An Autophagy Modifier Screen Identifies Small Molecules Capable of Reducing Autophagosome Accumulation in a Model of CLN3-Mediated Neurodegeneration.

Anton Petcherski,Susan Cotman,Stephen Haggarty,Elisabeth Butz,Surya Reis,Madeleine Klein,Mika Ruonala,Uma Chandrachud,Wen-Ning Zhao

doi:10.3390/cells8121531

Anton Petcherski, Susan Cotman + Show 7 more

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https://doi.org/10.3390/cells8121531

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Abstract

Alterations in the autophagosomal–lysosomal pathway are a major pathophysiological feature of CLN3 disease, which is the most common form of childhood-onset neurodegeneration. Accumulating autofluorescent lysosomal storage material in CLN3 disease, consisting of dolichols, lipids, biometals, and a protein that normally resides in the mitochondria, subunit c of the mitochondrial ATPase, provides evidence that autophagosomal–lysosomal turnover of cellular components is disrupted upon loss of CLN3 protein function. Using a murine neuronal cell model of the disease, which accurately mimics the major gene defect and the hallmark features of CLN3 disease, we conducted an unbiased search for modifiers of autophagy, extending previous work by further optimizing a GFP-LC3 based assay and performing a high-content screen on a library of ~2000 bioactive compounds. Here we corroborate our earlier screening results and identify expanded, independent sets of autophagy modifiers that increase or decrease the accumulation of autophagosomes in the CLN3 disease cells, highlighting several pathways of interest, including the regulation of calcium signaling, microtubule dynamics, and the mevalonate pathway. Follow-up analysis on fluspirilene, nicardipine, and verapamil, in particular, confirmed activity in reducing GFP-LC3 vesicle burden, while also demonstrating activity in normalizing lysosomal positioning and, for verapamil, in promoting storage material clearance in CLN3 disease neuronal cells. This study demonstrates the potential for cell-based screening studies to identify candidate molecules and pathways for further work to understand CLN3 disease pathogenesis and in drug development efforts.

Highlights

CLN3 disease, classified as a lysosomal disease, is the classical juvenile onset form of neuronal ceroid lipofuscinosis (NCL), or Batten disease, and it is the most common form of childhood-onset neurodegeneration
In the current study, we sought to expand upon our previous work to identify additional autophagy modifying compounds, especially those that would significantly decrease the burden of GFP-LC3-positive autophagic vesicles in CbCln3∆ex7/8/∆ex7/8 cells, reasoning that these compounds may alleviate the reduced flux seen in the absence of CLN3 function
Three of the top phenotype suppressors identified here were studied in further detail; fluspirilene, nicardipine, and verapamil were validated to induce autophagic flux and were shown to have activity in suppressing abnormal lysosomal positioning

Summary

Introduction

CLN3 disease, classified as a lysosomal disease, is the classical juvenile onset form of neuronal ceroid lipofuscinosis (NCL), or Batten disease, and it is the most common form of childhood-onset neurodegeneration. CLN3 disease is caused by mutations in the CLN3 gene, found on chromosome. In CLN3 disease patients, vision loss between ~4 and 8 years of age is typically the first recognized symptom, followed by cognitive impairment and onset of seizures. A progressive decline in cognition and motor function is seen over the decade of life, and late-onset cardiac symptoms can develop [2,3]. Palliative care to manage symptoms is the only treatment option, and CLN3 disease is fatal, with life expectancy not typically exceeding the early twenties [2]. Despite the identification of the CLN3 gene nearly

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