Respiratory dysfunction by AFG3L2 deficiency causes decreased mitochondrial calcium uptake via organellar network fragmentation

Abstract

The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with paraplegin in the inner mitochondrial membrane, named m-AAA proteases. These complexes are in charge of quality control of misfolded proteins and participate in the regulation of OPA1 proteolytic cleavage, required for mitochondrial fusion. Mutations in AFG3L2 cause spinocerebellar ataxia type 28 and a complex neurodegenerative syndrome of childhood. In this study, we demonstrated that the loss of AFG3L2 in mouse embryonic fibroblasts (MEFs) reduces mitochondrial Ca2+ uptake capacity. This defect is neither a consequence of global alteration in cellular Ca2+ homeostasis nor of the reduced driving force for Ca2+ internalization within mitochondria, since cytosolic Ca2+ transients and mitochondrial membrane potential remain unaffected. Moreover, experiments in permeabilized cells revealed unaltered mitochondrial Ca2+ uptake speed in Afg3l2−/− cells, indicating the presence of functional Ca2+ uptake machinery. Our results show that the defective Ca2+ handling in Afg3l2−/− cells is caused by fragmentation of the mitochondrial network, secondary to respiratory dysfunction and the consequent processing of OPA1. This leaves a number of mitochondria devoid of connections to the ER and thus without Ca2+ elevations, hampering the proper Ca2+ diffusion along the mitochondrial network. The recovery of mitochondrial fragmentation in Afg3l2−/− MEFs by overexpression of OPA1 rescues the impaired mitochondrial Ca2+ buffering, but fails to restore respiration. By linking mitochondrial morphology and Ca2+ homeostasis, these findings shed new light in the molecular mechanisms underlining neurodegeneration caused by AFG3L2 mutations.

Purkinje neuron Ca2+ influx reduction rescues ataxia in SCA28 model

“…We first performed IF and confocal microscopy using an Ab against both COX1 (mitochondrial marker) and calbindin (PC marker). In Afg3l2 -/-PCs, mitochondria were mostly round shaped, as was previously observed in MEFs (13), in contrast to the tubular-shaped organelles we observed in syngenic controls (Figure 3A). We also investigated whether alterations of mitochondrial morphology alter the trafficking of organelles to the distal dendritic branches of PCs.…”

“…In particular, the absence of AFG3L2 causes increased mitochondrial fragmentation due to enhanced OPA1 processing (12,13). Fragmentation of the mitochondrial network can modify organelle Ca 2+ uptake by changing the interaction with the Ca 2+ source (e.g., the ER and/or plasma membrane) and limiting proper diffusion of the Ca 2+ wave along the mitochondrial network (42,43).…”

“…The m-AAA complexes are crucial components of the quality-control system in the inner membrane, where they mediate the selective degradation of nonassembled and damaged proteins (6). In addition, they exert chaperone-like activity on respiratory chain complexes (7)(8)(9) and regulate the processing of the profusion protein OPA1 (10)(11)(12)(13) and the subunit of mitochondrial ribosomes MRPL32 (14).…”

Control of mitochondrial integrity in ageing and disease

“…Defective mitochondrial protein synthesis has been proposed as the molecular mechanism of fragmentation in Purkinje neurons [177]. The fragmentation leaves many mitochondria without ER connections limiting the Ca 2þ distribution along the mitochondrial network [176]. Mutations in the paraplegin gene cause axonal degeneration in hereditary spastic paraplegia (SPG7, MIM:607259) [178], with phenotypic consequences likely partially alleviated by the presence of AFG3L2 homo-oligomer and the presence of m-AAA protease activity [134].…”

Genetic landscape remodelling in spinocerebellar ataxias: the influence of next-generation sequencing

“…Knock-out of Afg3l2 induces mitochondrial network fragmentation and reduced mitochondrial uptake of Ca ?? in mouse embryonic fibroblasts [77]. In PC, early abnormal mitochondrial dynamics, respiratory dysfunction and neurodegeneration are imputed to decreased mitochondrial protein synthesis [78].…”