Precision Medicine, CRISPR, and Genome Engineering Moving from Association to Biology and Therapeutics

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oxidative phosphorylation and consequent oxidative damage. Excess mitochondrial

Ca2+ also can overload the buffering capacity, stimulate opening of the mitochon-

drial permeability transition pore (MPTP) and release mitochondrial components

[ 34 ]. As described below, new advances in genome editing make it straightforward

to generate tissue-specific deletions of important genes such as the MCU.

Other recent findings have further highlighted both the importance of regulation

of cellular Ca2+ levels in photoreceptors and the many unanswered questions in this

field. For example, knockout mice each specifically lacking the Na+/K+-Ca2+

exchanger in either rod or cone outer segments were able to slowly recover from

flashes of light [ 35 , 36 ]. This surprising result suggests an alternate and currently

unknown route of Ca2+ efflux from the outer segment. At the synapse, depolariza-

tion maintains high Ca2+ levels in the ER in rods due to diffusion from the soma

[ 22 ]. This mechanism allows for CICR-triggered synaptic release at non-ribbon

sites, which is thought to be significant in rods. Moreover, our recent work demon-

strates a role for Ca2+ uptake by mitochondria as critical for maintaining distinct

Ca2+ pools in the cell body and outer segment. Pharmacological inhibition of the

MCU leads to a redistribution of Ca2+ throughout the cell. This same study also

demonstrates large increases in mitochondrial Ca2+ due to overload in outer seg-

ment Ca2+ suggesting a critical role for mitochondrial Ca2+ buffering in prolonging

the health and survival of photoreceptors during degeneration due to disease-caus-

ing mutations [ 24 ].

Fig. 5.1 The many roles of Ca2+ in mitochondria. Ca2+ stimulates aerobic metabolism (yellow
stars) by activating proteins involved in substrate uptake, production of reducing equivalents and
electron transport. Ca2+ can enter mitochondria through the MCU at areas of high [Ca2+] such as
ER junctions (SER) and is extruded by Ca2+/Na+ (NCX) or Ca2+/H+ exchangers (HCX). Ca2+ binds
and activates AGC1, a major component of the malate aspartate shuttle [ 26 , 27 ]. This enhances
complete oxidation of glucose and increases O 2 consumption [ 3 ]. Reactive oxygen production and
MPTP opening occur when mitochondrial [Ca2+] is too high (not shown)

5 Genome Editing to Study Ca2+ Homeostasis in Zebrafish Cone Photoreceptors