Cytoprotection by a naturally occurring variant of ATP5G1 in Arctic ground squirrel neural progenitor cells
- Department of Neurology, University of California-San Francisco, United States
- Cardiovascular Research Institute, University of California-San Francisco, United States
- Department of Physiology, University of California-San Francisco, United States
- Innovative Genomics Institute, United States
Figures
Figure 1 with 1 supplement
Phenotypic characteristics of Mouse and AGS NPCs.
(A) Confocal image of mouse (top) and AGS (bottom) NPCs demonstrating similar morphology and expression of Nestin (red) and Ki-67 (teal) in nearly all cultured cells of both species. (B) Mouse and …
Figure 1—figure supplement 1
Mouse and AGS NPCs express Nestin and Ki-67 and additional metabolic phenotypic data.
(A–B) Representative live brightfield microscopy image of mouse and AGS NPCs. (C–D) Representative fluorescent microscopy images of fixed mouse and AGS NPCs immunolabeled with Nestin (green) and …
Figure 2 with 1 supplement
AGS cDNA library survival screen identifies AGS ATP5G1 as a cytoprotective factor.
(A) AGS NPC cDNA was introduced into mouse NPCs by nucleofection. Cells were screened for survival after exposure to hypoxia (1%, 48 hr), hypothermia (31°C, 72 hr), or rotenone (20 μM, 48 hr) to …
Figure 2—figure supplement 1
cDNA library construction and ATP5G expression in mouse and AGS NPCs.
(A) Agarose gel image of 11 randomly chosen AGS cDNA library clones prior to amplification demonstrating an average insert size of 2.4 kB. (B) Agarose gel image of amplified and normalized AGS cDNA …
Figure 3 with 3 supplements
Overexpression of AGS ATP5G1 in mouse NPCs recapitulates AGS metabolic phenotypes, which is dependent on the uniquely evolved leucine-32.
(A) Seahorse XF analyzer assay of cultured mouse NPCs expressing human ATP5G1, AGS ATP5G1, AGS ATP5G1L32P, human ATP5G1P32L, or empty vector and sequentially exposed to (i) oligomycin (1 μM), (ii) …
Figure 3—figure supplement 1
Additional data for overexpression of AGS ATP5G1 in mouse NPCs recapitulating AGS metabolic phenotypes, which is dependent on the uniquely evolved leucine-32.
(A) Mean branch length of mitochondrial networks of mouse NPCs expressing mCherry-ATP5G1 variants from Figure 3D–G. Bar graph represents mean ± SEM from automated processing of 30 cells/cell line. (B…
Figure 3—figure supplement 2
Human and AGS ATP5G1 constructs are appropriately targeted to the mitochondria of mouse NPCs.
(A-D) Confocal imaging of mouse NPCs transiently expressing mEmerald-mito7 and the indicated mCherry-ATP5G1 constructs demonstrate appropriate mitochondrial localization of the human, AGS, and …
Figure 3—figure supplement 3
Human and AGS ATP5G1 constructs are appropriately targeted to the mitochondria of AGS NPCs.
(A-D) Confocal imaging of AGS NPCs transiently expressing mEmerald-mito7 and the indicated mCherry-ATP5G1 constructs demonstrate appropriate mitochondrial localization of the human, AGS, and …
Figure 4 with 1 supplement
CRISPR base editing to generate ATP5G1L32P AGS NPCs results in a partial loss of AGS metabolic resilient phenotypes.
(A) AGS ATP5G1 CRISPR base editing strategy. To create AGS cells with the human amino acid substitution at leucine-32, AGS cells transiently expressing ABEmax were nucleofected with an sgRNA (blue …
Figure 4—figure supplement 1
Additional data for ABE cell lines.
(A) Sequencing data from an unsuccessfully edited clonal AGS cell line demonstrating the preservation of the wild-type sequence (* indicates wild-type thymidine). (B) qRT-PCR for ATP5G1, ATP5G2, and …
Tables
Table 1
Species-specific primers used in quantitative RT-PCR.
| Mouse | |
|---|---|
| Rpl27 Forward | ATA AGA ATG CGG CCG CAA GC |
| Rpl27 Reverse | ATC GAT TCG CTC CTC AAA CTT |
| Atp5g1 Forward | TGC AGA CCA CCA AGG CAC TG |
| Atp5g1 Reverse | GGC CTC TGG TCT GCT CAG GA |
| Atp5g2 Forward | CGT CTC TAC CCG CTC CCT GA |
| Atp5g2 Reverse | CTG CAG ACA GCG GAC GAC TC |
| Atp5g3 Forward | GGG CCC AGA ATG GTG TGT GT |
| Atp5g3 Reverse | TGC AGC ACC TGC ACC AAT GA |
| AGS | |
| Rpl27 Forward | CTG CCA TGG GCA AGA AGA AA |
| Rpl27 Reverse | AGC AGG GTC TCT GAA GAC AT |
| Atp5g1 Forward | TCC GGC TCT GAT CCG CTG TA |
| Atp5g1 Reverse | GGG AGC TGC TGC TGT AGG AA |
| Atp5g2 Forward | TGC CTG CTC CAG GTT CCT CT |
| Atp5g2 Reverse | GGG ACT GCC AAG CTG CTG AA |
| Atp5g3 Forward | TGA GGC CCA GAA TGG TGA ACG |
| Atp5g3 Reverse | CAG CAC CAG AAC CAG CCA CT |
Additional files
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Supplementary file 1
Cytoprotective AGS genes identified from AGS cDNA screens in mouse NPCs exposed to hypoxia, hypothermia, or rotenone.
- https://cdn.elifesciences.org/articles/55578/elife-55578-supp1-v2.xlsx
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Supplementary file 2
Ground squirrel-unique amino acid substitutions identified from the cytoprotective screened genes and associated Jensen-Shannon Divergence and BLOSUM-62 scores.
- https://cdn.elifesciences.org/articles/55578/elife-55578-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/55578/elife-55578-transrepform-v2.docx
