking at the cytoplasmaggregates was done in vitro. Im wondering whether this aspect has beeninvestigated through autopsies on deceased patients in physical human cells or ifit is only seen in a lab environment. Or could it be possible to take some cellbiopsy 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 infamilial 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 insporadic amyotrophic lateral sclerosis. J. Med. Genet 47:190-194, 2010.

[3] Ito, D., Seki, M., Tsunoda, Y., et al. Nuclear Transport Impairment ofAmyotrophic Lateral Sclerosis-Linked Mutations in FUS/TLS. Ann. Neurology69: 152-162, 2011.

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FUS on ALS Paper