Norbin is an endogenous regulator of metabotropic glutamate receptor 5 signaling

Abstract

Metabotropic glutamate receptor 5 (mGluR5) is highly expressed in the mammalian central nervous system (CNS). It is involved in multiple physiological functions and is a target for treatment of various CNS disorders, including schizophrenia. We report that Norbin, a neuron-specific protein, physically interacts with mGluR5 in vivo, increases the cell surface localization of the receptor, and positively regulates mGluR5 signaling. Genetic deletion of Norbin attenuates mGluR5-dependent stable changes in synaptic function measured as long-term depression or long-term potentiation of synaptic transmission in the hippocampus. As with mGluR5 knockout mice or mice treated with mGluR5-selective antagonists, Norbin knockout mice showed a behavioral phenotype associated with a rodent model of schizophrenia, as indexed by alterations both in sensorimotor gating and psychotomimetic-induced locomotor activity.

Keywords: LTD; LTP; NMDA; Norbin; calcium oscillation; mGluR5; neurochondrin; schizophrenia.

Figure 1. Analysis of the interaction between Norbin and group I mGluRs

(A). Upper panel: yeast two-hybrid screen using the cytoplasmic tail of mGluR5a (826–1171 a.a.) as bait identified three partial cDNA clones of Norbin (preys #32, 67, 69). Numbers represent corresponding amino acid residues. Lower panel: cytoplasmic tails of mGluR1–8 (as indicated) were co-expressed with Norbin C-terminus (499–729 a.a.) in yeast two-hybrid system and their interactions were tested by β-galactosidase assay. (B). Rat hippocampal homogenate was subjected to co-immunoprecipitation using purified antibody to Norbin or control normal rabbit IgG. Samples were analyzed by western blotting using either anti-mGluR5 (upper panel) or anti-Norbin (lower panel) antibodies. (C). Coronal slices (50 μm) of adult mouse brain were fluorescence immunostained with antibody to Norbin. Septum (Sep), nucleus accumbens (NAc), striatum (Str), hippocampus (Hip), dentate gyrus (DG) and amygdala (Amg) are shown with higher magnifications in the insets. (D). Synaptosomal fractions (Syn) from mouse brain homogenate (H) were analyzed by western blotting. S2 and P2, supernatant and pellet after 15,000g centrifugation. (E and F). Cultured DIV21 hippocampal neurons were fixed and double immunostained with antibodies to Norbin and to mGluR5 (labeled by Zenon Alexa Flour 568) (E); and with antibodies to Norbin, to dendritic marker MAP2, or to the spine marker spinophilin (F).

Figure 2. Effects of overexpression of Norbin on mGluR5 signaling

(A). HEK293 cells stably expressing mGluR5 were transiently transfected with Norbin and ratiometric calcium imaging following exposure to DHPG (10 μM) in cells not expressing (upper) or expressing (lower) Norbin is shown. (B). The relative distributions of the number of calcium peaks during 28 minutes of continuous exposure to 10 μM DHPG in cells not expressing (pink) or expressing (green) Norbin. The yellow and blue bars at the bottom of the figure show the number of cells that failed to respond to DHPG. The mean numbers of calcium peaks are displayed as dashed lines. (C). HEK293 cells were transiently transfected with mGluR5-WT, mGluR5-mut1 or mGluR5 mut1/2 alone (left panel) or together with pEGFP-Norbin (right panel). The number of calcium peaks during 20 minutes of continuous exposure to 10 μM DHPG was recorded. Only cells responding to treatment were included and the data were normalized to the mean number of peaks of cells transfected with mGluR5-mut1. Data represent means ± SEM (**p<0.01, Wilcoxon rank sum test).

Figure 3. Effects of Norbin on the abundance of mGluR5 at the cell surface

(A). Biotinylated cell surface proteins from N2a cells transfected with myc-mGluR5 (wild-type, mut1 or mut1/2) and vector or HA-Norbin (upper) and total lysates (lower) were analyzed by western blotting using antibodies to myc and HA. (B). shRNA-724 targeting Norbin or control shRNA was transfected into E18 cortical neurons using Nucleofector Kit. Five days after transfection, cell surface mGluR5 level was examined by surface biotinylation experiments. (C). Binding of [³H]MPEP to membrane fractions from forebrain of WT (○) and Norbin conditional knockout mice (●). Means ± SEM (n=4, *p<0.05 and **p< 0.01, Student's t-test). (D). E18 primary cortical neurons isolated from wild-type and Norbin knockout embyos were subjected to surface biotinylation experiments on DIV7. Biotinylated mGluR5 was detected by western blotting. Presenilin (PS1) was used as control surface protein and β-actin was used as loading control. Right panel: quantification of results from four independent cultures. Data represent means ± SEM (*p<0.05, Student's t-test).

Figure 4. Reduced DHPG-induced LTD, impaired LTP and schizophrenia-like behaviors in Norbin knockout mice

(A). Averaged slopes of fEPSPs were plotted (percentage of pre-DHPG baseline) as a function of time at Schaffer collateral–CA1 synapses before and after DHPG (100 μM, 5 min as indicated by the black bar) treatment for Norbin WT (n=8 slices) or Norbin KO (n=6 slices) mice. Data are means ± SEM. (B). LTP analyzed as averaged slopes of fEPSPs at Schaffer collateral–CA1 synapses before and after tetanic stimulation (blue arrows) for WT (n=12 slices), Norbin KO (n=16 slices) or heterozygous (Het) (n=10 slices) mice. Data are means ± SEM. (C). Upper panel: pre-pulse inhibition of startle in WT (n=19) and Norbin KO mice (n=20). Mice received prepulses of 74 dB and pulses of 120 dB. Data are means ± SEM. *p<0.05, Student's t-test. Lower panel: average startle magnitudes at 120 dB. (D). WT (n=8) and Norbin KO (n=8) mice were successively injected with saline, 0.1 mg/kg and 0.2 mg/kg MK-801 at the indicated times. Locomotor activity was measured by number of beam breaks. (E). WT and Norbin KO mice were injected with saline (WT n=10 and KO n=10) or MPEP (WT n=12 and KO n=10) and 0.2 mg/kg MK-801 successively at the indicated times. Locomotor activity was measured by number of beam breaks.