LPHN3, a presynaptic adhesion-GPCR implicated in ADHD, regulates the strength of neocortical layer 2/3 synaptic input to layer 5

Abstract

BackgroundLatrophilins (LPHNs) are a small family of neuronal adhesion-GPCRs originally discovered as receptors for the black widow spider toxin α-latrotoxin. Mutations in LPHN3 have recently been identified as risk factors for attention deficit hyperactivity disorder (ADHD) in humans, but their physiological function has remained elusive. In this study, we tested two hypotheses regarding LPHN3 function: (1) LPHN3 regulates synaptic transmission by modulating probability of release; and (2) LPHN3 controls synapse development and the abundance of synapses.ResultsWe manipulated LPHN3 expression in mouse layer 2/3 (L2/3) pyramidal neurons and examined the consequences on the L2/3 to L5 cortical microcircuit. Employing an optogenetic strategy combined with shRNA knockdown of LPHN3, we found that LPHN3 did not influence probability of release at synapses formed by L2/3 neurons onto L5 pyramidal cells. The strength of L2/3 afferent input to L5, however, was weakened by loss of LPHN3. Using Synaptophysin-GFP as an anatomical marker of presynaptic terminals, we found that the density of synapses formed by L2/3 axons in L5 was reduced when LPHN3 was lost. Finally, we investigated the structural organization of the extracellular domain of LPHN3. We used single particle negative stain electron microscopy to image the extracellular domain of LPHN3 and showed that the Olfactomedin and Lectin domains form a globular domain on an elongated stalk. Cell-based binding experiments with mutant proteins revealed that the Olfactomedin domain was required for binding to FLRT3, whereas both the Olfactomedin and Lectin domains were involved in binding to Teneurin 1. Mutant LPHN3 lacking the Olfactomedin domain was not capable of rescuing the deficit in presynaptic density following knockdown of endogenous LPHN3.ConclusionsWe find that LPHN3 regulates the number of synapses formed by L2/3 neurons in L5 and the strength of synaptic drive from the L2/3-L5 pathway. The Olfactomedin domain of LPHN3 is required for this effect on synapse number and binding to its postsynaptic ligand FLRT3. We propose that LPHN3 functions in synaptic development and is important in determining the connectivity rates between principal neurons in the cortex.

The role of G protein-coupled receptors in the early evolution of neurotransmission and the nervous system

“…Adhesion GPCRs are a growing research field, and several of these receptors have shown an important role in the CNS (Araç et al, 2012a; Langenhan et al, 2013;Strokes and Piao, 2010). For example, the brain-specific angiogenesis inhibitor (BAI) subfamily is involved in the control of synaptogenesis (Duman et al, 2013;Stephenson et al, 2014), whereas the latrophilins have been implicated in the control of synaptic transmission (O'Sullivan et al, 2012;O'Sullivan et al, 2014) and GPR56 is associated with brain development (Jeong et al, 2012;Luo et al, 2011;Singer et al, 2013).…”

“…In addition, latrophilin-1interacts with the postsynaptic neurexin I-α and -β, forming a trans-synaptic complex and thus modulating synaptic functions (Boucard et al, 2012;O'Sullivan et al, 2014;Südhof, 2001). The brain-specific angiogenesis inhibitor family (BAIs; subfamily VII adhesion GPCRs) members are well characterised for their roles in synaptogenesis, involving regulation of synapses and synaptic density (for a review, see Stephenson et al, 2014).…”

“…Adhesion GPCRs are a growing research field, and several of these receptors have shown an important role in the CNS (Araç et al, 2012a; Langenhan et al, 2013;Strokes and Piao, 2010). For example, the brain-specific angiogenesis inhibitor (BAI) subfamily is involved in the control of synaptogenesis (Duman et al, 2013;Stephenson et al, 2014), whereas the latrophilins have been implicated in the control of synaptic transmission (O'Sullivan et al, 2012;O'Sullivan et al, 2014) and GPR56 is associated with brain development (Jeong et al, 2012;Luo et al, 2011;Singer et al, 2013).GPCRs are well represented in several primitive metazoans, as well as in some pre-metazoan species (de Mendoza et al, 2014; Krishnan et al, 2012;Nordström et al, 2011). Interestingly, several of the pre-bilaterian metazoans, which lack most of the cell types that are commonly found in the bilaterians, still constitute a rich repertoire of GPCRs (Patel, 2012;Technau and Steele, 2011).…”

Deciphering the genomic toolkit underlying animal-bacteria interactions – insights through the demosponge Amphimedon queenslandica

“…It is tempting to speculate sponge feeding behaviour involves cell-cell communication mediated through a signalling system involving the glutamate family GPCRs and these chemical messengers. Studies in recent years have also begun to reveal the role of adhesion GPCRs in the nervous system (Silva, et al 2011;Boucard, et al 2012;O'Sullivan, et al 2014). In particular, the adhesion subfamily I latrophilins are involved in the development of the synapse and neurotransmission (Silva, et al 2011;Boucard, et al 2012;O'Sullivan, et al 2014).…”

“…Studies in recent years have also begun to reveal the role of adhesion GPCRs in the nervous system (Silva, et al 2011;Boucard, et al 2012;O'Sullivan, et al 2014). In particular, the adhesion subfamily I latrophilins are involved in the development of the synapse and neurotransmission (Silva, et al 2011;Boucard, et al 2012;O'Sullivan, et al 2014). The adhesion family GPCRs have undergone a large expansion in A. queenslandica, yet scarcely anything is known about their roles in sponges (Krishnan, et al 2014).…”