This web page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison.
Conclusions
This section of the website will be devoted to the experiments I designed to further this research, as well as future directions for more investigation of FRMD7. Below is a copy of my final presentation in PowerPoint format.
congenital_x-linked_nystagmus.pptx | |
File Size: | 2592 kb |
File Type: | pptx |
As I researched FRMD7 over the course of the semester, the biggest reoccurring theme I found was that there is a large lack of information about the role of the protein in normal motor neuronal cells in the eyes. One study suggested that FRMD7 was involved with the regulation of neuronal outgrowth and development in the mouse embryo, specifically in the axonal growth cone [1]. I became very interested in this component of FRMD7's function.
My first instinct was to look at the STRING interaction networks to find what other proteins FRMD7 is interacting with as the neurons are developing. This search proved to be rather unfruitful, since the human interaction network only provided proteins linked through "textmining." The mouse interaction network was even more disheartening, with only two proteins linked to FRMD7 through literature.
I chose mice as my model organism for several reasons, the first being that there has already been some experiments done using mice as a model for Nystagmus. Secondly, mouse FRMD7 is 85% homologous to the human copy, and mice have the same eye structure. Drosophila and C. elegans would have been faster, but I for my designed experiment it made more sense to me to use a phylogenically closer species.
As I hunted around the human network in STRING, I found TAC4. Short for tachyknin 4, TAC4 is a neurotransmitter encoding gene whose protein is cleaved into secreted peptides that can excite neuronal cells. TAC4 appears to form a complex with other proteins in the same tachyknin family (TAC1, TACR1, TACR2, TACR3, CHKL), that are involved with muscle contraction (both smooth and skeletal), calcium second messenger systems and G-coupled protein complexes. In the mice model, the link between FRMD7 and TAC4 had yet to be investigated, even though the TAC complex in mice was exactly the same. This led me to my first hypothesis:
Since the TAC complex is full of proteins that excite neurons, this complex interacts with FRMD7 during growth cone formation in mice.
To investigate this, I propose a Tap Tag analysis of FRMD7, and all the proteins in the TAC complex. I would start by creating a fusion protein of FRMD7 with the TAP tag and introducing it into a specific mice line with the fusion protein present. I would use mouse embryo brain cells harvested around embryo day 10 during development [2]. I would then lyse the cells and perform the affinity purifications. I would repeat the tagging with TAC4, and the other proteins in the TAC complex to ensure that they are all interacting and able to pull down each other.
I would then run a MudPit analysis which would result in protein mass spectrometry data. If my hypothesis is correct, each tagged protein in the TAC complex will pull down the rest of the proteins in the complex, including FRMD7.
The second piece I wanted to explore is whether or not FRMD7 and TAC4 are colocalizing in the axonal growth cone during mouse embryo development. If TAC4, FRMD7 and any other protein in the TAC complex is able to pull down one another, it is most likely they are colocalizing. This is my second hypothesis:
If TAC4 and FRMD7 are able to pull down each other, they most likely are colocalize in the mouse embryo motor neuron cells.
To explore this hypothesis, I would create a knock-in (KI) in the germline of mice with FRMD7 and TAC4 each fused to a different florescent protein. I would do this by extracting FRMD7 mRNA from cells, using reverse transcriptase to get a cDNA copy, amplifying this cDNA via PCR, integrating this gene into a bacterial plasmid along with the florescent protein, inserting the plasmid into bacterial cells an allowing them to grow and proliferate. Then I would isolate the plasmid, insert in in mouse ES cells, allow for recombination to occur and select for cells displaying the florescent phenotype. These cells would then be implanted in a surrogate. Then I would select for offspring with the fused gene, allow the F1 generation to mate and finally create a homologous KI line.
Then I would use this KI line to look for the colocalization of TAC4 and FRMD7 in axonal growth cones of day 10 mouse embryos. If my hypothesis is correct, I would expect FRMD7 and TAC4 florescence to overlap indicating colocalization.
So in summary, I'm looking to build the bridge between FRMD7 and the TAC complex in mice, and show that FRMD7 and TAC4 colocalize in developing axonal growth cones.
My first instinct was to look at the STRING interaction networks to find what other proteins FRMD7 is interacting with as the neurons are developing. This search proved to be rather unfruitful, since the human interaction network only provided proteins linked through "textmining." The mouse interaction network was even more disheartening, with only two proteins linked to FRMD7 through literature.
I chose mice as my model organism for several reasons, the first being that there has already been some experiments done using mice as a model for Nystagmus. Secondly, mouse FRMD7 is 85% homologous to the human copy, and mice have the same eye structure. Drosophila and C. elegans would have been faster, but I for my designed experiment it made more sense to me to use a phylogenically closer species.
As I hunted around the human network in STRING, I found TAC4. Short for tachyknin 4, TAC4 is a neurotransmitter encoding gene whose protein is cleaved into secreted peptides that can excite neuronal cells. TAC4 appears to form a complex with other proteins in the same tachyknin family (TAC1, TACR1, TACR2, TACR3, CHKL), that are involved with muscle contraction (both smooth and skeletal), calcium second messenger systems and G-coupled protein complexes. In the mice model, the link between FRMD7 and TAC4 had yet to be investigated, even though the TAC complex in mice was exactly the same. This led me to my first hypothesis:
Since the TAC complex is full of proteins that excite neurons, this complex interacts with FRMD7 during growth cone formation in mice.
To investigate this, I propose a Tap Tag analysis of FRMD7, and all the proteins in the TAC complex. I would start by creating a fusion protein of FRMD7 with the TAP tag and introducing it into a specific mice line with the fusion protein present. I would use mouse embryo brain cells harvested around embryo day 10 during development [2]. I would then lyse the cells and perform the affinity purifications. I would repeat the tagging with TAC4, and the other proteins in the TAC complex to ensure that they are all interacting and able to pull down each other.
I would then run a MudPit analysis which would result in protein mass spectrometry data. If my hypothesis is correct, each tagged protein in the TAC complex will pull down the rest of the proteins in the complex, including FRMD7.
The second piece I wanted to explore is whether or not FRMD7 and TAC4 are colocalizing in the axonal growth cone during mouse embryo development. If TAC4, FRMD7 and any other protein in the TAC complex is able to pull down one another, it is most likely they are colocalizing. This is my second hypothesis:
If TAC4 and FRMD7 are able to pull down each other, they most likely are colocalize in the mouse embryo motor neuron cells.
To explore this hypothesis, I would create a knock-in (KI) in the germline of mice with FRMD7 and TAC4 each fused to a different florescent protein. I would do this by extracting FRMD7 mRNA from cells, using reverse transcriptase to get a cDNA copy, amplifying this cDNA via PCR, integrating this gene into a bacterial plasmid along with the florescent protein, inserting the plasmid into bacterial cells an allowing them to grow and proliferate. Then I would isolate the plasmid, insert in in mouse ES cells, allow for recombination to occur and select for cells displaying the florescent phenotype. These cells would then be implanted in a surrogate. Then I would select for offspring with the fused gene, allow the F1 generation to mate and finally create a homologous KI line.
Then I would use this KI line to look for the colocalization of TAC4 and FRMD7 in axonal growth cones of day 10 mouse embryos. If my hypothesis is correct, I would expect FRMD7 and TAC4 florescence to overlap indicating colocalization.
So in summary, I'm looking to build the bridge between FRMD7 and the TAC complex in mice, and show that FRMD7 and TAC4 colocalize in developing axonal growth cones.
Future Directions
1. Further verification of the TAC complex colocalizing with FRMD7 in axonal growth cones - I would create a different fusion protein from the TAC complex, for example TACR1, along with the FRMD7 fusion protein and look for colocalization. I predict all the proteins in the TAC complex will colocalize with FRMD7.
2. RNAi in C. elegans - Since the FRMD7 protein did find a homolog in C. elegans, it's very likely human FRMD7 has other roles in cells than just motor neuron formation in the eyes. One study in particular showed that FRMD7 was present in heart and lung tissues during development in mice [2]. Through an investigation in WormBase, I found 8 genes (frm-5, max-1, frm-3, frm-10, frm-4, kin-32, kri-1, frm-8) that could be candidates for RNAi experiments related to Nystagmus. I think studying any of those genes independently would give further insight to FRMD7's other roles in humans.
3. Drug Discovery - To date there are several drugs that are used to treat Nystagmus, but they all have very varied and inconsistent results [3]. I also searched PubChem and found nothing that has been shown to bind to FRMD7. I think it would be beneficial to look into drug discovery research to find better compounds that will help treat symptoms. If a more potent drug was found, it may eliminate or at least reduce the need for invasive surgeries to treat the symptoms.
4. Connections to other nervous system disorders - As I was looking around on STRING, I noticed the mouse interaction network had Nystagmus only one link away from PARK7, a protein associated with Parkinson's disease. I also found that a few of the drugs prescribed to Nystagmus patients are also used as anti Parkinson's agents. This made me wonder if any other neurological disorders were also closely linked to Nystagmus. I think an analysis of FRMD7's connections to other neurological disorders would be beneficial, not only to Nystagmus, but other diseases like Parkinson's as well.
2. RNAi in C. elegans - Since the FRMD7 protein did find a homolog in C. elegans, it's very likely human FRMD7 has other roles in cells than just motor neuron formation in the eyes. One study in particular showed that FRMD7 was present in heart and lung tissues during development in mice [2]. Through an investigation in WormBase, I found 8 genes (frm-5, max-1, frm-3, frm-10, frm-4, kin-32, kri-1, frm-8) that could be candidates for RNAi experiments related to Nystagmus. I think studying any of those genes independently would give further insight to FRMD7's other roles in humans.
3. Drug Discovery - To date there are several drugs that are used to treat Nystagmus, but they all have very varied and inconsistent results [3]. I also searched PubChem and found nothing that has been shown to bind to FRMD7. I think it would be beneficial to look into drug discovery research to find better compounds that will help treat symptoms. If a more potent drug was found, it may eliminate or at least reduce the need for invasive surgeries to treat the symptoms.
4. Connections to other nervous system disorders - As I was looking around on STRING, I noticed the mouse interaction network had Nystagmus only one link away from PARK7, a protein associated with Parkinson's disease. I also found that a few of the drugs prescribed to Nystagmus patients are also used as anti Parkinson's agents. This made me wonder if any other neurological disorders were also closely linked to Nystagmus. I think an analysis of FRMD7's connections to other neurological disorders would be beneficial, not only to Nystagmus, but other diseases like Parkinson's as well.
References
[1] Betts-Henderson J., Bartesaghi, S., Crosier, M., Lindsay, S., Chen, H., Salomoni, P., Gottlob, I., Nicotera P. (2010)
The nystagmus-associated FRMD7 gene regulates neuronal outgrowth and development. Human Molecular Genetics, 19(2), 342. doi: 10.1093/hmg/ddp500
[2] Self, J. E., Haitchi, H. M., Griffiths, H., Holgate, S. T., Davies, D. E., Lotery A. (2010)
FRMD7 expression in developing mouse brain. Eye, 24, 165. doi: 10.1038/eye.2009.44
[3] Stahl, J. S., Plant, G. T., Leigh, R. J. (2002)
Medical treatment of nystagmus and its visual consequences. Royal Society of Medicine, 95(5), 235. Retrieved from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1279675/
The nystagmus-associated FRMD7 gene regulates neuronal outgrowth and development. Human Molecular Genetics, 19(2), 342. doi: 10.1093/hmg/ddp500
[2] Self, J. E., Haitchi, H. M., Griffiths, H., Holgate, S. T., Davies, D. E., Lotery A. (2010)
FRMD7 expression in developing mouse brain. Eye, 24, 165. doi: 10.1038/eye.2009.44
[3] Stahl, J. S., Plant, G. T., Leigh, R. J. (2002)
Medical treatment of nystagmus and its visual consequences. Royal Society of Medicine, 95(5), 235. Retrieved from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1279675/
Site created by: Kristen Klimo
Last updated: 5/11/2012
University of Wisconsin-Madison
Last updated: 5/11/2012
University of Wisconsin-Madison