This web page was produced as an assignment for Genetics 564, an undergraduate capstone course at UW-Madison.
Specific Aims
Amyotrophic lateral sclerosis (ALS) is a type of neurodegenerative disease characterized by motor neuron death, decreased muscle mass, and impaired movement [1]. Mutations in the gene FUS, which normally functions in DNA repair and splicing, lead to severe juvenile onset of ALS [2,3,4,5]. Altered FUS facilitates aggregate formation, which directly lead to neurodegenerative disease [5,6]. Although it is known that impaired DNA damage response mechanisms help facilitate FUS aggregates, it is unknown how defects in the DNA repair pathways lead to neuron defects [7,8].
The objective of this study is to determine how FUS-related DNA repair leads to proper motor neuron function. I hypothesize that FUS binding domains play a role in DNA repair mechanisms, due to their conserved nature and role in DNA binding. The long-term goal of this research is to determine how FUS-related DNA repair leads to neurodegeneration. To achieve this goal, Danio rerio will be used as a model due to homology, anatomical similarity, and simplistic motor movement screens [9].
Aim 1: Identify conserved amino acids of FUS critical for DNA repair and neurodegeneration
Approach: I will perform a protein alignment to generate CRISPR mutants in order to find conserved regions associated with DNA repair in FUS. To begin, I will align protein sequences via ClustalOmega to identify conserved amino acids in the RNA recognition motif, zinc-finger domain, and those that contain conserved pathogenic variants, like G187C2. I will then utilize CRISPR-Cas9 technology to induce specific mutations along those conserved regions. Sanger sequencing will then be used to confirm on target alterations. Next, I will screen for phenotypes showing defective motor movements. For those showing motor defects, I will measure the amount of single/double stranded breaks (SSBs/DSBs) via a comet assay when subjected to UV damage [6].
Hypothesis: I hypothesize that FUS mutations in the zinc finger domain will impact DNA repair and result in more SSBs/DSBs, due to zinc finger domains emerging roles in genome stability [10].
Rationale: Screening of D. rerio with these specific variants should result in a phenotype with increased SSBs/DSBs, which would define DNA repair binding regions important for genome stability and neuronal function.
Aim 2: Identify small molecules that rescue FUS mutant phenotype
Approach: I will perform a chemical screen by using a known CRISPR-Cas9 mutant, R521C, that shows increased SSBs/DSBs and motor movement impairments [9]. To begin, I will utilize a chemical library of small molecules that regulate DNA repair proteins involved with SSBs/DSBs, like those in homology directed repair and base excision repair [5,11]. I will perform a motor movement screen and comet assay in order to identify small molecules capable of restoring proper motor neuron function and DNA repair.
Hypothesis: Small molecules associated with the downregulation of proteins that terminate repair complexes will rescue the FUS mutant phenotype, due to increased retention time at SSBs/DSBs sites.
Rationale: By identifying small molecules that restore DNA repair and motor neuron function, FDA approved cancer drugs that target DNA repair pathways could easily be transitioned to ALS patients [12].
Aim 3: Identify new FUS protein-protein interactions in DNA repair
Approach: Utilizing lysed neuronal cells from D. rerio, I will subject samples to both wild type and mutant FUS baits (R521C). Tandem affinity purification and mass spectrometry will then be used to purify and obtain MS/MS data. Computer software would provide sequence coverage of these captured protein interactions. To confirm these interactions are legitimate, CRISPR-Cas9 will be used to make knockouts of potential FUS binding sites determined by BLAST. A comet assay and motor movement screen will identify DNA repair proteins as those with increased SSBs/DSBs and possible impacted neurodegeneration. Proteins will be sorted by GO terms, which would elucidate a more extensive protein interaction network of FUS in DNA repair.
Hypothesis: TAP-MS will provide new FUS interactions associated with DNA repair in D. rerio due to the presence of high levels of SSBs/DSBs commonly found in FUS mutants.
Rationale: Currently, databases like String show limited DNA repair protein interactions with FUS in D. rerio [14]. Eukaryotic DNA repair pathways are conserved and this data could suggest similar mechanisms in humans [13].
Through these approaches, the role that FUS plays in DNA repair and motor neuron function can be identified. As of today, there are no long term treatments that improve motor neuron function for FUS-ALS patients. By understanding targets of FUS in the DNA repair pathways, current FDA approved cancer drugs could easily be administered to those with FUS-ALS. This would not only help restore genomic stability, but also provide the potential for restored neuron function in patients. Ultimately, this understanding would highlight how DNA repair defects lead to neurodegeneration.
References:
The objective of this study is to determine how FUS-related DNA repair leads to proper motor neuron function. I hypothesize that FUS binding domains play a role in DNA repair mechanisms, due to their conserved nature and role in DNA binding. The long-term goal of this research is to determine how FUS-related DNA repair leads to neurodegeneration. To achieve this goal, Danio rerio will be used as a model due to homology, anatomical similarity, and simplistic motor movement screens [9].
Aim 1: Identify conserved amino acids of FUS critical for DNA repair and neurodegeneration
Approach: I will perform a protein alignment to generate CRISPR mutants in order to find conserved regions associated with DNA repair in FUS. To begin, I will align protein sequences via ClustalOmega to identify conserved amino acids in the RNA recognition motif, zinc-finger domain, and those that contain conserved pathogenic variants, like G187C2. I will then utilize CRISPR-Cas9 technology to induce specific mutations along those conserved regions. Sanger sequencing will then be used to confirm on target alterations. Next, I will screen for phenotypes showing defective motor movements. For those showing motor defects, I will measure the amount of single/double stranded breaks (SSBs/DSBs) via a comet assay when subjected to UV damage [6].
Hypothesis: I hypothesize that FUS mutations in the zinc finger domain will impact DNA repair and result in more SSBs/DSBs, due to zinc finger domains emerging roles in genome stability [10].
Rationale: Screening of D. rerio with these specific variants should result in a phenotype with increased SSBs/DSBs, which would define DNA repair binding regions important for genome stability and neuronal function.
Aim 2: Identify small molecules that rescue FUS mutant phenotype
Approach: I will perform a chemical screen by using a known CRISPR-Cas9 mutant, R521C, that shows increased SSBs/DSBs and motor movement impairments [9]. To begin, I will utilize a chemical library of small molecules that regulate DNA repair proteins involved with SSBs/DSBs, like those in homology directed repair and base excision repair [5,11]. I will perform a motor movement screen and comet assay in order to identify small molecules capable of restoring proper motor neuron function and DNA repair.
Hypothesis: Small molecules associated with the downregulation of proteins that terminate repair complexes will rescue the FUS mutant phenotype, due to increased retention time at SSBs/DSBs sites.
Rationale: By identifying small molecules that restore DNA repair and motor neuron function, FDA approved cancer drugs that target DNA repair pathways could easily be transitioned to ALS patients [12].
Aim 3: Identify new FUS protein-protein interactions in DNA repair
Approach: Utilizing lysed neuronal cells from D. rerio, I will subject samples to both wild type and mutant FUS baits (R521C). Tandem affinity purification and mass spectrometry will then be used to purify and obtain MS/MS data. Computer software would provide sequence coverage of these captured protein interactions. To confirm these interactions are legitimate, CRISPR-Cas9 will be used to make knockouts of potential FUS binding sites determined by BLAST. A comet assay and motor movement screen will identify DNA repair proteins as those with increased SSBs/DSBs and possible impacted neurodegeneration. Proteins will be sorted by GO terms, which would elucidate a more extensive protein interaction network of FUS in DNA repair.
Hypothesis: TAP-MS will provide new FUS interactions associated with DNA repair in D. rerio due to the presence of high levels of SSBs/DSBs commonly found in FUS mutants.
Rationale: Currently, databases like String show limited DNA repair protein interactions with FUS in D. rerio [14]. Eukaryotic DNA repair pathways are conserved and this data could suggest similar mechanisms in humans [13].
Through these approaches, the role that FUS plays in DNA repair and motor neuron function can be identified. As of today, there are no long term treatments that improve motor neuron function for FUS-ALS patients. By understanding targets of FUS in the DNA repair pathways, current FDA approved cancer drugs could easily be administered to those with FUS-ALS. This would not only help restore genomic stability, but also provide the potential for restored neuron function in patients. Ultimately, this understanding would highlight how DNA repair defects lead to neurodegeneration.
References:
- Shang, Y. & Huang E.J. (2016, September). Mechanisms of FUS mutations in familial amyotrophic lateral sclerosis. Brain Research 1647:65-78.
- Zou, Z.Y., Liu, M.S., Li, X.G., Cui, L.Y. (2015, September). Mutations in SOD1 and FUS caused juvenile-onset sporadic amyotrophic lateral sclerosis with aggressive progression. Ann Translation Medicine 3(15):221
- Conte, A., Lattante, S., et al. (2012, January). P525L FUS mutation is consistently associated with a severe form of juvenile Amyotrophic Lateral Sclerosis. Neurology Genetics 2:63
- Zhou, Y., Liu, S., et al. (2013, October). ALS-associated FUS mutations result in compromised FUS alternative splicing and autoregulation. Nature Communications 9:3683
- Wang, H., Guo, W., et. Al. (2018, September). Mutant FUS causes DNA ligation defects to inhibit oxidative damage repair in Amyotrophic Lateral Sclerosis. Nature Communications 9:3683
- Naumann, M., Pal, A., et al. (2018, January). Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation. Nature Communications 9:335
- Penndorf, D., Witte, O., et al. (2018, February). DNA plasticity and damage in amyotrophic lateral sclerosis. Neural Regeneration Research 3(2): 173–180.
- McGown, A., McDearmid, J.R., et al. (2012, October). Early interneuron dysfunction in ALS: Insights from a mutant sod1 zebrafish. Annals of Neurobiology 73(2):246-258.
- Qiu H., Lee S., Shang Y., Wang WY., et al. (2014, March). ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects. J Clin Invest 124(3):981-99
- Vilas C.K., Emery L.E., et. al. (2018, January). Caught with One’s Zinc Fingers in the Genomce Integrity Cookie Jar. Trends Genetics 34(4):313-325.
- Naumann M, Pal A, Goswami A, Lojewski X, et. al. (2018, January). Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation. Nature Communications 9(1):335.
- Dance, A. Targeting FUS: DNA Damage Control in ALS. The ALS Research Form. Retrieved on May 2, 2019 from https://www.alsresearchforum.org/targeting-fus-dna-damage-control-in-als/
- Taylor E.M., Lehmann, A.R. (1998, September). Conservation of eukayotic DNA repair mechanisms. Int J Radiat Biol 74(3):277-86.
- SMART. Retrieved on 4/11/2019 from https://version11.string-db.org/cgi/input.pl?sessionId=Odz7uTVCbz7d&input_page_show_search=on
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