Disrupted expression of mitochondrial NCLX sensitizes neuroglial networks to excitotoxic stimuli and renders synaptic activity toxic

Anna M Hagenston,Jing Yan,Carlos Bas-Orth,Yanwei Tan,Israel Sekler,Hilmar Bading

doi:10.1016/j.jbc.2021.101508

Abstract

The mitochondrial solute carrier family 8 sodium/calcium/lithium exchanger, member B1 (NCLX) is an important mediator of calcium extrusion from mitochondria. In this study, we tested the hypothesis that physiological expression levels of NCLX are essential for maintaining neuronal resilience in the face of excitotoxic challenge. Using an shRNA-mediated approach, we showed that reduced NCLX expression exacerbates neuronal mitochondrial calcium dysregulation, mitochondrial membrane potential (ΔΨm) breakdown, and reactive oxygen species generation during excitotoxic stimulation of primary hippocampal cultures. Moreover, NCLX knockdown—which affected both neurons and glia—resulted not only in enhanced neurodegeneration following an excitotoxic insult but also in neuronal and astrocytic cell death under basal conditions. Our data also revealed that synaptic activity, which promotes neuroprotective signaling, can become lethal upon NCLX depletion; expression of NCLX-targeted shRNA impaired the clearance of mitochondrial calcium following action potential bursts, and was associated both with ΔΨm breakdown and substantial neurodegeneration in hippocampal cultures undergoing synaptic activity. Finally, we showed that NCLX knockdown within the hippocampal cornu ammonis 1 region in vivo causes substantial neurodegeneration and astrodegeneration. In summary, we demonstrated that dysregulated NCLX expression not only sensitizes neuroglial networks to excitotoxic stimuli but also notably renders otherwise neuroprotective synaptic activity toxic. These findings may explain the emergence of neurodegeneration and astrodegeneration in patients with disorders characterized by disrupted NCLX expression or function, and suggest that treatments aimed at enhancing or restoring NCLX function may prevent central nervous system damage in these disease states.

Highlights

Mitochondrial dysfunction in general—and disturbed mitochondrial calcium signaling in particular—has been linked to death processes in affected both neurons and glia—resulted not numerous cell types from tissues throughout the only in enhanced neurodegeneration following body, including the central and peripheral nervous an excitotoxic insult, and in neuronal and systems [1,2,3]
We membrane potential (ΔΨm), disrupted energy showed that NCLX knockdown within the metabolism, mitochondrial permeability transition, hippocampal cornu ammonis 1 (CA1) region in and cell death is implicated in stroke, vivo causes substantial neuro- and traumatic brain and spinal cord injury, Huntington’s astrodegeneration
Grx1-roGFP 405/480 ratio, R was with recombinant adeno-associated viral vectors (rAAVs)-shCTRL or rAAV-shNCLX and challenged measured prior to and during NMDA application. with different concentrations of NMDA

Summary

Introduction

Mitochondrial dysfunction in general—and disturbed mitochondrial calcium signaling in particular—has been linked to death processes in affected both neurons and glia—resulted not numerous cell types from tissues throughout the only in enhanced neurodegeneration following body, including the central and peripheral nervous an excitotoxic insult, and in neuronal and systems [1,2,3]. Due to the lack of loss of astrocytes in nominally pure cultures was far appropriate commercially available anti-NCLX lower than that observed for these cells in a mixed antibodies, it was not possible in this study to culture system (Figure 4,Figure 5) It will be exciting directly examine NCLX protein expression at the to discover in future studies how pathologically single cell level, or to relate such parameters as reduced NCLX expression and/or function mitochondrial calcium recovery, ΔΨm breakdown, influences neuronal and glial or cellular viability to the degree of NCLX mitogenesis/mitophagy as well as both cell knockdown. Journal Pre-proof seems likely [85]

Experimental procedures

Findings

Methods