Overexpression of the mitochondrial Ca²⁺ uniporter in cones alters Ca²⁺ and mitochondrial homeostasis

Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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.