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. 2023 Aug 24;6(11):e202301936.
doi: 10.26508/lsa.202301936. Print 2023 Nov.

p62 and NBR1 functions are dispensable for aggrephagy in mouse ESCs and ESC-derived neurons

Riccardo Trapannone  1   2 , Julia Romanov  3   2 , Sascha Martens  4   2
Affiliations

p62 and NBR1 functions are dispensable for aggrephagy in mouse ESCs and ESC-derived neurons

Riccardo Trapannone et al. Life Sci Alliance. .

Abstract

Accumulation of protein aggregates is a hallmark of various neurodegenerative diseases. Selective autophagy mediates the delivery of specific cytoplasmic cargo material into lysosomes for degradation. In aggrephagy, which is the selective autophagy of protein aggregates, the cargo receptors p62 and NBR1 were shown to play important roles in cargo selection. They bind ubiquitinated cargo material via their ubiquitin-associated domains and tether it to autophagic membranes via their LC3-interacting regions. We used mouse embryonic stem cells (ESCs) in combination with genome editing to obtain further insights into the roles of p62 and NBR1 in aggrephagy. Unexpectedly, our data reveal that both ESCs and ESC-derived neurons do not show strong defects in the clearance of protein aggregates upon knockout of p62 or NBR1 and upon mutation of the p62 ubiquitin-associated domain and the LC3-interacting region motif. Taken together, our results show a robust aggregate clearance in ESCs and ESC-derived neurons. Thus, redundancy between the cargo receptors, other factors, and pathways, such as the ubiquitin-proteasome system, may compensate for the loss of function of p62 and NBR1.

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Conflict of interest statement

S Martens is member of the scientific advisory board of Casma Therapeutics.

Figures

Figure S1.
Figure S1.. Characterisation of the CRISPR cell lines.
(A) Immunostaining of pluripotent and differentiated (DIV21 neurons) mScarlet–p62 cells. (B) Western blot analysis of untagged Rex1GFP1ds2 and mScarlet–p62 pluripotent or differentiated (DIV21 neurons) cells. Two different neuronal differentiation protocols were tested: with or without valproic acid. (C) Live cell imaging of mScarlet-p62 DIV21 neurons. Zoom factor of the selected regions: 5x. (D) Sequencing data from the CRISPR cell line showing the successful mutation of the LC3-interacting region (LIR) motif (DDDW mutated into AAAA). (E) Western blot analysis of pluripotent and differentiated (DIV21 neurons) mScarlet–p62 WT and mScarlet–p62 LIR mutant. (F) Pluripotent Rex1GFP1ds2, p62 KO, mScarlet–p62 WT, mScarlet–p62 LIR mutant, and mScarlet–p62 F408V cells were treated with bafilomycin for 3 h. Lysates were collected afterwards and analysed by SDS–PAGE and Western blot, probing with the indicated antibodies. (G) Immunostaining of pluripotent and differentiated (DIV21 neurons) mScarlet–p62 WT and mScarlet–p62 LIR mutant cells. Scale bar: 20 μm.
Figure S2.
Figure S2.. Colocalisation of DAPGreen with autophagosomes, endosomes, and lysosomes.
(A) HeLa cells were transfected with RFP-LC3B and treated for 3 h with MG-132 before live cell imaging. (B) Hap1 cells were transfected with iRFP–Rab7 for 24 h and incubated with DAPGreen 1 h and 30 min before live cell imaging. (C) Hap1 cells were incubated with 1 h and 30 min with DAPGreen and 30 min with LysoTracker Blue. (D) mScarlet–p62 WT DIV21 neurons were incubated with 1 h and 30 min with DAPGreen and 30 min with LysoTracker Blue. The merge shows DAPGreen nad LysoTracker. Scale bar: 20 μm. Zoom factor of the selected regions: 5x.
Figure 1.
Figure 1.. Effect of p62 LC3-interacting region motif mutation on p62 condensate formation and endo-lysosomal colocalisation in neurons.
(A) mScarlet–p62 WT or LC3-interacting region mutant DIV21 neurons were incubated with DAPGreen staining for 1 h and 30 min and observed via live cell imaging. Zoom factor of the selected regions: 5x. Scale bar: 20 μm. Standard or dashed arrows indicate p62 particles in the cell body or in the processes, respectively. (B) Quantification of the live cell imaging data. All the quantifications were performed on single stacks. The graph shows the mean ± SEM from n = 4 independent biological replicates. A total of 67–70 cell bodies and 8,818–8,839 μm of processes were analysed throughout the replicates. The P-value was calculated with a two-tailed t test, and the statistical significance is shown on the graph (P > 0.05 non-significant, 0.01 < P <0.05 *, 0.001 < P <0.01 **, P < 0.001 ***).
Figure S3.
Figure S3.. Quantifications of the effect of p62 LC3-interacting region motif mutation on p62 aggregate formation and endo-lysosomal colocalisation.
(A) Quantification of the live cell imagine data from Fig 1. All the quantifications were performed on a single stack. The graph shows the mean ± SEM from n = 4 independent biological replicates. The P-value was calculated with a two-tailed t test (P > 0.05 non-significant [not shown], 0.01 < P < 0.05 *, 0.001 < P < 0.01 **, P < 0.001 ***). (B) Details (22 × 22 μm) of mScarlet–p62 WT and LC3-interacting region mutant neurons and ES cells using the “fire” lookup table.
Figure 2.
Figure 2.. Effect of the mutation in the LC3-interacting region motif of p62 in embryonic stem cells.
mScarlet–p62 WT and mScarlet–p62 LC3-interacting region mutant embryonic stem cells were treated for 3 h with either 400 nM bafilomycin or 10 μM MG-132 or a combination of the two drugs. A DMSO-treated sample served as a negative control. (A, B) Live cell imaging analysis and (B) quantification of the microscopy data. The graphs represent the mean ± SEM from n = 3 independent experiments. Zoom factor of the selected regions: 5x. Scale bar: 20 μm. For each treatment and cell line, between 60 and 150 cells were analysed. The analysis was performed on a single-stack image. P-values were calculated with a two-way ANOVA and Tukey’s multiple comparison test (P > 0.05 non-significant [not shown], 0.01 < P < 0.05 *, 0.001 < P < 0.01 **, P < 0.001 ***).
Figure 3.
Figure 3.. Effect of p62 knockout and the mutation in the LC3-interacting region motif of p62 in embryonic stem cells and neurons.
(A) mScarlet–p62 WT and mScarlet–p62 LC3-interacting region mutant embryonic stem cells were treated for 3 h with either 400 nM bafilomycin or 10 μM MG-132 or a combination of the two drugs. A DMSO-treated sample served as a negative control. Western blot analysis was performed using the antibodies indicated. (A, B) Quantification of the Western blot data from (A). p62 and NBR1 band intensity values were normalised to the GAPDH-loading control. The graphs represent the mean of the GAPDH-normalised values ± SEM from n = 3 independent experiments. P-values were calculated with a two-way ANOVA and Tukey’s multiple comparison test (P > 0.05 non-significant [not shown], 0.01 < P < 0.05 *, 0.001 < P < 0.01 **, P < 0.001 ***). (C) DIV21 neurons were treated for 5 h with 400 nM bafilomycin. Cell lysates were analysed by Western blot.
Figure S4.
Figure S4.. Effect of p62 KO on DAPGreen particle detection in neurons.
(A, B) WT and p62 KO DIV21 neurons (A) and embryonic stem cells (B) were incubated with DAPGreen staining for 1 h and 30 min and observed via live cell imaging. Scale bar: 20 μm. (B, C) Quantification of the data obtained in (B). A total of 346 WT and 334 p62 KO cells in three biological replicates (n = 3) were analysed. All of the quantifications were performed on single stacks. The P-value was calculated with a two-tailed t test (P > 0.05 non-significant [not shown], 0.01 < P < 0.05 *, 0.001 < P < 0.01 **, P < 0.001 ***).
Figure S5.
Figure S5.. Characterisation of the mScarlet–p62 F408V CRISPR cell line.
(A) Sequencing data from the CRISPR cell line showing the successful mutation of the F in position 408 with a V. (B) Immunostaining of pluripotent and differentiated (DIV21 neurons) mScarlet–p62 WT and mScarlet–p62 F408V cells. Scale bar: 20 μm.
Figure 4.
Figure 4.. Effect of p62 F408V mutation on p62 condensate formation and endo-lysosomal colocalisation in neurons.
(A) mScarlet–p62 WT or mScarlet–p62 F408V mutant DIV21 neurons were incubated for 1 h and 30 min with DAPGreen staining and observed via live cell imaging. Zoom factor of the selected regions: 5x. Scale bar: 20 μm. Standard or dashed arrows indicate p62 particles in the cell body or in the processes, respectively. (B) Quantification of the live cell imaging data. All the quantifications were performed on single stacks. The graphs show the mean ± SEM from n = 3 independent biological replicates. A total of 55–60 cell bodies and 5,223–4,841 μm of processes were analysed throughout the replicates. The P-value was calculated with a two-tailed t test and the statistical significance is shown on the graph (P > 0.05 non-significant [not shown], 0.01 < P < 0.05 *, 0.001 < P < 0.01 **, P < 0.001 ***).
Figure S6.
Figure S6.. mScarlet–p62 F408V does not bind ubiquitin chains in vitro.
GST (negative control) or GST-4x ubiquitin chains were bound to agarose beads. The beads were incubated with lysates from mScarlet–p62 WT, mScarlet–p62 LC3-interacting region mutant or mScarlet–p62 F408V DIV21 neurons. Confocal microscopy was used for mScarlet–p62 detection on the beads. Scale bar: 100 μm.
Figure S7.
Figure S7.. Western blot analysis of the NBR1 KO cell lines.
Cell lysates from different CRISPR clones, along with samples from the corresponding parental cell lines, were analysed by SDS–PAGE. The resulting blots were probed with anti-NBR1 and anti-GAPDH antibodies to show the loss of NBR1.
Figure 5.
Figure 5.. Effect of NBR1 KO and simultaneous p62 F408V mutation on p62 condensate formation and endo-lysosomal colocalisation in neurons.
(A) mScarlet–p62 WT or mScarlet–p62 F408V/NBR1 KO DIV21 neurons were incubated with DAPGreen staining for 1 h and 30 min and observed via live cell imaging (A). Zoom factor of the selected regions: 5x. Scale bar: 20 μm. Standard or dashed arrows indicate p62 particles in the cell body or in the processes, respectively. (B) Quantification of the live cell imaging data. All the quantifications were performed on single stacks. The graphs show the mean ± SEM from n = 3 independent biological replicates. A total of 49–50 cell bodies and 5,171–8,354 μm of processes were analysed throughout the replicates. The P-value was calculated with a two-tailed t test, and the statistical significance is shown on the graph (P > 0.05 non-significant [not shown], 0.01 < P < 0.05 *, 0.001 < P < 0.01 **, P < 0.001 ***).
Figure 6.
Figure 6.. Effect of NBR1 KO and p62 F408V mutation on p62 condensate formation in embryonic stem cells.
mScarlet–p62 WT, mScarlet–p62 F408V, mScarlet–p62 WT/NBR1 KO, and mScarlet–p62 F408V/NBR1 KO embryonic stem cells were treated for 3 h with either 400 nM bafilomycin or 10 μM MG-132 or a combination of the two drugs. A DMSO-treated sample served as a negative control. (A, B) Live cell imaging analysis and (B) quantification of the microscopy data. The graphs represent the mean ± SEM from n = 3 independent experiments. For each treatment and cell line, between 60 and 150 cells were analysed per replicate. The analysis was performed on a single-stack image. P-values were calculated with a two-way ANOVA and Tukey’s multiple comparison test (P > 0.05 non-significant [not shown], 0.01 < P < 0.05 *, 0.001 < P < 0.01 **, P < 0.001 ***).
Figure 7.
Figure 7.. Mutation of the p62 ubiquitin-associated domain (F408V) and knockout of NBR1 in embryonic stem cells and neurons.
(A) mScarlet–p62 WT, mScarlet–p62 F408V, mScarlet–p62 WT/NBR1 KO, and mScarlet–p62 F408V/NBR1 KO embryonic stem cells were treated for 3 h with either 400 nM bafilomycin or 10 μM MG-132 or a combination of the two drugs. A DMSO-treated sample served as a negative control. Western blot analysis was performed using the antibodies indicated. (A, B) Quantification of the Western blot data from (A). p62 and NBR1 band intensity values were normalised to the GAPDH-loading control. The graphs represent the mean of the GAPDH-normalised values ± SEM from n = 3 independent experiments. P-values were calculated with a two-way ANOVA and Tukey’s multiple comparison test (P > 0.05 non-significant [not shown], 0.01 < P <0.05 *, 0.001 < P < 0.01 **, P < 0.001 ***). (C) DIV21 neurons were treated for 5 h with 400 nM bafilomycin. Cell lysates were analysed by Western blot.
Figure 8.
Figure 8.. Puromycin pulse-chase experiment showing the effect of p62 mutations, p62 knock-out, and NBR1 knock-out in embryonic stem cells.
Cell lines were treated with 5 μg/ml puromycin for 2 h and either lysed immediately (T0) or let recover in absence of puromycin for the indicated time. A sample was pre-treated with 15 μM cycloheximide for 10 min and served as a negative control. Lysates were analysed by Western blot. (A, B, C, D, E) Comparison between mScarlet–p62 WT and mScarlet–p62 LC3-interacting region mutant (A), mScarlet–p62 WT/NBR1 KO (B), mScarlet–p62 F408V (C), mScarlet–p62 F408V/NBR1 KO (D), and p62 KO (E). (F) Quantification of the bands obtained on the Western blots (F). Puromycin band intensity values were normalised to the GAPDH loading control first and then adjusted to the T0 WT sample to get fold change values. The graphs represent the mean of the fold change ± SEM from n = 3 independent experiments.
Figure S8.
Figure S8.. p62 expression in the puromycin pulse-chase samples.
(A, B, C, D, E) Samples from one of the replicates quantified in Fig 8 were probed for p62 and GAPDH: mScarlet-p62 WT compared with LIR mutant (A), mScarlet-p62 WT/NBR1 KO (B), mScarlet-p62 F408V (C), mScarlet-p62 F408V/NBR1 KO (D) and p62 KO (E).
Figure S9.
Figure S9.. Controls for the puromycin pulse-chase experiment.
(A) mScarlet–p62 WT embryonic stem cells were treated with 5 μg/ml puromycin alone or in combination with the indicated treatment for 6 h before lysis and Western blot analysis. A sample was pre-treated with 15 μM cycloheximide for 10 min. (A, B) Quantification of the results obtained in (A). Puromycin and GAPDH band intensity values were normalised to the GAPDH-loading control. The graph shows the mean GAPDH-normalised values ± SEM from n = 3 independent biological replicates. The P-value was calculated with a one-way ANOVA and Tukey’s multiple comparison test (P > 0.05 non-significant [not shown], 0.01 < P < 0.05 *, 0.001 < P <0.01 **, P < 0.001 ***).
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