پروتئین پپتید پیشرو (HCNP-PP) مربوط به mRNA تحریک کننده مغز و اعصاب کولینرژیک هیپوکامپوس بالا در آمیگدال در اختلال افسردگی اساسی

The amygdala is innervated by the cholinergic system and is involved in major depressive disorder (MDD). Evidence suggests a hyper-activate cholinergic system in MDD. Hippocampal Cholinergic Neurostimulating Peptide (HCNP) regulates acetylcholine synthesis. The aim of the present work was to investigate expression levels of HCNP-precursor protein (HCNP-pp) mRNA and other cholinergic-related genes in the postmortem amygdala of MDD patients and matched controls (females: N = 16 pairs; males: N = 12 pairs), and in the mouse unpredictable chronic mild stress (UCMS) model that induced elevated anxiety-/depressive-like behaviors (females: N = 6 pairs; males: N = 6 pairs). Results indicate an up-regulation of HCNP-pp mRNA in the amygdala of women with MDD (p < 0.0001), but not males, and of UCMS-exposed mice (males and females; p = 0.037). HCNP-pp protein levels were investigated in the human female cohort, but no difference was found. There were no differences in gene expression of acetylcholinesterase (AChE), muscarinic (mAChRs) or nicotinic receptors (nAChRs) between MDD subjects and controls or UCMS and control mice, except for an up-regulation of AChE in UCMS-exposed mice (males and females; p = 0.044). Exploratory analyses revealed a baseline expression difference of cholinergic signaling-related genes between women and men (p < 0.0001). In conclusion, elevated amygdala HCNP-pp expression may contribute to mechanisms of MDD in women, potentially independently from regulating the cholinergic system. The differential expression of genes between women and men could also contribute to the increased vulnerability of females to develop MDD.

Major Depressive Disorder (MDD) is a severe mental disorder that is often chronic and recurrent and that leads to substantial impairments in an individual's ability to take care of everyday responsibilities. MDD is the leading cause of disability worldwide, as measured by years lost due to disability (WHO, 2008). The World Health Organization ranked MDD as the 3rd leading cause of burden of disease as of 2004, but importantly, projected that MDD would be the number one cause for burden of disease by 2030 (WHO, 2008). In the 1970s, Janowsky et al. first proposed a possible involvement of the cholinergic system in the etiology of MDD (Janowsky et al., 1974 and Janowsky et al., 1972). They hypothesized that a given affective state may represent a balance between central cholinergic and adrenergic neurotransmitter activity in those areas of the brain that regulate affect, with depression being a disease of cholinergic dominance and mania being a disease of adrenergic dominance (Janowsky et al., 1974 and Janowsky et al., 1972). This possible mechanism was recently revisited by Mineur and Piccioto (Mineur and Picciotto, 2010). Neurotransmission of the cholinergic system is carried out by acetylcholine (ACh), which is synthesized by cholineacetyltransferase (ChAT) and degraded by acetylcholinesterase (AChE). The main receptors for ACh are the nicotinic (nAChRs) and muscarinic (mAChRs) receptors. Several lines of evidence suggest involvement of the cholinergic system in MDD. Organophosphate poisoning inhibits AChE, resulting in increased ACh, and can cause depressive-like behavior in humans (Gershon and Shaw, 1961). Additionally, a neural nAChR antagonist reduces anxiety-like behavior in mice (Roni and Rahman, 2011) and an α4β2nAChR partial agonist elicits antidepressant properties in the forced swim test in mice (Zhang et al., 2012). Administration of scopolamine (a mAChR antagonist) showed antidepressant properties in unipolar and bipolar patients (Drevets and Furey, 2010, Furey and Drevets, 2006 and Furey et al., 2010), and MDD patients on both oral scopolamine and citalopram had better remission rates than with citalopram alone (Khajavi et al., 2012). Many MDD patients exhibit sleep disturbances, including a decrease in rapid eye movement (REM) latency. Interestingly, a cholinergic agonist produced a faster induction of REM sleep only in MDD patients and in subjects at high risk for psychiatric disorders (Palagini et al., 2013). Finally, knockdown of AChE in the hippocampus of adult mice increases anxiety- and depression-like behaviors and susceptibility to social stress, which was prevented by fluoxetine (Mineur et al., 2013). Taken together, these results support the idea that hyper-activation of the cholinergic system may be involved in MDD. Hippocampal Cholinergic Neurostimulating Peptide (HCNP) is involved in regulating ACh synthesis in a medial septal nucleus culture system (Ojika et al., 1992) by increasing the levels of ChAT in cholinergic neurons (Uematsu et al., 2009). HCNP is an undecapeptide cleaved from the precursor protein (HCNP-pp) (Otsuka and Ojika, 1996). HCNP-pp is also known as phosphatidylethanolamine-binding protein (PEBP) and Raf kinase inhibitor protein (RKIP) (Sedivy, 2011). The release of HCNP from hippocampal culture is specifically mediated by the NMDAR (Ojika et al., 1998). Results suggest that HCNP/HCNP-pp also acts as a key regulator for differentiation of cultured hippocampal progenitor cells (Sagisaka et al., 2010). At the neural network level, changes in the function of several cortical and subcortical brain regions are thought to underlie the mood regulation deficit in depression (Seminowicz et al., 2004). We previously found differential gene expression in the amygdala of men and women with MDD compared to controls, although with notable sex differences (Guilloux et al., 2012 and Sibille et al., 2009). This is in accordance with neuroimaging studies showing reduced volume or grey matter density of the amygdala in female MDD patients compared to control subjects, with no change in male MDD (Hastings et al., 2004 and Kong et al., 2013). The amygdala receives cholinergic input from the Nucleus Basalis of Meynert (Schafer et al., 1998) and expresses both muscarinic and nicotinic receptors (Klein and Yakel, 2006, McDonald and Mascagni, 2010 and McDonald and Mascagni, 2011). However, the cholinergic system in the amygdala has not been studied in detail in MDD subjects. Here, our working hypothesis is that HCNP-pp expression in the amygdala is involved in the pathogenesis of MDD by regulating the cholinergic system through HCNP. The aim of the present work was to investigate gene expression levels of HCNP-pp and genes involved in the cholinergic system in the postmortem amygdala of MDD patients and matched controls, and in a mouse model that elicits increased anxiety-/depressive-like behaviors.

In summary, we found an up-regulation of HCNP-pp mRNA in the postmortem amygdala of women with MDD but no change at the protein level. Since HCNP-pp is a precursor protein, changes in processing and post-translational modifications occur and may not have been measured here. Two alternate hypotheses/mechanisms are possible: First, even though we did not find a differential expression of acetylcholine receptors in the amygdala between MDD and control subjects, we cannot rule out a cholinergic deregulation in this structure (since different mechanisms can regulate receptor expression or function). In support, chronic administration of nicotine produced an up-regulation of β2α4nAChRs with no change at the mRNA level (Corringer et al., 2006). The amygdala receives cholinergic input from the Nucleus Basalis of Meynert located in the basal forebrain. ChAT is synthetized in the cytoplasm of the cholinergic neuron and is transported through the axon to the nerve terminals where it synthesizes ACh (Oda, 1999). At the same time, the amygdalar complex projects to the basal forebrain, in particular to the cholinergic neurons of the ventrolateral substantia innominata (Jolkkonen et al., 2002). Thus, it is possible that HCNP-pp, in particular HCNP, exerts its action in a different brain region since ChAT mRNA levels were too low in the amygdala in our cohort, and previous studies showed no detection of ChAT mRNA in the amygdala (Oda, 1999). A possible mechanism is that HCNP travels from the amygdala to the basal forebrain where it can regulate the expression of ChAT mRNA. This hypothesis is in accordance with previous experiments showing that an over-expression of HCNP-pp in the hippocampus increases the levels of ChAT in the septal nucleus (Uematsu et al., 2009), the main cholinergic projection to the hippocampus. However, further experiments are needed to test this hypothesis. Although regulation the cholinergic system is the more plausible hypothesis for the function of HCNP-pp, considering that a correlation between HCNP-pp and cholinergic-related genes is observed in men, an alternate hypothesis is that the differential expression of HCNP-pp mRNA is affecting the cellular population in the amygdala. In addition to its role in regulating the cholinergic system, HCNP/HCNP-pp is also involved in differentiation of cultured adult rat hippocampal progenitor cells. More specifically, a down-regulation of HCNP-pp was correlated with an up-regulation of GFAP (astrocyte marker) (Sagisaka et al., 2010). There is evidence of decreased GFAP in the amygdala of MDD patients, possibly indicating a reduction of astrocyte density (Altshuler et al., 2010). Also, a reduction in glia was found in MDD patients (Bowley et al., 2002). Taking this in consideration, the up-regulation of HCNP-pp we observed in the amygdala of women with MDD might be preventing progenitor cells from differentiating into astrocytes. Interestingly, reduced volume of the amygdala was reported in female MDD patients compared to control subjects, with no change in male MDD patients (Hastings et al., 2004). However, when analyzing the expression of GFAP in a previous study with the same samples used in our study, no difference between MDD and control postmortem female amygdala was found (Guilloux et al., 2012).