6 minute read
Understanding Effects of Chronic Methylphenidate Use on Neural Viability and Plasticity, Hannah Oakes, Biomedical Sciences, Pharmaceutical Sciences, Ph.D
UNDERSTANDING EFFECTS
of Chronic Methylphenidate Use on Neural Viability and Plasticity
Written by Hannah Warren
Hannah V. Oakes
Biomedical Sciences, Ph.D., concentration in Pharmaceutical Sciences
Dr. Brooks Pond, Faculty Advisor
Hannah Oakes transferred to ETSU as a first-generation college student from Northeast State Community College where she earned a total of three degrees with five majors. Originally intending to become an engineer, her initial degrees were in chemistry, mathematics, physics, general pre-engineering, and pre-engineering with a chemistry focus. Over the course of her education, she became particularly interested in cells and cell signaling, leading to a double major in biology with a biochemistry concentration and chemistry for her bachelor’s degree at ETSU. During the summer before her senior year, she was accepted into ETSU’s McNair program where she gained research experience within the Biochemistry department at the Quillen College of Medicine. After completion of her bachelor’s degree, she was accepted directly into ETSU’s Biomedical Sciences Ph.D. program.
Hannah’s dissertation advisor, Dr. Brooks Pond, earned her Ph.D. in Pharmacology from Duke University and completed a postdoctoral fellowship at St. Jude Children’s Research Hospital. Much of her work focuses on neuropharmacology, an area that Hannah was interested in. Hannah’s work with Dr. Pond has culminated with the completion of her dissertation on the effects of chronic use of the psychostimulant methylphenidate (Ritalin®) on neural growth and development in a multipart project. Over the past several decades, the number of individuals who have been prescribed methylphenidate for attention deficit hyperactivity disorder (ADHD) has been on the rise. Additionally, most take methylphenidate for many years, from childhood or adolescence to adulthood, which is an important time of brain development. Despite this, little is known about how this drug affects the brain over time.
Some antidepressants have been shown to increase neurogenesis or the “birth of new neurons” within the hippocampus, the area of the brain responsible for memory and learning, and Hannah wanted to know if chronic treatment with methylphenidate (which
shares some overlapping mechanisms with antidepressants) could also result in increased neurogenesis. Together, she and Dr. Pond created a mouse model where male mice were given intraperitoneal injections, twice daily, of methylphenidate replicating the dosing schedule of a child that is prescribed the drug for a total of twenty-eight days. Another group received higher dosages representative of an individual abusing methylphenidate. Additionally, all mice received injections of 5-ethynyl-2’-deoxyuridine (EdU), a thymidine analog that is incorporated into replicating DNA just prior to cell division. EdU allowed for the permanent “tagging” of newly created neurons, which can be labeled with a red fluorescent molecule through a simple chemical reaction. Then, tissue samples were collected and doublelabeled using immunohistochemistry for a general neuronal marker, causing all neurons to light up green. This enabled Hannah to count new neurons labeled with EdU (red) and create ratios of the number of new cells to total neurons (green). Analysis showed that chronic methylphenidate did increase neurogenesis within the hippocampus and that this effect was dose-dependent.
From other literature, Hannah knew that, even though drugs may stimulate neurogenesis, these new neurons do not always survive. Thus, in a second project, she repeated the model from the first study, but at the end of this twentyeight-day cycle, she further divided the mice into two more groups, one that received an additional month of treatment and one that received no drug during that time. Following the same analysis from the initial study, she found that if methylphenidate was continued, the new cells survived, but if it was discontinued, the new cells were less likely to survive. This relationship was also dose-
dependent, and the higher the dose of methylphenidate given, the greater the cell loss was when the drug was discontinued.
In an effort to examine the mechanism by which methylphenidate influences, neurogenesis, Hannah examined the expression of various neurotrophic factors. Neurotrophic factors are proteins in the body that promote neurogenesis and development. She suspected that brain derived neurotrophic factor (BDNF) and glial cell-line derived neurotrophic factor (GDNF) might be responsible since one or both are typically elevated during neurogenesis; however, she saw no significant elevation that would indicate they were being affected by the drug in this case, which was surprising. She went back to existing literature and
found other proteins to explore which included beta catenin, TrkB (the receptor for BDNF), and vascular endothelial growth factor (VEGF). Increases in these proteins have also been associated with increases in neurogenesis. Interestingly, these proteins were increased, and their patterns of expression seemed to align with the changes in neurogenesis she was observing. Hannah used this data to publish a paper in the Journal of Neural Transmission.
Hannah’s other study with methylphenidate examined neuron sensitivity to the neurotoxin 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP was discovered in the early eighties when a group of individuals attempted to illicitly synthesize meperidine, a synthetic opioid, and accidentally produced this major byproduct in their synthesis reaction. Within days of unknowingly injecting themselves with MPTP, users developed Parkinson’s disease (PD). MPTP is structurally similar to many pesticides, and PD tends to appear more frequently in agricultural regions. As such, MPTP is frequently used to model PD. Hannah built upon a previous study Dr. Pond was a part of that found that chronic methylphenidate use increased the sensitivity of neurons within the nigrostriatal pathway (the area of the brain affected by PD) making them more susceptible to the neurotoxic effects of MPTP. Hannah hypothesized that the sensitization could be caused by oxidative stress from excess dopamine, a pharmacologic effect of methylphenidate. Excess dopamine may be oxidized to form a quinone, which can lead to production of damaging free radicals. Fortunately, the brain has an antioxidant, glutathione, that can be conjugated to the quinone and prevent oxidative stress. However, over time, glutathione can be depleted, leaving neurons vulnerable. Interestingly, this pathway does appear to contribute to the effects of methylphenidate. Hannah found that long-term methylphenidate increased levels of dopamine quinones and subsequently depleted levels of glutathione within the nigrostriatal pathway. This work was published in Pharmacological Reports.
Finally, Hannah wanted to investigate differences in male and female responses to methylphenidate and subsequent MPTP challenge. Hannah wanted to understand why females who produce more estrogen (a neuroprotective agent) and are not, under normal conditions, as likely to develop Parkinson’s become more likely to develop it if they use methamphetamine, which increases dopamine, similarly to methylphenidate. She recreated the MPTP model, but utilized female mice in two different stages of cycling. Some mice were induced to be in anestrous (low estrogen), while others were induced to be in proestrous (high estrogen). Hannah is conducting neuronal counts of cells within the nigrostriatal pathway, as well as measuring quinone production and glutathione levels to examine this question. She is currently working to analyze this data.
Hannah hopes that her the research will draw attention to the need for female models in research, and that as more literature on this topic is produced, that individuals can be better informed of the potential long-term risks and benefits of chronic methylphenidate use. She plans to continue to publish findings from her data as analysis is complete. She is currently employed full time at Crown Laboratories and plans to continue working there after her graduation in May. Moving forward, she is interested in both industry driven research and academic research. Hannah encourages undergraduate students to take advantage of all the academic opportunities they are presented with. Look for scholarships, explore programs and services that are available to help them reach their full potential, and to take time to find a strong mentor for guidance along the way.
Hannah Oakes
