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Rachel Hutto, Celia M Bisbach, James Hurley, Susan E Brockerhoff; Overexpression of the mitochondrial Ca2+ uniporter in cones alters Ca2+ and mitochondrial homeostasis. Invest. Ophthalmol. Vis. Sci. 2018;59(9):988. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Cone photoreceptors rely on precise control of intracellular Ca2+ for function and survival. Mitochondria can buffer cytosolic Ca2+, and [Ca2+]mt can alter metabolism and trigger cell death. We hypothesized that increasing mitochondrial Ca2+ uptake in cones may influence cytosolic Ca2+ transients, mitochondrial function, and sensitivity to Ca2+-induced death. To test this, we generated transgenic zebrafish in which the mitochondrial Ca2+ uniporter (MCU) is overexpressed only in cones.
The Gateway-Tol2 system was used to generate MCU cDNA tagged with T2A-RFP or FLAG under control of a cone-specific promoter (gnat2). Constructs were injected into embryos to generate transgenic lines. MCU OE fish were compared to age-matched WT siblings, data reported as “AVG ± SEM” with two-tailed t-test result.For reporters of Ca2+ and autophagy we used Tg(gnat2:mito-GCaMP3), Tg(gnat2:GCaMP3), and Tg(gnat2:LC3-GFP) zebrafish. Larvae/eyes were embedded in 1% agarose or fixed in 4% PFA for cryosections then imaged via confocal microscopy.For scanning electron microscopy, samples were fixed in 4% glutaraldehyde, stained with 1% uranyl acetate and Walton’s lead aspartate, then imaged.
We found that cone-specific MCU overexpression increases basal [Ca2+]mt (mito-GCaMP3 fluorescence, 478% ± 12, p<0.05). Cytosolic Ca2+ transients also are altered in MCU OE models. Mitochondria normally are only in the ellipsoid region of WT cones, but surprisingly can be found closer to the synapse in MCU OE cones (14.1% ± 1.0 outside of ellipsoid region, p<0.05). MCU OE cone mitochondria display severely disrupted cristae, loss of electron density, and enhanced recruitment of LC3. Changes in cone mitochondrial morphology appear at 4 days of age, but cones are largely preserved until at least 3 months of age.
These findings demonstrate that cone mitochondria can alter cytosolic Ca2+ when MCU levels are elevated, which increases resting [Ca2+]mt. The delayed onset of cone loss despite the early emergence of mitochondrial morphological changes suggests cone mitochondria have a large buffering capacity for Ca2+ that is not easily overloaded to trigger cell death. The translocation of abnormal mitochondria away from the ellipsoid region raises the interesting possibility that this may be a precursor to mitochondrial turnover in cones.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
EM micrographs of WT cones (A) and MCU OE cones (B) at 5 days of age.
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