Compensatory redistribution of neuroligins and N-cadherin following deletion of synaptic β1-integrin

Abstract

β1-containing integrins are required for persistent synaptic potentiation in hippocampus and regulate hippocampal-dependent learning. Based largely on indirect evidence, there is a prevailing assumption that β1-integrins are localized at synapses, where they contribute to synapse adhesion and signaling, but this has not been examined directly. Here we investigate the fine localization of β1-integrin in adult mouse hippocampus using high-resolution immunogold labeling, with a particular emphasis on synaptic labeling patterns. We find that β1-integrins localize to synapses in CA1 and are concentrated postsynaptically. At the postsynaptic membrane, β1-integrins are found more commonly clustered near active zone centers rather than at the peripheral edges. In mice harboring a conditional deletion of β1-integrins, labeling for N-cadherin and neuroligins increases. Western blots show increased levels of N-cadherin in total lysates and neuroligins increase selectively in synaptosomes. These data suggest there is a dynamic, compensatory adjustment of synaptic adhesion. Such adjustment is specific only for certain cell adhesion molecules (CAMs), because labeling for SynCAM is unchanged. Together, our findings demonstrate unequivocally that β1-integrin is an integral synaptic adhesion protein, and suggest that adhesive function at the synapse reflects a cooperative and dynamic network of multiple CAM families.

Figure 1. Synaptic distribution of β1-integrin labeling

Immunogold labeling for β1-integrins in ultrathin sections using monoclonal antibody N29 (A, B) or p4C10 (C). Presynaptic terminals are shaded pink, and postsynaptic terminals, green. Gold particles are most commonly clustered at synaptic clefts (arrows) with a bias toward the postsynaptic density. Occasional particles are also found presynaptically (arrowheads in A). Dot plot (D) shows the distribution of β1-integrin immunogold labeling at synapses in all three control mice with respect to the peripheral edge (0) and center (0.5). Each synapse has been plotted along a line on the y axis. There is a distribution bias toward synapse centers. Magnification bar (A–C) = 500nm.

Figure 2. Immunogold labeling for synaptic CAMs at synapses lacking β1-integrins

Immunogold labeling for SynCAMs (A, D), N-cadherin (B, E) and Neuroligins (C, F) in control mice (CON, Cre/+; ++) (A–C) and conditional β1-integrin knockouts (cKO) (D–F). As expected immunogold labeling is largely synaptic for all of them and qualitatively, there is no effect of β1-integrin ablation. Magnification bar = 100nm.

Figure 3. Quantitative analysis of β1-integrin and other synaptic CAM labeling

(A) The percentage of synapses immunogold-labeled for β1-integrins, SynCAMs, N-cadherin and Neuroligins in CON mice (gray bars) and cKO mice (white bars) ± sem. Labeling in CON and cKO was compared using t-tests (100 synapses/mouse in 3 – 4 mice), *p ≤ 0.01. Histograms (B–D) show percentage of immunolabeled synapses having the given immunogold particle numbers in CON and cKO mice. Antibodies used are indicated in graph titles. Abbreviations: β1-integrins (βint), N-cadherin (Ncad), Neuroligins (Nlgs).

Figure 4

Increased levels of N-cadherin and Neuroligins in β1-integrin cKO. (A) Western blots of hippocampal lysates (L) and synaptosome fractions (S) taken from cKO, CON mice (Cre/+; ++) or floxed β1-integrin mice (floxβ1/floxβ1) (mixed littermates). N-cadherin appears in the lysates only in cKO, Neuroligin levels are increased in synaptosomes from cKOs only, and SynCAM remains unchanged (SynCAM 49 and SynCAM 100 refer to the sizes (kD) of the bands quantified and likely correspond to SynCAMs-3, and -1, respectively). PSD95 labeling was used to confirm the enrichment of synapses in the synaptosome fraction and tubulin was used as a loading control. (B) Film densitometry of synaptosome fractions shows Neuroligins to be significantly enriched in cKOs. Mean density was normalized to tubulin and expressed as a ratio to control. *p < .005 ANOVA, p<.05, Dunnett’s multiple comparison test.

Figure 5. Stable distribution of gold particles at synapses lacking β1-integrins

Dot plots permit qualitative comparison of immunogold particle distribution at synapses in con (Cre/+; ++) and cKO mice. Labeling convention is as in Figure 1D and antibodies used are indicated in titles. Neuroligin shows a modest decrease at the synapse periphery and an increase, centrally in cKOs.

Figure 6. Synaptic structural stability in absence of β1-integrins

Electron microscopic images of CA1 synapses in control mice (A–C) or conditional β1-integrin knockout mice (D–F) stained with ePTA (A, B, D, E) or BiUL (C, F). Arrows in A and C denote intercleft lines, and those in B and E point out presynaptic dense projections. All of the ePTA stained material shows prominent postsynaptic densities as well. Individual elements are more difficult to see with BiUL staining, but presynaptic densities, an intercleft line (arrow in C and F) and postsynaptic densities are clearly evident. In all images, the presynaptic terminal is oriented above the postsynaptic density. Magnification bar = 100nm.