A NEW HOPE FOR MULTIPLE SYSTEM ATROPHY: A TRANSLATIONAL RESEARCH OF A NOVEL NMDAR RECEPTOR MODULATOR FROM PRECLINICAL MODELS TO PATIENTS

Abstract Background NMDA receptor (NMDAR) is an essential glutamate receptor and as a contributor to neurodegenerative disorder1,2. Until now, NMDAR (especially glycine modulatory site (GMS) of the NMDARs) has been proposed to be a potential therapeutic target for the treatment of neurological disorders, such as multiple system atrophy (MSA)3. MSA, an atypical parkinsonism, is a fatal and rapidly progressive neurodegenerative disease with autonomic dysfunction and cerebellar deficits4-6. Up to now, the etiology and pathogenesis of MSA remains unclear4. One potential mechanism in MSA is accumulation of α-synuclein aggregated in oligodendrocytes7,8, and aggregation of α-synuclein promotes the NMDAR internalization9,10, contributing to NMDAR dysfunction and LTP impairment11,12. Unfortunately, currently available medications have not been proven efficacious in treating symptoms of MSA13. The need to develop novel therapeutic agents for the “unmet medical needs” in patients with MSA is of upmost importance. Take advantage of virtual high-throughput screening and AI-based predicted assay, a novel NMDAR modulator, RS-D7, was discovered with safety and efficacy profile. It is urgent and imperative to evaluate the therapeutic potentials of RS-D7 in MSA from basics to clinical trials. Aims & Objectives The aims of this study were to evaluation the therapeutic potential and mechanism of RS-D7 on the alleviation of MSA. Taking advantage of in vitro assays, in vivo efficacy tests, and proof- of-concept clinical trials in drug development, a series of experiments was conducted on RS-D7 in this study. Method In Experiment 1, enzymatic inhibition and cell-based inhibition assay was performed to examine the inhibition effect of RS-D7 on D-amino acid oxidase (DAO). To further determine the mechanism of RS-D7 on NMDAR, surface dynamics of NMDAR with RS-D7 was analyzed in primary neurons using single- particle tracking in Experiment 2, and fEPSP and LTP were recorded in hippocampal slices in Experiment 3. In Experiment 4 to 6, a series of in vivo animal experiments were performed with RS-D7 treatment using wild-type (WT), pharmacological (MK-801, a NMDAR antagonist) and genetic (TgM83 mice overexpressing human A53T α-synuclein) mouse models mimicking ataxia/MSA, respectively. In Experiment 7, an open-labeled, proof-of-concept clinical trial in MSA patients was conducted with RS-D7 prodrug (RS-D7pro). Results In the inhibition assay, RS-D7 acted as a potent DAO inhibitor (IC50 = 0.32 uM) with direct competitive inhibition. Then, surface dynamics of hippocampal NMDARs were decreased by MK-801but alleviated by RS-D7. Meanwhile, MK-801-induced NMDAR-mediated fEPSP and LTP deficits were also ameliorated after RS-D7 treatment. Next, RS-D7 demonstrated a high safety profile on WT mice and RS- D7 improved MK-801-induced ataxic behaviors in a dosage-dependent manner. Moreover, MSA-related behavioral deficits, reduction of membrane-bound NMDAR subunits, and abnormal inflammatory responses in TgM83 mice were also reversed by RS-D7. Final, the clinical rating results from MSA proof- of-concept clinical trial indicated that three ataxic scores (SARA, UMSARS and ICARS) were significantly improved in MSA patients after 12-week RS-D7pro treatment, and the symptoms rebounded and worsened 12 weeks after discontinuing RS-D7pro. Discussion & Conclusion Collectively, these promising results support the therapeutic potential of RS-D7 in MSA and offer new hope for MSA patients. References 1.HARDINGHAM, G. E. &BADING, H. 2010. Synaptic versus extrasynaptic NMDA receptor signalling: implications for neurodegenerative disorders. Nat Rev Neurosci, 11, 682-96. 2.MELDRUM, B. S. 2000. Glutamate as a neurotransmitter in the brain: review of physiology and pathology. J Nutr, 130, 1007s-15s. 3.MESZAROS, L., HOFFMANN, A., WIHAN, J. &WINKLER, J. 2020. Current Symptomatic and Disease- Modifying Treatments in Multiple System Atrophy. Int J Mol Sci, 21. 4.FANCIULLI, A., STANKOVIC, I., KRISMER, F., SEPPI, K., LEVIN, J. &WENNING, G. K. 2019. Multiple system atrophy. Int Rev Neurobiol, 149, 137-192. 5.GILMAN, S., WENNING, G. K., LOW, P. A., BROOKS, D. J., MATHIAS, C. J., TROJANOWSKI, J. Q., WOOD, N. W., COLOSIMO, C., DNeurology, 71, 670-6. 6.FANCIULLI, A. &WENNING, G. K. 2015. Multiple-system atrophy. N Engl J Med, 372, 249-63. 7.PAPP, M. I., KAHN, J. E. &LANTOS, P. L. 1989. Glial cytoplasmic inclusions in the CNS of patients with multiple system atrophy (striatonigral degeneration, olivopontocerebellar atrophy and Shy-Drager syndrome). J Neurol Sci, 94, 79-100. 8.JELLINGER, K. A. 2018. Multiple System Atrophy: An Oligodendroglioneural Synucleinopathy1. J Alzheimers Dis, 62, 1141-1179. 9.CHEN, Y., YANG, W., LI, X., LI, X., YANG, H., XU, Z. &YU, S. 2015. alpha-Synuclein-induced internalization of NMDA receptors in hippocampal neurons is associated with reduced inward current and Ca(2+) influx upon NMDA stimulation. Neuroscience, 300, 297-306. 10.YU, W., YANG, W., LI, X., LI, X. &YU, S. 2019. Alpha-synuclein oligomerization increases its effect on promoting NMDA receptor internalization. Int J Clin Exp Pathol, 12, 87-100. 11.DURANTE, V., DE IURE, A., LOFFREDO, V., VAIKATH, N., DE RISI, M., PACIOTTI, S., QUIROGA-VARELA, A., CHIASSERINI, D., MELLONE, M., MAZZOCCHETTI, P., CALABRESE, V., CAMPANELLI, F., MECHELLI, A., DI FILIPPO, M., GHIGLIERI, V., PICCONI, B., EL-AGNAF, O. M., DE LEONIBUS, E., GARDONI, F., TOZZI, A. &CALABRESI, P. 2019. Alpha-synuclein targets GluN2A NMDA receptor subunit causing striatal synaptic dysfunction and 12.TERAVSKIS, P. J., COVELO, A., MILLER, E. C., SINGH, B., MARTELL-MARTINEZ, H. A., BENNEYWORTH, M. A., GALLARDO, C., OXNARD, B. R., ARAQUE, A., LEE, M. K. &LIAO, D. 2018. A53T Mutant Alpha-Synuclein Induces Tau-Dependent Postsynaptic Impairment Independently of Neurodegenerative Changes. J Neurosci, 38, 9754-9767. 13.MEISSNER, W. G., FERNAGUT, P. O., DEHAY, B., PERAN, P., TRAON, A. P., FOUBERT-SAMIER, A., LOPEZ CUINA, M., BEZARD, E., TISON, F. &RASCOL, O. 2019. Multiple System Atrophy: Recent Developments and Future Perspectives. Mov Disord, 34, 1629-1642.