Abstract
Modulation of Ca2+ within cells is tightly regulated through complex and dynamic interactions between the plasma membrane and internal compartments. In this study, we exploit in vivo imaging strategies based on genetically encoded Ca2+ indicators to define changes in perikaryal Ca2+ concentration of intact photoreceptors. We developed double-transgenic zebrafish larvae expressing GCaMP3 in all cones and tdTomato in long-wavelength cones to test the hypothesis that photoreceptor degeneration induced by mutations in the phosphodiesterase-6 (Pde6) gene is driven by excessive [Ca2+]i levels within the cell body. Arguing against Ca2+ overload in Pde6 mutant photoreceptors, simultaneous analysis of cone photoreceptor morphology and Ca2+ fluxes revealed that degeneration of pde6cw59 mutant cones, which lack the cone-specific cGMP phosphodiesterase, is not associated with sustained increases in perikaryal [Ca2+]i. Analysis of [Ca2+]i in dissociated Pde6βrd1mouse rods shows conservation of this finding across vertebrates. In vivo, transient and Pde6-independent Ca2+ elevations (‘flashes') were detected throughout the inner segment and the synapse. As the mutant cells proceeded to degenerate, these Ca2+ fluxes diminished. This study thus provides insight into Ca2+ dynamics in a common form of inherited blindness and uncovers a dramatic, light-independent modulation of [Ca2+]i that occurs in normal cones.
Highlights
The most studied animal model for photoreceptor degeneration due to elevated cyclic guanosine-mono-phosphate (cGMP) is the rd[1] mouse, identified more than 90 years ago.[1,2] rd1 mice have a mutation in the beta subunit of rod cGMP Pde[6], a key mediator of the light response.[3,4] Normally, light activates a molecular cascade that causes Pde[6] to hydrolyze cGMP, resulting in closure of cation channels within the photoreceptor outer segment (OS)
Events associated with non-apoptotic cell death pathways such as poly-ADP-ribose-polymerase and calpain activation occur in rods dying due to elevated cGMP,[8,9] the molecular cascade activated in degenerating Pde[6] mutant photoreceptors is unknown
Double-transgenic fish (Tg(Trb2:tdTomato; transducin promoter (TaCP):GCaMP3)) were used for live imaging experiments, where we directed our observation of Ca2 þ dynamics to the cone photoreceptor cell body and synaptic terminal
Summary
The most studied animal model for photoreceptor degeneration due to elevated cGMP is the rd[1] mouse, identified more than 90 years ago.[1,2] rd mice have a mutation in the beta subunit of rod cGMP Pde[6], a key mediator of the light response.[3,4] Normally, light activates a molecular cascade that causes Pde[6] to hydrolyze cGMP, resulting in closure of cation channels within the photoreceptor outer segment (OS). In the absence of Pde[6] beta in rd[1] mice, cGMP levels remain elevated and rod photoreceptors start to degenerate after their differentiation and before eye opening.[3] Cone photoreceptors lacking Pde6c have elevated cGMP in their OSs.[5] Elevated cGMP is proposed to result in sustained increases in OS Ca2 þ , triggering death. Pde6cw[59] cones degenerate starting immediately after their differentiation at 4 days post fertilization (d.p.f.), and are mostly lost by 7 d.p.f.12,13 This mutation is analogous to the well-characterized rod Pde6brd1mutation.[3,4] To confirm our results in the mammalian model, we extended our approach to record intracellular Ca2 þ ([Ca2 þ ]i) levels in rd[1] mouse rods. Cell Death and Disease overload the endogenous Ca2 þ buffering and clearance mechanisms within the cell body, and have implications for the development of neuroprotection strategies in retinitis pigmentosa (RP) models of retinal degeneration
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