DOI: 10.5061/dryad.zkh1893nt
title: Glaucoma-associated optineurin mutations increase transcellular degradation of mitochondria in a vertebrate optic nerve

description: We previously described a process whereby mitochondria shed by retinal
 ganglion cell (RGC) axons are transferred to and degraded by surrounding
 astrocytes in the optic nerve head of mice. Since the mitophagy receptor
 Optineurin (OPTN) is one of the few large-effect glaucoma genes and axonal
 damage occurs at the optic nerve head in glaucoma, here we explored
 whether OPTN mutations perturb the transcellular degradation of
 mitochondria. Live-imaging of Xenopus laevis optic nerves revealed that
 diverse human mutant but not wildtype OPTN increase stationary
 mitochondria and mitophagy machinery and their colocalization within, and
 in the case of the glaucoma-associated OPTN mutations, also outside of,
 RGC axons. These extra-axonal mitochondria are degraded by astrocytes. Our
 studies demonstrate that expression of OPTN carrying a glaucoma-associated
 mutation results in increased transcellular degradation of axonal
 mitochondria.
description: # Glaucoma-associated optineurin mutations increase transcellular
 degradation of mitochondria in a vertebrate optic nerve Dataset DOI:
 [10.5061/dryad.zkh1893nt](10.5061/dryad.zkh1893nt) ## Description of the
 data and file structure This dataset contains 5 types of primary data,
 most of which are live imaging data of tadpole optic nerves. Most data are
 either time series(t-series) of single focal planes, or single-time
 sections spanning the thickness of the optic nerve (z-series); both data
 types were acquired using a spinning disc confocal microscope. There is
 one image acquired with a stereo-fluorescence microscope. There is one
 figure containing both a z-series and serial block-face scanning electron
 microscopy (SBEM)for the same nerve and including an x-ray image of the
 fixed and embedded tissue block, which was used in the registration of the
 fluorescence and SBEM data. T- and z-series fluorescence data were
 collected using an Andor Dragonfly Spinning Disc Confocal microscope.
 T-series were collected sequentially for different channels.  In the
 z-series, different channel data were collected at each individual
 z-position before moving to a new z-position. In z-series with two colors,
 green fluorescence was acquired in channel 1 and red was acquired in
 channel 2. In the z-series with three colors, green fluorescence was
 acquired in channel 1, red fluorescence in channel 2, and far-red
 fluorescence in channel 3. In the z-series with four colors, blue
 fluorescence was acquired in channel 1, green fluorescence in channel 2,
 red fluorescence in channel 3, and far-red fluorescence in channel 4.
 T-series analyses were performed using custom kymograph scripts written
 for IPLab software. Z-series analyses were performed using Imaris
 software. X-ray data were acquired with an X-ray microscope (XRM)
 instrument. SBEM data was collected with a 3View unit. Files attached
 contain both the raw data supporting primary and supplementary figures, as
 well as the analyses for each of these. Given the large number and size of
 files, they had to be organized and compressed to be able to be uploaded
 to Dryad. The raw data corresponding to each figure are provided
 separately for each figure, either all in one compressed file or
 subdivided in parts. All analysis files, also organized by figures, are
 provided also compressed and in 5 parts. Abbreviations | Ave | Average | |
 :---------- | :-------------------------------------------------- | | Avg
 | Average | | Ax | Axon | | Axo | Axon | | Chloro | Chloroquine | |
 Chloroq | Chloroquine | | Contro | Control | | D474N | OPTN^D474N^ | |
 diam | Diameter | | E/A | Evulsion / Axon | | E478G | OPTN^E478G^ | | E50K
 | OPTN^E50K^ | | EST | estimate | | EV | Evulsion | | Evul | Evulsion | |
 F178A | OPTN^F178A^ | | Fig | Figure | | FR | Far Red | | G | Green | |
 H486R | OPTN^H486R^ | | int | Mean Intensity | | IslGFP |
 *Tg(Isl2b:Tom20-mCherry)* | | IslTom20mCh | *Tg(Isl2b:GFP)* | | K |
 Kymograph | | Kymo | Kymograph | | LC3b | Microtubule-associated protein 1
 light chain 3 beta | | lw | line width | | M98K | OPTN^M98K^ | | mBFP |
 membrane blue fluorescent protein | | MinDis | Minimum Distance | | Mito |
 Mitochondria | | OPTN | Optineurin | | Ortho | Orthograde | | Prox |
 Proximal | | R | Red | | Retro | Retrograde | | ROI | Region of Interest |
 | Seg | Segment | | stdev | standard deviation | | V | Velocity | | Vol |
 Volume | | WT | Wild type | | YJ | Yaeram Jeong | | Ave | Average | |
 :---------- | :-------------------------------------------------- | | Avg
 | Average | | Ax | Axon | | Axo | Axon | | Chloro | Chloroquine | |
 Chloroq | Chloroquine | | Contro | Control | | D474N | OPTN^D474N^ | |
 diam | Diameter | | E/A | Evulsion / Axon | | E478G | OPTN^E478G^ | | E50K
 | OPTN^E50K^ | | EST | estimate | | EV | Evulsion | | Evul | Evulsion | |
 F178A | OPTN^F178A^ | | Fig | Figure | | FR | Far Red | | G | Green | |
 H486R | OPTN^H486R^ | | int | Mean Intensity | | IslGFP |
 Tg(Isl2b:Tom20-mCherry) | | IslTom20mCh | Tg(Isl2b:GFP) | | K | Kymograph
 | | Kymo | Kymograph | | LC3b  | Microtubule-associated protein 1 light
 chain 3 beta | | lw | line width | | M98K | OPTN^M98K^ | | mBFP | membrane
 blue fluorescent protein | | MinDis | Minimum Distance | | Mito |
 Mitochondria | | OPTN | Optineurin | | Ortho | Orthograde | | Prox |
 Proximal | | R | Red | | Retro | Retrograde | | ROI | Region of Interest |
 | Seg | Segment | | stdev | standard deviation | | V | Velocity | | Vol |
 Volume | | WT | Wild type | | YJ | Yaeram Jeong | ### Files and variables
 #### File: Figure_5_part_2.zip **Description:** Raw Data Files for Figure
 5, part 2. #### File: Figure_5_part_3.zip **Description:** Raw Data Files
 for Figure 5, part 3. #### File: Figure_1.zip **Description:** Raw Data
 Files for Figure 1. #### File: Figure_2_part_1.zip **Description:**  Raw
 Data Files of Figure 2, part 1. #### File:
 JeongEtAl_2025_Analysis_part_1.zip **Description:** Analysis Files, part
 1. Analysis files for Fig. 1, 2 and first part of Fig. 3.  Contain Excel
 files with data, GraphPad Prism files with data, graphs and statistics,
 and Imaris files containing the measured surfaces. #### File:
 JeongEtAl_2025_Analysis_part_2.zip **Description:**  Analysis Files, part
 2. Analysis files for the second part of Fig. 3. Contain Imaris files
 containing the measured surfaces. #### File:
 JeongEtAl_2025_Analysis_part_3.zip **Description:**  Analysis Files, part
 3. Analysis files for the third part of Fig. 3. Contain Excel files with
 data, GraphPad Prism files with data, graphs and statistics, and Imaris
 files containing the measured surfaces. #### File:
 JeongEtAl_2025_Analysis_part_4.zip **Description:** Analysis Files, part
 4. Analysis files for the first part of Fig. 4. Contain Excel files with
 data, GraphPad Prism files with data, graphs and statistics, and Imaris
 files containing the measured surfaces. #### File:
 JeongEtAl_2025_Analysis_part_5.zip **Description:** Analysis Files, part
 5. Analysis files for the second part of Fig. 4, Fig. 5, Fig. 6 and
 Supplemental Figs. 1-4.  Contain mainly Excel files with data, GraphPad
 Prism files with data, graphs and statistics, but Fig. 4 also Imaris files
 containing the measured surfaces, and for Fig. 5 two .txt files containing
 coordinate data for the models. #### File: Figure_2_part_2.zip
 **Description:** Raw Data Files for Figure 2, part 2. #### File:
 Figure_2_part_3.zip **Description:** Raw Data Files for Figure 3, part 3.
 #### File: Figure_4_part_1.zip **Description:** Raw Data Files for Figure
 4, part 1. #### File: Figure_5_part_1.zip **Description:** Raw Data Files
 for Figure 5, part 1. #### File: Figure_3.zip **Description:** Figure 3
 Raw Files. #### File: Movies.zip **Description:** All movies. #### File:
 Figure_4_part_2.zip **Description:** Raw Data Files for Figure 4, part 2.
 #### File: Figure_6.zip **Description:** Figure 6 Raw Files. #### File:
 Figure_4_part_3.zip **Description:** Raw Data Files for Figure 4, part 3.
 #### File: Sup_Figure_1.zip **Description:** Raw Data Files for
 Supplementary Figure 1. #### File: Sup_Figure_2_part_3.zip
 **Description:** Raw Data Files for Supplementary Figure 2, part 3. ####
 File: Sup_Figure_3_part_1.zip **Description:** Raw Data Files for
 Supplementary Figure 3, part 1. #### File: Sup_Figure_2_part_1.zip
 **Description:** Raw Data Files for Supplementary Figure 2, part 1. ####
 File: Sup_Figure_2_part_2.zip **Description:** Raw Data Files for
 Supplementary Figure 2, part 2. #### File: Sup_Figure_3_part_4.zip
 **Description:** Raw Data Files for Supplementary Figure 3, part 4. ####
 File: Sup_Figure_4_part_1.zip **Description:** Raw Data Files for
 Supplementary Figure 4, part 1. #### File: Sup_Figure_3_part_5.zip
 **Description:** Raw Data Files for Figure 3, part 5. #### File:
 Sup_Figure_2_part_4.zip **Description:** Raw Data Files for Supplementary
 Figure 2, part 4. #### File: Sup_Figure_3_part_2.zip **Description:** Raw
 Data Files for Supplementary Figure 3, part 2. #### File:
 Sup_Figure_3_part_3.zip **Description:** Raw Data Files for Supplementary
 Figure 3, part 3. #### File: Sup_Figure_4_part_2.zip **Description:** Raw
 Data Files for Supplementary Figure 4, part 2. #### File:
 JeongEtAl_2025_excel.csv **Description:** Table with both the file names
 as they appear in Dryad and the corresponding original name that contains
 a time stamp. #### Description of the data and file structure We have
 provided our raw data corresponding to each figure or supplementary figure
 in parts, to comply with the size limit requirements of the Dryad
 depository and similarly, all analysis files in five separate folders, all
 compressed in .zip formats. ##### Raw data Due to formatting requirements
 of the UC Davis Dryad data repository, some of the original Raw data in
 their original formats and naming schemes are not provided.  Rather, raw
 data is provided only in processed forms, as .tif formats. The processed
 files have been renamed to make explicit the Figure panel(s) for which
 they correspond. The naming convention list files as belonging to this
 study, by the prefix YJ, followed by the Figure and Panel to which they
 correspond, followed by the date the original data was acquired. An
 Image_Renaming table, YJ_rawdata_table.xlsx, is provided listing all image
 files in both their renamed formats as well as in their original formats;
 original data had unique identifier names which were based on date and
 time of acquisition. The data in their original formats and names are only
 available upon request. Some raw data files were used for multiple figures
 or panels, in which case the same original data raw file is presented
 duplicated with different processed names. In the Image_Renaming table,
 all such examples have been marked by a D in the 4th column. Folders for
 Figures 1, S1, 2, S2, and S4 contain exclusively t-series data, with the
 exception of Figure 1, which also contains a lower resolution image of the
 setup acquired with a stereo-fluorescence microscope. Folders for Figures
 3 and 4 contain exclusively z-series data. Folders for Figure S3 contain a
 combination of t-series and z-series data. The folder for Figure 5
 contains correlated light and SBEM. data. A lower resolution single
 channel image for the entire length of the optic nerve, presented as a
 single maximum intensity projection, was obtained by registering multiple
 overlapping z-series in 3-dimensions prior to the projection.  A higher
 resolution 4-color z-series was obtained for the same nerve, centered on a
 region of interest (ROI). An X-ray image for the fixed and embedded block
 containing the head of the same animal lived imaged was produced by
 rotating X-ray tomographic data to correspond with the rotation angle of
 the live imaging data, and is presented solely as a maximum intensity
 projection. The raw SBEM data for the region of the optic nerve is
 presented in .tif format, converted from its original .mrc formats using
 the IMOD mrc2tif command, as the original format was also not supported by
 the UCD dryad. It can be reconverted back to its original format by using
 the tif2mrc command in IMOD. Folder for Figures 6 contains a t- and
 z-series.  That is, a ROI imaged as a z-series was reimaged as a t-series
 every 30 seconds. ##### Analysis ###### Time-Series (t-series) Analysis –
 Figures 1, S1, 2, S2, S3, and S4 To analyze time-lapse microscopy data, we
 developed a custom script in IPLab, which is included in this dataset. The
 analysis began with manual tracing of the optic nerve’s contour in each
 image. Once outlined, the script automatically divided the nerve into a
 series of evenly spaced segments along its length. For each of these
 segments, a kymograph was generated. These images are powerful tools for
 visualizing the movement of particles over time. On each kymograph, we
 manually traced clear, continuous lines—each one corresponding to the path
 of a moving particle. From these traces, we extracted two key metrics: the
 proportion of stationary versus moving particles (in both directions), and
 the average velocity of those that moved. In experiments with sparsely
 labeled axons, we adjusted the approach slightly. Instead of dividing the
 nerve into equal parts, we manually traced individual axons before
 generating kymographs. These variants of the script are also included.
 While intermediate files such as kymographs and manual traces were
 generated during the analysis, they are not included here due to technical
 limitations. Many kymographs were processed using older software versions
 without standardized export options, and in some cases, tracing was
 performed live without generating separate files. However, all raw image
 sequences and the full analysis scripts are provided, allowing complete
 reproduction of the kymographs and quantitative analyses described in the
 manuscript. The quantitative results from kymograph analysis were first
 averaged at the nerve level, combining data across all segments within a
 single optic nerve. These nerve-level values were then summarized per
 animal to generate a final set of metrics for statistical comparison.
 These results are saved in Excel spreadsheets named according to the
 figure and panel they correspond to—for example, YJ_Fig1E_table.xlsx.
 These summary tables were then imported into GraphPad Prism for
 statistical analysis and figure generation. The Prism project files are
 also included and follow a matching naming convention (e.g.,
 YJ_Fig1E_graph.pzf). ###### Z-Series Analysis via Imaris – Figures 3, S3,
 and 4 For z-stack imaging data, we used Imaris software to perform 3D
 segmentation of particles within axons. Each dataset was first masked to
 isolate the optic nerve. Using fluorescent labeling, particles were
 segmented and their volumes calculated based on consistent processing
 parameters. Each Imaris analysis produced two main outputs: first,
 structured Excel spreadsheets listing individual particles, their volumes,
 and group assignments (e.g., Wt vs E50K). These files follow a consistent
 naming format, such as YJ_Fig3B_table.xlsx. Second, the Imaris .ims files
 are included to ensure full reproducibility. These files preserve all
 processing parameters—including thresholds, segmentation settings, and
 masking details—and can be opened in ImageJ or Fiji using the Bio-Formats
 plugin. These files follow a naming format like YJ_Fig3A_3DImage.ims. In
 some experiments, we distinguished particles located inside axons from
 those outside. This was achieved by creating additional masks for axons
 and for the nerve boundary within Imaris. As with the time-series
 analysis, summary Excel files were imported into GraphPad Prism for
 statistics and figure creation. The corresponding .pzfx or .pzf files are
 included in the same structured format (e.g., YJ_Fig3B_graph.pzfx). ######
 Electron Microscopy (EM) Analysis – Figure 5 Our EM analysis was primarily
 qualitative and focused on generating 3D models from correlated light EM
 datasets. These models were manually traced and represent individual
 astrocytes and axons. The included .txt files are converted versions of
 the original .mod models generated with IMOD software. These were created
 using the model2point command and can be converted back to .mod format
 using point2model. Once converted, they can be visualized alongside the
 corresponding .mrc files in IMOD. ###### Mito sphericity and fluorescence
 intensity analysis -Figure 6 Mitochondria sphericity in Figure 6C was
 determined using Imaris. Figure 6D–F show changes in fluorescence
 intensity within axon protrusions and the axon of origin, as well as the
 minimum distance between protrusions and the parent axon, supporting the
 interpretation of active cargo loading and mitophagy. Fluorescence
 intensity and localization for each label were measured at defined ROIs
 over time using IPLab software with a custom script, which is included in
 the dataset. The resulting data were compiled into summary spreadsheets
 (e.g., YJ_Fig6E_table.xlsx) and imported into GraphPad Prism for analysis.
 Corresponding Prism project files (.pzfx or .pzf) are also included,
 following the same structured naming format (e.g., YJ_Fig6E_graph.pzfx).
 ## Software * .tif image files can be opened in most standard image
 viewers, including ImageJ and Fiji. * .txt files are readable in Excel,
 Word, or any text editor. * .pzfx and .pzf files require GraphPad Prism. *
 .ims files can be opened in Imaris or in Fiji (ImageJ) with the
 Bio-Formats plugin.