Progress 10/01/17 to 09/30/19
Outputs
Target Audience:Our target audience is nutrition scientists, dietitians, and nutrition policy makers. We presented our data at Nutrition 2018, which is widely attended by a national (and international) audience of nutrition scientists, dietitians, and nutrition policy makers. Changes/Problems:We did not have enough time or animals to include primary neurons as a model of impaired ZIP12 function. We ran into difficulties with breeding mice, and we prioritized creating the transgenic strain of AD/ZIP12 KO mice and other associated experimental (or control) groups. Furthermore, the Neuro-2a cell model provided significant insight and flexibility in genetic and chemical manipulations for determining the role of zinc and ZIP12 in neuronal function. We were able to create enough mice to provide pilot data into how the loss of ZIP12 may affect Alzheimer's Disease pathogenesis, but we were unable to create enough mice for behavioral tests, which normally require n=12-16 per group. We, therefore, used our limited mice (about n=3) to examine neuronal architecture by Golgi staining. The brains have been stained and sectioned, and they are awaiting analyses. Because there was a recent development in this field, in which humans with ZIP12 polymorphisms were shown to have brain differences that are detectable by MRI in regions associated with Alzheimer's Disease and Parkinson's Disease, we have added some statistical genetics approaches to the cellular and animal approaches originally proposed in this project. What opportunities for training and professional development has the project provided?The graduate student supported by this OAES (Hatch) project, Morgan Strong, was able to present her research at Nutrition 2018 in Boston, MA. These conferences provided invaluable networking, training, and career development opportunities among nutrition scientists and dietitians. In recognition of the top caliber of research that Morgan Strong is performing, she was recognized as an Emerging Leaders in Nutrition Science Finalist and placed in the top 3. One of the undergraduates who was a significant participant in this project, Matthew Hart, received significant mentorship and training by the project director (PD) and the graduate student. He is now an MD/Ph.D. student, and this career development would not have been possible without this project and training opportunity. How have the results been disseminated to communities of interest?The research was presented by the graduate student, Morgan Strong, at an international conference (Nutrition 2018 in Boston, MA), which is attended by many of the target audience, such as dietitians, nutrition researchers, and nutrition policymakers. The presentation abstract was also published in Current Developments in Nutrition. We are currently working on publishing these results in a peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We identified that loss of ZIP12 function in Neuro-2a cells altered a select number of genes associated with mitochondrial function using RNA-sequencing and clustering analyses. We performed RNA-seq transcriptome profiling on cells transfected with a ZIP12-shRNA plasmid designed to inhibit ZIP12 function. We showed that genes associated with mitochondrial function are modestly altered by shRNA-mediated ZIP12 repression (largely 0.5 fold or less) but do reach statistical significance (p<0.0001). We developed an RNA-seq transcriptome pipeline using the Galaxy web interface connected to cloud computing in order to obtain these results. We determined that loss of ZIP12 function in Neuro-2a cells, by both CRISPR/Cas9-mediated deletion and shRNA-mediated repression, results in decreased neurite outgrowth which is compounded by both chemical and genetic models of Alzheimer's Disease and Parkinson's Disease. Furthermore, we determined that loss of ZIP12 by CRISPR-mediated deletion increases mitochondrial superoxide and reduced cellular respiration, which is an indicator of mitochondrial dysfunction. Genetic restoration of mitochondrial biogenesis by PGC-1alpha, genetic restoration of antioxidant gene protection by mitochondrial superoxide dismutase, and chemical antioxidants Vitamin E, MitoTEMPO, and Mitoquinone (mitochondrial Coenzyme Q10) restores neurite outgrowth in Neuro-2a cells without ZIP12. We were able to create a transgenic strain of mice with or without ZIP12 and with or without the Alzheimer's Disease transgene. However, our numbers were limited (about n=3 per group), so we focused on histological measures instead of behavioral assessments. We also determined that human ZIP12 polymorphisms that are associated with brain MRI differences commonly found in patients with neurodegeneration have reduced zinc transport activity. This use of human genetics data supports our findings in cellular and animal models about the role of zinc and ZIP12 in brain function and resilience to brain aging.
Publications
- Type:
Theses/Dissertations
Status:
Submitted
Year Published:
2019
Citation:
ZIP12 AS A POSSIBLE CANDIDATE GENE FOR NEURODEGENERATIVE DISORDERS. Morgan Strong. Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE July, 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Morgan D Strong, Matthew Hart, Tony Z Tang, Edralin A Lucas, Stephen L Clarke, Ahmed Bettaieb, Dingbo Lin, and Winyoo Chowanadisai. Loss of ZIP12 in Neuro-2A Cells Increases Mitochondrial Superoxide and Reduces Neurite Outgrowth, which Is Partially Restored by PGC-1alpha. Curr Dev Nutr. 2018 Nov; 2(11): nzy053.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Researchers presentedfindings from this study to the scientific community and individuals in relevant fields at the American Society for Nutrition's Nutrition 2018 conference. It also included the industrial personnel interested in health promotion and disease prevention. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The graduate student supported by this OAES (Hatch) project, Morgan Strong, was able to present her research at Nutrition 2018 in Boston, MA. This conference provided her invaluable networking, training, and career development opportunities among nutrition scientists and dietitians. In recognition of the top caliber of research she is performing, she was recognized as an Emerging Leaders in Nutrition Science Finalist and placed in the top 3. How have the results been disseminated to communities of interest?We presented our data at Nutrition 2018, which is widely attended by a national (and international) audience of nutrition scientists, dietitians, and nutrition policymakers. In addition, our research (supported by OAES) was recognized for its promising impact by the Emerging Leaders finalist award and the enhanced platform during the special presentation session. What do you plan to do during the next reporting period to accomplish the goals?We plan to assess how the loss of ZIP12 may affect increased sensitivity to mutated proteins associated with neurodegenerative diseases. The graduate student Morgan Strong is currently developing a cell viability assay with lactate dehydrogenase activity, a cell viability assay with greater sensitivity to cell death than trypan blue. She will then measure how different proteins associated with these diseases will affect cell viability in cells with and without ZIP12. We will also continue our experiments examining how cell and mitochondrial function are affected by the loss of ZIP12. We plan to continue to raise ZIP12 KO mice with and without the AD transgene to 10 months of age. We anticipate running experiments in May 2019. We have isolated RNA from Neuro-2a cells transfected with the minimal elements of the ZIP12 shRNA plasmid, and we are working with Dr. Peter Hoyt to determine transcriptome differences.
Impacts
What was accomplished under these goals?
We have created ZIP12 KO heterozygous (carrier) mice with the AD transgene and bred these mice to ZIP12 KO heterozygous mice to create a combination of mice with or without ZIP12 and with or without the AD gene where theyare currently being raised in the animal facility. We also performed RNA-seq transcriptome profiling on cells with CRISPR-mediated genome deletions in the ZIP12 gene. However, we discovered that clonal differences between the different CRISPR cells likely are responsible for the differences in transcriptome profiling. Clonal isolation is normally performed during CRISPR-mediate genome editing to ensure an identical population not sensitive to genetic drift, or change in genome population across experiments. As an alternate approach, we performed RNA-seq on across the cell population by transfection with a ZIP12-shRNA plasmid. We showed that genes associated with mitochondrial function are preferentially altered by shRNA-mediated ZIP12 repression, but the effect size (largely 0.5 fold or less) and statistical significance (p=0.1-0.2) were insufficient, likely due to the hypomorphic nature of shRNA repression. In order to increase the potency of the shRNA delivery, we stripped the ZIP12 shRNA (and control) plasmids of extraneous elements, thereby reducing the plasmid size by approximately two-thirds and presumably increasing the potency by about 2-3 times. Despite the need for an altered shRNA plasmid, we were able to develop an RNA-seq transcriptome pipeline using the Galaxy web interface connected to cloud computing.
Publications
|