Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS

Claire E Hall,Russell Burley,Zhi Yao,Nicholas M Luscombe,Giulia E Tyzack,L Miguel Martins,Sonia Gandhi,Elisavet Preza,Jasmine Harley,Christopher R Sibley,Rickie Patani,Selina Wray,Andrey Y Abramov,Andrea Serio,Molly M Stevens,Jernej Ule,Dimitri M Kullmann,Charles Arber ,Raphaëlle Luisier ,Samantha H Y Loh ,András Lakatos ,Sarah Crisp ,Patricia M Watson ,Minee L Choi

doi:10.1016/j.celrep.2017.05.024

Abstract

SummaryMotor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.

Highlights

Generation of induced pluripotent stem cells (iPSCs), Spinal Cord Motor neurons (MNs), and ACs Using established reprogramming methods (Okita et al, 2011), four clones of mutant iPSCs were generated from two patients with confirmed valosincontaining protein (VCP) mutations: R191Q (2 clones), R155C (2 clones) (Ludtmann et al, 2017)
We developed robust methods for generating highly enriched cultures of both MNs and ACs in feeder-free, chemically defined monolayer culture by adapting previously published protocols (Figure 1A) (Chen et al, 2014)
Noting that ER stress is associated with altered contact between ER and mitochondria, we quantified mitochondrial-ER contacts using an electron microscopy (EM) approach (Schneeberger et al, 2013), which confirmed a significant increase in the VCP-mutant d17 MNs compared to control (59.1% ± 2.4% versus 40.4% ± 4.1%, p < 0.05, unpaired t test; Figure 3E)

Summary

Graphical Abstract

Hall et al use iPSCs to examine the sequence of events by which motor neurons degenerate in a genetic form of ALS. They find that astrocytes, a type of supportive cell, degenerate under these conditions. The ALS-causing mutation disrupts the ability of astrocytes to promote survival of motor neurons. Highlights d Robust and enriched motor neurogenesis and astrogliogenesis from human iPSCs. Hall et al, 2017, Cell Reports 19, 1739–1749 May 30, 2017 a 2017 The Authors.

INTRODUCTION

RESULTS

30 Control VCP

DISCUSSION

EXPERIMENTAL PROCEDURES