An Unbiased Expression Screen for Synaptogenic Proteins Identifies the LRRTM Protein Family as Synaptic Organizers

Abstract

Summary A delineation of the molecular basis of synapse development is crucial for understanding brain function. Co-cultures of neurons with transfected fibroblastoid cells have been used to demonstrate the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the co-culture assay using custom-made full-length cDNA libraries. Re-isolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is far from complete. We identify LRRTM1 as a novel transmembrane protein capable of inducing presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially-induced clustering of LRRTMs mediates postsynaptic differentiation in dendrites. We generate LRRTM1 -/- mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the recently reported linkage of LRRTM1 to handedness and schizophrenia.

The Leucine-Rich Repeat Superfamily of Synaptic Adhesion Molecules: LRRTMs and Slitrks

“…All four LRRTM family members display synaptogenic activities in these assays, although the induction strength of LRRTM2 is the strongest (de Wit et al, 2009;Ko et al, 2009;Linhoff et al, 2009). LRRTM2 specifically localizes in excitatory synapses, and not in inhibitory synapses (Linhoff et al, 2009). The localizations of the other LRRTMs have not yet been determined due to a lack of reliable antibodies suitable for immunohistochemical analyses.…”

“…Four LRRTM genes are found only in vertebrates and share similar domain structures comprising 10 LRRs, a single transmembrane segment and a short cytoplasmic stretch, which ends in a type I PDZ-binding motif; this latter motif has been shown to mediate direct interactions with the postsynaptic scaffolding protein, PSD-95 (postsynaptic density 95), in heterologous cells (de Wit et al, 2009;Lauren et al, 2003;Linhoff et al, 2009). All four LRRTM mRNAs, determined by reverse transcription-polymerase chain reaction, are highly expressed in the brain and show overlapping distributions in various brain regions (Lauren et al, 2003).…”

“…A recent unbiased expression screen revealed that LRRTM proteins are synaptogenic in artificial synapse formation assays (Linhoff et al, 2009). All four LRRTM family members display synaptogenic activities in these assays, although the induction strength of LRRTM2 is the strongest (de Wit et al, 2009;Ko et al, 2009;Linhoff et al, 2009).…”

“…The localizations of the other LRRTMs have not yet been determined due to a lack of reliable antibodies suitable for immunohistochemical analyses. Overexpression of LRRTM1 or -2 specifically increases the number of excitatory synapses (de Wit et al, 2009;Ko et al, 2009;Linhoff et al, 2009). In addition, LRRTMs interact with neurexins, a well-known family of presynaptic adhesion molecules whose members bind to neuroligins (de Wit et al, 2009;Ko et al, 2009;Siddiqui et al, 2010).…”

“…Among the factors that regulate the development of neural circuits, proteins containing extracellular leucine-rich repeat (LRR) domains have recently emerged as key organizers of connectivity (de Wit et al, 2011;Ko and Kim, 2007). Artificial synapse formation assays have shown that a host of LRR-containing proteins, which are widely and highly expressed in the brain, are capable of recruiting various synaptic scaffolds and inducing synaptic differentiation in heterologous cells in which these LRR-containing proteins are exogenously expressed (de Wit et al, 2009;Kim et al, 2006;Ko et al, 2006;Linhoff et al, 2009;Mah et al, 2010;Takahashi et al, 2011;Woo et al, 2009). Selected LRR proteins that function at synapses are shown in Fig.…”