1 Neurexins: Genes and Proteins Structure

348



A.A. Chubykin



induces exhaustive release of neurotransmitters from presynaptic nerve terminals. This suggested a presynaptic localization for neurexins, which was later

confirmed by subsequent studies (Dean et al. 2003, Chubykin et al. 2005).

Three neurexin genes are expressed in mammals. Each gene produces two

principal forms, the longer a- and the shorter b-neurexin isoform (Rowen et al.

2002, Tabuchi and Suădhof 2002). These genes are highly conserved among

vertebrate species and exhibit identical exon/intron structures. With the sizes

of $1.1 and of $1.6 Mb, the neurexin-1 (NRXN1) and the neurexin-3

(NRXN3) genes are unusually large. In contrast, the neurexin-2 (NRXN2)

gene spans only $110 kb (Tabuchi and Suădhof 2002). The human neurexin

genes are located at 2p16 (NRXN1), 11q12 (NRXN2), and 14q32 (NRXN3),

respectively. Structurally, all neurexins are composed of alternatively spliced

extracellular domains, a single transmembrane region (TMR), and a short

cytoplasmic tail. Thus they resemble typical cell-surface receptors. a- and bneurexins differ only in their N-terminal extracellular sequences and share

identical C-termini, including the O-glycosylation site close to the TMR, the

TMR itself, and the cytoplasmic region. a-Neurexin contains six laminin/neurexin/sex hormone-binding globulin (LNS) domains, which are separated by

three epidermal growth factor-like (EGF) repeats, and five splice sites

SS#1–SS#5, while b-neurexin contains a small b-neurexin-specific sequence,

which is followed by only one LNS domain and two splice sites SS#4 and SS#5

(Fig. 17.1a).



17.2 Neurexin Genes Are Differentially Expressed

So far only a general characterization of the expression pattern of six major

neurexins (1a, 1b, 2a, 2b, 3a, and 3b) has been published (Ullrich et al. 1995).

The existing evidence suggests that these neurexin genes have differential pattern of expression. In situ hybridizations with oligonucleotides specific for the

six major neurexins have shown that neurexin 1a is expressed in all brain areas

with the highest level in the claustrum, anterior thalamic nuclei and deep

cerebellar nuclei. Neurexin 1b is expressed mostly in the cortical layers 2 and

3, the thalamus, and specific parts of the hippocampus. Neurexin 2a is

expressed only in specific subpopulations of the cortical layers 2, 4, and 6, the

thalamus and the cerebellum. The distribution of neurexin 2b is more uniform

with higher levels in the superficial layers of the cortex and the cerebellum.

Finally, neurexin 3a has low expression in most of the brain except for the very

superficial and the infragranular layers of the cortex, the striatum, septal nuclei,

and the reticular thalamic nucleus (RN). Neurexin 3b is expressed uniformly in

most brain regions (see Table 17.1) (Ullrich et al. 1995).

The distinctive anatomical features of the hippocampus and the olfactory

bulb provide a way to characterize expression of different neurexin genes in cells

forming highly specific synapses. The hippocampus is organized into two sheets



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349



Fig. 17.1 Neurexins and neuroligins. (a) The structure of neurexins and neuroligins.

SS–splice sites, EGF – epidermal growth factor-like repeats, LNS – laminin/neurexin/sex

hormone-binding globulin domains, pdz (small letters) – PSD-95-Dlg-ZO homology-binding

motif. (b) Binding specificities of neurexins to neuroligins. The binding affinity of neurexin

1b-SS#4 to neuroligin 1 is greater than to neuroligin 4 and much greater than to neuroligin 3.

The lowest binding affinity is to neuroligin 2. Binding of neurexins 1a-SS#4 or +SS#4 to

neuroligin 1-SS#B is weaker than neurexin 1b-SS#4, but stronger than neurexin 1b+SS#4.

(c) Scheme of a glutamatergic excitatory synapse. a- and b-neurexins lacking splice site SS#4

insert interact with neuroligin 1 with or without splice site #B insert. Presynaptically,

neurexins interact with CASK via their PDZ-binding motif and the CASK PDZ domain.

Postsynaptically, neuroligins interact with PSD-95 and S-SCAM via their PDZ-binding

motif and the corresponding PDZ domains. CaMK – Ca2+/calmodulin-dependent kinase

domain, L27–L27 domain, PDZ (capitalized)–PSD-95-Dlg-ZO homology domain, SH3–Src

homology 3 domain, GK–guanylate kinase domain, WW–WW domain, Ca2+ Ch–calcium

channel, AMPA R–AMPA receptor, NMDA R – NMDA receptor. (d) Scheme of a

GABAergic inhibitory synapse. a-neurexins with splice site SS#4 insert bind neuroligin 2

lacking splice site SS#B insert via their sixth LNS domain, and dystroglycan via their second

LNS domain. b-neurexins interact only with neuroligin 2, and not dystroglycan. GABAA R

– GABAA receptor



All brain; enriched in the claustrum,

anterior thalamic nuclei, deep

cerebellar nuclei

Cortical layers 2/3, the thalamus, parts

of the hippocampus

Subpopulations of cortical layers 2, 4, 6,

the thalamus, the cerebellum

All brain; enriched in the superficial

layers of the cortex, the cerebellum

Superficial and infragranular layers of

the cortex, the striatum, septal nuclei,

the reticular thalamic nucleus

All brain



Neurexin

1a



+++



À



À



+++

+++



+



++



+++



++



++



++



À



Glom



+



+



À



À

+++



++



+++



À



+++



MCL

À



EPL

À



++



+



+++



++



++



+



++



+



Neurexin

+++ +++ +++ ++

+++ +++ +

3b

DG, dentate gyrus; Glom, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; GCL, granular cell layer.

À, no expression; +, low expression; ++, medium expression; +++, high espression.



+



+



+



++



+++



++



+



–



+



Olfactory bulb



À



Neurexin

1b

Neurexin

2a

Neurexin

2b

Neurexin

3a



Brain



Genes



Table 17. 1 Distribution of neurexins in brain

Hippocampus

Pyramidal cells

Interneurons

CA1

CA3

DG

CA1–CA3 DG



GCL



+++



+++



+++



+



+



+



350

A.A. Chubykin



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351



of neurons folded into each other. They are called the dentate gyrus and

Ammon’s horn. Ammon’s horn consists of four regions called CA1–CA4,

where CA stands for cornu Ammonis, which means ‘Ammon’s horn’ in Latin.

The hippocampus is characterized by a unidirectional three-synaptic organization. Input from the entorhinal cortex (EC) is called perforant path (PP) and is

received in the Dentate Gyrus (DG) and the CA3. In addition to this input, the

CA3 neurons also receive input from the DG via the mossy fibers (MF). The

CA3 neurons send axons to CA1 via the Schaffer collateral pathway (SC). The

CA1 cells also receive direct input from the perforant path, although these

synapses are located on the distal apical dendrites.

In the hippocampus, there is a clear difference between CA1 and CA3 cells in

neurexin gene expression. CA1 pyramidal cells and interneurons have no

detectable neurexin 1b mRNA and CA1 pyramidal cells and dentate gyrus

granule cells lack neurexin 3a. In contrast, pyramidal cells of CA3 co-express

all six neurexin isoforms. Interestingly, interneurons express variable levels of

different neurexins in different areas, but neurexin 3a exhibits the highest

expression level (Table 17.1).

In the olfactory bulb, different neuronal cell types are organized in layers

making identification of these cells unambiguous. Neurexin 1a is expressed

mostly in periglomerular cells, but is almost absent from mitral and tufted

cells. In contrast, neurexin 1b is highly enriched in mitral and tufted cells.

Neurexin 2a has a very low level of expression in the olfactory bulb, whereas

neurexin 2b is highly expressed, particularly in inhibitory periglomerular and

granule cells. Neurexins 3a and 3b are both expressed in granule cells, but only

neurexin 3b is present in periglomerular cells (Table 17.1) (Ullrich et al. 1995).

The presence or absence of the splice site SS#4 determines the specificity of the

neurexin–neuroligin interaction. Thus, changes in the distribution of these isoforms may support the role of neurexin as a specific synaptic code. Preliminary

data using in situ hybridizations with the oligonucleotides specific for neurexins 1

and 2 with or without SS#4 indicate that expression levels of neurexins 1 and 2

with SS#4 are much higher in the striatum, substantia nigra, and cerebellar

nuclei, while in the CA3 region of the hippocampus the expression levels are

reversed with higher expression levels of neurexins 1 and 2 without SS#4. In the

hippocampus, the most prominent isoform is neurexin 3 without SS#4, which is

expressed mostly in the pyramidal cells of CA1–CA4 (Ichtchenko et al. 1995).



17.3 Dystroglycan and Neurexophilin – Neurexin-Interacting

Proteins with Unknown Functions

Neurexins have three different protein-binding partners: dystroglycans, neurexophilins, and neuroligins (Petrenko et al. 1996, Sugita et al. 2001). The

alternative splicing of neurexin transcripts determines the specificity of these

interactions.



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A.A. Chubykin



Dystroglycans represent a family of CAMs that associate with dystrophins

and are present at inhibitory central nervous synapses and neuromuscular

junctions (NMJs). Dystroglycans bind a-neurexins, but not b-neurexins, in a

Ca2+-dependent manner. The binding site for dystroglycans is localized to the

second LNS domain of a-neurexin (Fig. 17.1a). In addition to neurexin, dystroglycans are also receptors for agrin, laminin, and perlecan. Agrin also contains an LNS domain and is also alternatively spliced at amino acid residues

highly conserved between agrin and a-neurexin. Dystroglycans are responsible

for organizing the extracellular matrix (ECM) and facilitate the clustering of

acetylcholine receptors (AChR) at the NMJ. Impairments of dystroglycan

glycosylation abolish the interaction with its ligands and are causally implicated

in several muscular dystrophies, such as muscle–eye–brain disease (MEB),

Walker–Warburg syndrome (WWS), Fukuyama congenital muscular dystrophy (FCMD), and congenital muscular dystrophy 1C and 1D (MDC1C and

MDC1D), which are also often accompanied by mental retardation (Barresi

and Campbell 2006). Although much is already known about dystroglycan, the

importance of its interaction with neurexin for synaptic integrity or function

remains a mystery.

Neurexophilin was originally purified in a tight complex with neurexin 1a

(Petrenko et al. 1996). Based on in situ hybridization results, neurexophilin is a

secreted glycoprotein, which is processed from a pre-propeptide and expressed

at high levels primarily in interneurons containing gamma-aminobutyric acid

(GABA) neurotransmitter (Petrenko et al. 1996). The function of neurexophilin

is currently still unknown, but it might be related to its expression in inhibitory

interneurons.



17.4 Neuroligins: Genes and Proteins Structure

Neuroligins are postsynaptic CAMs that bind to both a- and b-neurexins

(Ichtchenko et al. 1995, 1996, Boucard et al. 2005). Neuroligins are composed of a large extracellular N-terminal sequence that is homologous to the

a/b-hydrolase fold domain of acetylcholinesterase (Ichtchenko et al. 1995),

an O-linked sugar-rich domain, a single transmembrane region, and a short

cytoplasmic tail. There are two differentially spliced sites, SS#A and SS#B,

in the esterase homology domain of neuroligins (Fig. 17.1a). Neuroligin 1

and neuroligin 3 have two alternatively used inserts for SS#A (A1 and A2),

whereas all other neuroligins (2, 4, 5, and 4*) have only one alternatively

used insert corresponding to A2 (Bolliger et al. 2008). Insert at SS#B has

been identified only in neuroligin 1, where it regulates the binding specificity

of a- and b-neurexins.

Rodents have four neuroligin genes (Ichtchenko et al. 1996) and five neuroligin genes have been identified in humans (Bolliger et al. 2001). Interestingly,

the three rodent neuroligin genes have more than 98% amino acid sequence