Elsevier

Experimental Neurology

Volume 335, January 2021, 113520
Experimental Neurology

Research paper
Proteomic analysis of human iPSC-derived sensory neurons implicates cell stress and microtubule dynamics dysfunction in bortezomib-induced peripheral neurotoxicity

https://doi.org/10.1016/j.expneurol.2020.113520Get rights and content

Highlights

  • Bortezomib exposure strongly induced the UPR and ISR in iSNs

  • Prolonged bortezomib exposure impairs axonal trafficking of mitochondria in iSNs

  • Bortezomib caused a system-wide decline in iSN cytoskeletal proteins and processes

  • Bortezomib promoted Rab6+ vesicle accumulation within iSN somata

  • Prolonged bortezomib exposure is associated with dramatic loss of MAP2 in iSNs

Abstract

The neurotoxic effects of the chemotherapeutic agent bortezomib on dorsal root ganglia sensory neurons are well documented, yet the mechanistic underpinnings that govern these cellular processes remain incompletely understood. In this study, system-wide proteomic changes were identified in human induced pluripotent stem cell-derived sensory neurons (iSNs) exposed to a clinically relevant dose of bortezomib. Label-free mass spectrometry facilitated the identification of approximately 2800 iSN proteins that exhibited differential levels in the setting of bortezomib. A significant proportion of these proteins affect the cellular processes of microtubule dynamics, cytoskeletal and cytoplasmic organization, and molecular transport, and pathway analysis revealed an enrichment of proteins in signaling pathways attributable to the unfolded protein response and the integrated stress response. Alterations in microtubule-associated proteins suggest a multifaceted relationship exists between bortezomib-induced proteotoxicity and microtubule cytoskeletal architecture, and MAP2 was prioritized as a topmost influential candidate. We observed a significant reduction in the overall levels of MAP2c in somata without discernable changes in neurites. As MAP2 is known to affect cellular processes including axonogenesis, neurite extension and branching, and neurite morphology, its altered levels are suggestive of a prominent role in bortezomib-induced neurotoxicity.

Introduction

Chemotherapy-induced peripheral neuropathy (CIPN) affects over 1/3 of the patient population undergoing treatment for cancer and its prevalence is anticipated to rise with improvements in survival rates (Shah et al. 2018). Symptoms of CIPN typically begin during the first 2 months of treatment and become more severe throughout its duration. Countermeasures are limited to dose reduction or a discontinuation of chemotherapy treatment, underscoring the need both to better understand the central pathobiology and to improve our repertoire of therapeutic agents (Staff et al., 2017).

The majority of CIPN signs and symptoms arise from damage to dorsal root ganglion (DRG) neurons or their axons in the form of a dying-back neuropathy, seemingly initiated through defects in axon transport, altered mitochondrial function, or altered calcium homeostasis (Staff et al., 2017), (Meregalli 2015). Although there are different mechanisms by which chemotherapeutic agents cause this effect, it is not entirely clear how proteasome inhibitors such as bortezomib facilitate axonal degeneration. Bortezomib inhibits the activity of the 26S proteasome, which recognizes ubiquinated proteins and selectively targets them for degradation in order to maintain cellular homeostasis. In tumorigenic cells, proteasome inhibitors lead to the accumulation of damaged and unfolded proteins, which ultimately results in cell cycle arrest and the upregulation of pro-apoptotic signaling proteins (Dou and Zonder 2014). Nontumorigenic cells rely on protein kinase B (Akt) upregulation, the endoplasmic reticulum (ER) stress response, the unfolded protein response (UPR), and autophagy to mitigate bortezomib's cytotoxic effects, and the degree to which any of these mechanisms are applicable to bortezomib neurotoxicity remains to be established.

Preclinical studies that yielded encouraging results for the prevention of CIPN failed in clinical trials and reinforced the need to refine our understanding of chemotherapeutic response (Fukuda et al. 2017). Towards that end, technological advancements in the stem cell field now allow for us to create iPSCs (induced pluripotent stem cells) from patients, and the directed differentiation of these cells into various tissue-specific subtypes has reliably recapitulated many disease phenotypes as well as drug intolerances that are not as readily detected in rodents or other species.

Due to the multifaceted impact of proteasome inhibition on cellular function, we employed a proteomics study to identify key processes responsible for bortezomib-induced neurotoxicity. We opted to investigate the proteomics signature due the discordance between RNA and protein levels (Wiita et al. 2013), (Rendleman et al. 2018). The study was performed on human iPSC-derived sensory neurons (iSNs) exposed to a clinically relevant dose of bortezomib to create a proteomics dataset that resembles the in vivo proteotoxicity of bortezomib.

Section snippets

Human iPSC cell culture and maintenance, iSN differentiation, and bortezomib treatment

Three iPSC lines created from the skin biopsies of individuals with no known health concerns were acquired from the Mayo Clinic Biotrust according to IRB approved guidelines. Fibroblasts were reprogrammed using Cytotune-iPS 2.0 (courtesy of Regen Theranostics) and karyotypically normal iPSC clones for each line that passed quality control testing for pluripotency and germ layer differentiation were selected. The lines used in this study were from 2 females (21-year-old, 6BT1; 77-year-old, 1BT1)

Bortezomib induces neurotoxic effects in iSNs

To ensure that the dose and duration of bortezomib exposure was appropriate for this model system, we compared the iSN model to our previous studies in rat E15 DRG neurons. A 24 h bortezomib exposure led to the accumulation of β3-tubulin (Tuj-1) proximal to the plasma membrane in the somata of rat E15 DRG neurons (Staff et al., 2013). This same effect was observed in iSNs at both 24 h and 48 h (Fig. 1A). Another similarity shared by both models was that bortezomib exposure had no effect on

Discussion

The central focus of this study was to better characterize the molecular underpinnings that predispose sensory neurons to bortezomib-induced neurotoxicity. Our approach was to use a model system based on iPSCs to recapitulate bortezomib's effects on human iSNs. Although iPSC-derived models possess immature characteristics, they have proven to be a useful model system for studying chemotherapeutic drug responses. Our iSN model reproduced a cascade of effects that have been observed in other

Acknowledgements

This publication was made possible by support from the Medical Genome Facility Proteomics Core, Mayo Clinic. The core is a shared resource of the Mayo Clinic Cancer Center (NCI P30 CA15083). We thank Dr. Cristine Charlesworth for her guidance on sample preparation for MS analysis.

NPS and SA conceived the study. NPS, SA, SCLH, and RFH contributed to experimental design. Experiments were completed by SA, RFH, SCLH, BA, JPK, BN, and IS. SCLH, RAM, NPS, and SD completed analyses of the

Funding

This work was supported by the National Institutes of Health [NPS: R01 CA211887] and the Mayo Foundation for Medical Education and Research.

Disclosure statement

The authors have no conflicting interests.

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