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

Abstract

Background 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. 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. Methods To 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. Results We 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. Conclusion These 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.

Selective Vulnerability of Tripartite Synapses in Amyotrophic Lateral Sclerosis

“…Pre-symptomatic and early-symptomatic changes in cortical dendritic spines, neuromuscular junctions and the synaptic inputs to spinal cord MNs have all been reported (Fogarty et al, 2015(Fogarty et al, , 2016Perkins et al, 2021;Sasaki & Iwata, 1995;Starr & Sattler, 2018;Tremblay et al, 2017;Van Zundert et al, 2008). Separate evidence indicates that astrocytes, the supportive glial cells of the CNS, may be responsible for non-cell autonomous degeneration of MNs (Devlin et al, 2015;Forsberg et al, 2011;Valori et al, 2014;Yamanaka et al, 2008;Yamanaka & Komine, 2018;Zhao et al, 2020).…”

Emerging Mechanisms Underpinning Neurophysiological Impairments in C9ORF72 Repeat Expansion-Mediated Amyotrophic Lateral Sclerosis/Frontotemporal Dementia

“… Benussi et al (2016) predict that synaptic/network plasticity impairments present 15 years before symptomatic onset making these pathological observations some of the earliest evidenced in ALS-FTD patients. Direct evidence of impaired synaptic potentiation of mini excitatory post-synaptic currents was recently confirmed in induced pluripotent stem cell (iPSC)-derived cortical neurons generated from C9ORF72 RE patients, a feature that was rescued in isogenic, gene-corrected lines ( Perkins et al, 2021 ). Beyond this, functional investigations of impaired synaptic plasticity in ALS and FTD have been determined in hippocampal murine preparations: UBQLN2 P 497 H ( Gorrie et al, 2014 ); SOD1 G 93 A ( Spalloni et al, 2011 ) and TDP-43 transgenic mice ( Koza et al, 2019 ), TDP-43 conditional knockout mice ( Wu et al, 2019 ).…”

“…Excitatory neurons represent approximately 80% of the adult cortex and numerous pieces of evidence are converging toward the contribution of these neurons to abnormalities in cortical excitability in ALS-FTD patients. Perkins et al (2021) demonstrated that cultures of excitatory cortical neurons derived from C9ORF72 RE iPSCs displayed an enhanced network burst frequency compared to control derived neurons. These properties appear to be explained by the fact that C9ORF72 RE excitatory neurons had an increased functional synaptic input due to increased synaptic density, but not altered intrinsic excitability.…”

“…Also, impaired plasticity has been observed at the neuromuscular junction of Drosophila over-expressing C9ORF72 RE ( Perry et al, 2017 ). Broad cellular disruption affecting molecules and signaling processes relevant to synaptic plasticity are highlighted by transcriptional disturbances in both C9ORF72 RE patient-derived cortical neurons ( Perkins et al, 2021 ) and C9ORF72 RE patient post-mortem cortex ( Prudencio et al, 2015 ). Defined molecular pathological mechanisms of altered cortical synaptic plasticity in ALS-FTD remain to be elucidated.…”

“…Mechanisms promoting cortical synaptic density remain unreported but are associated with transcriptional dysregulation consistent with modified expression of synaptic architecture proteins ( Prudencio et al, 2015 ; Perkins et al, 2021 ). Conversely, mechanisms supporting synaptic loss in C9ORF72 RE cortical neurons are now emerging.…”