Cytomatrix proteins CAST and ELKS regulate retinal photoreceptor development and maintenance

Abstract

At the presynaptic active zone (AZ), the related cytomatrix proteins CAST and ELKS organize the presynaptic release machinery. While CAST is known to regulate AZ size and neurotransmitter release, the role of ELKS and the integral system of CAST/ELKS together is poorly understood. Here, we show that CAST and ELKS have both redundant and unique roles in coordinating synaptic development, function, and maintenance of retinal photoreceptor ribbon synapses. A CAST/ELKS double knockout (dKO) mouse showed high levels of ectopic synapses and reduced responses to visual stimulation. Ectopic formation was not observed in ELKS conditional KO but progressively increased with age in CAST KO mice with higher rates in the dKO. Presynaptic calcium influx was strongly reduced in rod photoreceptors of CAST KO and dKO mice. Three-dimensional scanning EM reconstructions showed structural abnormalities in rod triads of CAST KO and dKO. Remarkably, AAV-mediated acute ELKS deletion after synapse maturation induced neurodegeneration and loss of ribbon synapses. These results suggest that CAST and ELKS work in concert to promote retinal synapse formation, transmission, and maintenance.

Presynaptic development is controlled by the core active zone proteins CAST/ELKS

“…To analyse the roles of CAST/ELKS we used the Cast KO/Elksfl/fl(CKO) transgenic mouse which constitutively ablates CAST expression and in the presence of Cre recombinase (Cre) ablates all Elks isoforms (α, β and γ) (Dong et al 2018;Hagiwara et al 2018). To do so, we injected our HdAd vector, which independently co-expresses Cre and EGFP, at P1.…”

“…However, initial loss of function studies on elks at C. elegans en passant synapses found no changes in synaptic morphology (Deken et al 2005). Genetic studies of mammalian retinal ribbon synapses in vivo which deleted both CAST/ELKS identified roles for CAST/ELKS regulation of synapse formation and AZ morphology (Hagiwara et al 2018). However, deletion of both CAST/ELKS at the mature calyx of Held in vivo, a large Ca V 2.1 exclusive glutamatergic presynapse with many conventional AZs in parallel (Baydyuk et al 2016), led to no changes in presynaptic morphology or AZ ultrastructure (Dong et al 2018).…”

“…During early development synaptic transmission at the calyx of Held relies on three subtypes of Ca V 2 channels which makes it ideal for studying the effects of CAST/ELKS on specific Ca 2+ subtypes. We used Cast/Erc2 knockout (KO) (Cast −/− ) and Elk1s/Erc1 conditional knockout (CKO) transgenic mice (Dong et al 2018;Hagiwara et al 2018) in conjunction with our ability to conditionally ablate ELKS expression in the calyx of Held (Dong et al 2018) and demonstrated that ELKS was significantly reduced by P7 and completely ablated by P9. Using confocal and electron microscopy we found that combined loss of CAST and ELKS resulted in significant reductions in calyx size.…”

Impaired experience-dependent maternal care in presynaptic active zone protein CAST-deficient dams

“…Here, we first expected that neglect in CAST KO dams might be caused by impaired OXT release. Indeed, previous studies indicated an essential role for CAST in recruiting Ca 2+ channels that can trigger neurotransmitter release at the active zone 21,22,25,[41][42][43] , as well as the OXT release depends on Ca 2+ influx into the axon terminal 44,45 . Our findings showed that magnocellular neuron terminals in the posterior pituitary were intensely labelled for CAST and OXT (Fig.…”

“…Previous work has shown that mutant forms of CAST/ELKS homologs in C. elegans (ELKS) and Drosophila (bruchpilot) exhibit distinct phenotypes, indicating they are required for synapse formation in C. elegans and for the promotion of active zone assembly in Drosophila 19,20 . Furthermore, analysis of CAST and/or ELKS deficient mice indicated that these proteins affect calcium channel-associated release machinery in retinal photoreceptor neurons, hippocampal neurons, calyx of the Held/MNTB synapses, and pancreatic β cells [21][22][23][24][25][26] . However, despite the importance of presynaptic events, only a few studies have reported the contribution of presynaptic neurotransmitter release to maternal behaviour 2,27 .…”