The human cognition-enhancing CORD7 mutation increases active zone number and synaptic release

Abstract

Humans carrying the CORD7 (cone-rod dystrophy 7) mutation possess increased verbal IQ and working memory. This autosomal dominant syndrome is caused by the single-amino acid R844H exchange (human numbering) located in the 310 helix of the C2A domain of RIMS1/RIM1 (Rab3-interacting molecule 1). RIM is an evolutionarily conserved multi-domain protein and essential component of presynaptic active zones, which is centrally involved in fast, Ca2+-triggered neurotransmitter release. How the CORD7 mutation affects synaptic function has remained unclear thus far. Here, we established Drosophila melanogaster as a disease model for clarifying the effects of the CORD7 mutation on RIM function and synaptic vesicle release. To this end, using protein expression and X-ray crystallography, we solved the molecular structure of the Drosophila C2A domain at 1.92 Å resolution and by comparison to its mammalian homolog ascertained that the location of the CORD7 mutation is structurally conserved in fly RIM. Further, CRISPR/Cas9-assisted genomic engineering was employed for the generation of rim alleles encoding the R915H CORD7 exchange or R915E,R916E substitutions (fly numbering) to effect local charge reversal at the 310 helix. Through electrophysiological characterization by two-electrode voltage clamp and focal recordings we determined that the CORD7 mutation exerts a semi-dominant rather than a dominant effect on synaptic transmission resulting in faster, more efficient synaptic release and increased size of the readily releasable pool but decreased sensitivity for the fast calcium chelator BAPTA. In addition, the rim CORD7 allele increased the number of presynaptic active zones but left their nanoscopic organization unperturbed as revealed by super-resolution microscopy of the presynaptic scaffold protein Bruchpilot/ELKS/CAST. We conclude that the CORD7 mutation leads to tighter release coupling, an increased readily releasable pool size and more release sites thereby promoting more efficient synaptic transmitter release. These results strongly suggest that similar mechanisms may underlie the CORD7 disease phenotype in patients and that enhanced synaptic transmission may contribute to their increased cognitive abilities.

Endogenous tagging of Unc-13 reveals nanocluster reorganization at active zones during presynaptic homeostatic potentiation

“…The animals are homozygous viable and appear healthy (e.g., emerging and effective flight). In the following, we focus on the detection of sfGFP using a combination of primary IgG antibodies and secondary F(ab’) 2 fragments to establish a protocol comparable to earlier applications of d STORM imaging at the Drosophila NMJ (Ehmann et al, 2014; Paul et al, 2015; Mrestani et al, 2021; Paul et al, 2022). We refer to the fusion protein and the genotype as Unc-13 GFSTF and unc-13 GFSTF , respectively.…”

“…d STORM imaging of the specimen was performed essentially as previously reported (Ehmann et al, 2014; Paul et al, 2015; Mrestani et al, 2021; Paul et al, 2022). Preparations were incubated with respective primary antibodies as described above.…”

“…TEVC recordings (Axoclamp 2B amplifier, Digidata 1440A; Molecular Devices) were obtained from abdominal muscle 6 in segments A2 and A3 as previously described (Paul et al, 2022). All measurements were obtained at room temperature in HL-3 (Stewart et al, 1994) with the following composition (in mM): NaCl 70, KCl 5, MgCl 2 20, NHCO 3 10, trehalose 5, sucrose 115, Hepes 5, and CaCl 2 1, pH adjusted to 7.2.…”

Ultrastructural analysis of wildtype and RIM1α knock-out active zones in a large cortical synapse

“…Rab3A-interacting molecules (RIM) form evolutionarily conserved, presynaptic scaffold complexes localized at the active zone (AZ) mesoscale (Emperador-Melero and Kaeser, 2020; Goodsell et al, 2020). In synaptic contacts of vertebrates and invertebrates, RIM and homologues facilitate synaptic transmission and its potentiation for short or long-term information storage (Castillo et al, 2002; Kintscher et al, 2013; Müller et al, 2012; Paul et al, 2022; Schoch et al, 2002; Stigloher et al, 2011).…”

Ultrastructural analysis of wild-type and RIM1α knockout active zones in a large cortical synapse

“…Rab3A-interacting molecules (RIMs) form evolutionarily conserved, presynaptic scaffold complexes at the active zone (AZ) mesoscale (Emperador-Melero and Kaeser, 2020;Goodsell et al, 2020). In vertebrates and invertebrates, RIMs and homologues facilitate synaptic transmission and information storage (Castillo et al, 2002;Kintscher et al, 2013;M€ uller et al, 2012;Paul et al, 2022;Schoch et al, 2002;Stigloher et al, 2011).…”