Contrasting roles of autophagy in cellular prion infection

Abstract

Autophagy is a cellular degradation program that can exert both beneficial and adverse effects in various neurodegenerative diseases. We tested the role of macroautophagy/autophagy in prion infection and how this machinery affects the life cycle of prions. In mouse embryonic fibroblasts, we found a pronounced dependence of prion replication on autophagy competence, suggesting that autophagy provides functions needed for prion propagation. However, in neuronal cells, autophagy had mostly the opposite role. Cells ablated for autophagy competence by gene editing harbored elevated amounts of misfolded prion protein, indicating that neuronal cells use autophagy for prion degradation. These data show that autophagy can have two functions in the replication of prions, and depending on the cellular context, this can be protective against or supportive of prion infection. These findings demonstrate that prions use cellular machineries to benefit propagation in certain cell types, whereas other cell types employ the same machinery as a defense mechanism.

Keywords: Atg5 knockout; autophagy; disaggregase; neurodegeneration; prion; prion infection.

Figure 1.

Infecting mouse embryonic fibroblasts with prions stimulates autophagy. Autophagy induction in MEF propagating prions. (A, B) MEFs were infected with Me7 (A) and RML (B) prions and analyzed for PRNP levels (either with or without PK digestion) by immunoblotting from passage 1 (P1) to passage 8 (P8) p.I., using anti-PRNP mAB 4H11. Blots were re-probed for autophagy marker LC3-I/II and ACTB/actin as a loading control. (C) immunoblots for different passages of N2a (K21) cells, either mock-infected or infected with 22 L prions. Immunoblots were probed with anti-4H11, anti-LC3, or anti-ACTB/actin as loading control. (D) immunoblot analysis of MEF cells either uninfected or infected with RML or Me7 prions before and after PK digestion. (E) immunoblot for uninfected MEF cells treated with either DMSO or with rapamycin (Rapa) or rapamycin+bafilomycin A₁ (Rapa+BA₁) for 4 h. Immunoblot was probed with LC3, SQSTM1/p62, or ACTB/actin as a loading control. (F and G) densitometric analysis for either total LC3 or SQSTM1/p62, respectively, from uninfected MEF cells (± S.D.) after treatment with rapamycin (Rapa) or rapamycin with bafilomycin A₁ (Rapa+BA₁) or DMSO. n = 3 experiments. *, p < 0.05. **, p < 0.01. (H) immunoblot for MEF cells infected with RML prion strain and treated with either DMSO or with rapamycin (Rapa) or rapamycin+bafilomycin (Rapa+BA1) for 4 h. Immunoblot was probed with LC3, SQSTM1/p62, or ACTB/actin as a loading control. (I and J) densitometric analysis for either total LC3 or SQSTM1/p62, respectively, from RML-infected MEF cells (± S.D.) after treatment with rapamycin (Rapa) or rapamycin with bafilomycin A₁ (Rapa+BA₁) or DMSO. n = 5 experiments. **, p < 0.01. ***, p < 0.001. (K) immunoblot for MEF cells infected with Me7 prion strain and treated with either DMSO or with rapamycin (Rapa) or rapamycin+bafilomycin (Rapa+BA₁) for 4 h. Immunoblot was probed with LC3, SQSTM1/p62, or ACTB/actin as a loading control. (L and M) densitometric analysis for either total LC3 or SQSTM1/p62, respectively, from Me7 MEF cells (± S.D.) after treatment with rapamycin (Rapa) or bafilomycin A₁ (Rapa+BA₁) or DMSO. n = 4 experiments. *, p < 0.05. **, p < 0.01.

Figure 2.

MEF cells with knockout in autophagy are not able to propagate prions efficiently. (A) immunoblot analysis showing complete loss of ATG5 and LC3-II signals in atg5 KO MEFs (atg5^-/-) compared to WT MEF cells (WT), verifying non-functional autophagy (B) non-PK digested lysates were probed for LC3-I/II and ACTB/actin for passages P3 and P5 after infection. (C) MEF-WT and MEF-atg5^-/- cells were infected with 22 L prions (22 L) or mock-infected with uninfected brain homogenates (mock). Cells were lysed at passage 3 (P3) and 5 (P5) p.I., and cell lysates were analyzed by immunoblot after PK digestion (mAb 4H11). Much lower amounts of PK-resistant PRNP were detected in autophagy-deficient cells (lanes 5 and 9) compared to WT MEFs (lanes 4 and 8). Lysate of persistently prion-infected ScN2a cells was used as a positive control for PK-resistant PRNP (cont, lane 3). (D) wild-type MEFs (MEF WT) and atg5 KO MEFs (atg5^-/-) were infected with 22 L prions and analyzed from day 1 to day 5 post-infection (before passage 1). Cell lysates were PK-digested or not (-/+ PK), and PrP was detected by immunoblotting. Both cell types took up and degraded the PK-resistant PRNP in the inoculum with a very similar pattern.

Figure 3.

Reintroduction of autophagy competence increases PK-resistant PRNP levels. (A) the atg5^-/- MEF cells transduced with ATG5-expressing lentivirus were analyzed for ATG5 protein expression in immunoblots. Whereas ATG5 protein signals are lacking in MEF-atg5^-/- cells, lentivirally transduced MEF-atg5^-/- (termed MEF-Atg5-r, lane 3) showed an ATG5 signal comparable to the WT. (B) MEF either WT, atg5^-/- or Atg5-r., were transfected with a construct coding for GFP-LC3, treated with imatinib/Gleevec to induce autophagy and autophagosome formation [89], and analyzed by confocal microscopy. Nuclei were stained with DAPI. GFP-LC3 puncta, representing autophagosomes, were observed in WT and reconstituted cells, but not in MEF-atg5^-/-, indicating that Atg5 gene reconstitution restored autophagy competence (C-E) prion infection of MEF cells. WT, atg5^-/- and Atg5-r. cells were infected with 22 L prions (22 L), or mock-brain (mock), and analyzed at passage 3 (P3) (C), 5 (P5) (D), and 10 (P10) (E) by immunoblotting (mAb 4H11; only +PK samples shown). MEF-atg5^-/- stably transduced with virus coding for GFP served as a negative control (MEFatg5^-/--GFP). Increased amounts of PK-resistant PRNP were detected in ATG5-reintroduced MEFs (lane 9) compared to KO (lane 7) and GFP-transduced MEFs (lane 8). (F and G) densitometric analysis of immunoblots in C and D. Significantly increased PK-resistant PRNP levels were detected in autophagy-reconstituted MEF-Atg5-r., compared to both MEF-atg5^-/- and MEF-atg5^-/--GFP at P3 and P5 after prion infection. Values represent the mean ± SEM of three independent experiments. **, p < 0.01. *, p < 0.05. (H and I) at passage 3 and passage 5 after infection, cells were analyzed for LC3 and GFP expression in immunoblot, using ACTB/actin as a loading control. Whereas LC3-II signals are lacking in MEF-atg5^-/--GFP expressing control cells, the LC3-II signal was present in lentivirally transduced MEF-atg5^-/- cells (termed MEF-Atg5-r, lane 3).

Figure 4.

Increase in Atg5 expression correlates with increased PK-resistant PRNP levels. MEF-atg5^-/- cells were left untreated (lanes 2 and 6) or transduced with different dilutions of recombinant virus (1:3, 1:2, or 1:1), encoding for GFP (transduction control; lanes 3–5) or ATG5 (lanes 7–9), respectively. Stably transduced cells were infected with prions (22 L) and analyzed for PK-resistant PRNP after passage 3 (A) and passage 5 (B), using anti-PRNP mAb 4H11. Only PK-treated samples are shown. Lysate of persistently prion-infected ScN2a cells was used as a positive control (cont, lane 1). (C and D) Non-PK digested samples were analyzed in immunoblot for LC3-II and ACTB/actin (loading control) (B, passage 3 and D, passage 5). Gradual reconstitution of autophagy competence rendered cells more susceptible to prion infection (compare lanes 6 to 9) and gradually elevated PK-resistant PRNP levels. (E, F) a transduction control MEF-atg5^-/- was stably transduced with lentiviruses expressing GFP only (no ATG5; termed MEF-atg5^-/--GFP). Expression of GFP in MEF-atg5^-/--GFP is shown in immunoblot analysis for P3 and P5, respectively.

Figure 5.

Stable knockdown of the Atg5 gene in mouse L929 fibroblasts does not affect prion propagation. (A) immunoblot for WT L929 mouse fibroblasts treated with either DMSO, rapamycin, or rapamycin and bafilomycin A₁ for 4 h. Specific antibodies against LC3 and SQSTM1/p62 were used to monitor the autophagic flux. Actin was used as a loading control. (B and C) densitometric analysis for either LC3-II or SQSTM1/p62, respectively, from L929 fibroblasts (± S.D.) after treatment with rapamycin (Rapa), rapamycin with bafilomycin A₁ (Rapa+BA₁), or DMSO. n = 4 experiments. (D) the Atg5 gene was knocked down using a lentivirus (sh-Atg5). Non-targeting lentivirus was used as a control (sh-CT). Cells were infected with 22 L prions and were passaged. P1, P3, P6 and P8 were shown. Immunoblots are showing the level of total PRNP (−PK) and PK-resistant PRNP. Specific antibodies against ATG5 and LC3 were used to confirm the knockdown and the effect on the autophagic machinery. ACTB/Actin was used as a loading control.

Figure 6.

Stable knockdown of Atg5 gene in mouse astrocytes does not affect prion propagation. (A) immunoblot for WT mouse astrocytes treated with DMSO, rapamycin, or rapamycin and bafilomycin A₁ for 4 h. Specific antibodies against LC3 and SQSTM1/p62 were used to monitor the autophagic flux. ACTB/Actin was used as a loading control. (B and C) densitometric analysis for either LC3-II or SQSTM1/p62, respectively, from mouse astrocytes (± S.D.) after treatment with rapamycin (Rapa), rapamycin with bafilomycin A₁ (Rapa+BA₁), or DMSO. n = 3 experiments. **, p < 0.001. (D) the Atg5 gene was knocked down using a lentivirus (sh-Atg5). Non-targeting lentivirus was used as a control (sh-CT). Cells were infected with 22 L prions and were passaged. P1, P2, P3 and P5 were shown. Immunoblots are showing the level of total PRNP (−PK) and PK-resistant PRNP. A specific antibody against the ATG5 protein was used to confirm the stable knockdown. ACTB/actin was used as a loading control.

Figure 7.

Stable knockdown of the Atg5 gene in neuronal CAD5 and N2a cells does not affect prion propagation. (A and E) immunoblot for CAD5 and N2a cells treated with DMSO, rapamycin, or rapamycin and bafilomycin A₁ for 4 h. Specific antibodies against LC3 and SQSTM1/p62 were used to monitor the autophagic flux. ACTB/Actin was used as a loading control. (B, C, F, and G) densitometric analysis for either LC3-II or SQSTM1/p62, respectively, from CAD-5 (B and C) and N2a (F and G) cells (± S.D.) after treatment with rapamycin (Rapa), rapamycin with bafilomycin A₁ (Rapa+BA₁), or DMSO. n = 3 experiments. **, p < 0.001. (D and H) the Atg5 gene was stably knocked down in CAD5 (D) and N2a (H) cells, using lentiviruses encoding shRNA targeting the Atg5 gene (sh-Atg5). Control cells were transduced in parallel with non-targeting shRNA viruses (sh-CT). Transduced cells were infected with 22 L prions, cells were lysed at every passage and lysates were subjected to PK digestion (or kept undigested), and analyzed by immunoblot (passages 2, 4, and 8 shown). Blots were probed for ATG5 protein, ACTB/actin, PRNP (-/+ PK), and LC3-I/II. Whereas both CAD5 and N2a cells showed a pronounced knockdown of Atg5, which translated into LC3-II reduction (less well for CAD5 cells), the PK-resistant PRNP levels appeared unchanged between mock and targeted cells.

Figure 8.

Knockout of the Atg5 gene in N2a cells increases the PK-resistant PRNP level. CRISPR-Cas9 gene editing is used to knockout Atg5 in N2a cells. N2a cells were co-transfected with CRISPR-Cas9 plasmids encoding sgRNA to target exons 2 and 3 combined, or exons 5 and 6 in the Atg5 gene. (A) parental cells were single-cell cloned, and individual clones were analyzed by immunoblot for ATG5 and LC3-II. Two representative clones are shown, targeting exons 2 and 3 (lane 2) and exons 5 and 6 (lane 3). (B) the genomic DNA of these cell clones was PCR amplified and cloned. Various plasmid DNA clones from each genomic DNA were sequenced. Gene editing resulted in ORF frameshifts and premature stop codons in all targeted exons, except exon 6, for which only WT alleles and in-frame deletions were found. (C) N2a cells with knockout in the Atg5 gene (KO) and control cells (WT) were infected with 22 L prions. Cells were lysed at passage 1 (P1) to passage 6 (P6), lysates subjected to PK digestion or not (-/+ PK), and cell lysates were analyzed by immunoblot for ATG5, PRNP with or without PK digestion, and LC3-I/II. Knockout of Atg5 was stable overall passages and resulted in complete loss of LC3-II signals, indicating non-functional autophagy. (D and E) densitometric analysis for 22 L-infected WT and KO N2a cells at P4 and P8, respectively (*p < .05). Data represented as mean ± SE.

Figure 9.

Colocalization of GnHCL-treated PRNP with lysosomes is higher in N2a cells. GnHCl treatment removes the soluble noninfectious PRNP and keeps only the insoluble infectious PRNP. Both 22 L-N2a and 22 L-MEF were fixed and stained with 4H11 anti-PRNP antibody (green) and LAMP1 antibody (red) (A), or 4H11 antibody (red) and transfected with GFP-LC3 plasmid (green) (B). (C) densitometric analysis for GnHCL-treated PRNP colocalized with LAMP1 or GFP-LC3. Data were represented as mean ± SD (*p < 0.001). (D and H) immunoblots for infected N2a (ScN2a) and infected MEF (ScMEF) cells, respectively, were treated with DMSO, rapamycin, or rapamycin and bafilomycin A₁ for 4 h. Specific antibodies against LC3 and SQSTM1/p62 were used to monitor the autophagic flux. ACTB/Actin was used as a loading control. (E and I) immunoblots for infected N2a (ScN2a) and infected MEF (ScMEF) cells, respectively, showing prion infection using 4H11 anti-PRNP antibody. ACTB/Actin was used as a loading control. (F and G) densitometric analysis for either LC3-II or SQSTM1/p62, respectively, from prion-infected N2a (ScN2a) cells (± S.D.) after treatment with rapamycin (Rapa), rapamycin with bafilomycin A₁ (Rapa+BA₁), or DMSO. n = 3 experiments. **, p < 0.01, ***, p < 0.001. (J and K) densitometric analysis for either LC3-II or SQSTM1/p62, respectively, from prion-infected MEFs (ScMEF) cells (± S.D.) after treatment with rapamycin (Rapa), rapamycin with bafilomycin A₁ (Rapa+BA₁), or DMSO. n = 4 experiments. *, p < 0.05, **p < .01.