Role Of Neuroligin And Pneurexin In Synapse Formation And Function In Vitro

The importance of a heterotypic adhesion complex such as the one formed by neuroligin and P-neurexin arises from the asymmetric nature of the synapse with presynaptic and postsynaptic specializations. Recently, experimental support for a role of neuroligin and P-neurexin in synapse formation and/or function has been provided by in vitro assays.

4.1. Effects on Synapse Formation

In 2000, a chimeric culture model between neuronal and non-neuronal cells was first used to address the role of neuroligin in synapse formation by Scheiffele and colleagues34, where pontine explants were co-cultured with non-neuronal HEK293 cells. This model has the advantage that the non-neuronal cell can be engineered to express proteins of interest by conventional transfection, and the resultant effect can be monitored to elucidate the function of these proteins. The chimeric synapses can also be easily distinguished from endogenous synapses between neurons due to the absence of other synaptic markers within the nonneuronal cell. Presenting neuroligin 1 on a non-neuronal cell was sufficient to instigate synapse formation, resulting in the recruitment and clustering of synapsin at the neuronal presynaptic contact surface with the transfected HEK293 cell. This was a specific effect, not elicited by other candidate molecules, such as A-cadherin, ephrinBl, TAG-1, agrin, or L134. More recently, analyses of the homophilic cell adhesion molecule SynCAM have revealed comparable synapse-inducing properties for an adhesion molecule with a structure completely unrelated to neuroligin domains35,36. Following the initial demonstration of neuroligin's potential to induce presynaptic specializations, the so-called reverse chimeric system has been used to demonstrate that P-neurexin can induce postsynaptic differentiation. P-Neurexin-expressing COS cells induced the clustering of several postsynaptic markers, including PSD-95 and gephyrin, as well as y aminobutyric acid (GABA)a and NMDA receptor subunits37. In addition, HEK293 cells co-transfected with both neuroligin and PSD-95 revealed that neuroligin was sufficient to anchor the postsynaptic PSD-95 with neuronal presynaptic synapsin, indicating that the neuroligin/p-neurexin complex can bridge the synaptic cleft38. Neuroligin and P-neurexin may nucleate the formation of both pre- and postsynaptic elements and are therefore a promising candidate pair for organizing the asymmetric nature of the synapse.

Consistent with Scheiffele's initial observations34, overexpression of neuroligin 1 caused an increase in the number and size of presynaptic terminals41. In other studies, neuroligin 1 overexpression resulted in increased postsynaptic differentiation and additional dendritic spines40, and a splice variant of neuroligin 1 (without an insert in B, see also Figure 7.1; Colorplate 6) predominantly caused changes in the size of spines and presynaptic terminal^. It appears that the actual effect observed not only depends on the molecules and their splice variants over-expressed but also on the availability and amount of their binding partner PSD-95, and possibly yet unknown other interacting molecules. It was hypothesized that limiting endogenous pools of PSD-95 may be responsible for the lack of postsynaptic changes in some cases, and co-transfection of PSD-95 with neuroligin did result in an enlargement and increased number of postsynaptic structures41. Although data from several recent investigations agree that neuroligins robustly modulate presynaptic maturation, these approaches and their most important results, which are compiled in Table 1, demonstrate that similar experiments sometimes yield quite different results on the regulation of postsynaptic protein clustering.

Differences between recent studies also exist with respect to the postsynaptic recruitment of neurotransmitter receptors (NTRs), which is an essential aspect of the differentiation of functional synapses (Table 1). Upon presentation of P-neurexin on COS cells37 or PC12 cells39, the NR1 subunit of NMDAR was detected within the resulting postsynaptic clusters, but no AMPAR subunit recruitment took place. However, upon overexpression of neuroligin in neurons, one study saw clustering of both NR1 and some AMPAR subunits, GluR2/340, whereas another did not observe any recruitment of the NTR subunits analyzed41. Interestingly, as with the postsynaptic effects of neuroligin addressed above, this latter study found that transfection of neurons with PSD-95 resulted in the recruitment of the AMPAR subunit, GluR1. Nam and Chen eventually demonstrated that in neurons, overexpression of P-neurexin only elicited the formation of AMPAR-negative postsynaptic clusters which subsequently could be induced to recruit the AMPAR subunit GluR1 by treatment with glutamate. The suggestion was that since these synapses already contained NMDAR glutamate receptors, glutamate treatment may represent an activity-dependent mechanism for initiating further maturation of the synapse. This mechanism has been proposed to occur

Table 1. Comparison of Overexpression Studies In Vitro to Analyze the Role of the Neuroligin/P-Neurexin Complex in Synapse Formation.

Method

Result

Reference

Overexpression in non-neuronal cell of

P-neurexin

Induces postsynaptic clustering in neurons

Neuroligins 1 and 2, PSD-95, gephyrin, NMDA (NR1) and GABAa receptor subunits PSD-95, NMDAR (NR2)

(37) (39)

Neuroligin 1 or 2

Induces presynaptic clustering in neurons Synapsin

(34)

Neuroligin 1 (mutated to abolish binding to P-neurexin

Fails to induce presynaptic clustering in neurons Synapsin

(58)

Overexpression in neurons of

Neuroligin 1

Induces presynaptic clustering VGLUT1 GAD65/VGAT P-neurexin, synaptobrevin

(17)

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