Arthur P.H. de Jong

Synaptic vesicle fusion in brain synapses occurs in phases that are either tightly coupled to action potentials (synchronous), immediately following action potentials (asynchronous), or as stochastic events in the absence of action potentials (spontaneous). Synaptotagmin-1, -2, and -9 are vesicle-associated Ca2+ sensors for synchronous release. Here we found that double C2 domain (Doc2) proteins act as Ca2+ sensors to trigger spontaneous release. Although Doc2 proteins are cytosolic, they function analogously to synaptotagmin-1 but with a higher Ca2+ sensitivity. Doc2 proteins bound to N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complexes in competition with synaptotagmin-1. Thus, different classes of multiple C2 domain-containing molecules trigger synchronous versus spontaneous fusion, which suggests a general mechanism for synaptic vesicle fusion triggered by the combined actions of SNAREs and multiple C2 domain-containing proteins.

The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.

Although cognitive ability is a highly heritable complex trait, only a few genes have been identified, explaining relatively low proportions of the observed trait variation. This implies that hundreds of genes of small effect may be of importance for cognitive ability. We applied an innovative method in which we tested for the effect of groups of genes defined according to cellular function (functional gene group analysis). Using an initial sample of 627 subjects, this functional gene group analysis detected that synaptic heterotrimeric guanine nucleotide binding proteins (G proteins) play an important role in cognitive ability (P(EMP) = 1.9 x 10(-4)). The association with heterotrimeric G proteins was validated in an independent population sample of 1507 subjects. Heterotrimeric G proteins are central relay factors between the activation of plasma membrane receptors by extracellular ligands and the cellular responses that these induce, and they can be considered a point of convergence, or a "signaling bottleneck." Although alterations in synaptic signaling processes may not be the exclusive explanation for the association of heterotrimeric G proteins with cognitive ability, such alterations may prominently affect the properties of neuronal networks in the brain in such a manner that impaired cognitive ability and lower intelligence are observed. The reported association of synaptic heterotrimeric G proteins with cognitive ability clearly points to a new direction in the study of the genetic basis of cognitive ability.