Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Oct 10;8(10):1233.
doi: 10.3390/cells8101233.

C9orf72 Proteins Regulate Autophagy and Undergo Autophagosomal or Proteasomal Degradation in a Cell Type-Dependent Manner

Affiliations
Free PMC article

C9orf72 Proteins Regulate Autophagy and Undergo Autophagosomal or Proteasomal Degradation in a Cell Type-Dependent Manner

Stina Leskelä et al. Cells. .
Free PMC article

Abstract

Dysfunctional autophagy or ubiquitin-proteasome system (UPS) are suggested to underlie abnormal protein aggregation in neurodegenerative diseases. Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS)-associated C9orf72 is implicated in autophagy, but whether it activates or inhibits autophagy is partially controversial. Here, we utilized knockdown or overexpression of C9orf72 in mouse N2a neuroblastoma cells or cultured neurons to elucidate the potential role of C9orf72 proteins in autophagy and UPS. Induction of autophagy in C9orf72 knockdown N2a cells led to decreased LC3BI to LC3BII conversion, p62 degradation, and formation of LC3-containing autophagosomes, suggesting compromised autophagy. Proteasomal activity was slightly decreased. No changes in autophagy nor proteasomal activity in C9orf72-overexpressing N2a cells were observed. However, in these cells, autophagy induction by serum starvation or rapamycin led to significantly decreased C9orf72 levels. The decreased levels of C9orf72 in serum-starved N2a cells were restored by the proteasomal inhibitor lactacystin, but not by the autophagy inhibitor bafilomycin A1 (BafA1) treatment. These data suggest that C9orf72 undergoes proteasomal degradation in N2a cells during autophagy. Lactacystin significantly elevated C9orf72 levels in N2a cells and neurons, further suggesting UPS-mediated regulation. In rapamycin and BafA1-treated neurons, C9orf72 levels were significantly increased. Altogether, these findings corroborate the previously suggested regulatory role for C9orf72 in autophagy and suggest cell type-dependent regulation of C9orf72 levels via UPS and/or autophagy.

Keywords: C9orf72; amyotrophic lateral sclerosis; autophagy; frontotemporal dementia; proteasomal degradation; ubiquitin-proteasome system.

Conflict of interest statement

The authors have no conflict of interest to report.

Figures

Figure 1
Figure 1
Knockdown of C9orf72 in N2a cells leads to decreased LC3BI to LC3BII conversion and p62 degradation during serum starvation-induced autophagy. (A) A representative Western blot of the total protein lysates of N2a cells transfected with control (Ctrl) or C9orf72 (C9) shRNA showing the levels of the endogenous C9orf72 isoform A, LC3BI, LC3BII, p62, LAMP2A, and GAPDH (loading control) under normal growth conditions, after serum starvation (ST) or ST and bafilomycin A1 (BafA1) treatment. Overnight ST leads to autophagy induction as indicated by the conversion of LC3BI to LC3BII and the decrease in p62 levels in control shRNA-transfected cells but not in C9orf72 shRNA-transfected cells. The levels of p62 are restored and LC3BII robustly accumulates upon 300 nM BafA1 treatment for 6 h. Lysosomal LAMP2A levels did not show statistically significant alterations. Molecular weight markers are indicated on the left as kDa. An unspecific band, detected by the C9orf72 antibody and not affected by the shRNA, is indicated by an asterisk (*). Quantification of (B) the endogenous levels of C9orf72 isoform A, indicating an approximately 40% decrease in C9orf72 shRNA-transfected cells as compared to cells transfected with control shRNA, (C) LC3BI levels, (D) LC3BII levels, (E) ratio of LC3BII/LC3BI, (F) p62 levels, and (G) LAMP2A (unspecific band is indicated by *) levels. The protein levels were normalized to GAPDH levels in each sample. Data are shown as mean % ± SEM of the protein levels compared to those in the control cells. n = 9 from three independent experiments, except in p62 quantification, Ctrl shRNA Normal, n = 8 and LAMP2A quantifications, Ctrl shRNA ST n = 8; one-way ANOVA, Newman‒Keuls, * p ≤ 0.05, ** p ≤ 0.01.
Figure 2
Figure 2
Knockdown of C9orf72 in N2a cells leads to decreased autophagosome formation during serum starvation-induced autophagy. (A) Representative fluorescent microscope images of the control or C9orf72 shRNA and GFP-LC3 plasmid co-transfected cells in normal growth medium, upon ST-induced autophagy or combined treatment with ST and BafA1. Scale bar: 10 µm. (B) Quantification of the LC3 puncta with ImageJ software from two independent experiments. Four coverslips were analyzed/each treatment group. Number of the analyzed cells in total: Ctrl shRNA Normal 21, C9 shRNA Normal 14, Ctrl shRNA ST 22, C9 shRNA ST 28, Ctrl shRNA ST + BafA1 48, C9 shRNA ST + BafA1 22. Data are shown as mean ± SEM, one-way ANOVA, Newman‒Keuls, * p ≤ 0.05, *** p ≤ 0.001.
Figure 3
Figure 3
N2a cells overexpressing the C9orf72 isoform A do not show alterations in autophagy but display decreased C9orf72 isoform A levels after serum starvation. (A) A representative Western blot of the total protein lysates of N2a cells transfected with the control pcDNA or C9orf72 isoform A-GFP (C9-A-GFP) plasmids showing the levels of C9-A-GFP, LC3BI, LC3BII, p62, and GAPDH (loading control) under normal growth conditions, after serum starvation (ST) or ST and BafA1 treatment. ST overnight leads to similarly increased LC3BII and decreased p62 levels in both the control and the C9-A-GFP overexpressing cells. The levels of p62 and LC3BII accumulate in a similar manner after 300 nM BafA1 treatment for 6 h in the pcDNA and C9-A-GFP-transfected cells. C9-A-GFP levels are decreased after ST treatment and are not restored after BafA1 treatment. Molecular weight markers are indicated on the left as kDa. An unspecific band detected by the C9orf72 antibody is indicated by an asterisk (*). Quantification of (B) C9-A-GFP, (C) LC3BI, (D) LC3BII, (E) LC3BII/LC3BI ratio, and (F) p62 levels. Data are shown as mean % ± SEM of the protein levels compared to those in the control cells from three independent experiments. n = 8, except C9-A-GFP ST + BafA1, n = 5. One-way ANOVA, Newman‒Keuls, * p ≤ 0.05, *** p ≤ 0.001.
Figure 4
Figure 4
Induction of autophagy by rapamycin leads to decreased C9orf72 protein isoform A levels, which are not restored in N2a cells but show a significant increase in mouse cortical neurons upon BafA1 treatment. (A) A representative Western blot of the total protein lysates extracted from transfected N2a cells showing the levels of C9-A-GFP, LC3BI, LC3BII, p62, and β-actin (loading control). Treatment with 200 nM rapamycin ± 50 nM Bafilomycin A1 (BafA1) overnight leads to decreased levels of C9-A-GFP and the levels are not restored after BafA1 treatment. Molecular weight markers are indicated on the left as kDa. An unspecific band, detected by the C9orf72 antibody, is indicated by an asterisk (*). (B) Quantification of the C9-A-GFP protein levels in N2a cells normalized to those of β-actin. Data are shown as mean % ± SEM of the protein levels compared to those in the vehicle-treated cells from two independent experiments. n = 6, except C9-A-GFP rapamycin, n = 5, one-way ANOVA, Newman‒Keuls, * p ≤ 0.05. (C) A representative Western blot of total protein lysates extracted from lentivirus-transduced primary cortical neurons showing the levels of the C9-A-GFP, LC3BI, LC3BII, p62, and β-actin (loading control). Molecular weight markers are indicated on the left as kDa. An unspecific band detected by the C9orf72 antibody is indicated by an asterisk (*). (D) Quantifications of C9-A-GFP protein levels normalized to those of β-actin. Data are shown as mean % ± SEM of the protein levels compared to those in the vehicle treated cells from three independent experiments. n = 11, one-way ANOVA, Newman‒Keuls, * p ≤ 0.05, ** p ≤ 0.01.
Figure 5
Figure 5
C9orf72 protein isoform A levels are elevated after proteasomal inhibition in N2a cells and mouse primary cortical neurons. (A) A representative Western blot of the total protein lysates extracted in T-PER and SDS extraction buffers from transfected N2a cells or (C) transduced mouse primary cortical neurons showing the levels of the C9-A-GFP, poly-ubiquitinated (Poly-Ub.) proteins, GAPDH or β-actin (loading control). Treatment with 10 µM lactacystin overnight leads to significantly increased levels of C9-A-GFP proteins and accumulation of poly-ubiquitinated proteins, especially to the SDS-soluble fraction in both N2a cells and cortical neurons. Molecular weight markers are indicated on the left as kDa. An unspecific band detected by the C9orf72 antibody is indicated by an asterisk (*). (B,D) Quantification of C9-A-GFP protein levels in the T-PER fraction of N2a cells or primary mouse cortical neurons normalized to those of GAPDH or β-actin. Data are shown as mean % ± SEM of the protein levels compared to those in the vehicle-treated cells from two independent experiments. n = 8 for all other samples, except n = 6 for C9-A-GFP-transduced neurons treated with vehicle, Mann‒Whitney U test, * p ≤ 0.05, *** p ≤ 0.001.
Figure 6
Figure 6
Decreased C9orf72 protein isoform A levels upon serum starvation-induced autophagy are restored by lactacystin treatment in N2a cells. (A) A representative Western blot of the total protein lysates extracted in T-PER and SDS extraction buffers from transfected N2a cells showing the levels of C9-A-GFP, LC3BI, LC3BII, p62, poly-ubiquitinated proteins (poly-Ub.), and GAPDH (loading control). Overnight serum starvation (ST) leads to decreased C9-A-GFP protein levels that are restored upon 10 µM lactacystin treatment. The levels of p62 also slightly increase, but LC3BI or II levels do not show major changes after lactacystin treatment in serum-starved cells. Molecular weight markers are indicated on the left as kDa. An unspecific band detected by the C9orf72 antibody is indicated by an asterisk (*). (B) Quantification of the C9-A-GFP protein levels normalized to those of GAPDH from two independent experiments. Data are shown as mean % ± SEM of the protein levels compared to those in the vehicle treated cells. n = 6 for all samples, one-way ANOVA, Newman‒Keuls, ** p ≤ 0.01, *** p ≤ 0.001.
Figure 7
Figure 7
C9orf72 knockdown mildly decreases whereas overexpression of the C9orf72 isoform A does not affect the proteasomal activity in N2a cells. Quantification of proteasomal activity (A) in cells transfected with the control (Ctrl) or the C9orf72 (C9) shRNA. Data are shown as mean % ± SEM of the activity in control shRNA samples. n = 6 from two independent experiments, Mann‒Whitney U test, * p ≤ 0.05. (B) in cells overexpressing the C9-A-GFP. Data are shown as mean % ± SEM of the activity in pcDNA samples. n = 6 from two independent experiments, one-way ANOVA, Newman‒Keuls, not significant.
Figure 8
Figure 8
Blockade of the basal autophagy with BafA1 does not significantly change the C9orf72 protein isoform A levels in N2a cells but leads to its decreased levels in mouse primary cortical neurons. (A) A representative Western blot of the total protein lysates from transfected N2a cells showing the levels of C9-A-GFP, LC3BI, LC3BII, p62, and β-actin (loading control). Treatment with 300 nM Bafilomycin A1 (BafA1) for 6 h does not alter C9-A-GFP protein levels but increases p62 levels, as expected. Molecular weight markers are indicated on the left as kDa. An unspecific band detected by the C9orf72 antibody is indicated by an asterisk (*). (B) Quantification of the C9-A-GFP protein levels normalized to those of β-actin. Data are shown as mean % ± SEM of the protein levels compared to those in the vehicle treated cells from three independent experiments. n = 9, except in vehicle n = 8, Mann‒Whitney U test, not significant. (C) A representative Western blot of the total protein lysates from lentivirus-transduced mouse primary cortical neurons showing the levels of C9-A-GFP, LC3BI, LC3BII, p62, and β-actin (loading control). Treatment with 300 nM BafA1 for 6 h leads to significantly decreased levels of C9-A-GFP proteins, while p62 levels are increased, as expected. Molecular weight markers are indicated on the left as kDa. An unspecific band detected by the C9orf72 antibody is indicated by an asterisk (*). (D) Quantification of the C9-A-GFP protein levels normalized to those of β-actin. Data are shown as mean % ± SEM of the protein levels compared to those in the vehicle treated cells from two independent experiments. n = 8, except C9-A-GFP BafA1 n = 7, Mann‒Whitney U test, *** p ≤ 0.001.

Similar articles

See all similar articles

Cited by 1 article

References

    1. Vieira R.T., Caixeta L., Machado S., Silva A.C., Nardi A.E., Arias-Carrión O., Carta M.G. Epidemiology of Early-Onset Dementia: A Review of the Literature. Clin. Pract. Epidemiol. Ment. Health. 2013;9:88–95. doi: 10.2174/1745017901309010088. - DOI - PMC - PubMed
    1. Bang J., Spina S., Miller B.L. Frontotemporal Dementia. Lancet. 2015;386:1672–1682. doi: 10.1016/S0140-6736(15)00461-4. - DOI - PMC - PubMed
    1. Swinnen B., Robberecht W. The Phenotypic Variability of Amyotrophic Lateral Sclerosis. Nat. Rev. Neurol. 2014;10:661–670. doi: 10.1038/nrneurol.2014.184. - DOI - PubMed
    1. Renton A.E., Majounie E., Waite A., Simón-Sánchez J., Rollinson S., Gibbs J.R., Schymick J.C., Laaksovirta H., van Swieten J.C., Myllykangas L., et al. A Hexanucleotide Repeat Expansion in C9ORF72 Is the Cause of Chromosome 9p21-Linked ALS-FTD. Neuron. 2011;72:257–268. doi: 10.1016/j.neuron.2011.09.010. - DOI - PMC - PubMed
    1. DeJesus-Hernandez M., Mackenzie I.R., Boeve B.F., Boxer A.L., Baker M., Rutherford N.J., Nicholson A.M., Finch N.A., Flynn H., Adamson J., et al. Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS. Neuron. 2011;72:245–256. doi: 10.1016/j.neuron.2011.09.011. - DOI - PMC - PubMed

Publication types

Feedback