Journal: iScience

Article Title: Light-evoked activity and BDNF regulate mitochondrial dynamics and mitochondrial localized translation in CNS axons

doi: 10.1016/j.isci.2025.113563

Figure Lengend Snippet: Axonal mitochondria increase in size, number, and area through eye development CFP+ mitochondria were imaged by confocal microscopy and analyzed in ImageJ. (A) Example maximum intensity projected image of a whole mounted P45 retina showing mitochondrial labeling within RGC cell bodies (circular objects) and axonal segments (linear segments) diving toward the optic nerve head (200 μm scale bar). (B) Example images of retinal, and (C) optic nerve axon mitochondria from postnatal day 9 (P9), P13, P15, and P45 mice (20 μm scale bar). (D–F) In both retinal and optic nerve RGC axons, the average mitochondrial size, number, and area (measured as percent of cross sections, representing fractional volume) increased from P9 to adulthood (Error bars indicate SEM; N = 4 replicate mice for P9 ages, N = 3 for P12 and P15, and N = 4 for P45 mice, 9 images analyzed per animal; one-way ANOVA with Holm-Sidak correction for multiple comparisons, ∗ p ≤ 0.05). See for image analysis procedures.

Article Snippet: The homogenate was then spun at 1000g and the supernatant was removed for subsequent magnet based mitochondrial isolation according to Milteny Biotec’s Mitochondrial Isolation Kit.

Techniques: Confocal Microscopy, Labeling

Journal: iScience

Article Title: Light-evoked activity and BDNF regulate mitochondrial dynamics and mitochondrial localized translation in CNS axons

doi: 10.1016/j.isci.2025.113563

Figure Lengend Snippet: Mitochondrial size and number increase at eye opening in RGC axons (A) Mitochondria were imaged and quantified in retinal nerve fiber layer RGC axons by transmission electron microscopy before (P12) and after (P15) eye opening. Increased magnification (insets, 500 nm scale bar) shows mitochondrial membrane enfolding and cristae, and representative mitochondrial size differences (full panel,1 μm scale bar). (B) Mitochondrial size, (C) number, and (D) area increased significantly between P12 and P15. (Error bars indicate SEM; n = 20 replicate sections per age, ≥150 traced mitochondria per age; significance identified by Student’s t test, ∗ p < 0.05).

Article Snippet: The homogenate was then spun at 1000g and the supernatant was removed for subsequent magnet based mitochondrial isolation according to Milteny Biotec’s Mitochondrial Isolation Kit.

Techniques: Transmission Assay, Electron Microscopy, Membrane

Journal: iScience

Article Title: Light-evoked activity and BDNF regulate mitochondrial dynamics and mitochondrial localized translation in CNS axons

doi: 10.1016/j.isci.2025.113563

Figure Lengend Snippet: Eye opening is sufficient and necessary for developmental changes in mitochondrial size and localization (A) Surgical model for premature eye opening and sutured eyelid closure. (B) Average mitochondrial size, (C) number, and (D) area increase with premature eye opening, and this developmental increase is inhibited by prolonged eye closure. (Points are averages from ≥18 images analyzed per condition; 9 images per replicate animal N = 3, except P15 sutured samples N = 2. Error bars indicate SEM. Student’s t test, ∗ p < 0.05.).

Article Snippet: The homogenate was then spun at 1000g and the supernatant was removed for subsequent magnet based mitochondrial isolation according to Milteny Biotec’s Mitochondrial Isolation Kit.

Techniques:

Journal: iScience

Article Title: Light-evoked activity and BDNF regulate mitochondrial dynamics and mitochondrial localized translation in CNS axons

doi: 10.1016/j.isci.2025.113563

Figure Lengend Snippet: Mitochondrial developmental changes are dependent on retinal electrical activity and are partially rescued by BDNF in optic nerve axons Vehicle Control, TTX- or TTX plus BDNF-treated mice analyzed at P15, along with vehicle Control injected mice analyzed at P12. (A) Measured changes in average mitochondrial size within retinal axons by confocal microscopy. (B) Representative images of mitochondria within optic nerve axons of Control, TTX, and TTX plus BDNF-treated eyes, fluorescence confocal microscopy images in upper panels (presented as a maximum intensity projection, scale bar 5 μm) and transmission electron microscopy in lower panels (TEM, presented as crop of a 4000x full field image, scale bar 500 nm). Axons were identified by myelination and neurofilament in TEM images. Corresponding mitochondrial (C) size, (D) number, and (E) area measurements from fluorescent microscopy and TEM (Error bars indicate SEM; N = 3 replicate eyes per condition, 9 images analyzed per eye in fluorescent imaging measurements; n > 60 sections from N = 2 replicated eyes per condition, >200 traced mitochondria in TEM measurements; significance detected using one-way ANOVA with Holm-Sidak corrections for multiple comparisons, ∗ p < 0.05). See for TEM tracings and quantification procedures.

Article Snippet: The homogenate was then spun at 1000g and the supernatant was removed for subsequent magnet based mitochondrial isolation according to Milteny Biotec’s Mitochondrial Isolation Kit.

Techniques: Activity Assay, Control, Injection, Confocal Microscopy, Fluorescence, Transmission Assay, Electron Microscopy, Microscopy, Imaging

Journal: iScience

Article Title: Light-evoked activity and BDNF regulate mitochondrial dynamics and mitochondrial localized translation in CNS axons

doi: 10.1016/j.isci.2025.113563

Figure Lengend Snippet: RGC nuclear-encoded mitochondrial gene expression in response to activity inhibition with TTX and/or BDNF is consistent with inhibition versus activation of mitochondrial dynamics and energetics (A) Model for filtering data acquired from RT-PCR gene array analysis of P15 acutely purified RGCs, after TTX and/or BDNF intravitreal injections at P11 and P13 ( N = 3 RGC preps per condition). Filters were placed to identify gene expression regulated in opposing directions by BDNF and TTX. The resulting genes were then passed through IPA pathway analysis software, which suggested 10 major upstream regulators, with PGC1-α and RICTOR at the top of the list. Downstream gene expression data modulated by these upstream regulators were transformed into Z score of activation. Up- or downregulated gene sets are denoted by color. (B) Genes identified in our array that are regulated by RICTOR represent mainly energetics genes. (C) Genes identified in our array that are regulated by PGC1-α represent mainly mitochondrial dynamics and biogenesis regulators. (D) Measuring the effect of BDNF on mitochondrial dependent oxygen consumption in purified RGCs shows an increase in the basal respiration rate and maximum respiration capacity (with FCCP addition) regardless of activity inhibition by TTX (introduced 35min after initial recording). TTX, Oligomycin, FCCP, and Rotenone/Antimycin A, were added sequentially at times points marked with vertical lines. Recorded values were acquired using the Seahorse XF96 instrument (Error bars indicate SEM; n = 6 replicates per condition, pooled from 3 separate RGC preps and assayed on one plate, ∗ indicate p ≤ 0.05, T-test). (E) Genes identified in our array that have been previously demonstrated as mitochondrial fission/fusion or mitochondrial size modifying are opposingly regulated by TTX and BNDF, with most genes upregulated by BDNF. (F) Model of the predicted mitochondrial events triggered by TTX or BDNF, based on gene pathway analysis and the identified mitochondrial changes in injected mice.

Article Snippet: The homogenate was then spun at 1000g and the supernatant was removed for subsequent magnet based mitochondrial isolation according to Milteny Biotec’s Mitochondrial Isolation Kit.

Techniques: Gene Expression, Activity Assay, Inhibition, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Purification, Software, Transformation Assay, Injection

Journal: iScience

Article Title: Light-evoked activity and BDNF regulate mitochondrial dynamics and mitochondrial localized translation in CNS axons

doi: 10.1016/j.isci.2025.113563

Figure Lengend Snippet: Activity regulates mitochondrial localized protein translation in axons (A) Representative images collected of P15 retinas, after in vivo intravitreal injections of TTX or BSS (control) at P11 and P13, and an injection of EU or OPP 2 h before tissue processing. (B) Cell viability of TTX treated RGCs, identified as Calcein-AM positive and Sytox negative, and normalized to total cell number by Hoescht. Graphed as a percent change relative to control cells (Error bars indicate SEM; 9 replicates; Student’s t test). (C) Confocal images of cultured RGCs, virally labeled with mitochondrial targeted dsRed and pulsed OPP for 15 min before fixation. OPP is detected in RGC cell bodies and in axons. (D) Axon tips demonstrate OPP puncta throughout, growth cones, and terminal axon domains. Axon tips labeled with phalloidin in far red. (E) Representative image of mitochondria, OPP, and ribosomal protein S3 colocalization within axons (scale bar 5 μm). (F) Mander’s coefficient values for colocalizations (error bars indicate SEM; n = 9 blind selected axons from N = 3 replicate RGC preps; one-way ANOVA with Holm-Sidak’s t-test). (G) TEM evidence of ribosomal protein S3 localization with mitochondria in axons, in vivo . The yellow circle represents an axonal area identified by ensheathing myelin structures, and the red arrows show all detectable S3 puncta identified by gold antibody labeling. (H) Correlation of OPP-mitochondrial colocalization relative to mitochondrial size (regression line and 95% confidence intervals are plotted, data points from n = 30 blind selected cells selected across N = 3 replicate RGC preps). (I) Representative images of mitochondrial and OPP-labeled axons in vehicle control, TTX, and TTX+BDNF treated RGCs (scale bar 5um). (J) Number of OPP puncta per 100 μm of axon termini, treated with BDNF, TTX, TTX+BDNF, cycloheximide, and vehicle controls (error bars indicate SEM; >10 blind selected axons from replicate RGC preps; one-way ANOVA with Tukey’s multiple comparisons test, ∗ p < 0.05). (K) Mean mitochondrial size in RGC axons incubated with TTX, BDNF, TTX+BDNF, or vehicle controls (error bars indicate SEM; >10 blind selected axons from replicate RGC preps; one-way ANOVA with Dunn’s multiple comparisons test, ∗ p < 0.05). See , for example, EU labeling and thresholding or masks used in mitochondrial area, OPP spot counts, colocalization, and TEM analyses.

Article Snippet: The homogenate was then spun at 1000g and the supernatant was removed for subsequent magnet based mitochondrial isolation according to Milteny Biotec’s Mitochondrial Isolation Kit.

Techniques: Activity Assay, In Vivo, Control, Injection, Cell Culture, Labeling, Antibody Labeling, Incubation

Journal: iScience

Article Title: Light-evoked activity and BDNF regulate mitochondrial dynamics and mitochondrial localized translation in CNS axons

doi: 10.1016/j.isci.2025.113563

Figure Lengend Snippet: Purified mitochondria retain their protein content and membrane integrity (A) Outline of mitochondrial isolation and subsequent assays. (B) TOM22-bound nanoparticles are visible bound to the outer mitochondrial membrane in both SEM (white spots) and TEM (black dots). Scale bars 50 nm and 100 nm. (C–E) Western blot analyses of purified mitochondria and supernatants. Magnetically isolated mitochondria retain (C) OXPHOS subunits, as well as (D) outer membrane (OM), inner membrane (IM), and inner membrane space (IMS) proteins. (E) B-Actin is detectable in cytoplasmic, 1000g fractions, but not mitochondrial isolate fractions. (F) FACS assayed mitochondria confirm the intact and functional state of mitochondria, which retain CFP and membrane potential-dependent mitotracker CMXROS. (G) Sorted CFP+ mitochondria also demonstrate polarization-dependent fluorescence with JC-1, and (H) lose membrane potential with FCCP depolarization to a greater degree than (I) vehicle-treated controls. See for uncut versions of blots and the cytoplasmic GAPDH blot.

Article Snippet: The homogenate was then spun at 1000g and the supernatant was removed for subsequent magnet based mitochondrial isolation according to Milteny Biotec’s Mitochondrial Isolation Kit.

Techniques: Purification, Membrane, Isolation, Western Blot, Functional Assay, Fluorescence

Journal: iScience

Article Title: Light-evoked activity and BDNF regulate mitochondrial dynamics and mitochondrial localized translation in CNS axons

doi: 10.1016/j.isci.2025.113563

Figure Lengend Snippet: Purified mitochondria bind nuclear-encoded mRNAs through outer membrane-associated proteins (A) Outline of mitochondrial isolation protocol and subsequent assays. For downstream blots and qPCR experiments, just prior to assigning group (i.e., control and proteinase K treated mitochondria), purified mitochondria were resuspended and split into equal volumes for equal starting material in subsequent work flow. (B) Relative to control mitochondrial pellets, proteinase K treated fractions retain inner membrane Comp. III and Comp. V proteins, but have reduced outer membrane (OM) TOM20 proteins and associated ribosomes (Ribo., Ribosomal Protein S3). (C) Proteinase K-treated mitochondria release bound RNA into supernatant fractions, as detected by qPCR of pelleted mitochondrial fractions and corresponding supernatant fractions. (D) Total retinal RNA extracts spiked into purified mitochondrial fractions lead to increased mitochondria-RNA binding and RNA in supernatant fractions. (E) Mitochondrial RNA binding decreases despite RNA spike-in, when proteinase K is also applied. Illustrated outcomes presented above all datasets. Data is normalized to RNR2 expression and graphed as a log2 fold change relative to stated fractional controls ( N = 3 replicate retina mitochondrial purifications, dotted vertical line is equivalent to a 2-fold change, statistical significance for individual gene fold changes were identified by Student’s t test against control fraction/baseline values, p∗<0.05). See for whole blots.

Article Snippet: The homogenate was then spun at 1000g and the supernatant was removed for subsequent magnet based mitochondrial isolation according to Milteny Biotec’s Mitochondrial Isolation Kit.

Techniques: Purification, Membrane, Isolation, Control, RNA Binding Assay, Expressing

Journal: iScience

Article Title: Light-evoked activity and BDNF regulate mitochondrial dynamics and mitochondrial localized translation in CNS axons

doi: 10.1016/j.isci.2025.113563

Figure Lengend Snippet: Proteomics mass spectrometry analysis reveals nuclear-encoded RNA binding proteins associated with purified mitochondria (A) Filtering used to identify mitochondria-specific proteins. (B) Venn diagram of total protein hits sorted by annotation in the MitoCarta database. (C and D) Candidate mitochondria-associated RNA binding proteins with cited evidence (PubMed ID shown) for their functional RNA binding role and mitochondrial interaction (displayed data is pooled from N = 3 replicate retina mitochondrial purifications and N = 3 replicate optic nerve mitochondrial purifications; all displayed proteins were considered significant/present as they were detected in one or more replicate preps and with one or more peptides mapped with >95% confidence.

Article Snippet: The homogenate was then spun at 1000g and the supernatant was removed for subsequent magnet based mitochondrial isolation according to Milteny Biotec’s Mitochondrial Isolation Kit.

Techniques: Mass Spectrometry, RNA Binding Assay, Purification, Functional Assay

Journal: iScience

Article Title: Light-evoked activity and BDNF regulate mitochondrial dynamics and mitochondrial localized translation in CNS axons

doi: 10.1016/j.isci.2025.113563

Figure Lengend Snippet: RNA-seq of purified axonal mitochondria reveals co-purifying nuclear-encoded RNAs with roles in axonal growth, maintenance, and mitochondrial regulation that are activity modulated (A) Schematic workflow of sample preparation. Intravitreal injection of AAV2 virus expressing Cre into the Mito-Tag mice drives the expression of a non-functional Cre-inducible 3xHA tagged mitochondrial outer membrane protein 25 (OMP25). Using the anatomical separation of the intravitreal retinal space and RGC axons passing through the optic nerve, mitochondria from axons are purified by pulling down HA-tagged mitochondria from dissected, homogenized, and HA magnetic antibody-incubated optic nerve lysates. (B) Heatmap shows the distribution of mitochondrial-encoded genes in isolated mitochondria and cytoplasm or nuclear fractions, demonstrating mitochondrial enrichment in the purifications used for analysis. The heatmap includes normalized expression values, with higher values indicating greater enrichment. (C) Dot plot of enriched cellular components for the top 200 nuclear encoded genes in the BSS-HA sample, indicating the -log10(FDR) significance and number of genes involved. Each dot represents a specific cellular component, with the size of the dot corresponding to the number of genes and the color intensity indicating the significance level. (D) Dot plot of enriched biological processes for the top 200 nuclear encoded genes in the BSS sample, showing -log10(FDR) significance and the number of genes involved. Each dot represents a specific biological process, with the size of the dot corresponding to the number of genes and the color intensity indicating the significance level. (E) Cumulative proportion of mitochondrial-related genes found on axonal mitochondria plotted against the percentile rank of overall gene expression. The plot shows the distribution of expression ranks, with higher percentiles indicating higher expression levels. (F) Volcano plot comparing TTX vs. Controls, highlighting mitochondrial-related genes. The plot shows log2 fold change on the x axis and -log10 p -value on the y axis, with significant genes ( p < 0.05 and logFC >1 or < −1) highlighted. (G) Volcano plot comparing BDNF+TTX vs. Controls, with mitochondrial-related genes labeled. The plot shows log2 fold change on the x axis and -log10 p -value on the y axis, with significant genes ( p < 0.05 and logFC >1 or < −1) highlighted. (H) Three bar graphs illustrate the pathways involved in different conditions and the corresponding number of genes implicated. Blue bars represent the total number of genes involved in each pathway, while orange bars indicate the number of mitochondrial genes. The percentage of mitochondrial genes is displayed at the end of each bar, with pathways ordered by the increasing proportion of mitochondrial genes.

Article Snippet: The homogenate was then spun at 1000g and the supernatant was removed for subsequent magnet based mitochondrial isolation according to Milteny Biotec’s Mitochondrial Isolation Kit.

Techniques: RNA Sequencing, Purification, Activity Assay, Sample Prep, Injection, Virus, Expressing, Functional Assay, Membrane, Incubation, Isolation, Gene Expression, Labeling

Journal: iScience

Article Title: Light-evoked activity and BDNF regulate mitochondrial dynamics and mitochondrial localized translation in CNS axons

doi: 10.1016/j.isci.2025.113563

Figure Lengend Snippet: A model for activity- and BDNF-regulated mitochondrial size, number, and associated protein translation (A) Neurons contain mitochondria, RNA, RNA binding and transport proteins, ribosomes, and newly synthesized proteins, throughout distal axon and dendrite compartments. (B) Electrical activity in RGCs, for example, driven by the light stimulation of retinal circuitry after eye opening, activates a signaling pathway that culminates in the activation of transcription factors such as CREB, and the expression of BDNF. BDNF signaling stimulates nuclear-encoded mitochondrial gene expression, coordinated by the activation of transcriptional regulators RICTOR and PGC1-α. (C) Neuronal activity and downstream BDNF signaling stimulate increases in mitochondrial size and number, reversed by activity inhibition. Changes in mitochondrial size triggered by activity modulation and BDNF also correlate with modifications in nuclear-encoded transcripts localization and mitochondrial localized translation events.

Article Snippet: The homogenate was then spun at 1000g and the supernatant was removed for subsequent magnet based mitochondrial isolation according to Milteny Biotec’s Mitochondrial Isolation Kit.

Techniques: Activity Assay, RNA Binding Assay, Synthesized, Activation Assay, Expressing, Gene Expression, Inhibition