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. 2024 Apr 16;10(1):178.
doi: 10.1038/s41420-024-01949-w.

AAV-mediated upregulation of VDAC1 rescues the mitochondrial respiration and sirtuins expression in a SOD1 mouse model of inherited ALS

Andrea Magrì #  1   2 , Cristiana Lucia Rita Lipari #  3 , Antonella Caccamo  4 , Giuseppe Battiato  3 , Stefano Conti Nibali  3 , Vito De Pinto  2   3 , Francesca Guarino  2   3 , Angela Messina  5   6
Affiliations

AAV-mediated upregulation of VDAC1 rescues the mitochondrial respiration and sirtuins expression in a SOD1 mouse model of inherited ALS

Andrea Magrì et al. Cell Death Discov. .

Abstract

Mitochondrial dysfunction represents one of the most common molecular hallmarks of both sporadic and familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by the selective degeneration and death of motor neurons. The accumulation of misfolded proteins on and within mitochondria, as observed for SOD1 G93A mutant, correlates with a drastic reduction of mitochondrial respiration and the inhibition of metabolites exchanges, including ADP/ATP and NAD+/NADH, across the Voltage-Dependent Anion-selective Channel 1 (VDAC1), the most abundant channel protein of the outer mitochondrial membrane. Here, we show that the AAV-mediated upregulation of VDAC1 in the spinal cord of transgenic mice expressing SOD1 G93A completely rescues the mitochondrial respiratory profile. This correlates with the increased activity and levels of key regulators of mitochondrial functions and maintenance, namely the respiratory chain Complex I and the sirtuins (Sirt), especially Sirt3. Furthermore, the selective increase of these mitochondrial proteins is associated with an increase in Tom20 levels, the receptor subunit of the TOM complex. Overall, our results indicate that the overexpression of VDAC1 has beneficial effects on ALS-affected tissue by stabilizing the Complex I-Sirt3 axis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. AAV2/5-mediated intraspinal injection of VDAC1 gene efficiently allows the overexpression of the relative protein.
A Schematization of the experimental plan followed in this work. To obtain four different experimental groups, AAV2/5 constructs, carrying VDAC1 or EGFP sequence, were delivered to wild-type and transgenic mice by intraspinal injection at P2. Then, body weight was monitored and mice were sacrificed at week 15, in concomitance with the occurrence of motor impairment. B Analysis of body weight from week 4 to 14 and body weight increase. Data are expressed as median or means ± SEM of n = 8 independent measurements per experimental group, and statistically analyzed by one-way ANOVA; ns, not significant. C Representative Western blots of spinal cord total homogenates extracted from transgenic mice previously injected with VDAC1 or EGFP construct aimed at investigating VDAC1 expression. Tubulin was used as loading control. D Relative quantification of VDAC1 expression of Western blot in (C). Data are expressed as mean ± SEM of n = 4 independent measurements and statistically analyzed by unpaired Student t-test, with ***p < 0.001.
Fig. 2
Fig. 2. VDAC1 upregulation in transgenic mice restores mitochondrial respiration in spinal cord.
A Representative trace of oxygen consumption by high-resolution respirometry achieved from fresh spinal cord homogenates of 15-week-old wild-type mice injected with AAV2/5 carrying EGFP. The scheme shows the oxygen consumption and concentration curves along with the SUIT protocol here applied to analyze the main respiratory states. PMG, pyruvate, malate and glutamate; S, succinate; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; Rot, rotenone; Ama, antimycin A. B Quantification and comparative analysis of oxygen consumption in wild-type and transgenic mice, either injected with AAV2/5 constructs carrying VDAC1 or EGFP, of spinal cord homogenates relative to the LEAK, N- and NS-pathway (OXPHOS state), ATP-linked OXPHOS respiration and maximal ET capacity. Data are expressed as pmol/s of oxygen per mg of tissue and as median ± SEM of n = 4 independent measurements. Data were statistically analyzed by one-way ANOVA, with *p < 0.05 and **p < 0.01; ns not significant.
Fig. 3
Fig. 3. VDAC1 upregulation drives the selective increase of activity and expression of specific ET complexes.
A, B Schematic representation of the experimental set up for the analysis of Complex I and II contribution to the maximal respiration applied in this work. In A, the electrons transfer from Complex I to III, thus excluding Complex II, was activated in the presence of ADP and of the NADH-linked substrates exclusively, namely pyruvate (P), malate (M) and glutamate (G). In B, activation of the electron transfer from Complex II to III was achieved in the presence of succinate (S) and rotenone (Rot), the last a specific inhibitor of Complex I. C Comparative analysis of the specific contribution of Complex I to the total OXPHOS respiration and Complex II to the maximal ET capacity in transgenic mice overexpressing or not VDAC1, calculated as a function of the maximal ET capacity (FCR). D Representative Western blots of spinal cord total homogenates extracted from transgenic mice previously injected with VDAC1 or EGFP AAVs showing the levels of the ET chain subunits NDUFV1, SDHA and COXIV. Tubulin was used as loading control. E Relative quantification of protein levels of Western blots in (D). All the data in the figure are expressed as mean ± SEM of n = 4 independent measurements and statistically analyzed by unpaired Student t-test, with *p < 0.05 and ***p < 0.001; ns not significant.
Fig. 4
Fig. 4. Increase of sirtuins levels and activity correlates with VDAC1 overexpression.
A Representative Western blots of spinal cord total homogenates extracted from transgenic mice previously injected with VDAC1 or EGFP AAVs showing the levels of Sirt3 and the relative quantification. B Representative western blot of spinal cord total homogenates as in A showing the levels of Sirt5 and the relative quantification. C Representative Western blot of spinal cord total homogenates as in A showing the levels of SOD2 and K68 acetylated SOD2. D Relative quantification of protein expression of Western blot in (C). In all the blots, Tubulin was used as loading control. All the data are expressed as mean ± SEM of n = 4 independent measurements and statistically analyzed by unpaired Student t-test, with **p < 0.01 and ***p < 0.001; ns not significant.
Fig. 5
Fig. 5. Downregulation of PGC-1α inversely correlates with Tom20 expression in transgenic mice injected with VDAC1.
A Representative Western blots of spinal cord total homogenates extracted from transgenic mice previously injected with VDAC1 or EGFP AAVs showing the levels of PGC-1α and Tom20. Tubulin was used as loading control. B Relative quantification of protein expression of Western blot in (A). Data are expressed as mean ± SEM of n = 4 independent measurements and statistically analyzed by unpaired Student t-test, with *p < 0.05 and **p < 0.01.
Fig. 6
Fig. 6. Proposed model on the role of VDAC1 upregulation in recover the mitochondrial dysfunction in ALS transgenic mice.
A, B The overexpression of VDAC1 in SOD1 G93A transgenic mice results in a general restore of the mitochondrial respiratory profile that possibly depends on two strictly interconnected mechanisms. The exogenous expression of VDAC1 mediated by AAV2/5 vector partially compensates for endogenous VDAC1 molecules whose functioning is compromised by the toxic interaction with SOD1 mutants. This promotes an increase of VDAC1 in the OMM and of the metabolic exchanges across the OMM, especially of ADP/ATP and NAD+/NADH. In particular, NADH feeds Complex I stimulating the activity of the N-pathway. At the same time, the reoxidation of NADH to NAD+ by Complex I stimulates the activity of Sirt3, a general regulator of the whole mitochondrial and ET chain activity (A). VDAC1 upregulation correlates with an increase in the levels of Tom20, a key component of TOM complex, that promotes the increase of mitochondrial proteins import within the organelle, as for Complex I and Sirt3 (B). The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.
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