A and B). As disease progressed, there was an
increase in transcript abundance that was most
pronounced for mitochondrial reads (Fig. 1B).
Emerging evidence suggests that imbalances in
the relative proportions of mitochondrial proteins
encoded by nuclear and mitochondrial genomes
negatively affect mitochondrial function (5). In D2
groups 2 to 4, differential expression of genes en-
coding mitochondrial proteins, as well as significant
sciencemag.org 17 FEBRUAR Y 2017 • VOL 355 ISSUE 6326 757
Fig. 1. Mitochondrial dysfunction is associated with progressive RGC
damage in glaucoma. (A) RGC samples were divided into molecularly distinct
groups by hierarchical clustering (HC) of RNA-seq–determined gene expression.
Control (D2-Gpnmb+) and young samples were molecularly similar (Spearman’s
rho; n = 63 samples; each sample was derived from a different mouse). Samples
from D2-Gpnmb+ RGCs, triangles; samples from D2 RGCs, circles. (Inset) Number
of DE genes (q < 0.05) between D2-Gpnmb+ and each group. D2 group 1 and
D2-Gpnmb+ represent no glaucoma at a molecular or transcriptomic level.
Transcriptomic sequencing was performed twice, and batch correction was
performed using RUVSeq (remove unwanted variation from RNA-seq data, see
Materials and Methods). (B) Mitochondrial:nuclear read total ratio increased
with greater HC distance from controls. Colors match key in (A). Error bars rep-
resent SEM. (C) Significantly enriched pathways between clusters and control
based on Ingenuity Pathway Analysis (there are no differentially expressed path-
ways in D2 group 1) (see also table S3). (D) Transcript expression primarily
increased for nuclear encoded mitochondrial proteins with increasing HC dis-
tance from controls. Dots represent individual genes; gray, not differentially
expressed; red, differentially expressed at q < 0.05. Genes taken from mouse
MitoCarta2.0 (28). (E) Oxidative phosphorylation genes were differentially ex-
pressed across all groups. Red, highest expression; blue, lowest expression; I to
V, mitochondrial complexes I to V (tabulated in table S1); G, D2-Gpnmb+; 1 to 4,
D2 groups 1 to 4. (F) RNA-seq identified increased mitochondrial fission gene
transcripts early in glaucoma and (G) suggests an early mitochondrial unfolded
protein response compared with that of controls. Data shown are for D2 group 4.
(H and I) Mitochondria of D2 mice have decreased cristae volume and decreased
cristae:total volume ratio (not shown) in RGC somal and dendritic mitochondria
(there was no significant difference in total mitochondrial size or volume) (n > 400
mitochondria from six retinas per group; pooled data are shown). Scale bar,
350 nm. All data are for mice at 9 months of age unless otherwise stated.
**P < 0.01; ***P < 0.001 (Student’s t test). See also tables S1 and S2.
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