CRISPR/Cas9 "CLN8 null" towards a dysfunctional autophagy in CLN8-associated neuronal ceroid lipofuscinosis diseases-ePoster # 4774

ePoster # 4774 Presenter: Rosaria Tinnirello The neuronal ceroid lipofuscinoses (NCLs) are autosomal recessive diseases with progressive dementia, motor disturbance, epilepsy, visual loss, affecting mainly children but also adults. Mutations in 13 distinct genes cause NCLs with marked phenotypic heterogeneity and undefined pathomechanism. CLN8 gene shows wide complexity with 31 different mutations leading to late-infantile NCL variants (vLINCL) and progressive epilepsy with mental retardation (EPMR). CLN8 encodes an ER-resident transmembrane protein with an ER-ERGIC retrieval signal and potential multiple functions (Passantino et al., 2013; di Ronza et al., 2018). How CLN8 mutations may lead to lysosomal dysfunction and accumulation of typical autofluorescent material of NCL diseases is an open question. Previously we reported that some proteins closely related to autophagic processes are potential interactors of CLN8 (1). We here show that CRISPR/Cas9 CLN8-null clones generated in our lab maintain survival and migration capacities similar to those of the "maternal" HeLa cell line but they grow as cells predispose to autophagic impairment. The CLN8-null clones show accumulation of auto-fluorescent material following treatment with the cell cycle blocker hidroxy-urea, spontaneous aggregates of ?-synuclein suggesting alterations in vesicle trafficking, an high amount of the immature unglycosylated Lamp1 and, finally, a higher defective autophagosomal/lysosomal response to modulators (thapsigargin, chloroquine or bafilomycin or starvation) as compared to the "maternal" cells. These data together with an impaired autophagosomal-lysosomal fusion reported in the cerebellum of the CLN8mnd mouse model suggest a direct CLN8 involvement in autophagic processes and indicate CRISPR/Cas9 CLN8-null cells as a platform to investigate intervention strategies helping lysosomal degradation system.