Journal: Neurobiology of disease

Article Title: FUDNC1-dependent mitophagy ameliorate motor neuron death in an amyotrophic lateral sclerosis mouse model.

doi: 10.1016/j.nbd.2024.106534

Figure Lengend Snippet: Fig. 2. Effects of FUNDC1 on motor function and survival in SOD1G93A mice. (A) Injection schematic and behavioral testing schedule. (B, C, J, and K) Immunoblot images (B and J) and quantitative analysis of FUNDC1 protein levels (C and K) in spinal cord tissues after intrathecal injection of shAAV9 or AAV9-FUNDC1 in SOD1G93A mice (n = 4). (D, E, L, and M) Kaplan–Meier survival curves (D, L) and body weight curves (E and M) of SOD1G93A mice infected with shAAV9 or AAV9- FUNDC1 (n = 12). (F, G, H, I, N, O, P, and Q) Behavioral changes at different time points(60 days (pre-symptomatic), 90 days (symptomatic), and 120 days (ter minal)) in SOD1G93A mice infected with shAAV9 or AAV9-FUNDC1: grip strength analysis (F and N), time on rotarod (G and O), and gait analysis (H, I, P, and Q) (n = 12). The results were analyzed using an unpaired two-tailed Student's t-test, and the data are presented as mean ± SD with significance indicated. *P < 0.05 or **P < 0.01, ***P < 0.001. NS indicates no significance (P > 0.05). shAAV9-EGFP: AAV knockdown virus negative control; shAAV9-FUNDC1: AAV knockdown FUNDC1 virus; AAV9-EGFP: AAV overexpression virus negative control; AAV9-FUNDC1: AAV overexpression FUNDC1 virus.

Article Snippet: The following primary antibodies were used for western blotting: FUNDC1 (1:1000, CST), LC3B-II (1:1000, CST, #49240), BAX (1:1000, Proteintech, Cat No. 60267-1-Ig), BCL-2 (1:1000, Proteintech, Cat No. 60178-1-Ig), GAPDH (1:5000, Proteintech, Cat No. 10494-1-AP), beta actin (1:5000, Cat No. 81115-1-RR), P62 (1:1000, Proteintech, Cat No. 18420-1-AP), HSP60 (1:1000, Zenbio, 222340), COXIV (1:1000, Zenbio, 200147), and FOXD3 (1:1000, Zenbio, 220508).

Techniques: Injection, Western Blot, Infection, Two Tailed Test, Knockdown, Virus, Negative Control, Over Expression

Journal: Neurobiology of disease

Article Title: FUDNC1-dependent mitophagy ameliorate motor neuron death in an amyotrophic lateral sclerosis mouse model.

doi: 10.1016/j.nbd.2024.106534

Figure Lengend Snippet: Fig. 3. The effect of FUNDC1 on neuronal apoptosis in the spinal cord tissue of SOD1G93A mice. (A, B, and C) shAAV9-EGFP and shAAV9-FUNDC1-infected spinal cord tissues of SOD1G93A mice were subjected to immunoblotting to determine the protein levels of BAX and BCL-2 (A), along with quantitative analysis results (B and C) (n = 4). (D, E, and F) AAV9-EGFP and AAV9-FUNDC1-infected spinal cord tissues of SOD1G93A mice were subjected to immunoblotting to determine the protein levels of BAX and BCL-2 (D), along with quantitative analysis results (E and F) (n = 4). (G, H, I, and J) In SOD1G93A mouse spinal cord tissues infected with AAV9/ shAAV9-FUNDC1, (G and H) representative Nissl-stained sections of the spinal cord anterior horn and quantitative analysis of surviving neurons, as well as (I and J), representative TUNEL-stained sections of the spinal cord anterior horn and quantitative analysis of the number of neurons undergoing apoptosis. (scale bar = 50 μm). Results are demonstrated as mean ± SD with statistics using an unpaired two-tailed Student's t-test. * P < 0.05, ** P < 0.01, *** P < 0.001. NS indicates no sig nificance (P > 0.05). AAV9-EGFP, AAV overexpression virus negative control; AAV9-FUNDC1, AAV overexpression FUNDC1 virus.

Article Snippet: The following primary antibodies were used for western blotting: FUNDC1 (1:1000, CST), LC3B-II (1:1000, CST, #49240), BAX (1:1000, Proteintech, Cat No. 60267-1-Ig), BCL-2 (1:1000, Proteintech, Cat No. 60178-1-Ig), GAPDH (1:5000, Proteintech, Cat No. 10494-1-AP), beta actin (1:5000, Cat No. 81115-1-RR), P62 (1:1000, Proteintech, Cat No. 18420-1-AP), HSP60 (1:1000, Zenbio, 222340), COXIV (1:1000, Zenbio, 200147), and FOXD3 (1:1000, Zenbio, 220508).

Techniques: Infection, Western Blot, Staining, TUNEL Assay, Two Tailed Test, Over Expression, Virus, Negative Control

Journal: Neurobiology of disease

Article Title: FUDNC1-dependent mitophagy ameliorate motor neuron death in an amyotrophic lateral sclerosis mouse model.

doi: 10.1016/j.nbd.2024.106534

Figure Lengend Snippet: Fig. 5. The effect of FUNDC1 on autophagy flux in hSOD1G93A N2a neuronal cells. (A, B, and C) Evaluation of autophagy flux in hSOD1G93AN2a cells using the lysosomal inhibitor bafilomycin A1 (Baf, 100 nM). Baf-induced accumulation of LC3B-II and P62 in siCtrl or siFUNDC1-transfected cells (n = 3) (D, E, F, G, H, I, and J) siCtrl and siFUNDC1-transfected hSOD1G93AN2a cells were subjected to immunoblotting to detect FUNDC1, TOM20, COXIV, HSP60, BAX, and BCL-2 protein levels (D), along with quantitative analysis results (E, F, I, J (n = 4)) (G, H(n = 3)). (K, L, and M)Evaluation of autophagy flux in hSOD1G93AN2a cells using the lysosomal inhibitor bafilomycin A1 (Baf, 100 nM). Baf-induced accumulation of LC3B-II and P62 in adctrl or adFUNDC1-transfected cells (n = 3). (N, O, P, Q, R, S, and T) hSOD1G93A N2a cells transfected with adctrl and adFUNDC1 were subjected to immunoblotting to detect FUNDC1, TOM20, COXIV, HSP60, BAX, and BCL-2 protein levels (H), along with quantitative analysis results O-T) (O, P, S and T(n = 4))(Q, R(n = 3)). (U, V, W, and X) hSOD1G93A N2a cells transfected with si/adFUNDC1 and GFP-LC3 were observed for the colocalization of autophagosomes GFP-LC3 (green fluorescence) and Mito-Tracker Red (red fluorescence) (U), along with quantitative analysis of average mitochondrial area, perimeter and mitophagosome (V, W, and X) (n = 20) (scale bar = 10 um). (Y, Z) hSOD1G93A N2a cells transfected with si/ adFUNDC1 were subjected to TUNEL staining (Z) and JC-1 staining (Y) (scale bar = 20 um). The results are presented using an unpaired, two-tailed Student's t-test and expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. NS indicates no significant difference (P > 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following primary antibodies were used for western blotting: FUNDC1 (1:1000, CST), LC3B-II (1:1000, CST, #49240), BAX (1:1000, Proteintech, Cat No. 60267-1-Ig), BCL-2 (1:1000, Proteintech, Cat No. 60178-1-Ig), GAPDH (1:5000, Proteintech, Cat No. 10494-1-AP), beta actin (1:5000, Cat No. 81115-1-RR), P62 (1:1000, Proteintech, Cat No. 18420-1-AP), HSP60 (1:1000, Zenbio, 222340), COXIV (1:1000, Zenbio, 200147), and FOXD3 (1:1000, Zenbio, 220508).

Techniques: Transfection, Western Blot, Fluorescence, TUNEL Assay, Staining, Two Tailed Test

Journal: Neurobiology of disease

Article Title: FUDNC1-dependent mitophagy ameliorate motor neuron death in an amyotrophic lateral sclerosis mouse model.

doi: 10.1016/j.nbd.2024.106534

Figure Lengend Snippet: Fig. 6. Effect of FUNDC1 on neuronal apoptosis and mitophagy in hSOD1G93A N2a cells. (A) Transfected hSOD1G93A N2a cells with adCtrl and adFUNDC1, treated with or without 10 μM CQ, and observed autophagic vesicles GFP-LC3 (green) and DAPI (blue) (scale bar = 10 μm). (B) Transfected hSOD1 G93A N2a cells with siCtrl and siFUNDC1, treated with or without 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP), colocalization of GFP-LC3 (green fluorescence) with Mito-Tracker Red (red fluorescence) was observed, along with quantitative analysis of average mitochondrial area, perimeter and mitophagosome (D, E, and F) (n = 15) (scale bar = 10 um). (C and G) Transfected hSOD1G93A N2a cells with siCtrl and siFUNDC1, treated with or without 10 μM CCCP, TUNEL-positive cells (green), DAPI (blue) (C), and results of quantitative analysis of the number of neuronal apoptosis positives (G) (scale bar = 50 um) were observed. (H, I, J, and K) Transfected hSOD1G93A N2a cells with adCtrl and adFUNDC1, treated with or without 10 μM CQ, and analyzed the protein levels of LC3B-II, TOM20, and BAX by immunoblotting (H), with quantitative analysis results (I, J, and K) (n = 4). (L, M, N, and O) Transfected hSOD1G93A N2a cells with siCtrl and siFUNDC1, treated with or without 10 μM CCCP, and analyzed the protein levels of LC3B-II, TOM20, and BAX by immunoblotting (L), with quantitative analysis results (M, N, and O) (n = 4). Results are demonstrated as mean ± SD with statistics using an unpaired two-tailed Student's t-test. * P < 0.05 or ** P < 0.01 indicates significance, whereas NS indicates no significant difference (P > 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following primary antibodies were used for western blotting: FUNDC1 (1:1000, CST), LC3B-II (1:1000, CST, #49240), BAX (1:1000, Proteintech, Cat No. 60267-1-Ig), BCL-2 (1:1000, Proteintech, Cat No. 60178-1-Ig), GAPDH (1:5000, Proteintech, Cat No. 10494-1-AP), beta actin (1:5000, Cat No. 81115-1-RR), P62 (1:1000, Proteintech, Cat No. 18420-1-AP), HSP60 (1:1000, Zenbio, 222340), COXIV (1:1000, Zenbio, 200147), and FOXD3 (1:1000, Zenbio, 220508).

Techniques: Transfection, Fluorescence, TUNEL Assay, Western Blot, Two Tailed Test

Journal: Neurobiology of disease

Article Title: FUDNC1-dependent mitophagy ameliorate motor neuron death in an amyotrophic lateral sclerosis mouse model.

doi: 10.1016/j.nbd.2024.106534

Figure Lengend Snippet: Fig. 7. Effects of FOXD3 on FUNDC1-associated neuroapoptosis in SOD1G93A mice and cells. (A and B) FOXD3 protein expression in the spinal cord tissues of SOD1G93A mice (A) Quantitative analysis of FOXD3 protein expression levels (B) (n = 4). (C) A dual fluorescein reporter gene assay to detect the transcriptional regulation of FUNDC1 by FOXD3 (n = 4). (D, E, and F) FOXD3, FUNDC1 protein expression in adCtrl and adFOXD3 transfected hSOD1G93A N2a cells (D), quantitative analysis of FOXD3, FUNDC1 protein expression levels (E and F) (n = 4). (G, H, I, J, and K) Protein levels of LC3B-II, P62, TOM20, and BAX in hSOD1G93AN2a cells transfected with adCtrl, adFOXD3, and adFOXD3 + siFUNDC1, respectively (G), and quantitatively analyzed for LC3B-II, P62, TOM20, and BAX protein expression levels (H, I, J, and K) (n = 4). Results are demonstrated as mean ± SD with statistics using an unpaired two-tailed Student's t-test. * P < 0.05 or ** P < 0.01. NS indicates no significance (P > 0.05).

Article Snippet: The following primary antibodies were used for western blotting: FUNDC1 (1:1000, CST), LC3B-II (1:1000, CST, #49240), BAX (1:1000, Proteintech, Cat No. 60267-1-Ig), BCL-2 (1:1000, Proteintech, Cat No. 60178-1-Ig), GAPDH (1:5000, Proteintech, Cat No. 10494-1-AP), beta actin (1:5000, Cat No. 81115-1-RR), P62 (1:1000, Proteintech, Cat No. 18420-1-AP), HSP60 (1:1000, Zenbio, 222340), COXIV (1:1000, Zenbio, 200147), and FOXD3 (1:1000, Zenbio, 220508).

Techniques: Expressing, Reporter Gene Assay, Transfection, Two Tailed Test