Journal: The EMBO Journal

Article Title: Longitudinal autophagy profiling of the mammalian brain reveals sustained mitophagy throughout healthy aging

doi: 10.1038/s44318-024-00241-y

Figure Lengend Snippet: ( A ) Schematic of a parasagittal brain section highlighting the cerebellum and Purkinje cells analyzed. ( B ) Mitophagy profiling in Purkinje neurons. Confocal photomicrographs from mito -QC brain sections showing representative instances of mitophagy throughout aging in cerebellar Purkinje neurons. Arrowheads indicate events of mitophagy. Scale bar = 20 µm. GCL granule cell layer, PCL Purkinje cell layer, ML molecular layer. ( C ) Macroautophagy profiling in Purkinje neurons. Confocal photomicrographs from auto -QC brain sections showing representative instances of nonselective macroautophagy throughout aging in cerebellar Purkinje neurons. Arrowheads indicate events of autophagy. Scale bar = 20 µm. ( D ) Quantitative analysis of Purkinje cells in mito -QC mice reveals a robust age-dependent increase in the number of acidified mitolysosomes in vivo, in addition to increases in mean mitolysosome size throughout time. One-way ANOVA with Bonferroni post hoc. **** P < 0.0001. n = 47. ( E ) Quantitative analysis of Purkinje cells in auto -QC mice demonstrates the numbers of acidified autolysosomes remain constant throughout aging and do not change in size. One-way ANOVA with Bonferroni post hoc. ns = not significant; P = > 0.9999. n = 27. ( F ) Identification of differentially acidified autolysosomes formed throughout aging within Purkinje cells in vivo. Immunohistochemical analysis of endolysosomal network using LAMP1 antibody identifies distinct lysosomal subpopulations. Shown is a 3D isosurface render generated using IMARIS, alongside confocal photomicrograph insets. Arrowheads indicate LAMP1-mCherry-positive (GFP-negative) colocalization, arrows indicate LAMP1-mCherry-GFP colocalization. Lysosomes are predominantly enriched at the pre-dendritic zones. Scale bar = 5 µm. ( G ) Quantitative analysis reveals an age-dependent increase in the number and mean size of differentially acidified lysosomes (LAMP1-mCherry-GFP-positive structures) over time. Although the overall number of LAMP1-positive endolysosomal structures remains constant throughout mammalian life, this compartment expands in size as a function of age. One-way ANOVA with Bonferroni post hoc. ns = not significant; P > 0.9999, *** P = 0.0002 and **** P = 0.0001. n = 27. ( H ) In vivo transmission electron microscopy (TEM) imaging of young and geriatric mito -QC mice. Scale bar = 1 µm. Quantitation of TEM images reveal an age-dependent increase in lysosomal (L) area, consistent with LAMP1 data in ( G ), with further TEM analysis demonstrating selective alterations in lysosomal area occurs at the somatodendritic zones of Purkinje neurons, while no change occurs at the axonal aspect. Mitochondria (Mit), Lipid droplet (LD). Student’s unpaired two-tailed t test. ** P = 0.0019 and 0.0017; ns = not significant; P = 0.1225. n = 5. Box plots extend from the 25th to the 75th percentiles, with a median line positioned inside the box. Whiskers denote the minimum and maximum values. .

Article Snippet: Shown is a 3D isosurface render generated using IMARIS, alongside confocal photomicrograph insets.

Techniques: In Vivo, Immunohistochemical staining, Generated, Transmission Assay, Electron Microscopy, Imaging, Quantitation Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: Longitudinal autophagy profiling of the mammalian brain reveals sustained mitophagy throughout healthy aging

doi: 10.1038/s44318-024-00241-y

Figure Lengend Snippet: ( A ) Simplified schematic of A9 and A10 DA circuits. Both A9 and A10 neurons reside in the ventral mesencephalon (VM) and have distinct targets within the brain. A9 DA neurons project to the dorsolateral striatum, forming the nigrostriatal pathway that modulates voluntary locomotion, whereas A10 DA neurons project to the ventromedial striatum and forebrain to control reward, motivation, and aversion. ( B , C ) Mitophagy in aging A9 DA neurons. 3D Isosurface renders of young and geriatric mito -QC A9 dopaminergic neurons (scale bar = 5 µm) and representative confocal images of mitophagy events throughout aging, scale bar = 20 µm. Quantitative analysis reveals increased mitophagy levels as a function of age. One-way ANOVA with Bonferroni post hoc. ** P = 0.0014. n = 43. ( D , E ) Macroautophagy in aging A9 DA neurons. Representative maximum projections of autophagy events throughout aging in A9 dopaminergic neurons (scale bar = 20 µm). Quantitative analysis of autophagy in A9 dopaminergic neurons shows no age-dependent alterations in autophagy levels. One-way ANOVA with Bonferroni post hoc. ns = not significant; P > 0.9999. n = 19. ( F , G ) Mitophagy in aging A10 DA neurons. Representative maximum projections of mitophagy events throughout aging in A10 dopaminergic neurons (scale bars = 20 µm). Quantitative analysis of mitophagy in A10 dopaminergic neurons reveals no change between young and geriatric mice. Some fluctuations in mitophagy levels were detected with a modest increase at 15 months. One-way ANOVA with Bonferroni post hoc. ns = not significant; P > 0.9999, * P = 0.024. n = 44. ( H , I ) Macroautophagy in aging A10 DA neurons. Isosurface renders of young and geriatric auto -QC A10 dopaminergic neurons (scale bar = 5 µm). Representative images of autophagy events throughout aging in A10 dopaminergic neurons (scale bar = 20 µm). Quantitative analysis of autophagy in A10 dopaminergic neurons reveals decreased levels of autophagy as a function of age. One-way ANOVA with Bonferroni post hoc. ** P = 0.0076. n = 26. Box plots extend from the 25th to the 75th percentiles, with a median line positioned inside the box. Whiskers denote the minimum and maximum values. .

Article Snippet: Shown is a 3D isosurface render generated using IMARIS, alongside confocal photomicrograph insets.

Techniques: Control