Neuroligin-Neurexin Interaction

       Neurexins (NRXNs) and Neuroligins (NLGNs) are synaptic cell-adhesion molecules that connect presynaptic and postsynaptic neurons at synapses. NLGNs, of which NLGN1-5 are the best characterized, are endogenous NRXN ligands.^[1] Upon binding NRXN, NLGNs mediate signaling across the synapse, and shape the properties of neural networks by specifying synaptic functions. Early experiments proved that NLGNs function at synapses by showing that NLGNs expressed in non-neuronal cells can induce co-cultured neurons to form presynaptic specializations onto the non-neuronal cell. The same is true for NRXNs which formed postsynaptic specializations.^[2] Subsequent analysis of gene-knockout mice surprisingly revealed that NLGNS and NRXNs are essential for synaptic function and organization, but not synapse formation.^[3] In effect, NRXNS and NLGNS shape synaptic efficacy and plasticity.^[2]

       Experimental results from the past 10 years have revealed that loss of Neuroligin function has a selective reductive effect on inhibitory synapse function indicating that functional inhibitory synapses rely more heavily on neuroligins than do excitatory synapses.^[1] This was an astonishing discovery as Autism Spectrum Disorders (ASDs) have long been attributed to perturbations in the Excitatory/Inhibitory (E/I) synaptic signal ratios within the brain. Knockdown studies have validated that the selective knockdown of NLGNs resulted in a significant imbalance of E/I transmission possibly altering oscillatory rhythms in the brain.^[3][1] As Sudhof et al. point out, a major reason for the difficitulties in understanding cognitive diseases is that they arise from subtle changes in a subset of synapses as opposed to obvious impairment of all synapses in all circuits.^[2] The altered E/I ratios associated with NLGN dysfunction may be an example of such a “subtle change”. Indeed, mice with loss-of function mutations in NLGN4 exhibit deficits in social interactions and communication that are reminiscent of ASDs.^[4] Of particular interest are those NLGN3 mutations like R451C in which mice have the impaired social ability but elevated learning capacity manifestations reminiscent of “savant” syndrome.^[3] In all, seven point mutations, two translocations and four large-scale deletions in the NRXN1 gene were detected in patients with autism.^[2] These mutations however were only found in a small percentage of Autism patients and are occasionally present in non-symptomatic siblings. Thus, these mutations may only increase the chance of autism rather than actually causing it.^[5]

       There are two types of NRXNS, alpha and beta. While alpha NRXNs are much larger than beta-NRXNs, both contain a single LNS (Laminin, NRXN, sex-hormone-binding globulin domains) domain. Extensive alternative splicing at five unique positions generates thousands of NRXN isoforms which likely specify a “code” of interactions at synapses that varies by location and activity of the neuron.^[2] All NRXN isoforms have the same “jelly roll fold” comprised of **14 beta sheets**. The alternative splicing only **impacts one end of the jelly roll**, including sheets 1, 2, 13 & 14 and the lone alpha helix in the LNS domain.^[6]

       The extracellular residues of NLGNs, which bind to the LNS domains of both alpha and beta NRXNs with nanomolar affinity, are composed of a single domain that is homologous with acetylcholinesterase (AChE).^[2] Neuroligin-4 consists of a twisted **12 stranded beta sheet surrounded by 14 alpha helices**. Three intramolecular **disulfide bridges** between residues Cys110-Cys146, Cys306-Cys317, and Cys476-Cys510, stabilize the structure. Neuroligins readily form a dimmer consisting of two neuroligin subunits. 100% of the dimer **interactions are hydrophobic** with the most unique feature being a **prominent four-helix bundle**.^[5] The **central pocket within Neuroligin**, which in Acetylcholinesterase contains the active center and oxyanion hole, is catalytically inactive due to a substitution of Gly for Ser **at position 254**, which is typically part of AChE’s hydrolytic catalytic triad. Within the central pocket however **lies a phosphate** bound by residues Gly254, Glu375, and His489, which provides the NLGN4 molecule with additional flexibility. The **so-called Cys-Loop** (Residues Cys110-Cys146) forms one side of the rim of the central pocket and is a homolog of the lid found in lipases of the α/β-hydrolase fold family. This Cys-Loop blocks the entry of substrate to the central pocket and provides stability to the NLGN structure.^[5]

       NRXN**-Beta1 is bound** through its hypervariable loop edge to the electronegative surface of the NLGN-4 molecule, opposite the Cys-loop. The NRXN-NLGN interface is established by both indirect and direct interactions. Indirect interactions include **coordination of a divalent calcium cation** by residues Asp 137, Asn 238, Val 154 and Ile 236 of NRXN-Beta1 and residues Gln 359 and Gly 360 of NLGN-4. Direct interactions between NRXN and NLGN include extensive hydrogen bonding and Van der Waals contacts as well as salt bridges between residues NRXN-Arg 109 & NLGN-Glu 270 and NRXN-Arg232 & NLGN Asp 351. The vast majority of these interactions are conserved among all neuroligin types.^[5]

       Several **point mutations** (R437C (451 in NLGN 3), G99S, K378R, & V403M) within NLGN have been positively linked with Autism Spectrum Disorders. Although these mutations are remote from the NLGN-NRXN interface, conclusions can be drawn as to their impact on the function of NLGN. **Arg437Cys**, a mutation shown to result in “savant” like attributes in mice, is believed to increase retention of NLGN in the endoplasmic reticulum preventing correct positioning at the cell surface. It could also disrupt a dense network of charged residues (Asp 388, Asp 486, Glu 434, and Lys 338) through Trp 484, which are believed to be important for processing events. **Val403Met** is believed to affect correct folding of the C-terminal domain of NLGN and prevent formation of the functional dimer. **The Lys378Arg mutation** which interacts with the Cys-loop Asp 122 through Van der Waals contacts could disrupt the Cys-loop reducing NLGN structural integrity. These mutations do not entirely disrupt NLGNs interaction with NRXN, but do impact NLGN in the subtle ways from which Autism likely arises.^[5]