This web page was produced as an assignment for Genetics 564, an undergraduate capstone course at UW-Madison.
Hereditary Spastic Paraplegia and KIF5A [1,2,3,5]
Hereditary Spastic Paraplegia (HPS) is a rare group of neurological disorders that is typically associated with progressive weakness and extreme spasticity of the lower extremities. These symptoms are a result of axonal degeneration that occurs in the lower motor neurons. Upon onset of HSP, patients typically experience leg stiffness that slowly progresses into a type of paralysis known as paraplegia. Pure HSP features these key symptoms while the complicated forms are accompanied by additional symptoms.
At this point, there are 70 different types of HSP resulting from various genes that are mutated. The pattern of inheritance for HSP varies form to form, either being autosomal dominant, autosomal recessive, or X-linked recessive. One type of autosomal dominant HSP is known as SPG10 which is due to mutations in the KIF5A gene.
At this point, there are 70 different types of HSP resulting from various genes that are mutated. The pattern of inheritance for HSP varies form to form, either being autosomal dominant, autosomal recessive, or X-linked recessive. One type of autosomal dominant HSP is known as SPG10 which is due to mutations in the KIF5A gene.
KIF5A: An Organelle Transport Protein [9,7]
KIF5A has one major domain known as the kinesin motor domain located between 7aa-333aa. This domain is a force producing region that plays a role in organelle transport. The domain has two separate binding sites: one for the microtubule and one for ATP. ATP hydrolysis at the kinesin motor domain results in mechanical work. This domain is well conserved amongst its homologs, indicating the value this region has to the functionality of the protein.
This protein has been found to carry mainly neurofilaments as well as organelles such as mitochondria. This gene is normally expressed in the cytoplasm of the neuronal body and then localizes to the end of the axon. In the case of HSP, the mutated KIF5A fails to localize and transport organelles properly resulting degradation of the axon.
KIF5A has one major domain known as the kinesin motor domain located between 7aa-333aa. This domain is a force producing region that plays a role in organelle transport. The domain has two separate binding sites: one for the microtubule and one for ATP. ATP hydrolysis at the kinesin motor domain results in mechanical work. This domain is well conserved amongst its homologs, indicating the value this region has to the functionality of the protein.
This protein has been found to carry mainly neurofilaments as well as organelles such as mitochondria. This gene is normally expressed in the cytoplasm of the neuronal body and then localizes to the end of the axon. In the case of HSP, the mutated KIF5A fails to localize and transport organelles properly resulting degradation of the axon.
Gap in Knowledge
When KIF5A is mutated, the protein fails to localize to the ends of the axons in lower motor neurons resulting in axonal degradation. It is believed that this degradation is what leads to the paralysis and spasticity in the lower extremities. Upon examining the protein interaction network for KIF5A, it has been observed that there are indirect associations with the Hox10 protein (also known as VSX2). Hox10 is a posterior body organizational protein that helps direct the localization and expression of different proteins to the posterior. This observation made then brought up the question of how might other protein interactions and modifications influence the localizing of KIF5A. Although it is known that KIF5A normally localizes to the extremities, it is unknown what influences this localization and whether mutations within this gene prevent this movement resulting in lower limb spasticity.
When KIF5A is mutated, the protein fails to localize to the ends of the axons in lower motor neurons resulting in axonal degradation. It is believed that this degradation is what leads to the paralysis and spasticity in the lower extremities. Upon examining the protein interaction network for KIF5A, it has been observed that there are indirect associations with the Hox10 protein (also known as VSX2). Hox10 is a posterior body organizational protein that helps direct the localization and expression of different proteins to the posterior. This observation made then brought up the question of how might other protein interactions and modifications influence the localizing of KIF5A. Although it is known that KIF5A normally localizes to the extremities, it is unknown what influences this localization and whether mutations within this gene prevent this movement resulting in lower limb spasticity.
Aims and Hypothesis
The objective of this study is to elucidate how KIF5A localizes to the lower extremities leading to proper neuron function. I hypothesis that both phosphorylation and certain protein interactions influence the localization of KIF5A to the axons of the lower extremities. The long term goal is to understand how KIF5A mutations leads to specific neurodegenerative effects of the lower extremities.
The objective of this study is to elucidate how KIF5A localizes to the lower extremities leading to proper neuron function. I hypothesis that both phosphorylation and certain protein interactions influence the localization of KIF5A to the axons of the lower extremities. The long term goal is to understand how KIF5A mutations leads to specific neurodegenerative effects of the lower extremities.
Model Organisms [10]
Danio rerio would be an ideal model organism for this project. Not only do danio rerio, also known as zebrafish, have a very conserved KIF5A protein but they are also ideal to use in neruodegenerative studies because of how similar their nervous system is to the human nervous system. In addition, it is very easy to observe changes in their behaviors and motor function that reflect motor neuron degeneration.
Danio rerio would be an ideal model organism for this project. Not only do danio rerio, also known as zebrafish, have a very conserved KIF5A protein but they are also ideal to use in neruodegenerative studies because of how similar their nervous system is to the human nervous system. In addition, it is very easy to observe changes in their behaviors and motor function that reflect motor neuron degeneration.
Aim 1: Find conserved amino acids of KIF5A that are associated with phosphorylation sites and see whether they have a role in KIF5A localization.
I will begin by using BLAST to find homologs for KIF5A and then Clustal Omega to perform sequence alignment. Additionally I will use NetPhos to view all the predicted phosphorylation sites and reference these to the conserved regions identified by ClustalOmega. I will then use CRISPR to create several mutants in zebrafish: S207A, S509A, S205A and S989A. I will then perform fluorescent tagging assay on all the mutants in addition to a positive control zebrafish, where there are no mutations, and a negative control, a zebrafish where KIF5A is completely knocked out. Using the results, I will be able to see where KIF5A and its altered counterparts localize within zebrafish. Additionally I will perform a phenotypic screen for spasticity with all zebrafish strains using high-throughput imaging. I hypothesis that mutations in phosphorylation sites in the kinesin motor domain (7aa-335aa) will impact KIF5A localization to various degrees, dependent on the mutation. The kinesin motor domain is the region in which KIF5A binds to microtubules (MT) for transportation, mutating this region could impact how far KIF5A travel along the MT or whether it binds at all without phosphorylation occurring.
I will begin by using BLAST to find homologs for KIF5A and then Clustal Omega to perform sequence alignment. Additionally I will use NetPhos to view all the predicted phosphorylation sites and reference these to the conserved regions identified by ClustalOmega. I will then use CRISPR to create several mutants in zebrafish: S207A, S509A, S205A and S989A. I will then perform fluorescent tagging assay on all the mutants in addition to a positive control zebrafish, where there are no mutations, and a negative control, a zebrafish where KIF5A is completely knocked out. Using the results, I will be able to see where KIF5A and its altered counterparts localize within zebrafish. Additionally I will perform a phenotypic screen for spasticity with all zebrafish strains using high-throughput imaging. I hypothesis that mutations in phosphorylation sites in the kinesin motor domain (7aa-335aa) will impact KIF5A localization to various degrees, dependent on the mutation. The kinesin motor domain is the region in which KIF5A binds to microtubules (MT) for transportation, mutating this region could impact how far KIF5A travel along the MT or whether it binds at all without phosphorylation occurring.
Aim 2: Perform a chemical screen in order to rescue mutated zebrafish and restore proper localization of KIF5A.
I will perform a chemical screen using the same experimental and control zebrafish from Aim 1 with the addition of a zebrafish with a known mutation that results in HSP (610C>T). I will utilize a diverse chemical library to perform the screen so I can I identify new small molecule protein interactions. I will then introduce the small molecules to each strain of zebrafish and observe for any changes that may occur and if any of the mutated strains of zebrafish with spasticity have recovered. I will then fluorescently tag KIF5A once again and observe its localization with each mutant and recovered mutant. I hypothesis that I will see a recovered phenotype in S207A, S205A, and the zebrafish with the known HSP mutation. Fluorescence assay will show that zebrafish with the recovered phenotype will have proper localization of KIF5A again. If the zebrafish do not have spasticity in their extremities then one of the small molecules in the library have help with proper localization.
I will perform a chemical screen using the same experimental and control zebrafish from Aim 1 with the addition of a zebrafish with a known mutation that results in HSP (610C>T). I will utilize a diverse chemical library to perform the screen so I can I identify new small molecule protein interactions. I will then introduce the small molecules to each strain of zebrafish and observe for any changes that may occur and if any of the mutated strains of zebrafish with spasticity have recovered. I will then fluorescently tag KIF5A once again and observe its localization with each mutant and recovered mutant. I hypothesis that I will see a recovered phenotype in S207A, S205A, and the zebrafish with the known HSP mutation. Fluorescence assay will show that zebrafish with the recovered phenotype will have proper localization of KIF5A again. If the zebrafish do not have spasticity in their extremities then one of the small molecules in the library have help with proper localization.
Aim 3: Identify novel proteins that interact with KIF5A by localizing the protein to the legs.
For this aim I will introduce two mutations into exon 1 of Hox13 to create a zebrafish with defective limbs, as this has been shown to occur in previous studies. I will also have a control zebrafish with no mutations and fully functional extremities. First I will perform a fluorescence screen with KIF5A for both strains of zebrafish for localization. I will then perform a TAP Tag analysis on both the mutant and the control to see what proteins interact with the bait, KIF5A. If a protein is identified I will knockout the gene that codes for the protein of interest using CRISPR Cas9 and insert a GFP tag creating my new mutant zebrafish. The control will have no knockout but a tag will be inserted. Localization of the protein will then be observed within the zebrafish. I will then create a protein interaction network to illustrate this proteins relationship to KIF5A. I hypothesis that I will be able to identify at least one protein that interacts with KIF5A in the control but not in the mutant zebrafish. If a protein is associated with zebrafish that has fins, but not with zebrafish without fins, than the protein must have some property that helps localize KIF5A to the extremities
For this aim I will introduce two mutations into exon 1 of Hox13 to create a zebrafish with defective limbs, as this has been shown to occur in previous studies. I will also have a control zebrafish with no mutations and fully functional extremities. First I will perform a fluorescence screen with KIF5A for both strains of zebrafish for localization. I will then perform a TAP Tag analysis on both the mutant and the control to see what proteins interact with the bait, KIF5A. If a protein is identified I will knockout the gene that codes for the protein of interest using CRISPR Cas9 and insert a GFP tag creating my new mutant zebrafish. The control will have no knockout but a tag will be inserted. Localization of the protein will then be observed within the zebrafish. I will then create a protein interaction network to illustrate this proteins relationship to KIF5A. I hypothesis that I will be able to identify at least one protein that interacts with KIF5A in the control but not in the mutant zebrafish. If a protein is associated with zebrafish that has fins, but not with zebrafish without fins, than the protein must have some property that helps localize KIF5A to the extremities
Future Directions
One topic that I failed to address in this project is how VSX2 interacts with KIF5A. Seeing this association in the protein interaction network helped me discover my gap in knowledge which was looking into what specific factors influence KIF5A directly when localizing to the extremities, but I never actually discussed the indirect relationship VSX2 had with KIF5A.
Another area that could be looked at is what cargo this protein actually transports. Scientists today know that KIF5A helps transport neurofilaments as well as mitochondria but the rest of the cargo's content is a mystery.
One topic that I failed to address in this project is how VSX2 interacts with KIF5A. Seeing this association in the protein interaction network helped me discover my gap in knowledge which was looking into what specific factors influence KIF5A directly when localizing to the extremities, but I never actually discussed the indirect relationship VSX2 had with KIF5A.
Another area that could be looked at is what cargo this protein actually transports. Scientists today know that KIF5A helps transport neurofilaments as well as mitochondria but the rest of the cargo's content is a mystery.
alex_myers_tuesday_final_talk.pptx | |
File Size: | 9142 kb |
File Type: | pptx |
References
[1] Hereditary Spastic Paraplegia (2016, April). Retrieved January 31, 2019, from https://rarediseases.info.nih.gov/diseases/6637/hereditary-spastic-paraplegia
[2] Spastic Paraplegia Foundation. Retrieved January 31, 2019, from https://sp-foundation-org.presencehost.net/who_we_are/overview.html
[3] Paik, N (2019, January). Hereditary Spastic Paraplegia. Retrieved January 31, 2019, from https://emedicine.medscape.com/article/306713-overview
[4] Reid, E (2002, November). A Kinesin Heavy Chain (KIF5A) Mutation in Hereditary Spastic Paraplegia (SPG10). Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC385095/
[5] Cuchanski, M., & Baldwin, K. J. (2018). Mutation in KIF5A c.610C>T Causing Hereditary Spastic Paraplegia with Axonal Sensorimotor Neuropathy. Case reports in neurology, 10(2), 165-168. doi:10.1159/000490456
[6] Musumeci, O., Bassi, M.T., Mazzeo, A. et al. Neurol Sci (2011) 32: 665. https://doi.org/10.1007/s10072-010-0445-8
[7] Filosto, M., Piccinelli, S. C., Palmieri, I., Necchini, N., Valente, M., Zanella, I., Biasiotto, G., Lorenzo, D. D., Cereda, C., … Padovani, A. (2018). A Novel Mutation in the Stalk Domain of KIF5A Causes a Slowly Progressive Atypical Motor Syndrome. Journal of clinical medicine, 8(1), 17. doi:10.3390/jcm8010017
[8] Kawaguchi, K. (2013). Role of Kinesin-1 in the Pathogenesis of SPG10, a Rare Form of Hereditary Spastic Paraplegia. The Neuroscientist, 19(4), 336–344. https://doi.org/10.1177/1073858412451655
[9]http://www.ebi.ac.uk/interpro/entry/IPR001752
[10] https://www.yourgenome.org/facts/why-use-the-zebrafish-in-research
[1] Hereditary Spastic Paraplegia (2016, April). Retrieved January 31, 2019, from https://rarediseases.info.nih.gov/diseases/6637/hereditary-spastic-paraplegia
[2] Spastic Paraplegia Foundation. Retrieved January 31, 2019, from https://sp-foundation-org.presencehost.net/who_we_are/overview.html
[3] Paik, N (2019, January). Hereditary Spastic Paraplegia. Retrieved January 31, 2019, from https://emedicine.medscape.com/article/306713-overview
[4] Reid, E (2002, November). A Kinesin Heavy Chain (KIF5A) Mutation in Hereditary Spastic Paraplegia (SPG10). Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC385095/
[5] Cuchanski, M., & Baldwin, K. J. (2018). Mutation in KIF5A c.610C>T Causing Hereditary Spastic Paraplegia with Axonal Sensorimotor Neuropathy. Case reports in neurology, 10(2), 165-168. doi:10.1159/000490456
[6] Musumeci, O., Bassi, M.T., Mazzeo, A. et al. Neurol Sci (2011) 32: 665. https://doi.org/10.1007/s10072-010-0445-8
[7] Filosto, M., Piccinelli, S. C., Palmieri, I., Necchini, N., Valente, M., Zanella, I., Biasiotto, G., Lorenzo, D. D., Cereda, C., … Padovani, A. (2018). A Novel Mutation in the Stalk Domain of KIF5A Causes a Slowly Progressive Atypical Motor Syndrome. Journal of clinical medicine, 8(1), 17. doi:10.3390/jcm8010017
[8] Kawaguchi, K. (2013). Role of Kinesin-1 in the Pathogenesis of SPG10, a Rare Form of Hereditary Spastic Paraplegia. The Neuroscientist, 19(4), 336–344. https://doi.org/10.1177/1073858412451655
[9]http://www.ebi.ac.uk/interpro/entry/IPR001752
[10] https://www.yourgenome.org/facts/why-use-the-zebrafish-in-research