Abstract—The in-trans delivery of protein therapeutics across the blood-brain barrier by K16ApoE peptide carrier has been demonstrated to improve the neurological symptoms and increase the life-span of late-infantile neuronal ceroid lipofuscinosis (LINCL) mice. However, acute toxicity of K16ApoE was observed in LINCL mice resulting in a narrow therapeutic index, limiting the potential of translating the K16ApoE into a viable drug delivery system. This study aims to unravel the toxic mechanism of action. We hypothesized that the toxic response towards the peptide was induced by inhibition of acetylcholinesterase (AChE) activity at neuro-muscular junction. Here, results from the dose-response study suggested that AChE activity was inhibited by K16ApoE at either low or high doses but not at the mid-dose where a significant increase in AChE activity was observed. Meanwhile, molecular docking simulations showed that the N-terminus of K16ApoE is capable of binding to the active site gorge of AChE. In addition to a favorable spatial orientation, this docking pose also revealed strong surface charge interactions which may account for the observed inhibitory effect. While statistical analysis of the dose response and survival ratio suggested that AChE is not the primary mechanism of action for the acute toxicity of K16ApoE, both biochemical evidence and structural analysis have assigned indirect but critical roles for AChE in the overall toxicity mechanism of this peptide carrier. I. INTRODUCTION Late-infantile neuronal ceroid lipofuscinosis (LINCL) is one of the most common forms of neuronal ceroid lipofuscinosis (NCL), which are a group of recessively-inherited neurodegenerative lysosomal storage diseases (LSDs) that primarily affect children [1]. LINCL is caused by mutations in TPP1 (the gene formally named CLN2), resulting in deficiencies in the lysosomal protease tripeptidyl peptidase I (TPP1) [2]. Manifestations of LINCL include seizures, loss of vision and locomotor function, progressive mental decline and premature death, typically less than 15-years. *Research supported by Zhejiang Provincial Natural Science Foundation of China under Grant number LY16H080008 and National Natural Science Foundation of China under Grant number: 81703410 and Wenzhou-Kean University Student-Partnering-with-Faculty (SpF) Research Funding. L. Lu is with Wenzhou-Kean University, Wenzhou, Zhejiang 325060 China (e-mail: lulu@kean.edu). T. M. Michelena is with Wenzhou-Kean University, Wenzhou, Zhejiang 325060 China (e-mail: tobmiche@kean.edu). A. Wong Author is with Wenzhou-Kean University, Wenzhou, Zhejiang 325060 China (e-mail: alwong@kean.edu). C. J. Zhang is with Wenzhou-Kean University, Wenzhou, Zhejiang 325060 China (e-mail: czhang@kean.edu). Y. Meng is with Wenzhou-Kean University, Wenzhou, Zhejiang 325060 China (phone: +86-0577-5587-0775; e-mail: ymeng@kean.edu). * Correspondence should be addressed to Y. Meng. (ymeng@kean.edu). There is currently no effective treatment for LINCL but progress is being made. In general, enzyme replacement therapy (ERT) is the most successful clinical approach for LSDs [3-5]. However, the blood-brain barrier (BBB) presents a major hurdle for the peripherally-administration of protein into the CNS and it limits the application of ERT in patients with neurological diseases. Direct administration of therapeutics via the cerebrospinal fluid has shown promise in LSD animal models, and one clinical trial has been initiated using intracerebroventricular delivery of ERT. However, while hopefully effective, this route is highly invasive and there are significant concerns whether it can represent a life-long solution to the disease [6-8]. Thus, there is continued interest in relatively non-invasive methods to help intravenously-administered recombinant proteins cross the fully-formed BBB, especially given that the extensive microvasculature of the brain potentially allows for widespread distribution. Previous studies have described ways to help recombinant lysosomal proteins cross the BBB but in animal models, results have been modest. Levels achievable in the brain were relatively low, typically <10% of normal, and therapeutic value in terms of increasing life-span remained to be demonstrated in a convincing way [9-14]. II. RELATED WORK We described a method that allows for extremely effective delivery of recombinant human TPP1 (rhTPP1) to lysosomes throughout the brain after intravenous administration, achieving up to 8-fold greater than normal wild-type TPP1 levels. Here, passage of rhTPP1 across the BBB is mediated by a co-injected, 36-residue peptide containing polylysine and apolipoprotein E sequences (K16ApoE). To our knowledge, this is the most effective demonstration to date of delivery of a therapeutic lysosomal protein to the brain by an intravenous route. Moreover, peptide-mediated delivery of TPP1 from the bloodstream into the brain is well-tolerated at therapeutic doses and translates into significantly increased lifespan and improved neurological function [15, 16]. However, K16ApoE exhibits the dose-dependent toxicity administered alone or in conjunction with TPP1 15 . When injected with moderate to high doses of the K16ApoE, mice exhibited significant difficulty breathing and muscle spasms ultimately leading to death at high doses [15, 16]. Therefore, K16ApoE has a very narrow therapeutic index. Characterization of peptide variants suggested that the toxicity and efficacy both are associated with the positive charge of K16ApoE. Eliminating the toxicity by removing the positive charges from K16ApoE resulted in diminishing the uptake of TPP1 to the brain [16]. The symptoms associated with toxicity are similar to those exhibited by organisms that are exposed to The inhibition of acetylcholinesterase by a brain-targeting polylysine-ApoE peptide: biochemical and structural characterizations L. Lu, T.M. Michelena, A. Wong, C.J. Zhang, Y. Meng * 978-1-5386-3646-6/18/$31.00 ©2018 IEEE 155