This web page was produced as an assignment for Genetics 564, an undergraduate course at UW-Madison.
Summary
Major depression is one of the most common mental disorders in the United States [1]. It is characterized by low mood and sadness for extended periods of time. Those affected experience interference with daily life, including their ability to work, eat, sleep, study, connect, and enjoy once-pleasurable activities [1]. Many studies have provided insight into the biological and genetic mechanisms of depression, including linking its symptoms to the circadian rhythm. Circadian rhythm is mediated by the daily light/dark cycle via cellular proteins called circadian clocks [3]. Circadian clocks can be modulated by antidepressant drugs that often help depressive symptoms [3]. Several studies show that natural products may act as antidepressants. An herb, valerian, has been shown to exhibit antidepressant effects by acting on neurotransmitters in the brain [7]. Depression has been studied in many laboratories using the model organism, zebrafish. Zebrafish exhibit depression-like behaviors, which include stopping swimming and hiding [4]. Although a study has been done to compare the gene expression of the light-responsive circadian clock genes in zebrafish and in blind cave fish, this study neglected to determine the gene expression of dopamine receptors during this time, for which PER2 is responsible [5, 6]. In addition, studies have not been conducted to determine how natural treatments such as valerian affect gene expression in zebrafish. Zebrafish and blind cavefish provide a unique approach to identify factors that regulate circadian rhythm via dopamine receptors.
Aim 1
Specific Aim 1: Determine how well conserved the human, zebrafish, and blind cavefish PER2 proteins are.
Approach: I will use online databases such as BLAST, Homologene, and Clustal Omega to investigate similarities and differences between said proteins.
Hypothesis: The proteins will share domains such as the PAS domain and the Period C domain.
Discussion: I chose to use zebrafish and blind cavefish as model organisms for my experiments because their PER2 proteins are highly conserved (77%), as well being highly similar to the human PER2 protein (51% and 72% respectively). I also chose to use the fish as models because they live in different light-dark environments. The zebrafish lives in environment with day and night fluctuations while the blind cavefish lives in constant darkness. In this way, we can determine how the light/dark cycle affects PER2 expression and depression-like symptoms.
Hypothesis: The proteins will share domains such as the PAS domain and the Period C domain.
Discussion: I chose to use zebrafish and blind cavefish as model organisms for my experiments because their PER2 proteins are highly conserved (77%), as well being highly similar to the human PER2 protein (51% and 72% respectively). I also chose to use the fish as models because they live in different light-dark environments. The zebrafish lives in environment with day and night fluctuations while the blind cavefish lives in constant darkness. In this way, we can determine how the light/dark cycle affects PER2 expression and depression-like symptoms.
Results: Though the blind cavefish PER2 protein is much smaller than the human or zebrafish PER2 protein, it is still highly conserved (as discussed above). I compared each organisms' PER2 sequence in Clustal Omega (at right) and hypothesize that the blind cavefish protein contains the same PAS_11 domain that the human and zebrafish protein share. This was determined by comparing regions of similarity (marked by an * under the sequences at right). The areas of similarity between the blind cavefish and the other two proteins fall in the region of the PAS_11 domain in the human PER2 protein. This domain is associated with light reception and regulation of circadian rhythms [8].
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Aim 2
Specific Aim 2: Determine the gene expression of PER2 and D2 dopamine receptors in light/dark environments.
Approach: I will use RNA-seq to identify gene transcripts that are found in the brains of wild type zebrafish and blind cavefish and PER2 mutant zebrafish and blind cavefish. I will also measure receptor levels using Western blots of postmortem fish brains. To determine if fish with abnormal levels of dopamine receptors indicate depression-like symptoms, we will observe the fish for behaviors like stillness and hiding.
Hypothesis: Fish with disrupted circadian rhythms will exhibit abnormal levels of PER2 expression and also dopamine receptors. We hypothesize genes with the following GO terms to be expressed, though at differing times in the circadian rhythm: signal transducer activity, transcription factor activity, circadian rhythm, transcription, nucleus, cytoplasm. Fish with abnormal dopamine receptors levels will exhibit depression-like behavior such as stillness and hiding.
Hypothesis: Fish with disrupted circadian rhythms will exhibit abnormal levels of PER2 expression and also dopamine receptors. We hypothesize genes with the following GO terms to be expressed, though at differing times in the circadian rhythm: signal transducer activity, transcription factor activity, circadian rhythm, transcription, nucleus, cytoplasm. Fish with abnormal dopamine receptors levels will exhibit depression-like behavior such as stillness and hiding.
Discussion: Fish will be reared in all light or all dark environments to determine how this disruption to the circadian rhythm (which is regulated by the light cycle) affects PER2 and dopamine receptor (DopR) expression. For the zebrafish reared in an all light environment, I hypothesize that PER2 expression will be elevated as PER2 expression peaks in the afternoon and thus a decrease in dopamine receptor expression will occur because PER2 decreases the expression of DopR (counter intuitive, I know). On the other hand, zebrafish reared in an all dark environment will see a decrease in expression of PER2 and and an increase in DopR expression as the expression of PER2 will not be triggered if there is no light. However, I hypothesize that in each environment, the blind cavefish will exhibit baseline expression of both PER2 and DopR. This is because even though the cavefish has no light sensing organs such as eye spots, it still maintains a normal circadian rhythm in constant darkness [9].
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Aim 3
Specific Aim 3: Determine how the natural product, valerian, affects protein expression in the brains of wild type and mutant zebrafish.
Approach: Label free quantification and mass spectrometry of proteins in the brains will be conducted to quantify valerian’s effect on the model organisms’ brains. Samples will also be subjected to LC-MS/MS to identify any phosphopeptides. These experiments will be compared to a control group, which is not fed valerian.
Hypothesis: Valerian fed fish and control fish brains will exhibit differences in protein expression and phosphorylation. Valerian fed fish may exhibit expression of proteins with the following GO terms: neurotransmission, GABA receptor activity (positive regulation), synaptic cleft
Hypothesis: Valerian fed fish and control fish brains will exhibit differences in protein expression and phosphorylation. Valerian fed fish may exhibit expression of proteins with the following GO terms: neurotransmission, GABA receptor activity (positive regulation), synaptic cleft
Discussion: Valerian root is an herb that has been historically used to treat anxiety and insomnia and was also used as a sedative in WWI [10]. When added to the diets of the zebrafish, I expect to see its calming effects in action, as well as a change in dopamine receptor availability. I hypothesize that WT zebrafish fed a normal fish diet will exhibit baseline (normal) levels of DopR expression. However, when valerian is added to their diet, we may see decreased expression of DopR along with relaxed behavior. These results differ greatly from the results I expect to see in the PER2 mutant zebrafish. For fish fed a normal diet, I expect to see an increase in DopR expression, as well as depressive like symptoms such as stillness and hiding. However, PER2 mutant fish fed valerian root may see their DopR levels return towards baseline levels (though levels may remain a little elevated). This change in expression may be coupled with a semi relief of depression symptoms such as stillness and hiding. This experiment would help us to determine if valerian root could be useful as a therapy for people experiencing depression.
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Objectives
Since light cycle and circadian rhythm studies have only investigated the psychological and behavioral effects in humans in relation to depression, this study will help to understand the biological and genetic effects of such cycles on dopamine receptors in the brain. This, as well as results from the valerian experiments could lead to future therapies for depression, including circadian rhythm synchronization, diet change, and pharmacological drugs.
Future Directions
Genetics:
The above experiments I developed could be instrumental to understanding the genetic basis of depression and PER2's role in the illness. To explore its role further, I would like to replicate Aim 1 and Aim 2, but this time with a depression trigger event. As someone who has no lab experience working with zebrafish or blind cavefish, I am unsure what this trigger would look like. One idea is separating a fish from its brood or school. [Any zebrafish people out there? If you have any ideas you'd like to share, you can email me at [email protected].] By adding a trigger, we can determine if a major negative life event has effect on the expression of PER2 and dopamine receptors. In general, the role of dopamine in depression should be further investigated so that we can design more effective experiments to study PER2 and depression. Social Work: One future direction for the field of social work is to educate the public about mental illnesses so we can fight the stigma that surrounds the mentally ill. Social workers should also advocate for equal access to mental health care for all, as there are currently huge disparities among demographics for health care. Lastly, we should examine our everyday language and be sensitive about the words we are using. Words such as "crazy," "loony," and "nuts" may carry extra weight and contribute to the mental illness stigma if not used responsibly. |
per2_final_presentation.pdf | |
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per2_final_presentation.pptx | |
File Size: | 2392 kb |
File Type: | pptx |
References
[1] National Institute of Mental Health http://www.nimh.nih.gov/health/topics/depression/index.shtml?utm_content=buffera397a&utm_source=buffer&utm_medium=twitter&utm_campaign=Buffer#part4
[2] WebMD. Causes of Depression. Retrieved January 27, 2014, from http://www.webmd.com/depression/guide/causes-depression
[3] Golombek, D., Bussi, I., and Agostino, P. (2014). Minutes, days, and years: molecular interactions among different scales of biological timing. Philosophical Transactions of the Royal Society. B 369. doi: 20120465
[4] Society for Neuroscience. (2010, November 18). Depression-like behavior identified in zebrafish; Inability to cope with stress may play role in depression. Science Daily. Retrieved March 10, 2014 from http://www.sciencedaily.com/releases/2010/11/101116101736.htm
[5] Mejia, R. (2011). Cave-dwelling Fish Provide Clues to the Circadian Cycle. PLos Biol. 9(9) e1001141. doi: 10.1371/journal.bio.1001141
[6] Cavallari, N., et al. A Blind Circadian Clock In Cavefish Reveals that Opsins Mediate Peripheral Clock Photoreception. PLoS Biol. doi: 10.1371/journal.pbio.1001142
[7] Müller, L. G. et al. (2011). Antidepressant-like effect of Valeriana glechomifolia Meyer (Valerianaceae) in mice. Progress in Neuro-Psychopharmacology & Biological Psychiatry 36(2012): 101-109. Doi: 10.1016/j.pnpbp.2011.08.015
[8] Pfam: http://pfam.sanger.ac.uk/family/PF08447
[9] Cavallari, N. et al. (2011). A Blind Circadian Clock in Cavefish Reveals that Opsins Mediate Peripheral Clock Photoreception. PLoS Biology. 9(9) e1001141. doi: 10.1371/journal.pbio.1001142
[10] Smith, Silense. Valerian Root: Its Uses, History, and Other Information. Yahoo Voices. http://voices.yahoo.com/valerian-root-its-uses-history-other-information-8653537.html?cat=5
Picture References
Sun http://www.clipartbest.com/cliparts/9cz/EMK/9czEMKnKi.gif
Moon http://bestclipartblog.com/clipart-pics/moon-clip-art-11.gif
Fish http://www.clker.com/cliparts/r/C/E/Y/M/x/simple-fish-md.png
Fish Food http://i.walmartimages.com/i/p/00/04/67/98/77/0004679877035_500X500.jpg
Valerian root http://www.herbalextractsplus.com/images/herbs/valerian-isp.jpg
Brain http://static.freepik.com/free-photo/cartoon-brain-outline-clip-art_411969.jpg
Group of people
http://1.bp.blogspot.com/_FY8xQMCUsY8/S_MFEQzaSNI/AAAAAAAAACo/aC1euzhdlfU/s1600/group%2520diverse%2520happy%2520people%2520black%2520white%2520brown%2520smile%2520old%2520young%2520teen.jpg
Zebrafish http://www.renalgenes.org/zebrafish.html
Blind cavefish http://www.ryanphotographic.com/characidae.htm
[1] National Institute of Mental Health http://www.nimh.nih.gov/health/topics/depression/index.shtml?utm_content=buffera397a&utm_source=buffer&utm_medium=twitter&utm_campaign=Buffer#part4
[2] WebMD. Causes of Depression. Retrieved January 27, 2014, from http://www.webmd.com/depression/guide/causes-depression
[3] Golombek, D., Bussi, I., and Agostino, P. (2014). Minutes, days, and years: molecular interactions among different scales of biological timing. Philosophical Transactions of the Royal Society. B 369. doi: 20120465
[4] Society for Neuroscience. (2010, November 18). Depression-like behavior identified in zebrafish; Inability to cope with stress may play role in depression. Science Daily. Retrieved March 10, 2014 from http://www.sciencedaily.com/releases/2010/11/101116101736.htm
[5] Mejia, R. (2011). Cave-dwelling Fish Provide Clues to the Circadian Cycle. PLos Biol. 9(9) e1001141. doi: 10.1371/journal.bio.1001141
[6] Cavallari, N., et al. A Blind Circadian Clock In Cavefish Reveals that Opsins Mediate Peripheral Clock Photoreception. PLoS Biol. doi: 10.1371/journal.pbio.1001142
[7] Müller, L. G. et al. (2011). Antidepressant-like effect of Valeriana glechomifolia Meyer (Valerianaceae) in mice. Progress in Neuro-Psychopharmacology & Biological Psychiatry 36(2012): 101-109. Doi: 10.1016/j.pnpbp.2011.08.015
[8] Pfam: http://pfam.sanger.ac.uk/family/PF08447
[9] Cavallari, N. et al. (2011). A Blind Circadian Clock in Cavefish Reveals that Opsins Mediate Peripheral Clock Photoreception. PLoS Biology. 9(9) e1001141. doi: 10.1371/journal.pbio.1001142
[10] Smith, Silense. Valerian Root: Its Uses, History, and Other Information. Yahoo Voices. http://voices.yahoo.com/valerian-root-its-uses-history-other-information-8653537.html?cat=5
Picture References
Sun http://www.clipartbest.com/cliparts/9cz/EMK/9czEMKnKi.gif
Moon http://bestclipartblog.com/clipart-pics/moon-clip-art-11.gif
Fish http://www.clker.com/cliparts/r/C/E/Y/M/x/simple-fish-md.png
Fish Food http://i.walmartimages.com/i/p/00/04/67/98/77/0004679877035_500X500.jpg
Valerian root http://www.herbalextractsplus.com/images/herbs/valerian-isp.jpg
Brain http://static.freepik.com/free-photo/cartoon-brain-outline-clip-art_411969.jpg
Group of people
http://1.bp.blogspot.com/_FY8xQMCUsY8/S_MFEQzaSNI/AAAAAAAAACo/aC1euzhdlfU/s1600/group%2520diverse%2520happy%2520people%2520black%2520white%2520brown%2520smile%2520old%2520young%2520teen.jpg
Zebrafish http://www.renalgenes.org/zebrafish.html
Blind cavefish http://www.ryanphotographic.com/characidae.htm