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Figure 2 Mitochondrial Dysfunction in Young and Old iNs Show full caption (A) Representative fluorescence images of mitochondria (hSyn::MitoEGFP) in SMI-312-positive axons in young (1 year) and old (71 years) donor-derived iNs (see also Figures S2 A and S2B). Scale bar, 20 μm. (B) Quantification of axonal mitochondrial morphologies for densities, mean lengths, and coverage in young and old iNs (n = 7 individuals, 6 replicates each). Boxplots show 25th to 75th percentiles, bars indicate medians, and whiskers show ranges. Significance values were calculated by unpaired t tests (see also Figures S2 A and S2B). (C and D) hSyn::BFP-tagged iNs were gated (D) and analyzed for MMPs using the JC-1 dye (C). (E) Representative JC-1 FACS density plots showing energized mitochondria in young iNs (1 year) and a loss of MMP in old iNs (71 years). (F) Quantification of the MMP in young and old hSyn::BFP-positive iNs (n = 7 individuals, 3 replicates). Bar graph shows means ± SD, and triangles indicate individual measurements. Significance values were calculated by Mann-Whitney test. (G) ATP levels in young and old purified iNs (6 individuals, 3 replicates). Bar graph shows means ± SD, and triangles indicate individual measurements. Significance values were calculated by Mann-Whitney test. (H) Quantification of oxidized proteins as assessed by quantitative western blot (OxyBlot; 6 individuals, 2 replicates, representative blot in Figure S2 C). Significance values in this figure: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.001.

We next asked whether the observed transcriptional changes corresponded to functional impairment of mitochondria in old iNs. Axonal localization of mitochondria is vital to their function, and reduced axonal densities and the appearance of fragmented mitochondria are associated with impaired mitochondria in neurons (). Using a lentiviral MitoEGFP reporter to label mitochondria, we detected significantly reduced mitochondrial densities in SMI-312-positive axons ( Figures 2 A, 2 B, S2 A, and S2B). Furthermore, we observed higher levels of mitochondrial fragmentation, as indicated by reduced mitochondrial length, as well as reduced mitochondrial coverage in the axons of old iNs ( Figures 2 B, S2 A, and S2B). These morphological features are indicative of general mitochondrial dysfunction in old iNs, so we decided to next test the mitochondrial MMP, which is known to determine many mitochondrial functions (). To measure MMPs specifically in iN cells, we used fluorescence-activated cell sorting (FACS) analysis of human synapsin-1 promoter-driven blue fluorescent protein (hSyn::BFP)-labeled iNs with the green fluorescent cationic dye JC-1, which accumulates in red fluorescent aggregates in mitochondria with high MMPs ( Figure 2 C) (). We first gated for the BFP-positive neuronal population ( Figure 2 D) and then measured green and red JC-1 fluorescence in this population ( Figure 2 E). Young iNs showed significantly higher MMPs than old iNs, which showed an average 43% drop ( Figure 2 F). Next, to test whether this age-dependent loss of MMP was consequential for the total neuronal energy levels in young and old iNs, we purified iNs by polysialylated-neural cell adhesion molecule (PSA-NCAM) FACS and then measured ATP level in FACS-purified iNs. In line with the gene expression and morphological and MMP defects, we detected significantly lower total ATP levels in old neurons ( Figure 2 G). Oxyblot analysis revealed significantly increased levels of oxidized protein damage in old iN cultures, further indicating mitochondrial defects ( Figures 2 H and S2 C). Furthermore, to test to what extent neuronal features downstream of mitochondria became impaired in old iNs, we assessed neurite beading and found that almost 50% of all neurites in old iN cultures showed axonal varicosities at 6 weeks in culture ( Figures S2 D and S2E). Consistently, and as early as 3 weeks in culture, synaptic transmission gene expression (GO: 0007270) was significantly decreased in old iNs ( Figure S2 F). While global antioxidant gene expression profiles of fibroblasts and iNs showed cell-type-specific differences, the major antioxidant genes NURR1 (NR4A2) and NRF2 (NFE2L2) did not show obvious expression differences between the cell types ( Figures S2 G–S2J). Interestingly, we found FOXO transcription factor transcripts to be slightly upregulated and FOXO3-repressed genes to be downregulated in old iNs ( Figures S2 K and S2L), whereas HIF1A and its target genes were not changed ( Figures S2 M and S2N). Together, these data show that directly converted iNs from young and old donor fibroblasts reflect broad functional defects associated with mitochondrial aging and provide a model to study bioenergetic aspects of human neuronal aging in vitro.