Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses
- University of California, San Francisco, United States
Figures
Figure 1 with 1 supplement
Neuroligin 2 is the critical neuroligin at inhibitory synapses.
(a) Knockdown of endogenous neuroligins 1–3 reduces inhibitory responses compared to untransfected control cells (*p=0.0391, n = 9). (b) Scatter plot showing reduction in IPSCs in NLGN2 …
Figure 1—figure supplement 1
Validation of NLGN2 knockdown construct and EPSCs for NLGN3 overexpression.
(a) qRT-PCR bar graph shows mean ± s.e.m. of NLGN2 mRNA remaining following NLGN2miR transduction normalized to control GFP transduction (n = 2 technical replicates). (b) Scatter plot showing …
Figure 2 with 1 supplement
Difference between NLGN2 and 3 function at inhibitory synapses resides in the extracellular region.
(a) To determine where this functional difference between NLGN2 and 3 resides, we made chimeric constructs and expressed them on a reduced endogenous neuroligin background. Black TM is transmembrane …
Figure 2—figure supplement 1
Distinct requirements for NLGN function between inhibitory and excitatory synapses.
While expression of a construct containing the NLGN3 extracellular region and NLGN2 cytoplasmic tail failed to rescue IPSCs, it was still able to enhance excitatory synaptic transmission …
Figure 3 with 1 supplement
Identification of critical domain to confer NLGN function at inhibitory synapses.
(a) Schematic of NLGN2 and 3 showing positions of chimera. Yellow are critical dimerization residues, pink is splice site A, grey SP is signal peptide, and black TM is transmembrane domain. (b) …
Figure 3—figure supplement 1
Chimeric extracellular mutants and effect on excitatory transmission.
(a) Alignment of the NLGN2 and NLGN3 extracellular regions. Sequence before residue 52 encompasses the highly dissimilar signal sequence and was not included in alignment. Highlighted in magenta is …
Figure 4 with 1 supplement
While NLGN c-tails do not confer specialization to inhibitory synapses, a c-tail is required for NLGN function.
(a) Alignment of NLGN1-4. Sequences correspond to mouse NLGN1, rat NLGN2, and human NLGN3 and NLGN4. Indicated at residue 133 is where most proximal c-tail truncation was performed. Highlighted in …
Figure 4—figure supplement 1
Further characterization of c-tail deletion mutant.
(a-b) Scatter plots showing overexpression on a wild-type background of (a) NLGN2 (**p=0.0039, n = 9) and (b) NLGN2Δ133 (*p=0.042, n = 11), both potentiate inhibitory responses. (c) Summary of a-b. …
Figure 5 with 1 supplement
Previously identified domains not required for NLGN2 function.
(a–c) Scatter plots showing replacement of endogenous neuroligin with truncation mutants (a) lacking the PDZ domain (NLGN2Δ4) (**p=0.0098, n = 11), (b) truncation of all regions downstream of the …
Figure 5—figure supplement 1
More detailed NLGN alignment showing where all truncations and point mutations were made.
Highlighted in gray is the transmembrane domain. In light red is a conserved residue that is a site of an autism-associated mutation in NLGN4. Highlighted in light purple is the critical region, …
Figure 6 with 1 supplement
Gephyrin-independent NLGN2 function.
(a) Scatter plot showing overexpression of a Gephyrin-miR construct to knockdown gephyrin expression significantly reduces inhibitory synaptic responses (#p=0.0156, n = 8). (b) Scatter plot showing …
Figure 6—figure supplement 1
Characterization of Gephyrin knockdown construct.
(a) Graph shows mean ± s.e.m. of gephyrin mRNA remaining following Gephyrin-miR transduction normalized to control (n = 3 technical replicates). (b) Western blot of lysates taken from dissociated …
Figure 7 with 1 supplement
Identification of critical residues in NLGN2 c-tail.
(a) Scatter plot showing deletion of a region from the most proximal c-tail truncation (NLGN2Δ98–133) failed to enhance inhibitory responses (p=0.1698, n = 17). (b) Further refining this area of …
Figure 7—figure supplement 1
Individual c-tail point mutations do not affect NLGN2 function.
(a–c) Scatter plots showing replacement of endogenous neuroligin with individual mutants of either (a) phospho-null mutation (*p=0.0195, n = 9), (b) phospho-mimic mutation (***p=0.0007, n = 13), or …
Figure 8
Separate gephyrin-dependent and gephyrin-independent mechanisms for neuroligin function at inhibitory synapses.
We propose separate gephyrin-dependent and gephyrin-independent mechanisms for NLGN function at inhibitory synapses. In our NLGN2-R705C-S714A manipulation, we block the gephyrin-independent pathway …
Author response image 1
Dissociated hippocampal rat cultures were prepared at E18.5 and transfected with NLmiRs-GFP and either NLGN2, NLGN2-R705C-S714A, or NLGN2-S714A-Y770A at DIV 8.
After 1 week cells were stained for surface HA (Abcam ab9110, 1:200). HA signal was normalized to GFP. (NLGN2 n=5 cells, NLGN2-R705C-S714A n=7 cells, NLGN2-S714A-Y770A n=7 cells; *p=0.0177, …
Author response image 2
Crystal structure of NLGN2 dimer (in blue) aligned with Neurexin1β structure (in orange).
In red is the critical extracellular domain in NLGN2 identified in our study.
Author response image 3
HEK293T cells were transfected with either GFP, GFP+NLGN2, GFP+NLGN3, or NLGN2+NLGN3.
After 2 days lysates were harvested and incubated in HA-conjugated agarose beads (Sigma-Aldrich, Saint Louis, MO) to immunoprecipitate HA-tagged protein complexes. Samples were run on a 4–12% …
Author response image 4
HEK293T cells were transfected with either GFP, NLGN2+gephyrin, NLGN2R705C+gephryin, or NLGN2S714A+gephyrin.
After 2 days lysates were harvested and incubated in HA-conjugated agarose beads (Sigma) to immunoprecipitate HA-tagged protein complexes. Samples were run on a 4–12% Bis-Tris gel and probed for HA …
Author response image 5
Scatterplot and bar graph showing expression of NLGN2S714A along with NLGN1-3miR and Gephyrin-miR fails to enhance inhibitory responses and is significantly different than expression of full-length NLGN2 on the NLGN1-3miR background (n = 8, **p=0.0023, ****p<0.0001).
Scale bar represents 25pA and 50ms.
