Using pathway-specific reporter and direct conversion constructs to investigate Juvenile Neuronal Ceroid Lipofuscinosis.

Abstract

Purpose : Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), or Batten disease, is a lysosomal storage disorder caused by mutations in CLN3 that cause blindness, central nervous system deterioration and death. Little is known about how genetic insults to CLN3 lead to accumulation of lysosomal aggregates or the mechanism of neuronal cell death. The purpose of this study is to generate fluorescent reporter constructs specific to pathways that we hypothesize to contribute to the pathophysiology of Batten disease.

Methods : We obtained dermal skin biopsies from two patients suspected clinically to have Batten disease. We isolated and cultured fibroblasts from each patient and used Sanger sequencing to molecularly confirm mutations in CLN3: patient 1 (1.02 kb del/Leu101del3CTC) and patient 2 (1.02 kb del/1.02 kb del). We cloned lentiviral constructs harboring fluorescent reporter constructs for investigation of autophagy (LC3-GFP/RFP; LC3 turns from green to red due to acidic pH within lysosomes) and endoplasmic reticulum (ER)-mediated stress (CHOP-tRFP). Each vector carries a blastocidin selection cassette that is suitable for CLN3 patient-specific stable cell line establishment. We also cloned a lentiviral plasmid carrying the transcription factors ASCL1, BRN2 and MYT1L, previously published for use in transdifferentiation of fibroblasts into neurons. Lentiviral plasmids were transiently transfected via electroporation into human control dermal fibroblasts to test their function and response to autophagy- and ER stress-inducing pharmacological agents.

Results : Stimulation of autophagy via amino acid starvation lead to an increase in the ratio of LC3-RFP:LC3-GFP-postive punctae. Inhibition of autophagic flux using either bafilomycin-A1 or chloroquine decreased the number of LC3-RFP-positive punctae localized to lysosomes as anticipated. Expression of CHOP-tRFP was restricted to the nuclei of fibroblasts. Transfection of the lentiviral plasmid carrying ASCL1, BRN2 and MYT1L did not induce aberrant cell death.

Conclusions : Fibroblasts from Batten disease patients are viable, and control fibroblasts tolerate transfection with ASCL1, BRN2 and MYT1L. Future experiments will test the ability of exogenous expression of these transcription factors to transdifferentiate CLN3 patient fibroblasts into neurons and will utilize pathway-specific reporters to evaluate Batten disease pathophysiology.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.