Figure 3. Splenic age-triggered iron deposits are rich in aggregation-prone proteins derived from damaged RPMs.

(A) Ultrastructural analyses of the spleen red pulp sections by transmission electron microscopy. Arrows indicate dark-colored dense deposits. (B) Volcano plot illustrating 3290 protein groups (in gray) that are significantly more abundant in magnetically-sorted, cell-free aggregates derived from aged versus young spleen. Based on the functional enrichment analyses, the red color denotes proteins linked with ‘pathways of neurodegeneration’, green - those associated with an ‘apoptotic process’ and blue - those related to the ‘immunoglobulin-like domain’ category. (C) Enriched functional categories among the top 387 protein hits that are more abundant in magnetically-sorted, cell-free aggregates derived from aged versus young spleen. (D) Venn diagram illustrating the number of common proteins identified in RPMs, erythrocytes (human), and aging-triggered splenic aggregates. (E) Enriched functional categories among differentially regulated genes in FACS-sorted RPMs derived from young versus aged mice (identified by RNA-seq). (F) Proteasomal activity was measured in RPMs derived from young, aged, and aged IR mice using a fluorescent proteasome activity probe with flow cytometry. (G) The total intracellular iron content in magnetically sorted RPMs derived from spleens of dextran- and iron dextran-injected mice (8 hr post-injection) was assessed using the Iron Assay Kit. (H) Venn diagram illustrating the number of common protein groups identified in aging-triggered and iron dextran-triggered splenic aggregates (log2 fold change >1.5 versus respective young and dextran-injected controls, respectively; n=2). The dextran-triggered aggregates were isolated 24 hr post-injection. Each dot represents one mouse; in (B) three biological replicates per group were analyzed. Data are represented as mean ± SEM. Welch’s unpaired t-test determined statistical significance between the two groups; statistical significance among the three groups was determined by One-Way ANOVA test with Tukey’s Multiple Comparison test. *p<0.05, **p<0.01.

Figure 3—figure supplement 1. Supplementary data related to RNA-seq analysis of RPMs derived from young, aged, and aged IR mice.

(A) Volcano plot of differentially regulated genes in aged versus young FACS-sorted RPMs, with top hits indicated.(B) Enriched functional categories among differentially regulated genes in FACS-sorted RPMs derived from young versus aged IR mice. Four biological replicates of FACS-sorted RPMs per group were analyzed by RNA-seq.

Figure 3—figure supplement 2. Components of lysosomes and ferritins are highly overrepresented in protein aggregates isolated from aged versus aged IR mice.

(A) Volcano plot illustrates protein groups (in gray) that are significantly more abundant in magnetically-sorted, cell-free aggregates derived from aged versus aged IR spleen. Based on the functional enrichment analyses, the red color denotes proteins linked with ‘lysosome’ and green – H and L ferritin. (B) Enriched functional categories among 50 protein hits that are more abundant in magnetically-sorted, cell-free aggregates derived from aged versus aged IR spleen. Three biological replicates per group were analyzed.