Neuropsin Inactivation Has Protective Effects against Depressive-Like Behaviours and Memory Impairment Induced by Chronic Stress

Mounting evidence suggests the interaction between stress and genetics contribute to the development of depressive symptoms. Currently, the molecular mechanisms mediating this process are poorly understood, hindering the development of new clinical interventions. Here, we investigate the interaction between neuropsin, a serine protease, and chronic stress on the development of depressive-like behaviours in mice. We found no difference in baseline behaviour between neuropsin knockout and wild-type mice. However, our results show that neuropsin knockout mice are protected against the development of depressive-like behaviours and memory impairment following chronic stress. We hypothesised that this difference in behaviour may be due to an interaction between neuropsin and elevated plasma corticosterone. To test this, we subjected mice to chronic corticosterone injections. These injections resulted in changes to hippocampal structure similar to that observed following chronic stress. We found that inactivation of neuropsin limits the extent of these anatomical changes in both the chronic stress and the corticosterone injection exposed cohorts. We next used viral vectors to knockdown or overexpress neuropsin in the hippocampus to confirm the results of the KO study. Additionally, we found that inactivation of neuropsin limited glutamate dysregulation, associated with increased generation of reactive oxygen species, resulting from prolonged elevated plasma corticosterone. In this study, we demonstrate that neuropsin inactivation protects against the impairment of hippocampal functions and the depressive-like behaviour induced by chronic stress or high levels of corticosterone. Consequently, we suggest neuropsin is a potential target for clinical interventions for the management of stress disorders.</p

Inactivation of neuropsin reduces the impairment of spatial memory performance following chronic stress or elevated corticosterone.

<p>(A) There is no significant difference in the water maze training curve between KO and WT mice. In the chronic stress groups, there is also no difference in the training curve. (B) Two weeks later, there is no significant difference in memory as measured by time spent in the target zone and the number of phantom platform crosses; however in the chronic stress group, neuropsin KO mice spent more time in the target zone and made a greater number of phantom platform crosses. (C) In both oil and corticosterone injection groups (20 mg/kg/day) there is no difference in the training curves. (D) KO mice performed significantly better in memory tests that WT following corticosterone injection. n = 10–11 in each group, values represent mean ± SEM. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Primers used for quantification of mRNA expression.

<p>Primers used for quantification of mRNA expression.</p

Neuropsin-deficient mice are resistant to chronic stress or elevated corticosterone induced depressive-like behaviour.

<p>(A, C, E) There is no significant difference between WT and KO mice in the novelty suppressed feeding, forced swim or sucrose preference tests without prior exposure to stress. After chronic stress, neuropsin KO mice approach food pellets more quickly than WT mice in the novelty suppressed feeding test. In the forced swim test, the immobility time of KO mice is shorter than WT mice. KO mice drink more sucrose than WT mice in the sucrose preference test. (B, D, F) There is no significant difference between WT and KO mice in the oil injection group. After chronic corticosterone injection (20 mg/kg/day), neuropsin KO mice show less depressive-like behaviour in novelty suppressed feeding, forced swim test, and sucrose preference test than WT mice. n = 8–10 in each group, values represent mean ± SEM. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Neuropsin-deficient mice are partially protected against the effect on hippocampal neurogenesis elicited by chronic stress or corticosterone injection.

<p>(A) Chronic stress (n = 7–10) or chronic corticosterone injections (n = 6) dramatically decrease hippocampal neurogenesis. Neuropsin KO mice have a higher cell count of DCX positive cells in the dentate gyrus. (B) There are more BrdU positive cells in the dentate gyrus in KO mice than WT mice. (C) There is no significant difference in KI67 cell counts in both groups; n = 7–10 for chronic stress groups, n = 6 for chronic corticosterone groups. Scale bar = 200μm, values represent mean ± SEM. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Inactivation of neuropsin blocks corticosterone induced hippocampal neuronal activity.

<p>(A) One day after chronic corticosterone or oil injection in WT and KO mice, c-Fos positive cells in the dentate gyrus (DG), CA1 and CA3. (B-D) Quantification of c-Fos positive cells in the DG, CA1, and CA3. KO/CORT mice display fewer c-Fos positive cells in the CA1 and CA3 compared to WT/CORT mice. n = 6 in each group, scale bar = 200 μm, values represent mean ± SEM. * <i>p</i> < 0.05, *** <i>p</i> < 0.001.</p

Neuropsin-deficient mice are resistant to glutamate transmission dysregulation induced by chronic elevated plasma corticosterone.

<p>(A) Gene expressions of <i>Klk8</i>, <i>GR</i>, <i>Sgk1</i> and <i>Fkbp5</i> in the hippocampus in WT and KO mice after chronic corticosterone or oil injection. (B) Gene expressions of glutamate receptors, <i>GluN2A</i>, <i>GluN2B</i> and <i>Camk2b</i> and glutamate reuptake (EAAT1), vesicle packing (VGluT1) in hippocampus. (C) Detection of NADPH induced reactive oxygen species in the hippocampus of WT and KO mice after chronic corticosterone or oil injection. n = 9–13 in each group. (D-I) Protein expressions of GR, SGK1, Fkbp5, NR2b, EAAT1 and CamKII measured by western blot assay. n = 6 in each group, values represent mean ± SEM. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Neuropsin interacts with corticosterone to influence dendritic spine density and myelination.

<p>(A, B) The hippocampal dendritic spine density decreases in both WT and KO mice following chronic corticosterone injections but KO mice preserve more dendritic spines compared to WT mice, n = 4, Scale bar = 10μm. (C, D) Immunohistochemistry staining of CNPase in cortex and hippocampus. Scale bar = 200μm. (E, F) After chronic corticosterone injections, KO mice retain a higher expression of <i>Cnp</i> and <i>Mog</i> compared to WT mice in the hippocampus but there is no significant difference in the cortex. n = 6 in each group, values represent mean ± SEM. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p