38 ARF6: A Novel Therapeutic Target for Neuroinflammatory Disease

Faculty Mentor: Weiquan (Wendy) Zhu (Ophthalmology & Visual Sciences, University of Utah)

Introduction

Alzheimer’s disease is a chronic, progressive, neurodegenerative disease that is estimated to affect 15 million Americans by the year 2060 (Brookmeyer et al., 2018). Typically arising at age 65 and older, this devastating illness starts with subtle working memory deficits — such as forgetting the names of family and friends — and can progress to deep confusion, sleep problems, delusions, loss of language, inability to recognize close relatives, and eventually, death. As can be imagined, such slow deterioration and memory loss of loved ones is devastating for all involved. By improving our mechanistic understanding of Alzheimer’s disease through research, we pave the way to more effective prevention and treatment strategies that relieve suffering and provide hope.

During the Summer Program for Undergraduate Research (SPUR), I was able to contribute to a project investigating the role of a molecular target called ARF6 in a potential signaling pathway at the blood-brain barrier (BBB) in Alzheimer’s disease. The underlying pathology of Alzheimer’s disease includes the buildup of proteins called amyloid-beta in the brain. When misfolded amyloid-beta protein fragments accumulate, they form plaques, which contribute to cognitive decline by interfering with neuronal function (Aziz et al., 2024). Amyloid-beta efflux transporters, such as lipoprotein receptor-related protein 1 (LRP1) and P-glycoprotein, are critical in clearing waste across the blood- brain barrier (BBB) and preventing amyloid-beta buildup (Chai et al., 2020; Erickson et al., 2012; Figure 1).

In addition to amyloid-beta buildup, another hallmark of Alzheimer’s disease is blood-brain barrier dysfunction (Erickson & Banks, 2013). Inflammatory conditions are known to drive a process called endothelial-to-mesenchymal transition, or EndoMT (Derada Troletti et al., 2016). During this process, endothelial cells that make up the BBB lose their “barrier” cell traits and adopt characteristics of mesenchymal cells, which are more loosely organized. This contributes to BBB breakdown and makes the BBB more permeable (Sun et al., 2022; Figure 2). Our hypothesis is that endothelial cells undergoing EndoMT may downregulate critical amyloid-beta efflux transporters, leading to decreased amyloid-beta clearance and increased amyloid-beta accumulation in Alzheimer’s disease.

In a mouse model of neuroinflammatory disease, inhibiting the function of ARF6 has been shown to reverse EndoMT, stabilize the BBB, attenuate inflammation, and improve symptoms of Multiple Sclerosis (MS) without compromising immune function (Sun et al., 2022). This evidence may have important implications for Alzheimer’s disease. Thus, we further hypothesize that inhibiting the function of ARF6 may help stabilize the BBB and possibly preserve amyloid-beta efflux transporter expression (Figure 3). Our results will reveal any links between EndoMT, efflux transporter expression, and amyloid-beta accumulation. They will also illuminate the role of ARF6 in the amyloid-beta transport pathway at the BBB to ultimately improve our mechanistic understanding of Alzheimer’s disease.

Methods

Using cell culture techniques, we treated human brain microvascular endothelial cells (HBMEC’s) with IL-1 to model inflammatory conditions of Alzheimer’s disease and induce EndoMT. We transduced the cells with adenoviruses to modulate ARF6 function. Recombinant adenoviruses are used as vectors to deliver a gene of interest into target cells, where the gene can be transcribed and translated and the exogenous protein expressed. Specifically, we used the “dominant negative” ARF6-T27N mutant to mimic the inactive form of ARF6, and the “constitutively active” (GTP-bound) ARF6-Q67L mutant to mimic the active form of ARF6 (Van Acker et al., 2019). As controls, we included a cell group that did not undergo transduction, a group that was transduced with an empty vector (Ad-Null), and a group that was transduced with the wild-type form of ARF6 (ARF6- WT). Following adenovirus transduction and IL-1 treatment, we harvested the cells, quantified the amount of total protein present, and performed western blotting to observe the expression of amyloid- beta efflux transporters under each experimental condition (Figure 4).

Results & Future Directions

This work is part of ongoing investigations. Our protein quantification assay indicated sufficient total protein yield, with all samples having a protein concentration of 0.92 mg/ml or higher. Using western blot, we were able to visualize exogenous ARF6 expression (Figure 5). Our antibodies were unable to detect LRP1 or P-glycoprotein, and efforts are ongoing to optimize antibody concentrations and test antibodies from a different supplier to visualize efflux-transporter expression. Other future directions include optimizing adenovirus concentration during adenoviral transduction to fine-tune the expression of exogenous ARF6, measuring key markers of EndoMT such as Snail and N- cadherin, performing a GTP-pulldown assay to assess ARF6 activation in addition to ARF6 expression, and testing ARF6 function in vivo in a mouse model of Alzheimer’s disease and Arf6 knockout mice.

Personal Reflection

This summer experience deepened my understanding of how scientists approach complex questions in molecular biology and cellular neuroscience. In addition to learning new techniques like cell culture and western blotting, I was able to expand my understanding of PCR, gain experience maintaining transgenic mouse lines (e.g., weaning mice, genotyping, injections for tamoxifen- inducible Cre-loxP knockout mice, etc.), improve my understanding of animal models used for specific disease applications, and learn more about statistics. I was also able to assist in efforts to improve a mouse model of Multiple Sclerosis (MS) to more closely reflect the relapsing-remitting form of the disease, which is the most common form in humans. I enjoyed being in an intellectually stimulating lab environment, where you are always learning and there are endless things to be curious about. It was a great experience to be part of collaborative efforts to troubleshoot, adjust, find solutions, and make progress. I would like to thank Dr. Wendy Zhu, Dr. Shannon Odelberg, Robert Pryor, and Dr. Guoxin Ying for their mentorship.

Bibliography

Aziz, N., Wal, P., Singh, Y. K., Kumar, P., & Singh, B. (2024). A Comprehensive and Concise Review on Significance of Potent Peptide; Amyloid Beta (Aβ) in Progression of Alzheimer’s Disease. Current Drug Therapy, 20, 1–13. https://doi.org/10.2174/0115748855299317241014051505

Brookmeyer, R., Abdalla, N., Kawas, C. H., & Corrada, M. M. (2018). Forecasting the prevalence of preclinical and clinical Alzheimer’s disease in the United States. Alzheimer’s & Dementia, 14(2), 121– 129. https://doi.org/10.1016/j.jalz.2017.10.009

Chai, A. B., Leung, G. K. F., Callaghan, R., & Gelissen, I. C. (2020). P-glycoprotein: A role in the export of amyloid-β in Alzheimer’s disease? The FEBS Journal, 287(4), 612–625. https://doi.org/10.1111/febs.15148

Derada Troletti, C., de Goede, P., Kamermans, A., & de Vries, H. E. (2016). Molecular alterations of the blood–brain barrier under inflammatory conditions: The role of endothelial to mesenchymal transition. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease, 1862(3), 452–460. https://doi.org/10.1016/j.bbadis. 2015.10.010

Erickson, M. A., & Banks, W. A. (2013). Blood–Brain Barrier Dysfunction as a Cause and Consequence of Alzheimer’s Disease. Journal of Cerebral Blood Flow & Metabolism, 33(10), 1500–1513. https://doi.org/10.1038/jcbfm.2013.135

Erickson, M. A., Hartvigson, P. E., Morofuji, Y., Owen, J. B., Butterfield, D. A., & Banks, W. A. (2012). Lipopolysaccharide impairs amyloid beta efflux from brain: Altered vascular sequestration, cerebrospinal fluid reabsorption, peripheral clearance and transporter function at the blood–brain barrier. Journal of Neuroinflammation, 9(1), 150. https://doi.org/10.1186/1742-2094-9-150

Sun, Z., Zhao, H., Fang, D., Davis, C. T., Shi, D. S., Lei, K., Rich, B. E., Winter, J. M., Guo, L., Sorensen, L. K., Pryor, R. J., Zhu, N., Lu, S., Dickey, L. L., Doty, D. J., Tong, Z., Thomas, K. R., Mueller, A. L., Grossmann, A. H., Zhu, W. (2022). Neuroinflammatory disease disrupts the blood-CNS barrier via crosstalk between proinflammatory and endothelial-to-mesenchymal-transition signaling. Neuron, 110(19), 3106-3120.e7. https://doi.org/10.1016/j.neuron.2022.07.015

Van Acker, T., Tavernier, J., & Peelman, F. (2019). The Small GTPase Arf6: An Overview of Its Mechanisms of Action and of Its Role in Host–Pathogen Interactions and Innate Immunity. International Journal of Molecular Sciences, 20(9), 2209. https://doi.org/10.3390/ijms20092209

Figures 1-4 were created with BioRender.com