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Differential Properties of the Synaptogenic Activities of the Neurexin Ligands Neuroligin1 and LRRTM2.

ABSTRACT: Synaptic cell adhesion molecules are well established to exhibit synaptogenic activity when overexpressed in target cells, indicating that they are involved in formation and functional maturation of synapses. The postsynaptic adhesion proteins Neuroligin1 and LRRTM2 both induce synaptic vesicle clusters in presynaptic axons in vitro by transsynaptically interacting with neurexins. In neurons, this is accompanied by the induction of glutamatergic, but not GABAergic synapses. Although the synaptogenic activity of Neuroligin1 has been well characterized, the properties of the synaptogenic activities of other synaptic adhesion molecules are largely unknown. In this paper, we now compared characteristics of the synaptogenic activities of Neuroligin1 and LRRTM2 upon overexpression in cultured mouse cortical neurons. Individual cortical neurons were transfected with Neuroligin1 and LRRTM2 expression plasmids, respectively, and synaptic vesicle clustering in contacting axons was examined by immunostaining for the vesicle membrane protein VAMP2. In immature neurons at 6-7 days in vitro (DIV) both Neuroligin1 and LRRTM2 exhibited strong synaptogenic activity. However, upon further neuronal differentiation only LRRTM2 retained significant synaptogenic activity at 12-13 DIV. A similar differential developmental maturation of the synaptogenic activities of Neuroligin1 and LRRTM2 was observed for the induction of glutamatergic synapses, which were detected by co-immunostaining for VGLUT1 and Homer1. Most interestingly, the synaptogenic activity of Neuroligin1 was strongly dependent on the expression and function of the synaptic adhesion molecule N-cadherin in immature neurons. In contrast, the synaptogenic activity of LRRTM2 was independent of N-cadherin expression and function in both immature (6-7 DIV) and more mature neurons (14-15 DIV). Taken together, our results with overexpression in cultured cortical neurons revealed striking differences in the properties of the synaptogenic activities of Neuroligin1 and LRRTM2, although both transsynaptically interact with presynaptic neurexins.

SUBMITTER: Dagar S

PROVIDER: S-EPMC6856695 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Differential Properties of the Synaptogenic Activities of the Neurexin Ligands Neuroligin1 and LRRTM2.

Dagar Sushma S Gottmann Kurt K

Frontiers in molecular neuroscience 20191108

Synaptic cell adhesion molecules are well established to exhibit synaptogenic activity when overexpressed in target cells, indicating that they are involved in formation and functional maturation of synapses. The postsynaptic adhesion proteins Neuroligin1 and LRRTM2 both induce synaptic vesicle clusters in presynaptic axons <i>in vitro</i> by transsynaptically interacting with neurexins. In neurons, this is accompanied by the induction of glutamatergic, but not GABAergic synapses. Although the s ...[more]

PMID: 31780894

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Project description:A new family of cyclometalated ruthenium(II) complexes [Ru(N^N)2(C^N)]+ derived from the ?-extended benzo[h]imidazo[4,5-f]quinolone ligand appended with thienyl groups (n = 1-4, compounds 1-4) was prepared and its members were characterized for their chemical, photophysical, and photobiological properties. The lipophilicities of 1-4, determined as octanol-water partition coefficients (log Po/w), were positive and increased with the number of thienyl units. The absorption and emission bands of the C^N compounds were red-shifted by up to 200 nm relative to the analogous Ru(II) diimine systems. All of the complexes exhibited dual emission with the intraligand fluorescence (1IL, C^N-based) shifting to lower energies with increasing n and the metal-to-ligand charge transfer phosphorescence (3MLCT, N^N-based) remaining unchanged. Compounds 1-3 exhibited excited state absorption (ESA) profiles consistent with lowest-lying 3MLCT states when probed by nanosecond transient absorption (TA) spectroscopy with 532 nm excitation and had contributions from 1IL(C^N) states with 355 nm excitation. These assignments were supported by the lifetimes observed (<10 ns for the 1IL states and around 20 ns for the 3MLCT states) as well as a noticeable ESA for 3 with 355 nm excitation that did not occur with 532 nm excitation. Compound 4 was the only member of the family with two 3MLCT emissive lifetimes (15, 110 ns), and the TA spectra collected with both 355 and 532 nm excitation was assigned to the 3IL state, which was corroborated by its 4-6 ?s lifetime. The ESA for 4 had a rise time of approximately 10 ns and an initial decay of 110 ns, which suggests a possible 3MLCT-3IL excited state equilibrium that results in delayed emission from the 3MLCT state. Compound 4 was nontoxic toward human skin melanoma cells (SKMEL28) in the dark (EC50 = >300 ?M); 1-3 were cytotoxic and yielded EC50 values between 1 and 20 ?M. The photocytotoxicites with visible light ranged from 87 nM with a phototherapeutic index (PI) of 13 for 1 to approximately 1 ?M (PI = >267) for 4. With red light, EC50 values varied from 270 nM (PI = 21) for 3 to 12 ?M for 4 (PI = >25). The larger PIs for 4, especially with visible light, were attributed to the much lower dark cytotoxicity for this compound. Because the dark cytotoxicity contributes substantially to the observed photocytotoxicity for 1-3, it was not possible to assess whether the 3IL state of 4 led to a much more potent phototoxic mechanism in the absence of dark toxicity. There was no stark contrast in cellular uptake and accumulation by laser scanning confocal and differential interference contrast microscopy to explain the large differences in dark toxicities between 1-3 and 4. Nevertheless, the study highlights a new family of Ru(II) C^N complexes where ?-conjugation beyond a certain point results in low dark cytotoxicity with high photocytotoxicity, opposing the notion that cyclometalated Ru(II) systems are too toxic to be phototherapeutic agents.

Dataset Information

Differential Properties of the Synaptogenic Activities of the Neurexin Ligands Neuroligin1 and LRRTM2.

Publications

Differential Properties of the Synaptogenic Activities of the Neurexin Ligands Neuroligin1 and LRRTM2.

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