8 Conclusions, the Concept of a Synaptic Code and Future Directions

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variations of different neurexin isoforms in different cell types and possibly even

individual cells.

Consequently, more detailed characterization of the various neurexin and

neuroligin isoforms and their expression patterns will be essential for our

understanding of the postulated neurexin–neuroligin-dependent synaptic

code. Understanding this neurexin–neuroligin-dependent code might help us

to learn how the brain is wired by specific connections between different cell

types and by individual neurexin–neuroligin pairs, which specify different types

of synapses. More modern, high-throughput, sensitive assays will shed more

light on synaptic complexity in vertebrate brains. One of the predictions would

be that the expression of neurexins–SS#4 is mostly in excitatory neurons, and

neurexins+SS#4 in inhibitory neurons. An individual neuron may express

either neurexin-SS#4 or neurexin+SS#4 isoform, but not both. The SS#4 splice

site could represent one of the potential markers of the neuronal type. Consequently, investigation of how the splicing is regulated becomes very important,

particularly, identification of the cis elements in the neurexin and neuroligin

genes, as well as the trans-acting splicing factors interacting with these elements.

A growing body of evidence suggests that the activity-dependent regulation

of neurexins and neuroligins at both excitatory and inhibitory synapses may be

involved in synaptic plasticity. However, we will need a considerably better and

more detailed knowledge of how synaptic activity is regulated by neurexins and

neuroligins at the molecular level, before we will understand the role of these

molecules in higher cognitive functions, such as memory formation and

behavior.

Acknowledgments The author thanks Thomas C. Suădhof and Dilja Krueger for their helpful

comments on the manuscript.



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Chapter 18



Synaptic Adhesion-Like Molecules (SALMs)

Philip Y. Wang and Robert J. Wenthold



Abstract The synaptic adhesion-like molecules (SALMs) are a newly discovered family of cell adhesion molecules that have a variety of functions

in neuronal development, including aspects of neurite outgrowth and synapse

formation (Ko et al. Neuron 50:233–245, 2006, Morimura et al. Gene

380:72–83, 2006, Wang et al. J Neurosci 26:2174–2183, 2006, Seabold et al.

J Biol Chem 283:8395–8405, 2008, Wang et al. Mol Cell Neurosci, 39:83–94,

2008). Also known as Lrfn (leucine-rich and fibronectin III domain-containing), five family members have been identified thus far: SALM1/Lrfn2,

SALM2/Lrfn1, SALM3/Lrfn4, SALM4/Lrfn3, and SALM5/Lrfn5. The

SALMs have been shown to interact with NMDA receptors and the PSD-95

family of MAGUK proteins. Recent studies also indicate that the individual

SALMs, while similar in structure, play distinct roles in heteromeric and

homomeric protein interactions and neurite outgrowth (Seabold et al. J Biol

Chem 283:8395–8405, 2008, Wang et al. Mol Cell Neurosci, 39:83–94, 2008).

Neurite outgrowth and synapse formation are fundamental mechanisms in the

development of the nervous system. While a considerable amount of information is known about both phenomena, the mechanism connecting the two is still

enigmatic. SALMs join a growing mosaic of synaptic proteins that contribute

to both neurite outgrowth and synapse formation during the course of development. Investigating SALMs and related proteins is essential for addressing

fundamental questions of neuronal development.

Keywords Synapse Á Dendrite Á Axon Á PDZ proteins Á LRR Á Growth cone Á

Heteromeric Á Homomeric Á Neurite outgrowth



R.J. Wenthold (*)

Laboratory of Neurochemistry, National Institute on Deafness and Other

Communication Disorders, National Institutes of Health, Bethesda, MD 20892, USA

e-mail: wenthold@nidcd.nih.gov



M. Hortsch, H. Umemori (eds.), The Sticky Synapse,

DOI 10.1007/978-0-387-92708-4_18, Ó Springer ScienceỵBusiness Media, LLC 2009



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18.1 SALM Family Structure and Expression

The synaptic adhesion-like molecules (SALMs) form a family of five adhesion

molecules. In the mouse, they range from 626 to 788 amino acid residues in

length. All SALMs contain a characteristic domain structure including six

leucine-rich repeat (LRR) regions, an immunoglobulin C2-like domain

(IgC2), a fibronectin type 3 domain (FN3), and a transmembrane region

(TM) (Fig. 18.1). Additionally, SALMs 1–3 contain a PDZ-binding domain

(PDZ-BD) at their distal C-termini, while SALMs 4–5 do not. The SALMs

share a considerable amount of sequence similarity, though there are regions of

high variability in both the N- and C-termini that could lend distinct characteristics to their individual functions (Fig. 18.2). Studies on SALMs and their

functions thus far have focused on the rat and mouse representatives of this

gene family. However, SALMs are present in a variety of mammalian species, as

well as in fish and amphibians (Morimura et al. 2006). Interestingly, additional

sequence analysis using the Ensembl database (www.ensembl.org) indicates

that the protein structure of the SALMs is quite conserved across these species.

As depicted in Fig. 18.3, the LRRs, IgC2, FN3, and PDZ-BD regions are all

conserved across various SALM1 sequences, including those of human, mouse,

platypus, chicken, and medaka (Japanese killifish). Analysis of the Drosophila

genome indicates that SALMs share the closest sequence identity with a family

of LRR and Ig-like domain containing transmembrane proteins called Kekkon

(Kekkon 1–5), which function in the developmental regulation of EGF receptor

activity during oogenesis (Ghiglione et al. 1999, 2003). Phylogenic analysis

reveals that SALMs are closely related to a variety of leucine-rich molecules

including the AMIGO, LINGO, NGL, PAL, and FLRT family of proteins (for

review of these proteins, see Chen et al. 2006). As we describe later in this

chapter, SALMs and these highly related proteins have one major characteristic

in common: their role in regulating neurite outgrowth.

Northern blot analysis indicates that all SALM transcripts are found in the

mouse and the rat brain, and transcripts for SALMs 2, 3, and 4 are seen to some

extent in testis (Ko et al. 2006, Morimura et al. 2006). Additionally, SALM4



Fig. 18.1 SALM protein domain structure. Schematic diagram illustrating the domain structure of the SALM family of proteins. The SALMs contain an N-terminal signal peptide, six

extracellular LRR regions flanked by N- and C-terminal LRR regions, an IgC2 domain, an

FN3 domain, a single TM region, and a PDZ-BD (present in SALMs 1–3)



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Fig. 18.2 SALM family sequence comparison. (A) Sequence analysis reveals that mouse

SALMs contain six LRR regions (flanked by N- and C-terminal LRR sequences), an IgC2



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transcripts are found in the gastrointenstinal tract and kidney (Morimura et al.

2006). Temporal expression profile blots indicate that transcript levels for

SALMs 2–4 show an incremental increase starting from E10.5, while SALM1

and SALM5 increase from around E11.5–E12.5 (Morimura et al. 2006). In situ

hybridization reveals that SALM transcripts are distinctly expressed in a variety

of brain regions, including the cerebral cortex, hippocampus, dentate gyrus,

and olfactory bulb (Ko et al. 2006, Morimura et al. 2006).

Western blot and subcellular fractionation experiments showed that

SALM proteins are highly expressed in the rat brain, and enriched in synaptosomal and postsynaptic density fractions (Ko et al. 2006, Wang et al. 2006).

SALM protein expression levels exhibit some differentiation among family

members. Protein levels for SALM1 are detectable from E18, display high

expression from P1 to P21, and then decrease at P28 (Wang et al. 2006).

Protein levels for SALM2 increase from P21 and remain high at 6 weeks

(Ko et al. 2006). SALM2 protein is widely distributed in brain, and for

example, has been detected in cortical pyramidal neurons, hippocampal

CA3 and CA1 neurons, and cerebellar Purkinje cells (Ko et al. 2006). At the

subcellular level, SALM2 proteins localize to cell bodies, neurites, and punctate structures that co-localize with the presynaptic protein, synapsin I (Ko

et al. 2006). Ultrastructural analysis using immunogold electron microscopy

shows that native SALM4 is present at a variety of presynaptic, postsynaptic,

and extrasynaptic sites in hippocampus, olfactory bulb, and cerebellar cortex

(Seabold et al. 2008). Overexpressed SALMs are localized throughout the cell

in the soma, axons, dendrites, and growth cones in young (
vitro 7) neuronal cultures, both on the cell surface and intracellularly (Wang

et al. 2006, 2008). In older (>DIV14) neuronal cultures, overexpressed

SALMs are localized throughout the cell, on the cell surface, and at synapses

(Ko et al. 2006, Wang et al. 2006, and unpublished observations). Additionally, Ko et al. (2006) demonstrated that SALM2 is localized to excitatory, but

not inhibitory synapses, and that perturbation of SALM2 expression leads to

aberrations in excitatory synaptic formation.



Fig 18.2 (continued) domain, an FN3 domain, a TM region, and a PDZ-BD at the distal Ctermini (present in SALMs 1–3). Illustrated domain locations based on the SALM1 sequence.

Similar amino acid residues are highlighted by the gray background, and the consensus

sequence/strength is shown above the alignment. Consensus strength correlates with the

length of the individual bars. Protein sequences for SALMs 1–5 (accession numbers

NM_027452, NM_030562, NM_153388, NM_175478, and NM_178714, respectively) were

aligned by the Clustal V method using MegAlign computer software. (B) Phylogenic tree

comparing the mouse SALMs was constructed with MegAlign based on the alignment

produced in (A)



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Fig. 18.3 SALM1 species comparison. SALM1 protein sequences from a variety of species

were acquired using the Ensembl database (www.ensembl.org). (A) The SALM1 sequence

structure is highly conserved among a variety of mammalian, avian, and fish species including



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18.2 SALM-Associated Proteins and Functional Significance

The known binding partners for the SALMs include the PSD-95 family of

membrane-associated guanylate kinase (MAGUK) proteins, the NR1 subunit

of the N-methyl-D-aspartate receptor (NMDAR), and the SALMs themselves –

through homomeric and heteromeric interactions (Ko et al. 2006, Morimura

et al. 2006, Wang et al. 2006, Seabold et al. 2008). The SALMs were independently identified in two different laboratories through yeast two-hybrid

screens using the PDZ domains of MAGUKs as bait (SAP97 in Wang et al.

2006, and PSD-95 in Ko et al. 2006). Classically described as scaffolding

proteins that assist in the tethering of receptors and associated proteins at

the postsynaptic density (PSD), MAGUKs have been linked to a variety of

functions in the CNS, including the trafficking of NMDARs to the synapse

and neurite outgrowth (Kim and Sheng 2004, Charych et al. 2006). Overexpression of PSD-95 decreases dendritic branching in immature neurons,

while knocking down PSD-95 increases it (Charych et al. 2006). Overexpression of SALMs in young neurons promotes neurite outgrowth, while overexpression of SALM 1–3 constructs lacking the PDZ-BD do not (Wang et al.

2008). This suggests that there may be a direct link in the process of neurite

outgrowth and SALM–MAGUK associations, at least for SALMs 1–3. Interestingly, SALMs 4, 5 lack a PDZ-BD, but still promote neurite outgrowth.

This may indicate that they act via a different mechanism or through heteromeric associations with other SALMs to induce MAGUK-associated neurite

outgrowth. In mature neurons, deletion of the PDZ-BD has effects on synapse

formation and morphology (Wang et al. 2006), further emphasizing the

importance of associated PDZ proteins in SALM function.

Additional evidence suggests that SALMs may interact, indirectly or

directly, with various other postsynaptic proteins. For example, beadinduced aggregation experiments revealed co-clustering of AMPARs and

GKAP with SALM2 (Ko et al. 2006). SALM1 has also been shown to

interact directly with NR1 in heterologous cells, and co-immunoprecipitates

with NR1 and NR2 subunits of NMDARs from brain (Wang et al. 2006).

The various protein domains that SALMs possess offer potential binding

sites for other proteins.



Fig 18.3 (continued) human (Homo sapiens), mouse (Mus musculus), platypus (Ornithorhynchus anatinus), chicken (Gallus gallus), and Japanese killifish/medaka (Oryzias latipes).

(B) Phylogenic tree comparing the SALM1 sequences of the various species. SALM1 proteins

sequences were aligned by the Clustal V method using MegAlign. Ensembl gene IDs used for

SALM1 human, mouse, platypus, chicken, and killifish were ENSG00000156564,

ENSOANG00000014625, ENSGALG00000010050, and ENSORLG00000018222, respectively. Accession number NM_027452 was used for the SALM1 mouse sequence



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18.3 Homomeric and Heteromeric SALM Interactions

Cell adhesion molecules (CAMs) are usually transmembrane proteins, and

participate in the formation of cellular junctions through interactions between

their extracellular protein domains. These CAM interactions can be cis, within

the same membrane, or trans, across the cell–cell junctions of adjacent cells.

These cis or trans interactions can be heteromeric (forming interactions with

other distinct proteins), or homomeric (forming dimers or higher order multimers with a single protein type). For example, nectins form homomeric cis

interactions (Takai and Nakanishi 2003), while L1-type CAMs and axonin-1

form heteromeric cis interactions (Buchstaller et al. 1996, Stoeckli et al. 1996).

Cadherins form homomeric trans interactions (Tepass et al. 2000), while neuroligin and neurexin form heteromeric trans interactions (Ichtchenko et al.

1995, Scheiffele et al. 2000).

Seabold et al. (2008) demonstrated that SALMs are able to form heteromeric

and homomeric interactions with each other, though with some distinctions

among the individual SALM family members. From brain extracts, SALMs

1–3 co-immunoprecipitate with each other, indicating the formation of heteromeric interactions, while SALMs 4 and 5 do not. When individually expressed

and co-plated in heterologous cells, only SALMs 4 and 5 are able to form

homomeric trans interactions. These interactions are due to the extracellular

N-terminus, as demonstrated through the use of chimera constructs made of the

N- and C-termini of SALMs 2 and 4. When transfected into heterologous cells,

a construct containing the N-terminus of SALM4 and the C-terminus of

SALM2 (SALM4/2) forms homomeric trans interactions, while the reverse

chimera construct (SALM2/4) does not. Application of antibodies directed to

the extracellular LRR of SALMs blocks this interaction, indicating a function

of the LRR region in these trans associations. Furthermore, when HeLa cells

and primary hippocampal neurons overexpressing SALM4 are co-cultured,

SALM4 is recruited to points of contact between the two different cell types.

These SALM4 accumulations are seen at both axonal and dendritic adhesions

between neurons and HeLa cells.

Interestingly, when co-transfected into heterologous cells in pairs, all five

SALMs appear to form both heteromeric and homomeric complexes, indicating that the potential for such complexes may exist in vivo under certain

conditions. The mechanism of SALM trafficking and the dynamics of their

early interactions in the secretory pathway are not yet known. A hypothetical

model of SALM interactions is depicted in Fig. 18.4. Early in the ER, SALMs

may form combinations of dimers or higher order multimers with each other or

other proteins. The constitution of such SALM multimers could contribute to

the distinctiveness of SALM function, including their cis/trans interactions. The

potential interplay between cis and trans interactions is highly complex, and

may contribute to the variability among the SALM function. Formation of cis

complexes may regulate the formation of trans complexes. For example,