Fig 2: Impaired synaptic transmission and decreased surface AMPAR levels of Snx6-/- neurons.(A) Hippocampal slice culture from Snx6fl/fl mouse was partially infected with AAV-EGFP-2A-Cre. Lower panel shows a CA1 neuron infected with AAV-EGFP-2A-Cre and an adjacent control neuron (indicated with red arrows) that were chosen to be recorded simultaneously. (B) Dual recording analysis of AMPAR-mediated synaptic responses (n = 18 pairs). Scatterplots show amplitudes of AMPAR eEPSCs (absolute values) for single pairs of neurons (open circles) and mean ± SEM (filled circle) across all neuron pairs collected. The current amplitudes of infected neurons were plotted on the ordinate and the current amplitudes of the control neurons were plotted on the abscissa. Inset shows sample current traces from an infected (green) and a control (black) neurons. Bar graph shows mean ± SEM of AMPAR amplitudes represented in the scatterplots. (C) NMDAR-mediated eEPSCs (n = 12 pairs). (D) Paired-pulse recording of AMPAR eEPSCs (n = 11 pairs). Two identical stimulus pulses were delivered in an interval of 50 ms and AMPAR eEPSCs were recorded at -70 mV. Left are sample traces of eEPSCs from a pair of infected (green) and control neurons. The paired-pulse ratio (PPR) was the enhancement of the second eEPSC relative to the first eEPSC. Bar graph shows mean ± SEM of PPRs. (E) Hippocampal neurons transfected with pLL3.7.1 were fixed on DIV18 and immunostained with antibodies to surface GluN2B. (F) Quantification of puncta number per 100 µm dendrite length and fluorescence mean intensity in spines for surface GluN2B (mean ± SEM, n = 30, N = 3). (G) DIV13 neurons were co-transfected with constructs expressing DsRed and EGFP, EGFP-SNX6, mEmerald-Homer1c-FL or EGFP-Homer1c-C, fixed on DIV18 and immunostained with antibodies to surface GluA1. (H) Quantification of signal intensity and spine distribution of surface GluA1 (mean ± SEM, n = 32, N = 3). (I) Same as (G), except that neurons were immunostained with antibodies to surface GluA2. (J) Quantification of surface GluA2 (mean ± SEM, n = 33, N = 3). (K) Hippocampal neurons from Snx6fl/fl (WT) and Nestin-Cre; Snx6fl/fl (KO) littermates were cultured till DIV16. Surface levels of mGluR5, GluA1 and GluA2 were then measured by cleavable surface biotinylation followed by immunoblotting with antibodies to mGluR5, GluA1 and GluA2. SNX6 serves as a negative control for surface proteins. Shown are immunoblots from two pairs of Snx6fl/fl and Nestin-Cre; Snx6fl/fl littermates. (L) Antibody uptake assay was performed on neurons transfected with pLL3.7.1. Shown are representative images of dendrites immunostained for internalized and surface AMPAR signals. (M) Quantification of AMPAR endocytosis rate (mean ± SEM, 45 dendritic segments, n = 15, N = 3). (N) DIV14 neurons were treated with inverse agonists for mGluRs for 48 hr, fixed and immunostained with antibodies to surface GluA1. (O) Quantification of surface GluA1 (mean ± SEM, n = 30, N = 3). (P) Same as (N), except that neurons were immunostained with antibodies to surface GluA2. (Q) Quantification of surface GluA2 (mean ± SEM, n = 30, N = 3). Bar: 2 µm.DOI: http://dx.doi.org/10.7554/eLife.20991.032

Fig 3: HIP14L deficiency reduces GluN2B palmitoylation and increases 2B-NMDAR surface expression and NMDA-induced cell death in striatal neurons. Each of the representative western blot panels is an example from one blot; the dividing line in (B) indicates where some lanes on the original blot were removed. (A) GluN2B palmitoylation was significantly increased (41.2 ± 5.32%) in striatum from Hip14–/– mice vs. Hip14+/+ control. Data are presented from 8 independent experiments in which tissue from HIP14–/– and wild-type control were paired; lines connect the points from paired data. Bars represent means ± SD *p < 0.05 (Paired t-test, p = 0.0411). (B) GluN2B palmitoylation was significantly decreased in striatum from Hip14L–/– mice (19.1 ± 1.05%) when compared with Hip14L+/+ control. Data are presented from 9 independent experiments, as described in (A). *p < 0.05 (Paired t-test, p = 0.0336). (C) The surface expression of 2B-NMDARs in striatal neurons of MSN-CTX co-cultures from FVB/N mice showed a trend toward an increase with 250 nM Hip14 ASO treatment for 10 days (N = 6/38 cells; n.s. p = 0.0676, Non-parametric test, Mann–Whitney U). Representative images are shown in Supplementary Figure S3A. (D) A trend toward decreased surface 2B-NMDARs was observed in MSN-CTX co-cultured striatal neurons from YAC128 mice with 250 nM Hip14 ASO treatment for 10 days (N = 5/42 cells; n.sp = 0.0770, Non-parametric test, Mann–Whitney U). However, these trends did not reach statistical significance for either genotype. Representative images are shown in Supplementary Figure S3B. (E,F) The surface expression of GFP-GluN2B measured at DIV 18 in striatal neurons of MSN-CTX co-cultures from FVB/N mice was significantly increased (22.6 ± 5.72%) when GFP-GluN2B WT was co-transfected with pSuper-Hip14L siRNA (N = 5/51 cells; **p = 0.0058, non-parametric test, Mann–Whitney U). However, knockdown of HIP14L had no effect on surface expression of GFP-GluN2B in striatal neurons from YAC128 MSN-CTX co-cultures. Representative images are shown in Supplementary Figures 3C,D. (G,H) Neurons were transfected with pSuper-GFP Hip14L siRNA (siRNA+) or pSuper-GFP Scrambled siRNA (control–), treated at DIV 14 with vehicle or 50 µM NMDA for 15 min, returned to conditioned medium at 37°C for 1 h, then fixed and stained with GFP antibody (green, siRNA construct), red stain for TUNEL-positive neurons and DAPI (blue) to identify nuclei. (G) Representative images from cells transfected with pSuper-GFP Hip14L siRNA show a typical TUNEL-positive neuron in the bottom panel (NMDA treated), in which the nucleus is condensed and homogeneously stained with DAPI, and the red TUNEL stain fills the nucleus. (H) The percentage of TUNEL-positive cells among all siRNA-transfected (green) neurons (representing apoptotic cell death) after NMDA treatment was significantly increased upon knockdown of HIP14L in striatal neurons of MSN-CTX co-cultures from FVB/N mice. Experiments were repeated five times and 100–200 cells in each experiment were counted. The results are presented as mean ± SD (one-way ANOVA, *p < 0.05, ***p < 0.001, Bonferroni’s multiple comparison test).

Fig 4: CIE does not impact the expression levels of proteins commonly associated with alterations in excitatory neurotransmission in the vHC. However, in the dHC, the GluA1 subunit is upregulated. Representative western blot examples illustrating the expression of (A) the GluA1 and (B) GluA2 subunit of the AMPA receptor, (E) the GluN1, (F) and GluN2 subunit of the NMDA receptor and (G) SK2. In panels (A,B,E–G) the corresponding expression of actin (normalization protein) is shown in the lower blot panel of each protein of interest. (C,D,H–J) Group data, normalized to actin and the internal control group (Air), for the (C) GluA1 (Air n = 4, CIE n = 4), (D) GluA2 (Air n = 4, CIE n = 4), (H) GluN1 (Air n = 4, CIE n = 4), (I) GluN2B subunit (Air n = 4, CIE n = 4) and (J) the SK2 channel (Air n = 4, CIE n = 4) illustrating an increase in the expression of the GluA1 subunit (Air n = 4, CIE n = 4) in the dHC without changes in other proteins in the dHC and vHC of Air and CIE-treated rats. All data are expressed as the mean ± SEM, *p < 0.05.

Fig 5: Calpain cleavage of GluN2B to isolate Cys cluster II-containing C-terminal fragment reveals reduced palmitoylation in YAC128 striatum. (A) Cartoon indicating relative location of GluN2B Cys clusters I and II and the major calpain cleavage site. Each panel of image data shows results from one representative gel and the dividing line(s) indicate where lanes were removed for ease of comparing genotypes under identical experimental conditions. (B–D) Palmitoylation of Cys cluster II-containing C-terminal fragment was examined by IP/Btn-BMCC method. Representative blots show that palmitoylation (detected by probing with streptavidin Alexa 680, B) of GluN2B Cys cluster II-containing C-terminal fragment as a ratio to total GluN2B levels (detected by C-terminal specific antibody MA1-2014, as shown in C) was significantly decreased in striatum from YAC128 compared to FVB/N wild-type mice. Black arrow = ~62 kDa cluster II-containing C-terminal fragment. (D) Summary graph indicates the corresponding ratio of palmitoylation signal (calculated as in Figure 1) for YAC128 normalized to the FVB/N control. Data presented for striatal tissue from 10 independent experiments; bars represent means ± SD *p < 0.05 (paired t-test) and data points from same experiment are connected by lines. (E–G) Palmitoylation of Cys cluster I-containing N-terminal fragment was examined by ABE/western blotting method. (E) Purified palmitoylated GluN2B was detected with GluN2B N-terminal specific antibody (AGC-003); total GluN2B expression is shown in representative blot in (F), using the same ACG-003 antibody. Black arrow = ~115 kDa cluster I-containing N-terminal fragment. (G) Palmitoylation level of cluster I-containing fragment of GluN2B was calculated as ratio of palmitoylation signal (shown in E) to the total protein signal (in F); summary graph indicates corresponding palmitoylation level in YAC128 normalized to FVB/N, as in Figure 1. Data presented for striatal tissue from 7 independent experiments; bars represent means ± SD and data points from same experiment are connected by a line.