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

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.

The SARS-CoV-2 cytopathic effect is blocked with autophagy modulators

“…ROC-325 (38)(39)(40) and clomipramine (41,42) display autophagy inhibitor activity that can completely prevent SARS-CoV-2 CPE without any significant inherent cytotoxicity. Hycanthone, an FDA-approved schistosomicide and oxidative metabolite of lucanthone (43)(44)(45), and mefloquine (46-48) both showed moderate levels of activity against SARS-CoV-2 CPE, but did exhibit drug-induced cell toxicity at the highest drug concentration tested (up to 30 µM) . The autophagy inhibitor verteporfin, a benzoporphyrin derivative used in the clinic as a photosensitizer (49) (58).…”

Succinic Semialdehyde Dehydrogenase Deficiency: An Update

“…Modulation of autophagy is expected to show high potential for diseases as diverse as metabolic disorders, cancers, neurodegenerative, and infectious diseases [81,82], and drugs that modulate the autophagic flux and exhibit therapeutic potential for various diseases have been identified in drug screens [82]. Disease-specific autophagy drug screens may also reveal novel pathways to be targeted for autophagy induction, as has recently been shown for juvenile neuronal ceroid lipofuscinosis, where pathways, such as calcium signaling, isoprenoid pathway, and microtubule dynamics, were identified as potential drug targets for ameliorating autophagy defects [83]. Therefore, drug screens for further potential autophagy modulators could also be carried out for SSADH-D to identify novel disease-specific pathways that could be targeted to enhance autophagy.…”

The rapidly evolving view of lysosomal storage diseases

“…Furthermore, a cell‐based assay was used to identify three compounds that enhance galactocerebrosidase activity (Jang et al , 2016). Another phenotypic‐based approach was used to identify modulators of autophagy in a murine neuronal cell model of CLN3 disease and led to the identification of compounds that normalized lysosomal positioning and promote clearance of storage material (Petcherski et al , 2019).…”