Parkin is a disease modifier in the mutant SOD1 mouse model of familial ALS (2018)
Figure 8

Figure 8. Parkin knockout improves the decline of mitochondrial dynamics proteins in SOD1-G93A spinal cord, but mitochondrial protein ubiquitination profiles and March5 and Mul1 levels are unaffected

A, B) Western blots (A) and quantification (B) of Miro1 in spinal cord homogenates at disease end stage. β-actin is used as loading reference. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (4 males and 4 females) mice per group; *p= 0.011 by paired Student's t test (for comparison G93A vs. PKO/G93A) and **p= 0.003 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A). Parkin knockout increases the levels of Miro1 in SOD1-G93A mice at disease end stage.

C, D) Western blots (C) and quantification (D) of Mfn2 in spinal cord homogenates at disease end stage. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females) mice per group; No statistically significant differences were found between G93A and PKO/G93A (p=0.078 by paired Wilcoxon's test); ***p= 0.0007 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A) and *p= 0.037 by paired Friedman's test with Dunn's correction (for PKO vs. PKO/G93A). Parkin knockout increases the levels of Mfn2 in SOD1-G93A mice at disease end stage.

E) Western blots of spinal cord mitochondria at disease end stage, probed for lysine 48 (left panel) and lysine 63 (right panel) ubiquitin chains. Citrate synthase is used as loading control. Asterisks indicate ubiquitinated proteins with different abundance in SOD1-G93A samples.

F, G) Representative Western blot (F) and quantification (G) of LC3 II in spinal cord mitochondria at end stage. Protein levels are normalized by Complex V. LC3 II is increased in both G93A and PKO/G93A mitochondria and Parkin knockout does not affect LC3 II levels. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (4 males and 4 females) mice per group; No statistically significant differences were found between G93A and PKO/G93A (p=0.504 by paired Student's t test). *p= 0.035 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A) and **p= 0.0014 by paired one-way ANOVA with Tukey's correction (for PKO vs. PKO/G93A)

H) Representative Western blot of March5 and Mul1 in spinal cord mitochondria at end stage.

I) Quantification of March5 at disease end stage. Results are expressed as mean ± SEM and percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.742). Protein levels are normalized by Complex V. March5 levels are unaffected by Parkin knockout.

J) Quantification of Mul1 at disease end stage. Results are expressed as mean ± SEM and percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.94). Protein levels are normalized by Complex V. Mul1 levels are unaffected by Parkin knockout.

Expanded view Figure legends

Expanded View 1. Parkin protein levels are unaffected in liver while a small decrease is measured in G93A cerebellum.

A) Representative Western blot for Parkin and p62 in cerebellum (Cb) homogenates at 130 days.

B) Quantification indicates that Parkin is decreased in G93A mice relative to Non Tg at 130 days. Protein levels were normalized by β-actin. Results are expressed as mean ± SEM and percent of Non Tg; n = 8 (4 males and 4 females) mice per group. *p= 0.039 by paired Wilcoxon's test.

C) Quantification of p62 in the homogenates indicates that p62 is increased in PKO and PKO/G93A mice, independent of SOD1-G93A expression, at 130 days of age. β-actin was used for normalization. Results are expressed as mean ± SEM and percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between Non Tg and G93A by paired Friedman's test with Dunn's correction (p= 0.99). ***p= 0.0001 by paired Student's t test (G93A vs. PKO/G93A).

D) Representative Western blot of Parkin and p62 in liver homogenates at 130 days.

(E) Parkin protein levels were quantified at 130 days. β-actin was used for normalization. Results are expressed as mean ± SEM and percent of Non Tg; n = 8 (4 males and 4 females) mice per group. No statistically significant differences were found between Non Tg and G93A (p= 0.546 by paired Wilcoxon's test).

F) Quantification of p62 protein levels in liver at 130 days. Results are expressed as mean ± SEM and percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between Non Tg and G93A (p= 0.546 by paired Wilcoxon's test). No statistically significant differences were found among the other groups.

Expanded View 2. SOD1 aggregates similarly in spinal cord mitochondria of G93A and PKO/G93A mice.

A) Representative filter trap blot for detection of SOD1 aggregates in spinal cord mitochondria at 130 days.

B) Quantification of the SOD1 signal intensity at 130 days of age revealed no statistically significant differences between G93A and PKO/G93A, indicative of similar amount of aggregates in mitochondria. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A (p= 0.987 by paired Student's t test). **p= 0.0058 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A), ***p= 0.0002, by paired one-way ANOVA with Tukey's correction (for PKO vs. PKO/G93A).

Expanded View 3. Parkin knockout mitigates the accumulation of mitophagy adaptors in G93A mitochondria at disease end stage.

A, B) Representative Western blots of p62 (A), OPTN and VCP (B) in spinal cord mitochondria from end stage mice.

C) p62 quantification, using Complex V as normalizer, shows a strong accumulation of p62 in SOD1-G93A mitochondria at end stage. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A (p= 0.078 by paired Wilcoxon's test); ***p= 0.0007 (Non Tg vs. G93A) and *p= 0.037 (PKO and PKO/G93A) both by paired Friedman's test with Dunn's correction.

D) Quantification of OPTN accumulation in end stage mitochondria, with Complex V as protein loading control. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females) mice per group. **p= 0.0078 (for G93A and PKO/G93A) by paired Wilcoxon's test; ***p= 0.0003 (Non Tg vs. G93A) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found.

E) Quantification of VCP in mitochondria at disease end stage. Complex V was used as normalizer. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females) mice per group. *p= 0.039 (for G93A and PKO/G93A) by paired Wilcoxon's test; ***p= 0.0007 (Non Tg vs. G93A) and *p= 0.037 (PKO and PKO/G93A) both by paired Friedman's test with Dunn's correction.

Expanded View 4. Increased mitochondrial protein turnover in G93A mice is attenuated by Parkin knockout.

A, B) Western blots of COXI (A) and Tim23 (B) in spinal cord homogenates at 130 days.

C) Quantification of COXI at 130 days with β-actin as a normalizer. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A (p= 0.493 by paired Student's t test).

D) Quantification of Tim23 at 130 days, using β-actin as loading control, showed decreased levels of Tim23 in G93A mice. Results are expressed as mean ± SEM and as a percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A (p= 0.921 by paired Student's t test). *p= 0.035 (Non Tg Vs. G93A) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found.

E, F) Representative Western blots of COXI (E) and Tim23 (F) in disease end stage homogenates.

G) Quantification of COXI at disease end stage using β-actin as loading control. Results are expressed as mean ± SEM and as percent of Non Tg; n = 5 (3 males and 2 females) mice per group. *p= 0.047 for (G93A and PKO/G93A) by paired Student's t test; **p= 0.0018 by paired Friedman's test with Dunn's correction (Non Tg vs. G93A).

H) Quantification of Tim23 with β-actin as normalizer at end stage, showed that Parkin knockout can alleviate the turnover of mitochondrial proteins in G93A spinal cords. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A (p= 0.190 by paired Student's t test). *p= 0.037 (for PKO vs. PKO/G93A) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found.

Expanded view 5. Parkin overexpression increases vulnerability to mitochondrial oxidative stress in mutant SOD1-expressing NSC34 motor neuron-like cells.

A) Western blot of Parkin in lysates from NSC34 motor neuron-like cells, stably transfected with either human SOD1-G93A or empty vector (Mock). Mouse spinal cord homogenate was used as a positive control for Parkin. β-actin was the loading control.

B) Representative images of NSC34 cells transfected with mtDsRed and YFP-Parkin or GFP and treated with 10 μM mtPQ or vehicle (DMSO) for 24 hours. Scale bar, 10 μm. Parkin-YFP clusters with mitochondria upon oxidative stress.

C) Viability of NSC34 cells after transfection with YFP-Parkin or pEGFP-N1 and treatment with DMSO (vehicle) or 10μM mtPQ for 24 hours. Results are expressed as mean ± SEM and as percent of vehicle-treated cells (for each plasmid transfection). n= 40 fields per condition (from 2 independent experiments); for Mock NSC34 cells, GFP vs. Parkin transfected, treated with mtPQ, *p= 0.013, by unpaired Student's t test; for G93A NSC34 cells, GFP vs. Parkin transfected, treated with mtPQ, *p= 0.029, by unpaired Mann-Whitney t test.

Validation Status

(Antibodypedia)

WB

IHC

Rabbit

Polyclonal

Mouse

Monoclonal

COXI (Complex IV,

subunit 1)

Mouse

Monoclonal

Sigma

WB

AB_1848699

N/A

Rabbit

Polyclonal

PARIS (Parkin Interacting Substrate

(ZNF746))

AB_1587578

AB_11213655

Rabbit

Polyclonal

Rabbit

Polyclonal

AB_11212727

AB_91202http://antibodyregistry.org/AB_91202

Expanded view Table 1

All the antibodies used are described in the table. N/A, not assessed; IHC, immunohistochemistry; WB, western blot; RRID, Research Resource Identifiers (https://scicrunch.org/resources).

Expanded View Table 2

Primer sequences used for qPCR. For, forward; Rev, reverse; m, mouse.