Abstract
Purpose :
Our goal was to determine if mitochondrial biogenesis in zebrafish cone photoreceptors is circadian and/or regulated by light. Photoreceptors use more energy in darkness compared to light, and at night the number of mitochondria in zebrafish cones increases. We hypothesized that mitochondrial biogenesis would correlate with increased energy needs and increased mitochondrial number.
Methods :
Wild-type and transgenic zebrafish retinas from light/dark and dark-only cohorts were collected over a 24-hour cycle. Transgenic gnat2:mito-cpYFP retinas expressing YFP in cone mitochondria were fixed and sectioned at 20 µm for immunohistochemistry. Sections were incubated with primary antibodies against GFP and MTCO-1 and imaged with confocal microscopy. Length measurements were taken from the center of single cone mitochondrial clusters. Transgenic retinas were also analyzed by SDS-PAGE followed by Western blotting to assess cone mitochondrial protein content. Lastly, mRNA extraction followed by cDNA synthesis was conducted using intact wild-type retinas. 18 genes involved in mitochondrial biogenesis were analyzed via qPCR, in addition to ratios of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA). Experiments were conducted in triplicate with three biological replicates and the ΔΔCt method was used to determine relative expression.
Results :
Immunohistochemistry revealed that cone mitochondrial clusters elongate 30% following dark onset. Western blot analysis is ongoing. mRNA transcripts for two genes involved with mitochondrial biogenesis, PolG1 and TFAM, increased 6-fold and 3-fold respectively in the hours leading up to dark onset; transcripts for the mitochondrial fusion factor, Mfn2, increased 3-fold just prior to light onset. These effects were independent of light exposure. Just following light onset, mtDNA:nDNA ratios in the light/dark cohort trended lower than in the dark-only cohort.
Conclusions :
Cone mitochondrial clusters enlarge at night, and this is preceded by upregulation of mitochondrial biogenesis genes. These effects occurred independently of light exposure. Mitochondrial fusion prior to light onset may be followed by a decrease in mtDNA number. These results highlight a possible mechanism in photoreceptors to increase energy production in the dark.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.