A differential autophagy-dependent response to DNA double-strand breaks in bone marrow mesenchymal stem cells from sporadic ALS patients.

Shane Wald-Altman,Or Kakhlon,Miguel Weil,Edward Pichinuk

doi:10.1242/dmm.027938

Abstract

ABSTRACTAmyotrophic lateral sclerosis (ALS) is an incurable motor neurodegenerative disease caused by a diversity of genetic and environmental factors that leads to neuromuscular degeneration and has pathophysiological implications in non-neural systems. Our previous work showed abnormal levels of mRNA expression for biomarker genes in non-neuronal cell samples from ALS patients. The same genes proved to be differentially expressed in the brain, spinal cord and muscle of the SOD1G93A ALS mouse model. These observations support the idea that there is a pathophysiological relevance for the ALS biomarkers discovered in human mesenchymal stem cells (hMSCs) isolated from bone marrow samples of ALS patients (ALS-hMSCs). Here, we demonstrate that ALS-hMSCs are also a useful patient-based model to study intrinsic cell molecular mechanisms of the disease. We investigated the ALS-hMSC response to oxidative DNA damage exerted by neocarzinostatin (NCS)-induced DNA double-strand breaks (DSBs). We found that the ALS-hMSCs responded to this stress differently from cells taken from healthy controls (HC-hMSCs). Interestingly, we found that ALS-hMSC death in response to induction of DSBs was dependent on autophagy, which was initialized by an increase of phosphorylated (p)AMPK, and blocked by the class III phosphoinositide 3-kinase (PI3K) and autophagy inhibitor 3-methyladenine (3MeA). ALS-hMSC death in response to DSBs was not apoptotic as it was caspase independent. This unique ALS-hMSC-specific response to DNA damage emphasizes the possibility that an intrinsic abnormal regulatory mechanism controlling autophagy initiation exists in ALS-patient-derived hMSCs. This mechanism may also be relevant to the most-affected tissues in ALS. Hence, our approach might open avenues for new personalized therapies for ALS.

Highlights

To test whether DNA damage has a role in Amyotrophic lateral sclerosis (ALS), we investigated the response of the ALShMSCs to oxidative DNA damage, adopting the well-established effect of neocarzinostatin (NCS) to induce double-strand breaks (DSBs) to cause stress; this leads to similar biological effects to those induced by ionizing radiation and involves the known DNA repair molecular cascade (Kuo et al, 1984; Banin et al, 1998)
The apoptotic cell death pathway has been studied as the major cellular response to DNA damage, recent reports on ionizing radiation (IR) and NCS-induced DNA damage on cancer cells suggest that autophagy plays an important role in determining cell fate (Rodriguez-Rocha et al, 2011; Guerra, 2012), by acting either as a death mediator through autosis (Liu and Levine, 2015) or as a cytoprotective mechanism exerting futile attempts for cell rescue in face of cell injury and cell death
We found in our NCS-mediated DNA damage ALS-human mesenchymal stem cells (hMSCs) model an overall increase in the autophagy marker LC3 by western blot (WB) analysis at 24 h post NCS induction, representing more than 30% of the cell population, as judged by cell-based high-content analysis (HCA) of the LC3 integrated intensity levels

Summary

Introduction

This unique ALShMSC-specific response to DNA damage indicates that an intrinsic abnormal regulatory mechanism controlling cytotoxic autophagy initiation exists in sALS-patient-derived hMSCs. This result emphasizes the viability of using ALS-patient-derived nonneuronal cells, like hMSCs, in searching for intrinsic diseaserelated biomarkers and disease molecular pathways that may be relevant to the most-affected tissues in ALS. These results indicate that there is no significant difference in the response that senses DNA damage induced by NCS between HC-hMSCs and ALS-hMSCs, which implies that the observed decrease in cell viability of ALS-hMSCs at 24 h post DNA damage induction is not due to failure in the early sensing-stage machinery, but rather that there is a later signal transduction event that mediates the cell death pathway in the diseased cells and not in HC cells.

Results

Conclusion