In this study, we identify for the first time that the antidepressant-induced changes in neurogenesis are dependent on the GR. Specifically, the selective serotonin reuptake inhibitor antidepressant, sertraline, increases neuronal differentiation and promotes neuronal maturation of human hippocampal progenitor cells via a GR-dependent mechanism that is associated with GR phosphorylation via PKA signaling. Interestingly, this effect is only observed when sertraline is present during the proliferation phase, and it is accompanied by exit of cells from the cell cycle, as shown by reduced proliferation and increased GR-dependent expression of the CDK2 inhibitors, p27Kip1 and p57Kip2.
We and others have previously shown that antidepressants, including selective serotonin reuptake inhibitors, induce GR nuclear translocation,50, 21, 22
modulate GR-dependent gene transcription,21, 24, 50, 30
and change GR expression in cell culture,51, 52, 53, 54, 55, 19
animals19, 52, 56, 57, 58, 59, 60, 61, 36
and humans.19, 62, 63, 64
In this study, we identify for the first time a critical role for the GR in the antidepressant effects on neurogenesis: first, the sertraline-induced changes in neuronal differentiation and progenitor cell proliferation are abolished by the GR antagonist, RU486; and second, sertraline modulates GR phosphorylation, induces GR transactivation and changes the expression of GR-regulated genes relevant to neurogenesis (see below).
Surprisingly, sertraline (like amitriptyline and clomipramine) increases proliferation only in the presence of glucocorticoids (dexamethasone or cortisol), but not by itself. The strength of our in vitro
model is that it separates the direct effects of antidepressants on progenitor cells from the indirect effects, which occur in vivo
. Notably, increased hippocampal cell proliferation by antidepressant treatment in animals occurs always in the context of endogenous glucocorticoid production—which is not present in our in vitro
system, unless when cells are co-treated. Indeed, some animal studies have reported that administration of exogenous glucocorticoids, or circadian fluctuations of endogenous glucocorticoids, are required for increased hippocampal cell proliferation upon antidepressant treatment.12, 17
Moreover, a study on human post-mortem brain tissue has found that antidepressants increase the number of neural progenitor cells in patients with major depression to levels above those present in controls, and depressed patients are generally characterized by elevated endogenous levels of glucocorticoids.4
Taken together, our in vitro
findings and the above mentioned in vivo
studies indicate a pivotal role of the GR in the effects of antidepressants on hippocampal progenitor cells. It is important, however, to emphasize that our findings also demonstrate that the molecular processes that lead to increased neuronal differentiation are activated directly by antidepressants alone and do not require glucocorticoids. Our data suggest that such complex GR-dependent regulation of cell fate is the result of differential GR phosphorylation and GR-dependent gene expression by antidepressants, glucocorticoids and by antidepressant and glucocorticoid co-treatment. Indeed, different GR phosphoisoforms have been reported to selectively occupy promoters of different GR target genes, which may therefore explain the diverse GR-dependent effects on gene expression and neurogenesis that we observed in our study (see below).26, 65, 66, 67, 68
Further investigation of the molecular signaling mechanisms, which underlie the effect of antidepressants on neurogenesis showed that sertraline increases GR transactivation after 12
h of treatment. At this time-point, expression of the CDK2 inhibitors, p27Kip1
, was strongly increased, and this increase was blocked by RU486. p27Kip1
are GR-target genes that promote cell cycle exit and increase neuronal differentiation in the developing rat brain.39, 38, 69
Therefore, increased expression of these genes is consistent with our findings (discussed above), which show that sertraline decreases proliferation and initiates neuronal differentiation already during the proliferation phase. It is also of note that sertraline decreased GR expression (both at the mRNA and the protein level) at the same time point at which it induced GR transactivation. This is in line with reports from the literature in both cell culture and animal studies,23, 51, 61
and probably results from the increased GR transactivation.19, 23, 24
Finally, sertraline increased the expression of p11 and ß-arrestin-2, which have both recently been implicated in the antidepressant effects on neurogenesis in vivo
.12, 40, 42, 43
Interestingly, the cell cycle-promoting genes, CCND1 and HDM2, were upregulated only by sertraline and dexamethasone co-treatment, the only condition which increases cell proliferation. In contrast, dexamethasone increased expression of the cell cycle-inhibiting genes, FOXO1 and GADD45B, which may explain the reduced cell proliferation and reduced neuronal differentiation with this treatment. See also Supplementary Discussion (‘Gene interactions') and Supplementary Figure 13 for further discussion and a summary model.
Previous studies have proposed that antidepressants modulate GR-function by cAMP/PKA signaling.30, 31
Our data confirm and extend this model, by showing that the GR-dependent changes in neurogenesis upon sertraline treatment are mediated by PKA. Specifically, the PDE4-inhibitor, rolipram, which increases cAMP levels and thus leads to higher PKA activity, mimics and enhances the effects of sertraline on neuronal differentiation and progenitor proliferation. Accordingly, the PKA inhibitor, H89, inhibits the effects of sertraline. Furthermore, the sertraline-induced changes in GR phosphorylation and GR transactivation are also abolished by inhibition of PKA. One potential mechanism by which sertraline may cause this PKA activation is by liberating G-protein alpha(s) subunits from membrane-associated lipid rafts.70, 71, 72, 73
The subsequent cAMP production and PKA activation may then ultimately induce the GR phosphorylation, GR transactivation and GR-dependent gene transcription, which we observed in our study.19, 66, 74, 25, 26
Interestingly, such a mechanism may be independent of monoamine reuptake systems, and could thus explain how neurogenesis is modulated by antidepressants of different pharmacological classes3
(and, in our experiments, by both the selective serotonin reuptake inhibitor, sertraline and the tricyclic antidepressants, amitriptyline and clomipramine).
In conclusion, our data suggest that anti-depressants regulate differentiation and proliferation of human hippocampal progenitor cells by a GR-dependent mechanism that requires PKA signaling. This is accompanied by changes in GR phosphorylation and GR-dependent gene transcription, including increased expression of p27Kip1 and p57Kip2. Of note, our findings point toward a complex regulation of neurogenesis by antidepressants, with different GR-dependent mechanisms that lead to enhanced cell proliferation without changes in neuronal differentiation, or enhanced neuronal differentiation in the presence of decreased cell proliferation. It will be important for future studies to elucidate the impact of progenitor proliferation vs neuronal differentiation on depression and other mental illnesses. Moreover, modulation of GR phosphorylation and GR-dependent gene transcription may represent a future strategy of antidepressant drug treatment to overcome neurogenesis-related disturbances in depression.