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Eukaryon

Huntingtin Knockout Leaves Mice Shaking

Matthew McMahon
Department of Biology
Lake Forest College
Lake Forest, Illinois 60045

Mice who had the mutated Huntingtin gene removed from their DNA 21 days after birth continued to show signs of Huntington’s disease progression. Similar cellular changes and physical symptoms were found in mice regardless of the continued presence of the mutant gene.

 

The Huntingtin gene plays a crucial role in the production of Huntingtin proteins, which are necessary for proper early brain development1. Mutations to this gene, which causes excessive repeats of glutamine, a building block of certain proteins, have been linked to the creation of abnormally long mutant Huntingtin proteins, as well as the development of Huntington’s disease (HD), an incurable genetic degenerative disorder. Studies of HD often observe the effects of mutant proteins on neurons, and many have found mechanisms by which these proteins cause cellular dysfunction and death2,3. Some recent studies, however, have found that the mutated Huntingtin gene causes alterations in the creation and formation of several types of brain cells during development4,5. A new paper by Molero et al.6 details an effort to determine whether or not these changes are sufficient to cause HD progression, without the presence of mutant Huntingtin proteins.

The researchers hypothesized that developmental changes due to the mutant Huntingtin gene are enough to cause HD development and progression. To test this, Molero et al. had to study symptoms and brain activity of mice whose brain cells underwent the developmental changes caused by the mutant Huntingtin gene but did continue to produce mutant Huntingtin proteins throughout life. To solve this issue, the researchers used three groups of mice. One group, the control, consisted of mice that never had the mutant Huntingtin gene. A second group, labelled Q97 mice, had the mutant Huntingtin gene throughout their lives. A third group, called Q97CRE mice, had the mutant Huntingtin gene in their DNA at conception, but then had it removed 21 days after birth. As the mutant Huntingtin gene is necessary to produce mutant Huntingtin proteins, the presence or absence of the gene also indicates whether or not the mutant proteins will accumulate in each group of mice. The researchers were able to remove the mutant Huntingtin gene from the Q97CRE mice post-birth using the Cre-lox recombination method7, which is analogous to cutting a piece of string with a pair of scissors and tying the remaining string back together. The scissors in this example are the protein Cre recombinase, the string is DNA, and the cutting is done at two recognizable, palindromic, sections of DNA, called loxP sites (Fig. 1).

 

Figure 1: Cre-Lox Recombination

As worsening motor function is the most Mrackova Fig 1apparent characteristic of HD, Molero et al. administered motor function tests to each group at 3 and 9 months to compare their performances. The data from tests, involving grip strength and motor coordination, showed that control mice performed best, the Q97 mice performed worst, and the Q97CRE mice performed similarly to the control mice at 3 months, but more similarly to the Q97 mice by 9 months, showing a drastic decrease in performance with age. In balance tests, however, the Q97CRE mice broke this trend, and improved in performance at 9 months, becoming more similar to the control mice than the Q97 mice. From these results, the researchers concluded that cell-related developmental alterations due to the mutant Huntingtin gene can cause most of the motor function loss associated with HD.

 

While motor symptoms are the most visible component of HD, they must be caused by changes in cellular activity in the brain. HD and other neurodegenerative diseases are known to cause alterations in both neural plasticity and cell signaling activity,8,9,10 so Molero et al. set out to determine if these changes were also present in the Q97CRE mice. Neural plasticity refers to 2 different ways a neuron can be changed for a long period of time. In long-term depression, neurons send significantly weaker signals in response to a stimulus, and in long-term potentiation, neurons send significantly stronger signals in response to a stimulus. Using microarrays, devices made up of a series of electrodes which can measure brain activity, Molero et al. found that a greater number of synapses - connections between neurons - in the Q97 and Q97CRE mice showed long-term potentiation compared to the control mice. Additionally, the synapses of the Q97 and Q97CRE mice showed less frequent and more irregular sending of signals, which slowed brain cell activity. Due to similarities between the Q97 and Q97CRE mice in the data collected, Molero et al. concluded that developmental alterations due to the mutant Huntingtin gene are able to cause the changes in neural plasticity and cell signaling activity associated with HD.

 

Two more significant cellular changes associated with HD are a general increase in neuron death and an increase in neuron vulnerability to excitotoxicity, the damage caused by overactivity11. To determine whether the Q97CRE mice displayed higher instances of age-related cell death, Molero et al. stained brain sections of 9-month-old mice from each of the three groups with a stain that colors dying neurons, and then counted the colored cells. The brains of Q97 mice showed the greatest number of dying neurons, followed closely by those of the Q97CRE mice. The control mice brains showed, by far, the fewest number of dying neurons. To test vulnerability to excitotoxicity, Molero et al. injected mouse brains with a substance that overexcites the neurons and then measured the size of the resulting lesions (damaged areas of the brains) in each group. Following in the trend of the neuron death data, the Q97 mice had the largest lesions, followed closely by the Q97CRE mice, with the control mice having significantly smaller lesions than the other two groups. Due to similarities in the data between the Q97 and Q97CRE mice, Molero et al. concluded that developmental changes due to the mutant Huntingtin gene are able to cause the cell vulnerabilities and death associated with HD.

 

Due to the many similarities in motor symptoms and cellular changes between the Q97 and Q97CRE mice found in this study, Molero et al. made the conclusion that developmental alterations caused by the mutated Huntingtin gene can cause the development and progress of HD. (Fig. 2). By showing a new way in which HD can progress, Molero et al.’s study has the potential to drastically impact the direction of future HD treatment research. If medications that combat the effects of proteins will have little effect on HD progression, a potential implication of this study, then there is little point in developing those therapies. Continued research into this topic could look into comparing the motor function and neuron activity of mice that have the mutant Huntingtin gene removed post birth with mice that have undergone promising Huntingtin protein-focused HD treatments, such as one outlined by Ochaba et al.12 It would also be interesting to determine if cellular structures and pathways that have been shown to be disrupted by mutant Huntingtin proteins (in studies such as one by Gasset-Rosa et al.2) are also disrupted in mice that have the mutant Huntingtin gene removed post-birth. If not, it would be interesting to see what impact the preservation of these structures and processes have on the progression of HD. Regardless, Molero et al.’s study shines a new light on a disease which affects more than 300,000 Americans every year, and perhaps through a better understanding of the mechanisms by which HD effects the brain, more comprehensive and effective treatments can be made.

 

 Figure 2: New Mechanism of HD ProgressionMcMahon Fig 2

Molero et al. showed in their study that the production and accumulation of mutant Huntingtin proteins is not necessary to the progression of HD. Instead it is shown that developmental alterations cause by the mutant Huntingtin gene, which effect the creation and formation of several types of brain cell, are able to cause most of the same symptoms and changes occur in HD normally, without the presence of mutant Huntingtin proteins.

 

References

  1. Reiner, A., Dragatsis, I., Zeitlin, S., & Goldowitz, D. (2003). Wild-type Huntingtin plays a role in brain development and neuronal survival. Molecular Neurobiology, 28, 259-275. doi:10.1385/mn:28:3:259
  2. Gasset-Rosa, F., Chillon-Marinas, C., Goginashvili, A., Atwal, R. S., Artates, J. W., Tabet, R., … Lagier-Tourenne, C. (2017). Polyglutamine-expanded Huntingtin exacerbates age-related disruption of nuclear integrity and nucleocytoplasmic transport. Neuron, 94, 48-57.e44. doi:10.1016/j.neuron.2017.03.027
  3. Enokido, Y., Tamura, T., Ito, H., Arumughan, A., Komuro, A., Shiwaku, H., … Okazawa, H. (2010). Mutant Huntingtin impairs ku70-mediated dna repair. The Journal of Cell Biology, 189, 425.
  4. Molero, A. E., Gokhan, S., Gonzalez, S., Feig, J. L., Alexandre, L. C., & Mehler, M. F. (2009). Impairment of developmental stem cell-mediated striatal neurogenesis and pluripotency genes in a knock-in model of huntington’s disease. Proceedings of the National Academy of Sciences, 106, 21900.
  5. Molina-Calavita, M., Barnat, M., Elias, S., Aparicio, E., Piel, M., & Humbert, S. (2014). Mutant Huntingtin affects cortical progenitor cell division and development of the mouse neocortex. The Journal of Neuroscience, 34, 10034.
  6. Molero, A. E., Arteaga-Bracho, E. E., Chen, C. H., Gulinello, M., Winchester, M. L., Pichamoorthy, N., … Mehler, M. F. (2016). Selective expression of mutant Huntingtin during development recapitulates characteristic features of huntington’s disease. Proceedings of the National Academy of Sciences, 113, 5736-5741. doi:10.1073/pnas.1603871113
  7. Hayashi, S., & McMahon, A. P. (2002). Efficient recombination in diverse tissues by a tamoxifen-inducible form of cre: A tool for temporally regulated gene activation/inactivation in the mouse. Developmental Biology, 244, 305-318. doi:https://doi.org/10.1006/dbio.2002.0597
  8. Picconi, B., Centonze, D., Håkansson, K., Bernardi, G., Greengard, P., Fisone, G., … Calabresi, P. (2003). Loss of bidirectional striatal synaptic plasticity in l-dopa–induced dyskinesia. Nature Neuroscience, 6, 501. doi:10.1038/nn1040
  9. Thiele, S. L., Chen, B., Lo, C., Gertler, T. S., Warre, R., Surmeier, J. D., … Nash, J. E. (2014). Selective loss of bi-directional synaptic plasticity in the direct and indirect striatal output pathways accompanies generation of parkinsonism and l-dopa induced dyskinesia in mouse models. Neurobiology of Disease, 71, 334-344. doi:https://doi.org/10.1016/j.nbd.2014.08.006
  10. André, V. M., Cepeda, C., Fisher, Y. E., Huynh, M., Bardakjian, N., Singh, S., … Levine, M. S. (2011). Differential electrophysiological changes in striatal output neurons in huntington’s disease. The Journal of Neuroscience, 31, 1170.
  11. Zeron, M. M., Hansson, O., Chen, N., Wellington, C. L., Leavitt, B. R., Brundin, P., … Raymond, L. A. (2002). Increased sensitivity to n-methyl-d-aspartate receptor-mediated excitotoxicity in a mouse model of huntington’s disease. Neuron, 33, 849-860. doi:10.1016/S0896-6273(02)00615-3
  12. Ochaba, J., Monteys, Alex M., O’Rourke, Jacqueline G., Reidling, Jack C., Steffan, Joan S., Davidson, Beverly L., & Thompson, Leslie M. (2016). Pias1 regulates mutant Huntingtin accumulation and huntington’s disease-associated phenotypes in vivo. Neuron, 90, 507-520. doi:10.1016/j.neuron.2016.03.016

Process:

Before I began writing this paper, I spent somewhere around an hour brainstorming ideas for the title of this paper. None of my ideas excited me too greatly, and I settled on the current title of this paper, which I hope plays on some connection between the removal of a gene being called knockout, and knockouts occurring in boxing, the latter of which likely leave those who experience them shaking (and a major symptom of HD is involuntary jerking movements, which could be described as shaking). After the title was decided, I wrote the summary sentences under the title, and began to write the paper. Throughout the writing process I referred back to the paper I read for my capsule presentation. About half way through the paper, I spent a couple of hours creating a new figure (the one depicting Cre-lox recombination) and making some edits to the figure I used for my capsule presentation, adding a legend, and making some of the word choice more in line with what was written in my paper. After finishing a first draft of the paper, I went to sleep, and the next day I came back to it, and began to rewrite it paragraph by paragraph. I am much happier with both the clarity and brevity of the second, and current, draft of this paper, and the rewriting process also allowed me to find several more places in which to add references. After finishing the rewrite of the paper, I went back and edited the whole thing for word length and cleared up a couple of spelling and capitalization mistakes. Throughout the process of writing the first and second draft of this paper, I also referred back to the News and Views article I found for the workshop, as well as the one modelled and given out during the workshop.

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