On 2016 Oct 16, Thomas Langer commented:

Loss of m-AAA proteases increases mitochondrial Ca2+ influx at low cytosolic [Ca2+]

We demonstrate in our paper that the m-AAA protease (AFG3L2/SPG7) degrades EMRE, an essential subunit of the mitochondrial Ca2+ uniporter MCU. Loss or decrease of m-AAA protease activity, as observed in SCA28, impairs the assembly of MCU with the gatekeeper subunits MICU1/2 and results in the formation of unregulated, open MCU. This causes an increased mitochondrial Ca2+ influx at low cytosolic [Ca2+] and renders neurons more susceptible to Ca2+ overload, opening of the mitochondrial permeability transition pore (MPTP) and cell death. Thus, we do not propose in our manuscript that the formation of deregulated MCU causes an increase in cytosolic [Ca2+], as suggested in the comment by Casari et al.. Our findings explain the striking observation by the Casari group that reduced Ca2+ influx into AFG3L2-deficient neurons (by pharmacological inhibition or genetic ablation of mGluR1) protects against neuronal death (Maltecca et al., 2015): lowered cytosolic [Ca2+] in these settings result in decreased mitochondrial Ca2+ influx via deregulated MCU lacking gatekeeper subunits in AFG3L2-deficient neurons, thus preventing mitochondrial Ca2+ overload. Of note, our findings are also in agreement with two recent studies in MICU1-deficient mice demonstrating that deregulated Ca2+ influx causes MPTP opening-induced cell death (Antony et al., Nat. Com., 2016) and ataxia by specifically affecting Purkinje cells (Liu et al., Cell Reports, 2016). Strikingly, reduced EMRE expression was found to suppress ataxia (Liu et al., Cell Reports, 2016).

Casari et al. have suggested that other (yet poorly understood) functions of the m-AAA protease lower the mitochondrial membrane potential (Maltecca et al., 2015) and impair mitochondrial morphology (Maltecca et al., 2012), resulting in decreased mitochondrial Ca2+ influx. Our results do not support a major role of disturbed mitochondrial morphology (Fig. 7), but we agree (and confirm in Fig. S6) that lowering the mitochondrial membrane potential decreases mitochondrial Ca2+ influx after histamine stimulation. We therefore have assessed mitochondrial Ca2+ influx upon mild increase of cytosolic [Ca2+] and observed an increased Ca2+ influx into m-AAA protease-deficient mitochondria (Fig. 6). The rationale of this protocol relies on the sigmoidal relationship between mitochondrial Ca2+ influx and extramitochondrial [Ca2+]. In resting conditions, mitochondrial Ca2+ accumulation is negligible when cytosolic [Ca2+] is below a threshold (~500 nM). Inhibition of SERCA leads to ER Ca2+ leaks, thus causing a slow and small increase of cytosolic [Ca2+]. In this experimental setup (low cytoplasmic [Ca2+]), mitochondrial Ca2+ influx is less hampered by a reduced mitochondrial membrane potential and indeed we observed an increased mitochondrial Ca2+ influx in AFG3L2-deficient mitochondria. We therefore suggest (and discuss in our manuscript) that m-AAA protease-deficient mitochondria show increased Ca2+ influx at resting [Ca2+] but decreased Ca2+ influx at high Ca2+ concentrations (due to the lowered membrane potential).

Casari et al. also raise doubts about the relative role of Ca2+ and mtROS for MPTP opening. We demonstrate a reduced Ca2+ retention capacity of AFG3L2-deficient mitochondria in vitro and in vivo, which correlates with the increased mitochondrial Ca2+ influx (observed upon SERCA inhibition) and the increased ROS levels in AFG3L2-deficient mitochondria. Increased mitochondrial Ca2+ influx under resting conditions is known to trigger MPTP opening (Antony et al., Nat. Com., 2016) and to cause increased mtROS production (Hoffman et al., Cell Reports, 2013; Mallilankaraman et al., Cell, 2012). Thus, both events are interdependent and their relative contribution to MPTP opening is difficult to dissect. We have not addressed this issue in the present manuscript and, by no means, exclude a contribution of mtROS to MPTP opening.

Together, our results provide compelling evidence that m-AAA protease deficiency causes the accumulation of MCU-EMRE complexes lacking gatekeeper subunits and impairs mitochondrial Ca2+ handling, sensitizing neurons for MPTP opening. The relative contribution of deregulated mitochondrial Ca2+ influx and lowered mitochondrial membrane potential for disease pathogenesis is currently difficult to assess and certainly warrants further studies in appropriate mouse models. However, we would like to point out that other mitochondrial diseases affecting respiration and the formation of the mitochondrial membrane potential do not show the striking vulnerability of Purkinje cells seen in SCA28. At the same time, MCU-dependent mitochondrial Ca2+ influx is a crucial determinant of excitotoxicity in neurons (Qui et al., Nat. Com., 2013). This study also demonstrates that synaptic activity transcriptionally suppresses MCU expression thereby counteracting mitochondrial Ca2+ overload at high cytosolic [Ca2+] and preventing induction of excitotoxicity. Our results thus open up the attractive possibility that increased Ca2+ influx under resting conditions and the accompanying mild stress increases progressively the vulnerability of Purkinje cells, causing late-onset neurodegeneration in SCA28 patients, which are only heterozygous for mutations in AFG3L2.

On 2016 Oct 13, Giorgio Casari commented:

Increased or decreased calcium influx?

In this elegant paper the authors propose that loss of m-AAA (i.e. the depletion of both SPG7 and AFG3L2) facilitates the formation of active MCU complexes through the increased availability of EMRE, thus (i) increasing calcium influx into mitochondria, (ii) triggering MPTP opening and (iii) causing the consequent increase of neuronal cytoplasmic calcium leading to neurodegeneration. We previously reported that loss or reduction of AFG3L2 causes (i) decreased mitochondrial potential and fission, thus (ii) decreased calcium entry and (iii) the consequent augmented neuronal cytoplasmic calcium leading to neurodegeneration. While the functional link of m-AAA with MAIP and MCU-EMRE represents a new milestone in the characterization of the roles this multifaceted protease complex, we would like to comment on the conclusions pertaining to the calcium dynamics. 1. In SPG7/AFG3L2 knock-down HeLa cells (Figure S6A) mitochondrial matrix calcium is dramatically reduced (approx. from 100 to 50 microM) following histamine stimulation, which triggers IP3-mediated calcium release from ER. This reduction is in complete agreement with the one we previously detected in Afg3l2 ko MEFs (Maltecca et al., 2012), and that we also confirmed in Afg3l2 knock-out primary Purkinje neurons (the cells that are primarily affected in SCA28) upon challenge with KCl (Maltecca et al., 2015). The decreased mitochondrial calcium uptake correlates with the 40% reduction of mitochondrial membrane potential in SPG7/AFG3L2 knock-down cells (Figure S6B), as expected since the mitochondrial potential is the major component of the driving force for calcium uptake by MCU. Accordingly, these data are in line with the decreased mitochondrial membrane potential observed in Afg3l2 knock-out Purkinje neurons (Maltecca et al., 2015). We think that this aspect is central, because the respiratory defect is the primary event associated to m-AAA deficiency and neurodegeneration. So, the data of König et al. agree with our own findings that mitochondrial matrix calcium is reduced after m-AAA depletion. 2. By a different protocol (SERCA pumps inhibition and ER calcium leakage; Figure 6 C-F), the authors detected a small increase of mitochondrial calcium concentration in SPG7/AFG3L2 knock-down HeLa cells (from approx. 3 to 6 microM). The huge difference in calcium concentration detected in the two experiments (100 to 50 microM in Figure S6A and 3 to 6 microM in Figure 6 C) possibly reflects the stimulated (histamine) vs. unstimulated (calcium leakage) conditions, this latter being more difficult to correlate to physiologic neuronal situation. 3. The authors show increased sensitivity to MPTP opening in the absence of m-AAA and they propose the consequent calcium release as the cause of calcium deregulation and neuronal cell death. ROS are strong sensitizers of MPTP to calcium and thus favor its opening. It is well known that m-AAA loss massively increases intramitochondrial ROS production. Thus, higher ROS levels, rather than high calcium concentrations, can be the trigger of MPTP opening. Taking all this into consideration, we think that mitochondrial depolarization (as shown in Figure S6B) and decreased mitochondrial calcium entry (Fig S6A), even in the presence of increased amount of MCU-EMRE complexes, may lead to inefficient mitochondrial calcium buffering and, finally, to cytoplasmic calcium deregulation. ROS dependent MPTP opening, which may occur irrespective of a low matrix calcium concentration, may additionally contribute to this final event.

Minor comment At page 7 we read: “Notably, these experiments likely underestimate the effect on mitochondrial Ca2+ influx observed upon loss of the m-AAA protease, since the loss of the m-AAA protease also decreases ΔΨ (i.e., the main force driving mitochondrial Ca2+ influx), as revealed by the significant impairment of mitochondrial Ca2+ influx triggered by histamine stimulation (Maltecca et al., 2015) (Figures S6A–S6E)”. The reference is not appropriate, since in Maltecca et al., 2015 the reduced mitochondrial calcium uptake has been demonstrated in Afg3l2 knock-out Purkinje neurons upon challenge with KCl and not with histamine. We used histamine stimulation, which triggers IP3-mediated calcium release from ER, in Afg3l2 ko MEF in a previous publication (Maltecca F, De Stefani D, Cassina L, Consolato F, Wasilewski M, Scorrano L, Rizzuto R, Casari G. Respiratory dysfunction by AFG3L2 deficiency causes decreased mitochondrial calcium uptake via organellar network fragmentation. Hum Mol Genet. 2012, 21:3858-70. doi: 10.1093/hmg/dds214).

On 2016 Oct 13, Giorgio Casari commented:

Increased or decreased calcium influx?

In this elegant paper the authors propose that loss of m-AAA (i.e. the depletion of both SPG7 and AFG3L2) facilitates the formation of active MCU complexes through the increased availability of EMRE, thus (i) increasing calcium influx into mitochondria, (ii) triggering MPTP opening and (iii) causing the consequent increase of neuronal cytoplasmic calcium leading to neurodegeneration. We previously reported that loss or reduction of AFG3L2 causes (i) decreased mitochondrial potential and fission, thus (ii) decreased calcium entry and (iii) the consequent augmented neuronal cytoplasmic calcium leading to neurodegeneration. While the functional link of m-AAA with MAIP and MCU-EMRE represents a new milestone in the characterization of the roles this multifaceted protease complex, we would like to comment on the conclusions pertaining to the calcium dynamics. 1. In SPG7/AFG3L2 knock-down HeLa cells (Figure S6A) mitochondrial matrix calcium is dramatically reduced (approx. from 100 to 50 microM) following histamine stimulation, which triggers IP3-mediated calcium release from ER. This reduction is in complete agreement with the one we previously detected in Afg3l2 ko MEFs (Maltecca et al., 2012), and that we also confirmed in Afg3l2 knock-out primary Purkinje neurons (the cells that are primarily affected in SCA28) upon challenge with KCl (Maltecca et al., 2015). The decreased mitochondrial calcium uptake correlates with the 40% reduction of mitochondrial membrane potential in SPG7/AFG3L2 knock-down cells (Figure S6B), as expected since the mitochondrial potential is the major component of the driving force for calcium uptake by MCU. Accordingly, these data are in line with the decreased mitochondrial membrane potential observed in Afg3l2 knock-out Purkinje neurons (Maltecca et al., 2015). We think that this aspect is central, because the respiratory defect is the primary event associated to m-AAA deficiency and neurodegeneration. So, the data of König et al. agree with our own findings that mitochondrial matrix calcium is reduced after m-AAA depletion. 2. By a different protocol (SERCA pumps inhibition and ER calcium leakage; Figure 6 C-F), the authors detected a small increase of mitochondrial calcium concentration in SPG7/AFG3L2 knock-down HeLa cells (from approx. 3 to 6 microM). The huge difference in calcium concentration detected in the two experiments (100 to 50 microM in Figure S6A and 3 to 6 microM in Figure 6 C) possibly reflects the stimulated (histamine) vs. unstimulated (calcium leakage) conditions, this latter being more difficult to correlate to physiologic neuronal situation. 3. The authors show increased sensitivity to MPTP opening in the absence of m-AAA and they propose the consequent calcium release as the cause of calcium deregulation and neuronal cell death. ROS are strong sensitizers of MPTP to calcium and thus favor its opening. It is well known that m-AAA loss massively increases intramitochondrial ROS production. Thus, higher ROS levels, rather than high calcium concentrations, can be the trigger of MPTP opening. Taking all this into consideration, we think that mitochondrial depolarization (as shown in Figure S6B) and decreased mitochondrial calcium entry (Fig S6A), even in the presence of increased amount of MCU-EMRE complexes, may lead to inefficient mitochondrial calcium buffering and, finally, to cytoplasmic calcium deregulation. ROS dependent MPTP opening, which may occur irrespective of a low matrix calcium concentration, may additionally contribute to this final event.

Minor comment At page 7 we read: “Notably, these experiments likely underestimate the effect on mitochondrial Ca2+ influx observed upon loss of the m-AAA protease, since the loss of the m-AAA protease also decreases ΔΨ (i.e., the main force driving mitochondrial Ca2+ influx), as revealed by the significant impairment of mitochondrial Ca2+ influx triggered by histamine stimulation (Maltecca et al., 2015) (Figures S6A–S6E)”. The reference is not appropriate, since in Maltecca et al., 2015 the reduced mitochondrial calcium uptake has been demonstrated in Afg3l2 knock-out Purkinje neurons upon challenge with KCl and not with histamine. We used histamine stimulation, which triggers IP3-mediated calcium release from ER, in Afg3l2 ko MEF in a previous publication (Maltecca F, De Stefani D, Cassina L, Consolato F, Wasilewski M, Scorrano L, Rizzuto R, Casari G. Respiratory dysfunction by AFG3L2 deficiency causes decreased mitochondrial calcium uptake via organellar network fragmentation. Hum Mol Genet. 2012, 21:3858-70. doi: 10.1093/hmg/dds214).

On 2016 Oct 16, Thomas Langer commented:

Loss of m-AAA proteases increases mitochondrial Ca2+ influx at low cytosolic [Ca2+]

We demonstrate in our paper that the m-AAA protease (AFG3L2/SPG7) degrades EMRE, an essential subunit of the mitochondrial Ca2+ uniporter MCU. Loss or decrease of m-AAA protease activity, as observed in SCA28, impairs the assembly of MCU with the gatekeeper subunits MICU1/2 and results in the formation of unregulated, open MCU. This causes an increased mitochondrial Ca2+ influx at low cytosolic [Ca2+] and renders neurons more susceptible to Ca2+ overload, opening of the mitochondrial permeability transition pore (MPTP) and cell death. Thus, we do not propose in our manuscript that the formation of deregulated MCU causes an increase in cytosolic [Ca2+], as suggested in the comment by Casari et al.. Our findings explain the striking observation by the Casari group that reduced Ca2+ influx into AFG3L2-deficient neurons (by pharmacological inhibition or genetic ablation of mGluR1) protects against neuronal death (Maltecca et al., 2015): lowered cytosolic [Ca2+] in these settings result in decreased mitochondrial Ca2+ influx via deregulated MCU lacking gatekeeper subunits in AFG3L2-deficient neurons, thus preventing mitochondrial Ca2+ overload. Of note, our findings are also in agreement with two recent studies in MICU1-deficient mice demonstrating that deregulated Ca2+ influx causes MPTP opening-induced cell death (Antony et al., Nat. Com., 2016) and ataxia by specifically affecting Purkinje cells (Liu et al., Cell Reports, 2016). Strikingly, reduced EMRE expression was found to suppress ataxia (Liu et al., Cell Reports, 2016).

Casari et al. have suggested that other (yet poorly understood) functions of the m-AAA protease lower the mitochondrial membrane potential (Maltecca et al., 2015) and impair mitochondrial morphology (Maltecca et al., 2012), resulting in decreased mitochondrial Ca2+ influx. Our results do not support a major role of disturbed mitochondrial morphology (Fig. 7), but we agree (and confirm in Fig. S6) that lowering the mitochondrial membrane potential decreases mitochondrial Ca2+ influx after histamine stimulation. We therefore have assessed mitochondrial Ca2+ influx upon mild increase of cytosolic [Ca2+] and observed an increased Ca2+ influx into m-AAA protease-deficient mitochondria (Fig. 6). The rationale of this protocol relies on the sigmoidal relationship between mitochondrial Ca2+ influx and extramitochondrial [Ca2+]. In resting conditions, mitochondrial Ca2+ accumulation is negligible when cytosolic [Ca2+] is below a threshold (~500 nM). Inhibition of SERCA leads to ER Ca2+ leaks, thus causing a slow and small increase of cytosolic [Ca2+]. In this experimental setup (low cytoplasmic [Ca2+]), mitochondrial Ca2+ influx is less hampered by a reduced mitochondrial membrane potential and indeed we observed an increased mitochondrial Ca2+ influx in AFG3L2-deficient mitochondria. We therefore suggest (and discuss in our manuscript) that m-AAA protease-deficient mitochondria show increased Ca2+ influx at resting [Ca2+] but decreased Ca2+ influx at high Ca2+ concentrations (due to the lowered membrane potential).

Casari et al. also raise doubts about the relative role of Ca2+ and mtROS for MPTP opening. We demonstrate a reduced Ca2+ retention capacity of AFG3L2-deficient mitochondria in vitro and in vivo, which correlates with the increased mitochondrial Ca2+ influx (observed upon SERCA inhibition) and the increased ROS levels in AFG3L2-deficient mitochondria. Increased mitochondrial Ca2+ influx under resting conditions is known to trigger MPTP opening (Antony et al., Nat. Com., 2016) and to cause increased mtROS production (Hoffman et al., Cell Reports, 2013; Mallilankaraman et al., Cell, 2012). Thus, both events are interdependent and their relative contribution to MPTP opening is difficult to dissect. We have not addressed this issue in the present manuscript and, by no means, exclude a contribution of mtROS to MPTP opening.

Together, our results provide compelling evidence that m-AAA protease deficiency causes the accumulation of MCU-EMRE complexes lacking gatekeeper subunits and impairs mitochondrial Ca2+ handling, sensitizing neurons for MPTP opening. The relative contribution of deregulated mitochondrial Ca2+ influx and lowered mitochondrial membrane potential for disease pathogenesis is currently difficult to assess and certainly warrants further studies in appropriate mouse models. However, we would like to point out that other mitochondrial diseases affecting respiration and the formation of the mitochondrial membrane potential do not show the striking vulnerability of Purkinje cells seen in SCA28. At the same time, MCU-dependent mitochondrial Ca2+ influx is a crucial determinant of excitotoxicity in neurons (Qui et al., Nat. Com., 2013). This study also demonstrates that synaptic activity transcriptionally suppresses MCU expression thereby counteracting mitochondrial Ca2+ overload at high cytosolic [Ca2+] and preventing induction of excitotoxicity. Our results thus open up the attractive possibility that increased Ca2+ influx under resting conditions and the accompanying mild stress increases progressively the vulnerability of Purkinje cells, causing late-onset neurodegeneration in SCA28 patients, which are only heterozygous for mutations in AFG3L2.