Neuropsin cleaves EphB2 in the amygdala to control anxiety

Abstract

A minority of individuals experiencing traumatic events develop anxiety disorders. The reason for the lack of correspondence between the prevalence of exposure to psychological trauma and the development of anxiety is unknown. Extracellular proteolysis contributes to fear-associated responses by facilitating neuronal plasticity at the neuron-matrix interface. Here we show in mice that the serine protease neuropsin is critical for stress-related plasticity in the amygdala by regulating the dynamics of the EphB2-NMDA-receptor interaction, the expression of Fkbp5 and anxiety-like behaviour. Stress results in neuropsin-dependent cleavage of EphB2 in the amygdala causing dissociation of EphB2 from the NR1 subunit of the NMDA receptor and promoting membrane turnover of EphB2 receptors. Dynamic EphB2-NR1 interaction enhances NMDA receptor current, induces Fkbp5 gene expression and enhances behavioural signatures of anxiety. On stress, neuropsin-deficient mice do not show EphB2 cleavage and its dissociation from NR1 resulting in a static EphB2-NR1 interaction, attenuated induction of the Fkbp5 gene and low anxiety. The behavioural response to stress can be restored by intra-amygdala injection of neuropsin into neuropsin-deficient mice and disrupted by the injection of either anti-EphB2 antibodies or silencing the Fkbp5 gene in the amygdala of wild-type mice. Our findings establish a novel neuronal pathway linking stress-induced proteolysis of EphB2 in the amygdala to anxiety.

Figure 1. Neuropsin and EphB2 colocalize in neurons of the basolateral complex of the amygdala

(a) Double immunohistochemistry showed neuropsin (green) and EphB2 (red) co-localize in lateral amygdala neurons (arrows show EphB2-rich clusters at neuropsin detection sites). Cells were highlighted with TOTO-3 stain. (b) Triple immunohistochemistry confirmed the presence of neuropsin/EphB2-rich clusters on the neuronal surface and low degree of co-localization with cytoplasmic Fkbp51. (c, d) Western blotting revealed amydala neuropsin upregulation following 6 hour restraint stress (F_{(2, 7)} =8.81; p<0.05). Digits inside columns indicate n number. *p<0.05. Results are shown as mean±SEM.

Figure 2. Neuropsin cleaves EphB2 and regulates its expression both in vitro and in the amygdala after stress

(a, b) EphB2-S band density in SH-SY5Y cells decreased upon 15 min neuropsin treatment (F_(3,18)=11.24; p<0.001). Neuropsin did not cleave other molecules of the same class (b and Suppl. Fig. 3a). (c) Exposure of EphB2-GFP-transfected SH-SY5Y or HEK293 cells to neuropsin (15 or 45 min) resulted in the appearance of a ~70kDa N-terminal EphB2 fragment in the medium (Suppl. Fig. 6). (d, e) A 2-fold increase in the membrane-associated EphB2 in neuropsin−/− (F_{(3, 12)}=6.4; p<0.05 vs non-stressed) but not wild-type mice was observed after stress (p<0.05 vs stressed neuropsin−/− mice). (f) qRT-PCR revealed a 2-fold upregulation of the EphB2 gene expression following 6 hour stress (F_{(3, 23)}=13.48; p<0.001), not observed in neuropsin-deficient animals (p<0.001 vs. stressed wild-type mice). EphB2-S and EphB2-L describe short and long splice variants, respectively. NP – neuropsin. Digits inside columns indicate n number. *p<0.05; **p<0.01; ***p<0.001. Results shown as mean±SEM.

Figure 3. Neuropsin regulates the dynamics of EphB2/NR1 interaction and controls the expression of Fkbp5

(a, b) EphB2 immunoprecipitation (before and after 15 minutes of restraint stress) from amygdalae revealed dissociation of EphB2/NR1 complexes in wild-type (F_{(3, 19)}=4.2; p<0.05), but not neuropsin−/− mice. EphB2/NR1 dissociation was restored in neuropsin−/− mice by intraamygdalar neuropsin injections (F_{(3, 13)}=4.7; p<0.05). Microarray analysis of wild-type and neuropsin−/− amygdalae revealed differential expression of Fkbp5 (heatmap in c, Suppl. Fig. 10). Exon-specific Fkbp5 probes showed an upregulation of the whole transcript (d) confirmed by qRT-PCR (e; F_{(3, 12)}=72.15; p<0.001). qRT-PCR revealed attenuated stress-induced upregulation of Fkbp5 in neuropsin−/− mice (e; p<0.01 for wild-type after stress vs. neuropsin−/− after stress) rescued by intraamygdalar neuropsin injections (F_{(3, 14)}=9.2; p<0.01). (f, g) Fkbp51 protein levels were upregulated in wild-type mice (F_{(3, 14)}=8.95; p<0.001) but not in neuropsin−/− mice by stress. (h) Neuropsin-mediated upregulation of Fkbp5 in amygdala neuronal cultures (F_{(4, 29)}=19.04; p<0.0001) was blocked by anti-EphB2 antibody and mimicked by stimulation of NMDA receptors (i; p<0.05). *p<0.05; **p<0.01; ***p<0.001. Results are shown as mean±SEM. NP- neuropsin. Digits inside columns indicate n number.

Figure 4. Neuropsin controls NMDA receptor current, E-LTP and stress-induced anxiety

(a-c) Whole-cell recordings from basal nucleus neurons of neuropsin −/− animals demonstrated lower NMDA currents compared to wild-type mice. Induction of LTP in the lateral-basal pathway (d) using a strong (e) or weak (f) protocol revealed an impairment of E-LTP in neuropsin−/− mice. The elevated plus maze test following acute or chronic restraint stress demonstrated lack of anxiety in neuropsin −/− mice as indicated by the number of entries into open arms (g). General locomotor activity was similar in both genotypes (h, i). The behavioural phenotype was reversed by bilaterally injecting neuropsin back into the amygdala of neuropsin−/− mice (j). Stress-induced anxiety in wild-type animals was disrupted by blocking EphB2 (k) or silencing the Fkbp5 gene (l) in the amygdala. NP – neuropsin; LA-lateral amygdala; BLA – basal amygdala; CA – central amygdala; MeA – medial amygdala. Digits inside columns or near symbols indicate n number. 6hS – six hour stress, 21dS – 21 days of daily restraint. *p<0.05, **p<0.01, ***p<0.001. Results shown as mean±SEM.