Restoration of defective oxidative phosphorylation to a subset of neurons prevents mitochondrial encephalopathy
Abstract
Synopsis
![image](/cms/10.1038/s44321-024-00111-4/asset/d275b30f-d88c-44f5-9010-d196b601836a/assets/graphic/44321_2024_111_figa_html.jpg)
The paper explained
Problem
Results
Impact
Introduction
Results
Retro‐orbital injections of recombinant AAV-PHP.eB restored deleted OXPHOS genes, prolonged lifespan and maintained weight in OXPHOS nKO mice
![](/cms/10.1038/s44321-024-00111-4/asset/9ba43203-4747-42ca-9871-b4dbb07a3eb8/assets/graphic/44321_2024_111_fig1_html.png)
![](/cms/10.1038/s44321-024-00111-4/asset/f3acc58a-281d-4d4c-9f2c-11d2f8c6ec77/assets/graphic/44321_2024_111_fig2_html.png)
AAV-mediated gene therapy improved motor function, balance, and coordination in OXPHOS-deficient mice
NDUFS3 levels in the CNS were increased in Ndufs3-nKO mice injected with AAV-PHP.eB-Ndufs3
![](/cms/10.1038/s44321-024-00111-4/asset/76e2817e-2a5f-4339-9026-10daf9ae3f6e/assets/graphic/44321_2024_111_fig3_html.png)
![](/cms/10.1038/s44321-024-00111-4/asset/386caf83-054d-4610-b417-445c9f6a5536/assets/graphic/44321_2024_111_fig9_esm.jpg)
COX1 levels are increased in the CNS of COX10-nKO mice injected with AAV-PHP.eB-Cox10
![](/cms/10.1038/s44321-024-00111-4/asset/3a7ae347-5293-49b4-bab6-210733514890/assets/graphic/44321_2024_111_fig4_html.png)
Reintroduction of deleted OXPHOS proteins leads to proper assembly and function of affected complexes
![](/cms/10.1038/s44321-024-00111-4/asset/33c0f357-996b-4327-8f95-68d2986294eb/assets/graphic/44321_2024_111_fig5_html.png)
AAV-PHP.eB-mediated OXPHOS restoration reduces neuroinflammation and neuronal loss in cortex and hippocampus of treated OXPHOS nKO mice
![](/cms/10.1038/s44321-024-00111-4/asset/bb7b38cb-106d-46ca-bdbe-801809910bc8/assets/graphic/44321_2024_111_fig6_html.png)
![](/cms/10.1038/s44321-024-00111-4/asset/473e1078-a8c2-46d7-aacc-e55d60c7d914/assets/graphic/44321_2024_111_fig10_esm.jpg)
![](/cms/10.1038/s44321-024-00111-4/asset/ae2d102e-4623-4027-9d3d-b1ef6f561cec/assets/graphic/44321_2024_111_fig7_html.png)
Early therapeutic intervention improves quality of life and extends survival to at least 15 months of age in nKO mice
![](/cms/10.1038/s44321-024-00111-4/asset/20eb7b1f-b3eb-4b92-b08d-97bbe8061273/assets/graphic/44321_2024_111_fig11_esm.jpg)
![](/cms/10.1038/s44321-024-00111-4/asset/5e6c0f56-77f3-4c29-9f30-09c5c50fc348/assets/graphic/44321_2024_111_fig12_esm.jpg)
Expression from AAV-PHP.eB-hSYN is limited to a subset of neurons but promotes robust CNS phenotypic correction
![](/cms/10.1038/s44321-024-00111-4/asset/d92d028f-4128-4b03-9ea0-c4c52f8fc741/assets/graphic/44321_2024_111_fig8_html.png)
Ndufs3 gene deletion in more than half of CamKIIα+ neurons does not cause detectable CNS phenotypes
![](/cms/10.1038/s44321-024-00111-4/asset/8ace1b15-bde4-491f-b48b-f2fd736d900f/assets/graphic/44321_2024_111_fig13_esm.jpg)
Discussion
Methods
Reagent/Resource | Reference or Source | Identifier or Catalog Number |
---|---|---|
Experimental Models | ||
Cox10fl/fl-CaMKIIα-Cre+/– | Diaz et al (2012) | N/A |
Ndufs3fl/fl-CaMKIIα-Cre+/– | Peralta et al (2020) | N/A |
Recombinant DNA | ||
pAAV-hSYN-eGFP | Addgene | 50465 |
pAAV-hSYN-Ndufs3 | This study | N/A |
pAAV-hSYN-Cox10 | This study | N/A |
Antibodies | ||
NDUFS3 | Abcam | ab14711 |
NDUFA9 | Abcam | ab14713 |
NDUFB8 | Abcam | ab110242 |
MTCO1 | Abcam | ab14705 |
COX IV | Abcam | ab14744 |
SDHA | Abcam | ab14715 |
UQCRC1 | Abcam | ab110252 |
VDAC | Abcam | ab14734 |
GFAP | Cell Signaling | 3680 |
Proteintech | 16825-1-AP | |
TUJI | Abcam | ab18207 |
IBA1 | Wako (WB) | 016-20001 |
Wako (ICC) | 019-19741 | |
GFP | UC Davis | 75-131 |
NeuN | Cell Signaling | 12943 |
Anti-mouse Alexa 488 | Invitrogen | A11001 |
Anti-rabbit Alexa 568 | Invitrogen | A11036 |
Anti-mouse Alexa 594 | Invitrogen | A11032 |
Anti-rabbit Alexa 647 | Invitrogen | A31573 |
Anti-mouse IgG | Cell Signaling | 7076 |
Anti-rabbit IgG | Cell Signaling | 7074 |
Oligonucleotides and other sequence-based reagents | ||
Genotyping primers | Appendix Table S1 | |
dPCR primer/probe sets | Appendix Table S2 | |
Chemicals, Enzymes and other reagents | ||
Paraformaldehyde (PFA) | Sigma | 441244 |
Sucrose | Sigma | S-0389 |
Tissue-Tek OCT | Sakura | 4583 |
OmniPur 10X PBS | Calbiotech | 6505 |
Triton X-100 | Sigma | T9284 |
Bovine Serum Albumin (BSA) | Sigma | A7030 |
Vectashield | Biotium | 23004 |
cOmplete™ Protease Inhibitor Cocktail | Roche | 04693116001 |
PhosSTOP™ | Roche | 04906845001 |
Trizma base | Sigma | T1503 |
Glycine | Sigma | 1.00590 |
Sodium dodecyl sulfate (SDS) | Sigma | L3771 |
Trans-Blot Turbo 5X Transfer Buffer | Bio-Rad | 10026938 |
Ponceau S Solution | Sigma | P7170-1L |
Tween 20 | Sigma | P1379-500ML |
SuperSignal Pico West | ThermoFisher Scientific | 34578 |
Digitonin | Sigma | 300410 |
20X Running Buffer | Invitrogen | BN2001 |
20X Cathode Additive | Invitrogen | BN2002 |
NADH | Calbiotech | 481913 |
Nitrobluetetrazolium | Sigma | N6876 |
DAB | ThermoScientific | 34001 |
catalase | Sigma | C9322 |
Cytochrome c | Sigma | 2506 |
Proteinase K | Sigma | P2308 |
Software | ||
Activity Monitor | N/A | |
ImageLab | N/A | |
FIJI | N/A | |
QuPath | N/A | |
Prism | N/A | |
Other | ||
5X In-Fusion Snap Assembly Master Mix | Takada | ST2320 |
One-Shot Stbl3 Chemically Competent E. coli | Invitrogen | C737303 |
NucleoBond Xtra Maxi EF | Macherey-Nagel | 740424 |
DC Protein Assay | Bio-Rad | 5000113, 5000114 |
Mini-PROTEAN TGX Stain-Free 4–20% gel, 10-well | Bio-Rad | 4568094 |
0.2 μm PVDF membrane | Bio-Rad | 10026934 |
0.2 μM nitrocellulose membrane | Bio-Rad | 162-0097 |
Mini-PROTEAN Tetra Cell | Bio-Rad | 1658004EDU |
NativePAGE 3–12% Bis-Tris gel, 10 well | Invitrogen | BN2011BX10 |
XCell SureLock Mini-Cell | Invitrogen | EI0001 |
QIAcuity Probe PCR kit | Qiagen | 250101 |
QIAcuity NanoPlate 8.5k 24-well plate | Qiagen | 250011 |
Open Field Analysis | Med Associates Inc | N/A |
RotaRod | Ugo Basile | 47650 |
Cryostat | Leica | CM 1850-3-1 |
Synergy H1 Hybrid Reader | Biotek | N/A |
Trans-Blot Turbo Transfer System | Bio-Rad | 1704150 |
ChemiDoc Touch Imaging System | Bio-Rad | 1708370 |
QIAcuity One, 5plex | Qiagen | 911020 |
Animals
Adeno-associated viral (AAV) vector production and injection
Open field and rotarod testing
Tissue staining and microscopy
Antibody | Company | Catalog # | Dilution | Species |
---|---|---|---|---|
NDUFS3 | Abcam | ab14711 | 1:500 | Mouse |
MTCO1 | Abcam | ab14705 | 1:100 | Mouse |
GFAP | Cell Signaling | 3680 | 1:500 | Mouse |
Proteintech | 16825-1-AP | 1:500 | Rabbit | |
NeuN | Cell Signaling | 12943 | 1:500 | Rabbit |
IBA1 | Wako | 019-19741 | 1:1000 | Rabbit |
GFP | UC Davis | 75-131 | 1:500 | Mouse |
Alexa 488 | Invitrogen | A11001 | 1:500 | Mouse |
Alexa 568 | Invitrogen | A11036 | 1:500 | Rabbit |
Alexa 594 | Invitrogen | A11032 | 1:500 | Mouse |
Alexa 647 | Invitrogen | A31573 | 1:500 | Rabbit |
Western blotting
Antibody | Company | Catalog # | Dilution | Species |
---|---|---|---|---|
NDUFS3 | Abcam | Ab14711 | 1:1000 | Mouse |
NDUFB8 | Abcam | Ab110242 | 1:1000 | Mouse |
MTCO1 | Abcam | Ab14705 | 1:1000 | Mouse |
UQCRC1 | Abcam | Ab110252 | 1:1000 | Mouse |
SDHA | Abcam | Ab14715 | 1:1000 | Mouse |
IBA1 | Wako | 016-20001 | 1:500 | Rabbit |
GFAP | Proteintech | 16825-1-AP | 1:4000 | Rabbit |
TUJI | Abcam | Ab18207 | 1:20,000 | Rabbit |
GFP | UC Davis | 75-131 | 1:1000 | Mouse |
Anti-mouse IgG | Cell Signaling | 7076 | 1:5000 | Horse |
Anti-rabbit IgG | Cell Signaling | 7074 | 1:5000 | Goat |
BN-PAGE and in-gel activity assay
Antibody | Company | Catalog # | Dilution | Species |
---|---|---|---|---|
NDUFA9 | Abcam | Ab14713 | 1:1000 | Mouse |
MTCO1 | Abcam | Ab14705 | 1:1000 | Mouse |
UQCRC1 | Abcam | Ab110252 | 1:1000 | Mouse |
SDHA | Abcam | Ab14715 | 1:1000 | Mouse |
Anti-mouse IgG | Cell Signaling | 7076 | 1:5000 | Horse |
DNA extraction and viral titer quantification
Statistics and reproducibility
Data availability
Author contributions
Disclosure and competing interests statement
Acknowledgements
Supporting Information
References
Information & Authors
Information
Published In
This month's cover highlights the article Experimental vaccination by single dose sporozoite injection of blood-stage attenuated malaria parasites by Julia M. Sattler, Friedrich Frischknecht and colleagues. The image shows an artistic rendering of a Plasmodium infected red blood cell amongst healthy erythrocytes. Sattler et al use genetic attenuation to slow the development of parasites in red cells thereby giving the immune system sufficient time to raise an effective response. In mice, this leads to the clearance of the acute and protection from subsequent infections.
Scientific illustration by Marco Binder, German Cancer Research Center Heidelberg (DKFZ)
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