The Role of Muscarinic Acetylcholine Signaling in Oligodendrocyte Maturation Brittany Hayden* and Jennifer Ness-Myers* *Department of Biology, Messiah University, Mechanicsburg, PA..

Abstract Results The myelin sheath is a protective insulating layer that covers neurons in the 1.6 nervous system. Myelin supports the generation of rapid electrical signals to ensure efficient communication between neurons. Within the central nervous 1.4 system, myelin is produced by specialized cells called oligodendrocytes. Unfortunately, in neurodegenerative diseases such as multiple sclerosis, the 1.2 myelin sheath undergoes damage which leads to neuron degradation and disruption in neuron communication. In order to prevent and treat 1 neurodegenerative diseases, current research seeks to further understand 0.8 the mechanism underlying myelination and identify treatments that could RQ promote remyelination of neurons. One pathway known to be involved in regulating myelination in oligodendrocytes is the muscarinic acetylcholine 0.6 signaling pathway. Research suggests inhibiting specific receptors in this A B 0.4 pathway promotes the generation of new myelin in humans. Our lab sought to Figure 2. Primary rat OPC cultures. (A,B) Phase contrast micrographs of viable OPCs, of further investigate the impact of several antagonists on oligodendrocyte 3 bipolar or tripolar morphology, originating from neonatal rat cortices . 0.2 precursor cells (OPCs) differentiation and remyelination. OPCs were isolated from neonatal rat brains, cultured, and treated with specific antagonists. Gene 0 expression analysis was performed in order to assess oligodendrocyte Methods MAG MBP MAG MBP MAG MBP maturation. Control D+P Clem Mixed Glial Preparation: Forebrain cortices from rat newborns were dissected, meninges were removed, and the tissue was digested to obtain glial cells. Cells were Figure 3. Myelin protein expression in treated OPCs. Quantitative PCR was Introduction plated at a density of 1.5x10 7 cells per flask until confluent, about 10-12 days. Media performed to analyze gene expression levels of MAG and MBP of three different was changed every two days. treatments: control (DMSO), Darifenacin + Pirenzepine, and Clemastine. The RQ • Neurons send electrical signals along their axons that are more rapidly was determined based on the expression compared to the levels of GADPH. transmitted with the presence of myelin. Oligodendrocytes are the myelinating OPC Isolation and Culture: Once phase-bright OPCs were visible on a confluent bed of astrocytes, a differential 1 hour shake was used to remove the microglia, and 18 neuroglia cells of the CNS and as such produce the myelin that encloses the 1.8 axon of a neuron 1. Unfortunately, in the case of neurodegenerative diseases hours to detach the OPCs. The OPCs were then plated on Poly-D-lysine coated plates (NDs), myelin undergoes degradation. at a density of 2x10 4 cells per cm 2. The cultures were incubated and fed with media 1.6 every two days until they were 80-90% confluent. 1.4 • One cell signaling pathway involved in myelination is the muscarinic Passaging OPCs: Papain was used to selectively detach the OPCs from the plates. acetylcholine signaling pathway. Inhibition of this pathway with antagonists have The cells were collected via centrifugation and plated for treatment. 1.2 been shown to promote oligodendrocyte differentiation and remyelination1. Treatment of OPCs with Muscarinic Antagonists: OPCs were treated everyday for 2-4 days with muscarinic antagonists including Pirenzepene, Darifenacin, Clemastine, 1 • The goal of this study was to investigate the treatment paradigm of MS and Cevimeline, and DMSO (control). RQ other NDs through the analysis of myelin protein gene regulation in primary 0.8 oligodendrocyte cultures. We aimed to compare specific muscarinic antagonists RNA Isolation and Reverse Transcription: Following treatment, RNA was isolated 0.6 and the downstream effects of maturation in oligodendrocytes. from the OPCs using PureLink RNA Mini Kit per the manufacturers protocol. To perform this, Trizol was added to the plates and cells were scraped off. Isolated RNA 0.4 was reverse transcribed to cDNA using a High-Capacity RNA-to-cDNA Kit per manufacturers protocol. 0.2 Real-time PCR: Taqman primers were used for qPCR to analyze gene expression Figure 1. Oligodendrocytes and 0 myelin. (A) Schematic of levels of certain targets such as MBP, MAG, and c-fos, that are indicative of cFOS cFOS cFOS cFOS cFOS oligodendrocytes (in purple) and myelin myelination. Control Cev Pir + Cev Dar + Cev Clem + Cev internodes (in blue). Oligodendrocytes can myelinate multiple axons (in light Treatment Target Figure 4. cFOS expression in treated OPCs. Quantitative PCR was performed to gray) by extending various processes analyze gene expression levels of cFOS of five different OPC treatments: DMSO Darifenacin M3 antagonist that contact myelin internodes. (B) (control), Cevimeline only, Pirenzepine + Cevimeline, Darifenacin + Cevimeline, Vertical and longitudinal slices through Pirenzepine M1 antagonist and Clemastine + Cevimeline. The RQ was determined based on the expression a myelin sheath. Here, the inner and compared to the levels of GADPH. outer tongues of the myelin are visible, Clemastine M1 and M3 antagonist rich in cytoplasm (indicated by pink). Paranodal loops, also rich in cytoplasm, Cevimeline M1 and M3 agonist Conclusions are present and function to anchor the • Treatment with antagonists should be performed for 3 or more days in order to myelin membrane to the axon 2. Table 1. Target muscarinic receptor of treatments. There are five different subtypes ensure adequate and detectable gene expression of downstream signals. of receptors in the muscarinic pathway (M1-M5). Each antagonist or agonist used as • The gene expression of MAG (myelin-associated glycoprotein) and MBP an OPC treatment targets one or more of these receptor subtypes. (myelin basic protein) in OPCs decreased with the treatment of Darifenacin + Pirenzepine, which differs from previous research. References 1Mao L-M, Faris HJ, Wang JQ. 2018. Muscarinic Acetylcholine Receptors Inhibit Fyn Activity in Acknowledgments • cFOS is an immediate early response gene involved in cell proliferation and the Rat Striatum In Vivo. J Mol Neurosci 64:523–532. I would like to thank Messiah University and the Department of Biological Sciences for providing the differentiation. The upregulation in gene expression of cFOS in OPCs observed 2Michalski J-P, Kothary R. 2015. Oligodendrocytes in a Nutshell. Front Cell Neurosci [Internet] resources and laboratory space throughout this research. I would also like to thank Dr. Ness-Myers for with the treatment of Darifenacin + Cevimeline indicates initiation of cellular 9. Available from: https://www.frontiersin.org/articles/10.3389/fncel.2015.00340/ful her continued guidance and support, as well as the other members of her lab Kylee Kimbel, Halle processes. 3Chen Y, Balasubramaniyan V, Peng J, Hurlock EC, Tallquist M, Li J, Lu QR. 2007. Isolation Becker, and Ronnie Woodring for their encouragement and assistance. and culture of rat and mouse oligodendrocyte precursor cells. Nat Protoc 2:1044–1051.l