EMBO/soda-nel huggingface dataset:
Complex IV subunit isoform COX6A2 protects fast-spiking interneurons from oxidative stress and supports their function (2020)
Figure 1

Figure 1. Cox6a2 expression is restricted to PV+ interneurons in the mouse brain.

(A) Analysis of the single-cell transcriptomics dataset for the mouse visual cortex (Tasic et al, 2016) revealed that the expression of the metabolism-associated gene Cox6a2 is highly restricted to PV+ interneurons. Cox6a2 is expressed in all subtypes of PV+ interneurons (marked in blue color) but rarely detected in other neuronal subtypes from the mouse visual cortex. Cox6a1, which encodes the other protein isoform of COX6A, is expressed in all subtypes.

(B,C) RT-PCR and Western-blot, respectively, showing expression of Cox6a2 in the brain and heart.

(D) Western-blot analysis showed that COX6A2 is first detected in the brain at postnatal day 0 (P0). Then, COX6A2 protein levels gradually increase until P60.

(E-H) COX6A2 immunoreactivity is detected in PV+ interneurons in different brain areas of adult mice, including the cortex (general overview in E, and single PV+ interneuron magnification in F), hippocampus (G) and striatum (H). White arrowheads indicate PV/Cox6a2 double positive cells.

(I,J) Quantifications showing almost 100% of co-localization of COX6A2+ cells and PV+ cells in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult mice (n animals=4, 2 months old, n sections=24 SS, 16 MT, 8 HI).

(K) COX6A2 immunoreactivity in the cortex at P9 (n animals=2, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate Cox6a2 positive cells.

(L-N) As soon as parvalbumin immunoreactivity becomes visible at P13, almost 100% of PV+ cells are co-labelled with COX6A2 immunoreactivity in the SS and MT cortices, and the HI of P13 mice (n=2).

Data information: All error bars represent SD. In all quantifications the data are shown per section analyzed. Scale bars: 20 μm (E,G,H,K,L) and 10 μm (F,K inset). COX4: cytochrome c oxidase subunit 4; GADPH: glyceraldehyde 3-phosphate dehydrogenase.
Complex IV subunit isoform COX6A2 protects fast-spiking interneurons from oxidative stress and supports their function (2020)
Figure 2

Figure 2. Cox6a2 expression in PV+ interneurons is conserved from rodents up to humans.

(A,B) COX6A2 immunoreactivity is virtually restricted to PV+ interneurons in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult rat (n animals=1, 2.5 months old, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate PV/Cox6a2 double positive cells.

(C,D) In the SS of adult rhesus monkey, COX6A2 immunoreactivity is present exclusively in PV+ cells (n animals=1, 1-year-old, n sections=6). White arrowheads indicate PV/Cox6a2 double positive cells.

(E,F) In adult humans, all cortical PV+ interneurons from Brodmann areas 9 and 10 (BA9 and BA10) are co-labelled with COX6A2 immunoreactivity. However, PV+ interneurons only represent 60% of the cells in which COX6A2 is detected (n human subjects=2, 59- and 69-year-old, n sections=6 BA9, 6 BA10). White arrowheads indicate PV/Cox6a2 double positive cells.

Data information: All error bars represent SD. In all quantifications the data are shown per section analyzed. Scale bars: 50 μm (A,C,E).
Complex IV subunit isoform COX6A2 protects fast-spiking interneurons from oxidative stress and supports their function (2020)
Figure 3

Figure 3. Cox6a2-/- alters essential cellular and extracellular characteristics of PV+ interneurons.

(A) Images showing absence of COX6A2 immunoreactivity in the cortex of Cox6a2-/- mice.

(B,C) Parvalbumin immunoreactivity in PV+ interneurons was significantly decreased in the somatosensory cortex of Cox6a2-/- mice compared to wild-types (n=4 WT, 4 Cox6a2-/-; 2-month-old). C: two-tailed unpaired Student's t-test.

(D) In Cox6a2-/- mice, cortical PV+ interneurons showed a fewer number of synapses innervating principal neurons (n=3 WT, 3 Cox6a2-/-; 2-month-old). Two-tailed unpaired Student's t-test.

(E) To analyze changes in oxidative stress in PV+ interneurons, the somatosensory and motor cortices of WT and Cox6a2-/- mice were stained with an antibody against 8-OHdG, a marker of oxidative DNA damage.

(F) 8-OHdG immunoreactivity was significantly higher in PV+ interneurons in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests.

(G) The density of PNNs around PV+ interneurons residing in the somatosensory and motor cortices was analyzed by measuring the immunoreactivity of WFA that stains PNNs.

(H) WFA immunoreactivity around PV+ cells was lower in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests.

Data information: All error bars represent SD. For detailed statistical information, see Appendix Table S5B-D. Scale bars: 50 μm (E,G), 20 μm (A,B) and 10 μm (E inset,G inset).
Complex IV subunit isoform COX6A2 protects fast-spiking interneurons from oxidative stress and supports their function (2020)
Figure 4

Figure 4. Intracellular signaling in PV+ interneurons is affected by Cox6a2 knockout.

(A) PV+ interneurons were sorted from cortices of PV-EGFP WT and Cox6a2-/- mice at P15 and processed for bulk RNA sequencing (n= 4 WT, 6 Cox6a2-/-; P15). Heatmap shows expression for top down- (red) and upregulated (blue) genes in Cox6a2-/- PV+ interneurons relative to their WT counterparts.

(B) Cox6a2 expression was effectively abolished in Cox6a2-/- PV+ interneurons. In addition, Cox6a2 knockout led to a decreased expression of Gad1 and Gad2 genes (n= 4 WT, 6 Cox6a2-/-). Two-tailed unpaired Mann-Whitney tests.

(C) Changes in expression of oxidative stress-related genes (green), genes coding for AMPA receptors (orange), sodium channels (red), potassium channels (magenta), and internal calcium signaling proteins (blue). Color scale bars show normalized read counts.

(D) Significantly down- (red) and upregulated (blue) gene ontology (GO) terms based on top 200 down-/upregulated genes in Cox6a2-/- PV+ interneurons (Benjamini-Hochberg-adjusted p-values). See also Appendix Tables S1, S2 and S3. For detailed statistical information, see Appendix Table S5E.

Data information: All error bars represent SD.
Complex IV subunit isoform COX6A2 protects fast-spiking interneurons from oxidative stress and supports their function (2020)
Figure 5

Figure 5. Ex vivo live-cell imaging shows that COX6A2 regulates energy balance in PV+ interneurons.

(A) Experimental scheme. The selective expression of CRE-recombinase in PV-CRE and PV-CRE;Cox6a2fl/fl mice ensures the expression of PercevalHR only in PV+ interneurons.

(B) Experiment timeline. AAVs expressing PercevalHR were injected in the somatosensory (SS) cortex of mice at postnatal day 15 (P15). Approximately, 3 weeks after the injections, the brains were sliced into coronal sections for live-cell imaging of ATP-to-ADP ratio by 2-photon microscopy.

(C) PercevalHR is a ratiometric fluorescent protein that changes its excitation spectrum based on its binding to ATP (930 nm) or ADP (840 nm).

(D) Representative images of PV+ interneurons from PV-CRE and PV-CRE;Cox6a2fl/fl mice showing PercevalHR fluorescence after excitation at 930 nm and 840 nm. White arrowheads indicate PercevalHR expressing cells. Scale bar: 20 μm.

(E) PV+ interneurons from PV-CRE;Cox6a2fl/fl mice exhibited significantly lower ATP-to-ADP ratios than PV+ interneurons from PV-CRE mice throughout the entire duration of the recordings (23 cells from 4 PV-CRE mice; 15 cells from 6 PV-CRE;Cox6a2-/- mice). Data were analyzed by two-way ANOVA. Error bars represent SD.
Complex IV subunit isoform COX6A2 protects fast-spiking interneurons from oxidative stress and supports their function (2020)
Figure 6

Figure 6. Cox6a2 knockout in PV+ interneurons delays their functional and morphological maturation.

(A,B) The resting membrane potential and amplitude of action potentials in WT and Cox6a2-/- PV+ interneurons, respectively. Two-tailed unpaired Student's t-tests.

(C,D) The threshold for action potentials increased over maturation in WT but not in Cox6a2-/- PV+ interneurons. As a result, the threshold for action potentials was significantly lower in P34-45 Cox6a2-/- PV+ interneurons when compared to their WT counterparts. D: two-tailed unpaired Student's t-tests.

(E,F) Trains of action potentials were evoked by 2 sec depolarizing current pulses. The amplitude difference (difference between the mean amplitude of 1st-10th action potentials and 190th-200th action potentials) was significantly higher in P34-45 Cox6a2-/- compared to WT PV+ interneurons. In addition, the amplitude difference decreased with maturation in WT but not in Cox6a2-/- PV+ interneurons. F: two-tailed unpaired Mann-Whitney tests.

(G) While in WT PV+ interneurons the maximal firing frequency increased during maturation, such increase was abolished by Cox6a2 knockout. Thus, maximal firing frequency reached during 2 sec depolarizing current pulses was lower in P34-45 Cox6a2-/- compared to WT PV+ interneurons. Two-tailed unpaired Student's t-tests.

(H) Recorded PV+ interneurons were filled with biocytin and their dendritic morphology was reconstructed using Imaris software. Scale bars: 30 μm.

(I,J) Cox6a2-/- PV+ interneurons showed a negative correlation between age and dendritic length and between age and full branch depth (maximal number of branches between the soma and a terminal dendritic point), which contrasted with the expected positive correlation between these parameters in WT PV+ interneurons. Linear regression of Pearson correlation.

(K,L) WT and Cox6a2-/- PV+ interneurons showed similar values for dendritic length and full branch depth at P22-P33. However, at P34-P45, Cox6a2-/- PV+ interneurons showed lower dendritic length and full branch depth than WT PV+ interneurons. Two-way ANOVA followed by Tukey's multiple comparisons tests.

(M,N) While younger WT and Cox6a2-/- PV+ interneurons have similar dendritic tree complexities (M), at the later stage of maturation Cox6a2-/- PV+ interneurons show a reduction in dendritic tree complexity in comparison to WT PV+ interneurons (N). Multiple t-tests, two-stage step-up method of Benjamini, Krieger and Yekutieli.

Data information: For amplitude and maximal firing frequency, n=37 WT (n=22 P22-P33, n=15 P34-P45) and 37 Cox6a2-/- (n=11 P22-P33, n=26 P34-P45) PV+ interneurons. For the rest of the electrophysiological parameters analyzed, n=39 WT (n=24 P22-P33, n=15 P34-P45) and 43 Cox6a2-/- (n=14 P22-P33, n=29 P34-P45) PV+ interneurons. For all morphological comparisons, n= 24 WT (n=19 P22-P33, n=5 P34-P45) and 19 Cox6a2-/- (n=7 P22-P33, n=12 P34-P45) PV+ interneurons. All error bars represent SD. For detailed statistical information, see Appendix Tables S5F-H.
Complex IV subunit isoform COX6A2 protects fast-spiking interneurons from oxidative stress and supports their function (2020)
Figure 7

Figure 7. Cox6a2-/- mice show behavioral alterations.

(A) Illustration of the arena used for the open field test (OFT).

(B) In the OFT, Cox6a2-/- mice travelled a longer distance and moved faster than WT mice. Cox6a2-/- mice also moved more often from the outer zone to the inner zone of the arena. Two-tailed unpaired Student's t-tests.

(C) Illustration of the arena used for the social interaction test (SIT).

(D) In the SIT, Cox6a2-/- mice also travelled a longer distance with higher mean velocity than WT mice. Additionally, both Cox6a2-/- and WT mice spent more time exploring the container with the stranger mouse than the empty container. Total distance moved, mean velocity, and f [number of crossings] outer to inner zone: two-tailed unpaired Student's t-test. Social interaction: two-way ANOVA followed by Tukey's multiple comparisons test.

(E) Sensory gating was tested by the pre-pulse inhibition (PPI) of the acoustic startle response (ASR) test.

(F) The startle responses were significantly lower in Cox6a2-/- mice when the loud stimulus (120 dB) was presented alone the first 5 times. Two-way ANOVA followed by Tukey's multiple comparisons tests.

(G) The PPI of the acoustic startle response was similar at all pre-pulses for Cox6a2-/- and WT mice. Two-way ANOVA followed by Sidak's multiple comparisons tests.

Data information: For all behavioral tests, n=13 WT and 13 Cox6a2-/- mice (~6-10 months old), all females. All error bars represent SD. For detailed statistical information, see Appendix Tables S5I-K.
Progesterone and Wnt4 control mammary stem cells via myoepithelial crosstalk (2014)
Figure 1

PR, RANKL, and Wnt4 and their role in the regenerative capacity of the mammary gland

A Experimental scheme. Mammary tissue fragments dissected from wild‐type (WT) or mutant (MT) donor mice were engrafted to contralateral mammary fat pads of RAG1−/− recipient mice surgically divested of the endogenous epithelium. Between 8 and 12 weeks later, the engrafted glands were assessed by fluorescent stereomicroscopy and new fragments were dissected for serial engraftment.
B, C Serial transplantations of PR+/+ and PR−/− mammary epithelia. (B) Fluorescence stereo micrographs of third‐generation mammary outgrowths derived from 8‐week‐old PR+/+; EGFP and PR−/−; EGFP donor mice. (C) Table summarizing 3 independent serial transplant experiments with PR+/+; EGFP and PR−/−; EGFP. Each engrafted gland is represented by a micrograph; black sectors represent area of fat pad filled by engrafted epithelium. Scale bar: 200 μm.
D, E Serial transplantation of RANKL+/+ and RANKL−/− mammary epithelia. (D) Fluorescence stereo micrographs of third‐generation mammary outgrowths derived from 5‐week‐old RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Insets: higher magnification showing side branches present in the WT control (arrowheads) absent from RANKL−/−; EGFP epithelium. (E) Table summarizing three independent serial transplant experiments with RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Scale bar: 200 μm.
F, G Serial transplantation of Wnt4+/+ and Wnt4−/− mammary epithelia. (F) Fluorescence stereo micrographs of third‐generation mammary outgrowths derived from mammary buds of E12.5 and E13.5 Wnt4+/+; EGFP and Wnt4−/−; EGFP embryos. Scale bar: 200 μm. (G) Table summarizing three independent serial transplant experiments with Wnt4+/+; EGFP donor mice. Scale bar: 200 μm.
H Box plot showing the difference between percentage of reconstitution between WT and MT contralateral grafts in each transplant generation. P values were determined by Mann–Whitney U‐test.
Progesterone and Wnt4 control mammary stem cells via myoepithelial crosstalk (2014)
Figure 2

Control of Wnt4 expression

A Epifluorescence stereo micrograph of inguinal mammary gland from a 5‐day‐old Wnt4::Cre; mT/mG female (n = 7). Scale bars: 0.5 mm and 0.1 mm (inset).
B, C Histological sections of mammary glands from a 5‐day‐old (B; n = 7) and a 10‐day‐old (C; n = 5) Wnt4::Cre; mT/mG female stained by double immunofluorescence for EGFP (green) and PR (magenta, not detected), counterstained with DAPI (blue). Scale bar: 50 mm.
D–F EGFP (green) and PR (magenta) co‐immunofluorescence counterstained with DAPI (blue) on histological sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (D) TEB of a 4‐week‐old female (n = 4); scale bar: 30 μm. (E) Ducts of an 8‐week‐old female (n = 3); scale bar: 100 μm; inset, scale bar: 20 μm. (F) Duct of a female at day 10.5 of pregnancy (n = 3); scale bar: 150 μm; inset, scale bar: 30 μm.
G–I EGFP (green), PR (magenta), and p63 (white) triple co‐immunofluorescence counterstained with DAPI (blue) on histological sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (G) Ducts of 5‐day‐old female (n = 3). (H) TEB of a 4‐week‐old female (n = 3). (I) Duct of an 8‐week‐old female (n = 3). Scale bars: 30 μm.
J–O Epifluorescence stereo micrographs of mammary glands harvested from 15‐day‐old Wnt4::GFP females either WT (n = 18) (J, M), ERα−/− (n = 4) (K, N), or PR−/− (n = 3) (L, O). dTomato expression (J–L); EGFP expression (M–O) is not abrogated in ERα−/− nor PR−/− epithelia. Arrowheads mark the main duct originating from the nipple. Scale bar: 50 μm.
P Scheme of ex vivo hormone stimulation of mammary organoids.
Q Bar plots showing relative PR and Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal (6 weeks old) and 3 adult (11 weeks old) mice exposed for 6 h to vehicle (C), 17β‐estradiol (20 nmol) (E2), or R5020 (20 nmol) (P). Bars represent the mean ± SD of 3 independent experiments.
R–X Epifluorescence stereo micrographs of contralateral mammary glands that were engrafted with Wnt4::GFP epithelium from 8‐week‐old females, either PRWT (R, T, V, X) or PR−/− (S, U, W). dTomato expression (R, S); EGFP expression (T, U) double epifluorescence (V, W, X) on contralateral engrafted glands 3 weeks after surgery when recipients were 6 weeks old. Representative result from three independent experiments. Arrowheads point to TEBs (V, X) or to origin of growth (W). Scale bar (R–W): 5 mm, (X): 1 mm.
Progesterone and Wnt4 control mammary stem cells via myoepithelial crosstalk (2014)
Figure 3

Conditional deletion of Wnt4 in the mammary epithelium and its consequences

A–D Representative epifluorescence stereo micrographs of inguinal mammary glands from a 10‐day‐old mT/mG (A, C) and MMTV::Cre; mT/mG (B, D) female (n = 7). Scale bar: 1 mm.
E Higher magnification of inguinal mammary gland from a 10‐day‐old MMTV::Cre; mT/mG female. Scale bar: 0.2 mm.
F Immunofluorescence for EGFP (green) on a mammary gland section from a 10‐day‐old MMTV::Cre; mT/mG female counterstained with DAPI (blue) (n = 7). Scale bar: 30 μm.
G Epifluorescence stereo micrographs of mammary glands from 10‐day‐old littermates either MMTV::Cre; mT/mG Wnt4fl/+ or MMTV::Cre; mT/mG; Wnt4fl/fl. Scale bar: 1 mm. Arrowhead marks the main duct originating from the nipple.
H Bar plot showing ratio of branching points in prepubertal Cre+;Wnt4fl/fl (n = 12) relative to Cre+;Wnt4fl/+ littermates (n = 10). Two‐tailed, paired Student's t‐test was used to calculate statistical significance.
I Fluorescence stereo micrographs of mammary glands from 35‐day‐old littermates either MMTV::Cre; mT/m; Wnt4fl/+ or MMTV::Cre; mT/m;Wnt4fl/fl. Note ductal elongation is delayed in Wnt4‐deficient mammary epithelium compared to Wnt4fl/+ control. LN: lymph node. Scale bar: 4 mm.
J, K Bar plots showing number of terminal end buds (TEBs) (J) and area of mammary fat pad filled by the ductal system (K). Ctrl (Wnt4fl/wt or Wnt4fl/fl; Cre−): n = 6, fl/+; Cre+: n = 4, and fl/fl; Cre+: n = 7). Data are presented as the mean ± SD. Two‐tailed Student's t‐test was used to calculate statistical significance.
L, M Bar plots showing BrdU incorporation index in inguinal mammary glands of 4‐week‐old Wnt4fl/+; Cre+ (n = 3) or Wnt4fl/fl; Cre+ (n = 4) in the ducts (L) and in the TEBs (M). Data are presented as the mean ± SD. Two‐tailed Student's t‐test was used to calculate statistical significance.
Progesterone and Wnt4 control mammary stem cells via myoepithelial crosstalk (2014)
Figure 4

Canonical Wnt signaling activity during mammary gland development

A Whole‐mount micrograph of X‐gal‐stained Axin2+/lacZ in E12.5 embryo showing β‐galactosidase expression in the mammary buds (arrows) (n = 8). Scale bar: 1 mm. Arrowheads mark mammary buds.
B–F Whole‐mount micrographs of X‐gal (blue)‐ and carmine alum (red)‐stained mammary glands harvested from Axin2+/lacZ mice at distinct developmental stages. (B) At postnatal day 1, β‐galactosidase activity detected in the nipple area (n = 6). Scale bar: 1 mm. (C, D) In 5‐week‐old mammary glands, reporter activity was detected around the ducts (small arrows) and in the neck region of the terminal end buds (TEBs) (large arrows) (C) (n = 8). Scale bars: 400 μm (C) and 100 μm (D). (E) At 8.5 day of pregnancy, reporter expression was detected in the ducts. Higher magnification (inset) suggests myoepithelial expression (n = 10). Scale bar: 1 mm. (F) Whole‐mount at day 14.5 of pregnancy: reporter activity is limited to ducts (n = 5). Scale bar: 200 μm.
G Histological section of Axin2::LacZ mammary gland at day 8.5 of pregnancy counterstained with nuclear red; luminal epithelial cells show no detectable β‐galactosidase activity but myoepithelial cells do. Scale bar: 200 μm.
H β‐galactosidase activity (blue) colocalizes with the myoepithelial marker p63 (green) detected by immunofluorescence. Arrows point to myoepithelial cells. L, lumen. Scale bar: 100 μm.
I Representative whole‐mount stereo micrographs of X‐gal (blue)‐ and carmine alum (magenta)‐stained mammary gland biopsies taken from 14‐week‐old Axin2::LacZ females collected at diestrus and estrus, respectively (n = 3). Scale bar: 200 μm.
J Relative Wnt4 and Axin2 mRNA expression in mammary glands from three mice in estrus versus diestrus assessed by semiquantitative qRT–PCR normalized to 18S rRNA. Two‐tailed, paired Student's t‐test was used to calculate statistical significance.
K Stereo micrographs of X‐gal‐ and carmine alum‐stained mammary glands from ovariectomized Axin2::LacZ females treated for 72 h with vehicle (n = 4) (left), 17‐β‐estradiol (E2) (n = 6) (center), 17‐β‐estradiol and progesterone (E2 and P) (n = 8) (right). Scale bar: 200 μm.
L, M Stereo micrographs of contralateral glands whole‐mounted and X‐gal stained after engraftment with mammary buds from Axin2::LacZ transgenic and Wnt4+/+ or Wnt4−/− female E12.5 and E13.5 embryos (L) or Axin2::LacZ transgenic and either PR+/− or PR−/− 8‐week‐old females (M), at day 8.5 of pregnancy. β‐galactosidase expression reflecting Axin2 transcription is readily detected in PR+/− as well as Wnt4+/+ mammary epithelia but not in the PR−/− (n = 6) and Wnt4−/− counterparts (n = 6). Blue: X‐gal staining; magenta: carmine alum counterstain. Scale bars: 200 μm.
Progesterone and Wnt4 control mammary stem cells via myoepithelial crosstalk (2014)
Figure 5

Wnt4‐independent canonical Wnt signaling during mammary gland development

X‐gal‐stained whole‐mounts of Axin2::LacZ embryos at E14.5 either Wnt4+/+ or Wnt4−/− (n = 5 or 4, respectively). Arrows mark mammary buds. Scale bar: 5 mm.
Whole‐mount analysis of X‐gal‐ and carmine alum‐stained mammary glands harvested from 6‐week‐old littermates either Wnt4fl/wt;MMTV::Cre+;Axin2+/lacZ (n = 5) or Wnt4fl/fl MMTV::Cre+;Axin2+/lacZ (n = 4). Left insets: higher magnification; right insets: histological section of the same gland. Note expression of reporter detected around the neck of TEBs in Wnt4‐deficient and littermate control is comparable. LN, lymph node. Scale bars: 1 mm, and inset scale bar: 100 μm.