Human coronavirus-induced neuronal programmed cell death is cyclophilin d dependent and potentially caspase dispensable

Abstract

Human coronaviruses (HCoV) are recognized respiratory pathogens. Some HCoV strains, including HCoV-OC43, can invade the central nervous system, where they infect neurons, with unclear consequences. We have previously reported that HCoV-OC43 infection of human neurons activates the unfolded-protein response and caspase-3 and induces cell death and that the viral spike (S) glycoprotein is involved in the process. We now report on underlying mechanisms associated with the induction of programmed cell death (PCD) after infection by the reference HCoV-OC43 virus (rOC/ATCC) and a more neurovirulent and cytotoxic HCoV-OC43 variant harboring two point mutations in the S glycoprotein (rOC/U(S183-241)). Even though caspase-3 and caspase-9 were both activated after infection, the use of caspase inhibitors neither reduced nor delayed virus-induced PCD, suggesting that these proteases are not essential in the process. On the other hand, the proapoptotic proteins BAX, cytochrome c (CytC), and apoptosis-inducing factor (AIF) were relocalized toward the mitochondria, cytosol, and nucleus, respectively, after infection by both virus variants. Moreover, LA-N-5 neuronal cells treated with cyclosporine (CsA), an inhibitor of the mitochondrial permeabilization transition pore (mPTP), or knocked down for cyclophilin D (CypD) were completely protected from rOC/ATCC-induced neuronal PCD, underlining the involvement of CypD in the process. On the other hand, CsA and CypD knockdown had moderate effects on rOC/U(S183-241)-induced PCD. In conclusion, our results are consistent with mitochondrial AIF and cyclophilin D being central in HCoV-OC43-induced PCD, while caspases appear not to be essential.

Fig 1

rOC/ATCC and rOC/U_S183-241 induce programmed cell death in human neurons. Differentiated LA-N-5 cells were infected with rOC/ATCC or rOC/U_S183-241 for 24, 48, or 72 h. (A) Viability of infected human neurons. Cell viability was evaluated by the MTS-PMS assay and is expressed as relative percentage compared to that of mock-infected cells. (B) Fragmentation of DNA in infected neurons. Fragmented DNA was quantified by sandwich ELISA against histone and DNA and is expressed as a relative fold increase compared to that in mock-infected cells. (C) Fluorescence-activated cell sorting (FACS) TUNEL/7-AAD labeling of infected neurons. Cells stained with 7-AAD and TUNEL labeled were analyzed by FACS. The graph represent the percentage of quadrants. Nonsignificant percentages of 7-AAD-positive/TUNEL-negative cells were omitted. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 2

Infection by rOC/ATCC and rOC/U_S183-241 activates caspase-3 and -9 in human neurons. Differentiated LA-N-5 cells were infected with rOC/ATCC or rOC/U_S183-241 for 24, 48, or 72 h. The relative activity of caspase-9 was measured using LEHD-pNA (A), and the relative activity of caspase-3 was measured using DEVD-pNA (B); results are expressed as a relative fold increase compared to mock-infected cells. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 3

Caspases are activated following infection of human neurons by rOC/ATCC and rOC/U_S183-241, but the induced programmed cell death is not inhibited by Z-VAD-FMK. (A and B) Differentiated LA-N-5 cells were infected with rOC/ATCC or rOC/U_S183-241 and treated with either the pan-caspase inhibitor Z-VAD-FMK (VAD) (A) or the caspase-9 inhibitor Z-LEHD-FMK (LEHD) (B) for 24, 48, or 72 h. Viability of infected human neurons was evaluated with the MTS-PMS assay and expressed as relative percentage compared to that of mock-infected cells. (C) Immunofluorescence of active caspase-3. Vehicle-treated (left column) and Z-VAD-FMK-treated (right column) cells were incubated with anti-activated caspase-3 antibody (green). DAPI (blue) insets demonstrate equivalent cell density in the microscope fields analyzed. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 4

Infections by rOC/ATCC and rOC/U_S183-241 promote BAX, CytC, and AIF relocalization in human neurons. Differentiated LA-N-5 cells were infected with rOC/ATCC or rOC/U_S183-241. (A) Immunofluorescent detection of activated BAX. Cells were incubated with the MitoTracker Red CMXROS (red), fixed, and incubated with an anti-activated BAX antibody (green). Colocalization is represented by merged BAX and MitoTracker Red CMXROS signals (yellow) as indicated by white arrows. (B) Western immunoblotting of mitochondrial BAX and CytC. Mitochondrial protein fractions were subjected to Western immunoblotting analysis using antibodies directed against BAX or CytC. VDAC served as a loading control. (C) Immunofluorescent detection of AIF. Cells were incubated with the MitoTracker Red CMXROS (red), fixed, and incubated with an anti-AIF antibody (green) and DRAQ5 (blue). Nuclear colocalization is represented by merged AIF and DRAQ5 signals (turquoise) as indicated by white arrows, and residual mitochondrial colocalization is represented by merged AIF and MitoTracker (yellow) as indicated by white arrowheads. (D) Western immunoblotting of nuclear AIF. Nuclear protein fractions were subjected to Western immunoblotting analysis using antibodies directed against AIF. p84 served as a loading control.

Fig 5

Cyclosporine treatment and cyclophilin D knockdown protects human neurons from rOC/ATCC-induced programmed cell death. Wild-type LA-N-5 cells were treated with cyclosporine (CsA), and two LA-N-5 populations knocked down for CypD (pop K CypD-kd and pop M CypD-kd) were infected with rOC/ATCC or rOC/U_S183-241. Images represent phase-contrast microscopy pictures taken at 48 h postinfection. The control LA-N-5 empty cell population is not presented since these cells were comparable to nontreated CsA wild-type LA-N-5 cells. Arrows indicates dendrites and axons. Arrowheads indicate rounding and shrinking bodies.

Fig 6

Human neuronal PCD induced by rOC/ATCC is cyclophilin D dependent. Wild-type LA-N-5 cells, treated with cyclosporine (CsA) or mock treated (vehicle) for 24, 48, or 72 h, and two LA-N-5 populations knocked down for CypD, pop K CypD-kd (pop K) and pop M CypD-kd (pop M) and the LA-N-5 empty vector population (LAN5 empty) were infected with rOC/ATCC or rOC/U_S183-241. (A and B) Cell viability of infected wild-type LA-N-5 cells treated with cyclosporine (A) and of infected LA-N-5 K and M populations knocked down for CypD (B) was evaluated with the MTS-PMS assay and is expressed as relative percentage compared to that of mock-infected cells. (C and D) Fragmentation of DNA in infected wild-type LA-N-5 cells treated with cyclosporine (C) and of infected LA-N-5 K and M populations knocked down for CypD (D) was evaluated and expressed as a relative fold increase compared to that in mock-infected cells. (E) FACS TUNEL labeling/7-AAD staining of infected wild-type LA-N-5 cells treated with cyclosporine. Cells were analyzed by FACS at 72 h postinfection. The graph represents percentages of quadrants. Nonsignificant percentages of 7-AAD-positive/TUNEL-negative cells were omitted. (F) Quantification of CypD expression in LA-N-5 knockdown populations. Expression of CypD in the two populations of LA-N-5 neurons knocked down for CypD was evaluated by quantitative PCR and is expressed as a relative percentage compared to that in the LA-N-5 empty vector population. Nonsignificant percentages of 7-AAD-positive/TUNEL-negative cells were omitted. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to corresponding noninfected LA-N-5 population). #, P < 0.05 compared to LA-N-5 empty vector infected with the same virus.

Fig 7

Cyclophilin D inhibition alters AIF nuclear translocation in infected neurons. Differentiated LA-N-5 cells were infected with rOC/ATCC or rOC/U_S183-241 and treated with cyclosporine (CsA). (A) Detection of AIF by immunofluorescence. Cells were fixed and incubated with an anti-AIF antibody (green) and DRAQ5 (blue). Colocalization is represented by merged AIF and DRAQ5 signals (turquoise) as indicated by white arrows. (B) Western immunoblotting of mitochondrial AIF and CytC. Mitochondrial proteins fractions were subjected to Western immunoblotting analysis using antibodies directed against AIF or CytC. VDAC served as a loading control. Intensities of the bands were evaluated with ChemiGenius2 Syngene software and expressed as percentage relative to the loading control. Results are representative of two independent experiments.