Human iPSC models of neuronal ceroid lipofuscinosis capture distinct effects of TPP1 and CLN3 mutations on the endocytic pathway

Abstract

Neuronal ceroid lipofuscinosis (NCL) comprises ∼13 genetically distinct lysosomal disorders primarily affecting the central nervous system. Here we report successful reprograming of patient fibroblasts into induced pluripotent stem cells (iPSCs) for the two most common NCL subtypes: classic late-infantile NCL, caused by TPP1(CLN2) mutation, and juvenile NCL, caused by CLN3 mutation. CLN2/TPP1- and CLN3-iPSCs displayed overlapping but distinct biochemical and morphological abnormalities within the endosomal-lysosomal system. In neuronal derivatives, further abnormalities were observed in mitochondria, Golgi and endoplasmic reticulum. While lysosomal storage was undetectable in iPSCs, progressive disease subtype-specific storage material was evident upon neural differentiation and was rescued by reintroducing the non-mutated NCL proteins. In proof-of-concept studies, we further documented differential effects of potential small molecule TPP1 activity inducers. Fenofibrate and gemfibrozil, previously reported to induce TPP1 activity in control cells, failed to increase TPP1 activity in patient iPSC-derived neural progenitor cells. Conversely, nonsense suppression by PTC124 resulted in both an increase of TPP1 activity and attenuation of neuropathology in patient iPSC-derived neural progenitor cells. This study therefore documents the high value of this powerful new set of tools for improved drug screening and for investigating early mechanisms driving NCL pathogenesis.

Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function

“…20). We have also recently reported that human CLN3 ⌬ex7/8/⌬ex7/8 NPCs display normal ER morphology, yet upon differentiation into mature neurons, which causes progression of other NCL-related phenotypes such as accumulation of storage material, the ER was dilated (11). These observations combined with the key observation in this study that neuronal lineage cells bearing the common JNCL mutation in Cln3/CLN3 are more sensitive to the effects of thapsigargin-mediated Ca 2ϩ release, might suggest that despite the lack of overt ER stress, JNCL cells may be more poised to elicit such a response to further stressors.…”

“…Human NPCs were derived and maintained as described previously (11). The control line was derived from GM8330-8 patient fibroblasts.…”

“…ab51520, used at 1:500 dilution), cells were fixed in an ice-cold 50:50 (v/v) mixture of methanol/acetone for 10 min. The immunostaining procedure was performed as described previously (11,20). Briefly, 0.05% Triton X-100 diluted in PBS, pH 7.4, was used for cell permeabilization, and 5% bovine serum albumin (BSA) diluted in PBS, pH 7.4, was used for blocking.…”

“…Human CLN3 Patient Neural Progenitor Cells Display Thapsigargin-responsive Abnormal Autophagosome Accumulation-We have recently established a set of human neural progenitor cell lines (NPCs) by reprogramming of JNCL (CLN3) patient skin fibroblasts and unaffected control skin fibroblasts to generate iPSCs, which were then differentiated into a neurally committed lineage (11). Importantly, we therefore next sought to test whether the effects of thapsigargin treatment on autophagosome accumulation in the CbCln3 ⌬ex7/8/⌬ex7/8 murine cells would also be observed in CLN3 patient NPCs.…”

“…Although the primary function of CLN3 has not yet been fully uncovered it is proposed to play a role in vesicular trafficking because its deficiency leads to altered dis-tribution of endosomal and lysosomal proteins and phospholipids (7)(8)(9)(10)(11)(12), abnormal morphology of endocytic and lysosomal organelles (7,11), lysosomal pH dyshomeostasis (13)(14)(15), and amino acid transport defects (16). The hallmark JNCL storage material containing subunit c accumulates in both autophagosomes and lysosomes implicating further impact of CLN3 deficiency on the autophagy pathway (17).…”