Altered network properties in C9ORF72 repeat expansion cortical neurons are due to synaptic dysfunction

Emma M Perkins,Siddharthan Chandran,Poulomi Banerjee,Arpan R Mehta,Bhuvaneish T Selvaraj,David J A Wyllie,Matthew R Livesey,Owen Dando,Thomas H Gillingwater,Daumante Suminaite,Christopher M Henstridge,Jyoti Nanda,Karen Burr,Giles E Hardingham

doi:10.1186/s13024-021-00433-8

Abstract

BackgroundPhysiological disturbances in cortical network excitability and plasticity are established and widespread in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those harbouring the C9ORF72 repeat expansion (C9ORF72RE) mutation – the most common genetic impairment causal to ALS and FTD. Noting that perturbations in cortical function are evidenced pre-symptomatically, and that the cortex is associated with widespread pathology, cortical dysfunction is thought to be an early driver of neurodegenerative disease progression. However, our understanding of how altered network function manifests at the cellular and molecular level is not clear.MethodsTo address this we have generated cortical neurons from patient-derived iPSCs harbouring C9ORF72RE mutations, as well as from their isogenic expansion-corrected controls. We have established a model of network activity in these neurons using multi-electrode array electrophysiology. We have then mechanistically examined the physiological processes underpinning network dysfunction using a combination of patch-clamp electrophysiology, immunocytochemistry, pharmacology and transcriptomic profiling.ResultsWe find that C9ORF72RE causes elevated network burst activity, associated with enhanced synaptic input, yet lower burst duration, attributable to impaired pre-synaptic vesicle dynamics. We also show that the C9ORF72RE is associated with impaired synaptic plasticity. Moreover, RNA-seq analysis revealed dysregulated molecular pathways impacting on synaptic function. All molecular, cellular and network deficits are rescued by CRISPR/Cas9 correction of C9ORF72RE. Our study provides a mechanistic view of the early dysregulated processes that underpin cortical network dysfunction in ALS-FTD.ConclusionThese findings suggest synaptic pathophysiology is widespread in ALS-FTD and has an early and fundamental role in driving altered network function that is thought to contribute to neurodegenerative processes in these patients. The overall importance is the identification of previously unidentified defects in pre and postsynaptic compartments affecting synaptic plasticity, synaptic vesicle stores, and network propagation, which directly impact upon cortical function.

Highlights

Physiological disturbances in cortical network excitability and plasticity are established and widespread in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those harbouring the C9ORF72 repeat expansion (C9ORF72RE) mutation – the most common genetic impairment causal to ALS and FTD
C9ORF72RE-derived cortical neurons display network dysfunction induced pluripotent stem cell (iPSC) from three patients harbouring C9ORF72RE mutations (C9–1,2,3), three paired isogenic control lines (C9Δ1,2,3) in which the C9ORF72RE mutation had been selectively excised by CRISPR/Cas9-mediated gene-editing [52], and an unrelated healthy individual (Con) were used to generate cultures of excitatory cortical neurons using an established protocol [6, 52]
Shorter inter-burst lengths are typically associated with increased glutamate-mediated excitatory network activity, the reduced network burst duration in C9ORF72RE cortical neurons (Fig. 1a) is inconsistent with this [3, 43]

Summary

Introduction

Physiological disturbances in cortical network excitability and plasticity are established and widespread in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those harbouring the C9ORF72 repeat expansion (C9ORF72RE) mutation – the most common genetic impairment causal to ALS and FTD. Our study provides a mechanistic view of the early dysregulated processes that underpin cortical network dysfunction in ALS-FTD. For many progressive neurodegenerative diseases, including ALS-FTD, functional impairments in plasticity are thought to manifest early in disease progression, being representative of altered synaptic homeostasis that precede and potentially cause neuronal dysfunction and/or loss, and lead to cognitive impairments [38, 55, 56]. Our understanding of the potential synaptic plasticity dysregulation that may occur in ALS-FTD has come from studies that use ex vivo brain slice preparations from rodent models of rare genetic mutations [24, 54]. Despite its proposed pathogenicity and early prominence, cortical dysfunction in ALS-FTD remains poorly defined at both the synaptic and network level

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