Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Antioxidant removal damages mitochondria and activates mitophagy in iPSC-derived i3Neurons (A) WT i3Neurons expressing mito-EBFP2 were untreated or cultured in antioxidant (AOx) removal media for 3-4 h and stained with TMRM and SiR-tubulin. White arrowheads indicate mitochondria lacking membrane potential. (B) WT i3Neurons expressing mito-Keima (mt-Keima) were untreated, cultured in AOx removal media for 4-5 h or 6-7 h and stained with SiR-Tubulin. (C and D) WT i3Neurons were untreated or cultured in AOx removal media for 4 h and immunostained for phospho-Serine 65 ubiquitin (S65 UB), HSP60 and tubulin (C) and the percentage of mitochondrial volume positive for S65 UB was quantified (D). S65 UB is a product of PINK1/Parkin activity. White arrowheads point to S65 UB located on mitochondria. (E and F) WT i3Neurons were cultured in AOx removal media for 4 h and immunostained for ATG13, HSP60 and Tubulin (E) and the percentage of mitochondrial volume positive for ATG13 was quantified (F). Untreated samples shown in . White arrowheads indicate ATG13 puncta localised to mitochondria. (G and H) WT i3Neurons were untreated or cultured in AOx removal media for 4 h and immunostained for ATG9A, HSP60 and Tubulin (G) and the ATG9A intensity (Arbitrary unit (A.U.)) at axon was quantified (H). Data in (D), (F) and (H) are mean ± SD from three independent experiments. Two-way ANOVA. **p<0.005, ***p<0.001. n.s., not significant. Scale bars, 15 µm.

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Derivative Assay, Expressing, Cell Culture, Staining, Membrane, Ubiquitin Proteomics, Activity Assay

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Antioxidant removal damages mitochondria and promotes mitophagy in iPSC-derived i3Neurons (A) Representative confocal images of untreated WT i3Neurons immunostained for S65 UB, HSP60 and Tubulin. Related to and . (B) Representative confocal images of untreated WT i3Neurons immunostained for ATG13, HSP60 and Tubulin. Related to and . Scale bars, 15 µm.

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Presynapse is a hotspot for axonal PINK1/Parkin dependent mitophagosome formation (A and B) WT i3Neurons were immunostained for Synapsin I (A) or Synaptophysin (B) and Tubulin. The Synapsin I and Synaptophysin stainings were displayed as RGB thermal spectrum. (C) Immunoblot (IB) for autophagy adaptors including OPTN, NDP52, TAX1BP1, NBR1 and p62 in iPSCs, day 3 pre-differentiated (Pre-Diff) iPSCs, i3Neurons and WT Hela cells. kDa, kilodaltons. (D and E) WT i3Neurons expressing EGFP-OPTN were cultured in AOx removal media for 4 h and immunostained for Synapsin I, EGFP, HSP60 and Tubulin (D) and the percentage of mitochondrial volume positive for OPTN was quantified (E). Untreated samples are shown in . The Synapsin I staining were displayed as RGB thermal spectrum. White arrowheads indicate OPTN puncta localised to mitochondria. (F and G) WT and PINK1 KO iNeurons were cultured in AOx removal media for 6 h and immunostained for ATG13, HSP60 and Tubulin (F) and the percentage of mitochondrial volume positive for ATG13 was quantified (G). Untreated samples shown in . White arrowheads point to ATG13 puncta localised to mitochondria. Data in (E) and (G) are mean ± SD from three independent experiments. Two-way ANOVA. **p<0.005, ***p<0.001. n.s., not significant. Scale bars, 15 µm.

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Western Blot, Expressing, Cell Culture, Staining

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: The initiation of PINK1/Parkin dependent mitophagosome formation primarily occurs in presynapses (A) The expression of presynaptic marker Synapsin I and postsynaptic marker PSD95 were examined in iPSCs, iPSC-derived i3Neurons, ESCs and ESC-derived iNeurons using immunoblotting (IB). (B) Immunoblot for indicated autophagy adaptors and regulators in iPSCs, day 3 pre-differentiated (Pre-Diff) iPSCs, iPSC-derived i3Neurons, ESCs, day 7 Pre-Diff ESCs, ESC-derived iNeurons and WT Hela cells. Second from the bottom actin panel is the loading control for p62, FIP200, ULK1, TBK1 and GABARAPL1. The VCP panel is the loading control for NAP1, SINTBAD and LC3A. kDa, kilodaltons. Related to . (C) The expression of EGFP-OPTN in WT i3Neurons was confirmed by IB for OPTN and EGFP. (D) Representative images of untreated WT i3Neurons expressing EGFP-OPTN that were immunostained for Synapsin I, EGFP, HSP60 and Tubulin. The Synapsin I staining is displayed as RGB thermal spectrum. (E) Confirmation of PINK1 KO ESC-derived iNeurons by IB. (F) WT ESC-derived iNeurons were immunostained for Synapsin I. (G) Untreated WT and PINK1 KO iNeurons were immunostained for ATG13, HSP60 and Tubulin. Related to and . Scale bars, 15 µm.

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Expressing, Marker, Derivative Assay, Western Blot, Control, Staining

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Confirmation that AOx removal-induced PINK1/Parkin mitophagy is OPTN dependent, and PINK1/Parkin mitophagy prevents axon degeneration. (A) Confirmation of OPTN KO ESC-derived iNeurons by immunoblotting. (B) Representative confocal images of WT and OPTN KO iNeurons with or without EGFP-OPTN expression cultured in AOx removal media for 6 h and immunostained for ATG13, HSP60 and TUJ1. White arrowheads point to ATG13 puncta localised to mitochondria. (C) Representative confocal images of untreated WT, PINK1 KO and OPTN KO iNeurons stained with MitoTracker Green (MTG), MitoSOX red (MSR) and SiR-Tubulin. Related to and . (D) Representative confocal images of untreated WT, PINK1 KO and OPTN KO iNeurons immunostained for Cleaved Caspase 3 (CC3), HSP60 and Tubulin. Related to . Scale bars, 15 µm.

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Derivative Assay, Western Blot, Expressing, Cell Culture, Staining

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Evidence of presynaptic mitophagy in highly active and mature iPSC-derived midbrain neurons and human brain (A) Representative images (left) and quantification (right) of DIV90-DIV120 human iPSC-derived neuronal cultures expressing dopaminergic (first and third row) and substantia nigra pars compacta (second row) and neurons (MAP2+) versus astrocyte (GFP+; bottom row) identity markers (after 90-120 days in BrainPhys maturation medium. Means ± SEM. Each data point represents one replicate well containing an average of 5600 neurons (MAP2+) across 25 fields of view. (B-D) Whole-cell patch-clamping of healthy human iPSC-derived midbrain neurons after 142-164 days in BrainPhys maturation medium. Mean ± SEM. Each data point represents a single neuron patched. (B) Example of a live human neuron that has been filled with rhodamine from the patch pipette. Midbrain iPSC-derived neurons develop functional voltage-gated sodium channels. (C) Midbrain iPSC-derived neurons develop mature, repetitive action potentials. (D) Midbrain iPSC-derived neurons develop functional excitatory (AMPA) and inhibitory (GABA) postsynaptic circuits. (E) Representative images of DIV157 iPSC-derived midbrain neurons cultured in basal (left) or 6 h of AOx removal condition (middle and right) and immunostained for Synapsin I, WIPI2, HSP60 and Tubulin. White arrowheads indicate WIPI2 puncta localised to mitochondria. (F) Electron microscopy (EM) and the 3D rendered structures images were obtained from the open access dataset generated by Shapson-Coe et al., featuring high-resolution serial section EM of 1 mm of human temporal cortex . The presynapses were identified by the distinct bouton morphology along the axon (shown in 3D rendered panel) and the presence of presynaptic density (shown in EM overview or insets, indicated by orange arrowhead). Several examples of mitophagy events were observed in the presynapses, indicated by the autophagosomal membrane forming around the mitochondrion (white arrowhead). Different Z-plane of the EM images (bottom right panel) were examined to identify the completion of the autophagosomal membrane structure.

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Derivative Assay, Expressing, Transferring, Functional Assay, Cell Culture, Electron Microscopy, Generated, Membrane

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Evidence of presynaptic mitophagy in highly active and mature iPSC-derived midbrain neurons and human brain (A) Representative images of DIV157 midbrain iPSC-derived neurons cultured in basal condition and immunostained for Synapsin I, WIPI2, HSP60 and Tubulin. White arrowhead indicates WIPI2 puncta localised to mitochondria. Related to . (B) Another example electron microscopy (EM) images of presynaptic mitophagy shown in the open access dataset generated by Shapson-Coe et al., featuring high-resolution serial section EM of 1 mm of human temporal cortex. The presynapses were identified by the distinct bouton morphology along axon (shown in 3D rendered panel) and the presence of presynaptic density (shown in EM overview or insets, indicated by orange arrowhead). Mitophagy was indicated by the autophagosomal membrane forming around the mitochondrion (white arrowhead). Different Z-plane of the EM images were examined to identify the completion of the autophagosomal membrane structure. Related to .

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Derivative Assay, Cell Culture, Electron Microscopy, Generated, Membrane

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: PINK1/Parkin mitophagy protects neurons from oxidative stress-induced mitochondrial collapse and neuronal degeneration (A and B) WT, PINK1 KO and OPTN KO iNeurons were cultured in AOx removal media for 5-6 h and stained with MitoTracker Green (MTG), MitoSOX red (MSR) and SiR-Tubulin (A) and the percentage change in MSR to MTG ratio compared to untreated control was quantified (B). Untreated samples are shown in . (C-E, F-G) WT, PINK1 KO and OPTN KO iNeurons were treated with pan-caspase inhibitor QVD-OPh (QVD) and cultured in untreated or AOx removal conditions for 4 days, and were immunostained for Synapsin I, TOM20, Cytochrome c (Cyt c ) and Tubulin (C, F). The percentage of mitochondrial volume lacking Cyt c (D), the percentage of the number of presynapse with damaged mitochondria lacking Cyt c (E) and the average density of total mitochondria per presynapse (G) were quantified. (H) Schematic of the mechanism of reactive oxygen species (ROS) activating apoptosis. (I and J) WT, PINK1 KO and OPTN KO iNeurons were cultured in AOx removal media for 4 days and were immunostained for Cleaved Caspase 3 (CC3), HSP60 and Tubulin (I). The fold change of CC3 intensity per axon volume (J) was quantified. Untreated samples are shown in . White arrowheads point to axons with caspase 3 activation. Data in (B), (D), (E), (G) and (J) are mean ± SD from three independent experiments. Two-way ANOVA. p*<0.05, **p<0.005, ***p<0.001, ****p<0.0001. n.s., not significant. Scale bars, 15 µm.

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Cell Culture, Staining, Control, Activation Assay

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Long term AOx removal does not induce mitochondria herniation or cGAS-STING activation. (A) Representative confocal images of WT and PINK1 KO iNeurons that were untreated, treated with 500 nM ABT737 for 4 h or AOx removal for 4 days and immunostained for TOM20, DNA and Tubulin. White arrowheads point to herniated mitochondria with mtDNA leakage. The overview images were captured using 40X objective with 2.5X zoom and the inset images were captured separately using the same objective with 6.5X zoom. Scale bars, 15 µm. (B) WT, PINK1 KO, OPTN KO iNeurons and WT HeLa cells were untreated, treated with AOx removal for 4 days, 500 nM ABT737 for 4 h, 1 µg/ml diABZi for 2 h or 60 µg/ml cGAMP for 4 h, and immunoblotted for regulators of cGAS-STING pathway including p-TBK1 (Ser172), STING, p-STING (Ser366), p-IRF3 (Ser396), p-P65 (S536).

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Roxadustat stimulates PINK1-independent mitophagy in PINK1/Parkin-mitophagy defective neurons in response to oxidatively damaged mitochondria. (A and B) WT, PINK1 KO and OPTN KO iNeurons expressing mito-mKeima XL (mt-mKeima XL ) were treated with pan-caspase inhibitor QVD-OPh (QVD) and cultured in either AOx removal media for 4 days, 40 µM of Roxadustat for 48 h, or a combination of AOx removal for 4 days with Roxadustat treatment in the final 48 h. These neurons were stained with SiR-Tubulin and Hoechst33342 and underwent live cell imaging. Untreated samples are shown in . The relative fold change in average number of mitolysosomes per cell were quantified in (B). Data in (B) is mean ± SD from three independent experiments. Two-way ANOVA. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. n.s., not significant. Scale bars, 15 µm.

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Expressing, Cell Culture, Staining, Live Cell Imaging

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Roxadustat protects PINK1/Parkin-mitophagy defective neurons from axon degeneration by restoring mitochondrial health. (A) Representative live cell confocal images of QVD treated control WT, PINK1 KO and OPTN KO iNeurons expressing mt-mKeima XL stained with SiR-Tubulin and Hoechst33342. Related to and . (B) Representative confocal images of untreated and 48 h 40 µM Roxadustat treated WT, PINK1 KO and OPTN KO iNeurons immunostained for Cleaved Caspase 3 (CC3), HSP60 and Tubulin. Related to and Scale bars, 15 µm. (C) Experimental overview of how the 4 days AOx removal, a combination of 4 days AOx removal with 40 µM Roxadustat treatment in the final 48 h and the 48 h 40 µM Roxadustat treatment were performed.

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Control, Expressing, Staining

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Roxadustat restores mitochondrial health in PINK1/Parkin-mitophagy defective neurons and prevents axon degeneration. (A and B) WT, PINK1 KO and OPTN KO iNeurons were treated with either AOx removal media for 4 days or a combination of AOx removal for 4 days with 40 µM Roxadustat treatment in the final 48 h. The neurons were immunostained for Cleaved Caspase 3 (CC3), HSP60 and Tubulin (A). The percentage change of CC3 intensity per axon volume (B) was quantified. Untreated and 48 h 40 µM Roxadustat treated samples shown in . (C and D) The activation of Caspase-3, levels of synaptic proteins Synapsin I and PSD95, and the expression of PINK1/Parkin-independent mitophagy adaptor proteins BNIP3 and NIX were analysed through immunoblotting in WT, PINK1 KO and OPTN KO iNeurons that were untreated, treated with either AOx removal condition for 4 days, 40 µM Roxadustat for 48 h, a combination of AOx removal for 4 days with 40 µM Roxadustat treatment in the final 48 h, or QVD. The ratio of Cleaved Caspase-3 to total full length Caspase-3 were quantified (D). Data in (B) and (D) were mean ± SD from three independent experiments. Two-way ANOVA. ***p<0.001, ****p<0.0001. n.s., not significant. Scale bars, 15 µm.

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Activation Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Roxadustat restores mitochondrial health and presynaptic mitochondrial density in PINK1/Parkin-mitophagy defective neurons and prevents cytochrome c leakage. (A-E) WT, PINK1 KO and OPTN KO iNeurons were treated pan-caspase inhibitor Q-VD-OPh (QVD) and cultured with either AOx removal media for 4 days or a combination of AOx removal for 4 days with 40 µM Roxadustat treatment in the final 48 h. The neurons were immunostained for Synapsin I, TOM20, Cytochrome c (Cyt c ) and Tubulin (A and D). The percentage of mitochondrial volume lacking cyt c (B), the percentage of the number of presynapse with damaged mitochondria lacking cyt c (C) and the average density of total mitochondria per presynapse (E) were quantified. Data in (B), (C) and (E) are mean ± SD from three independent experiments. 30-60 presynapses were analysed per sample for each independent experiments. Two-way ANOVA. ****p<0.0001. n.s., not significant. Scale bars, 15 µm.

Article Snippet: Cells were washed three times with DPBS then incubated with conjugated antibody staining buffer comprising β-Tubulin (9F3) Rabbit mAb (AF647 Conjugate) (CST; 3624S).

Techniques: Cell Culture