Most neurotransmitters exert their effects by binding to the corresponding receptor, so we thus paid attention to the changes in related receptors in 3×Tg-AD mice here. Compared to WT, the expression of ADRA1A tended to be decreased (Fig. 5A, B), while the expression of 5HT1A and GABRA2 was significantly down regulated in the hippocampus of 3×Tg-AD mice (Fig. 5A, C, D). The ratios of pGluA1(Ser831)/GluA1 and pCREB/CREB were significantly lower in the hippocampus of 3×Tg-AD mice relative to the corresponding WT controls (Fig. 5E, F, G, and H). In contrast, the expression of H3R was significantly higher in the hippocampus of 3×Tg-AD mice compared to WT (Fig. 5I, J).
DISCUSSION
AD is usually accompanied by depression, leading to exacerbation of this complex and intractable disease. However, their causal relationship and mechanism are still not fully understood. In the present study, we demonstrated that elderly female 3×Tg AD mice showed obvious depression-like behavior, which was involved by the changes in monoamine neurotransmitters and receptors in the hippocampus.
Studies have found that depressive-like behavior is induced in a variety of AD animal models (Kosel et al. 2020). 3×Tg AD mice, a widely used animal model of AD, can simulate cognition deficits and pathological changes of human AD. It has been proved that 18-month-old male 3×Tg AD mice also show depression-like behavior (Romano et al. 2014). However, there are gender differences in AD (Nebel et al. 2018), and the relationship between AD-related depression-like behavior and gender has not been reported. Therefore, female 24-month-old 3×Tg AD mice were used to address this issue. Aged female 3×Tg AD mice displayed impairment of learning and memory in the PAT and locomotor activity in the OFT. Behavioral despair and anhedonia are the characteristics of depression, the former of which is most commonly evaluated using the TST and forced swimming test (FST) (Bogdanova et al. 2013). However, only the TST was performed in this study as the mice drowned in the FST, probably due to physical limitations associated with old age. Another possibility was that the mice lacked a strong will to survive. Regardless, it was observed that the elderly female 3×Tg AD mice showed significantly prolonged immobility time in the TST. These results suggest that aged female 3×Tg AD mice exhibit both cognitive impairment and depression-like behavior.
Depression, a psychiatric disorder with multifactorial etiology, is contributed by the imbalance of neurotransmitters, including the monoaminergic, glutamatergic, and GABAergic signaling systems (Oakes et al. 2017; Chi et al. 2014). Neurotransmitters mediate biological activity in emotional processes through their receptors. In the present study, we detected the neurotransmitters and corresponding receptors related to depression to investigate the mechanism involved in the comorbidity of AD and depression. NE and 5-HT are major monoamine neurotransmitters in the brain; their depletion has been implicated in the pathogenesis of depressive disorders, which has been confirmed in patients and animal models (Hirschfeld 2000; Delgado and Moreno 2000). We demonstrated here that aged female 3×Tg-AD mice displayed significantly lower NE and 5-HT levels in the hippocampus, a key brain region related to cognition and depression (Campbell and Macqueen 2004). Meanwhile, the levels of tyrosine, the precursor of NE synthesis, and VMA, an NE metabolite, were also significantly decreased. These are supported by previous findings that aged male 3×Tg-AD mice displayed significant decreases in NA and 5-HT levels in the prefrontal cortex (Romano et al. 2014). In contrast to NE, the acceleration of 5-HT metabolism led to decreases in 5-HT and increases in its primary metabolite 5-HIAA in the AD mice, while L-tryptophan, the precursor of 5-HT synthesis, was not significantly changed. There appears to be a decrease in NE production while promoting the metabolism of serotonin in AD mice with depression. Both 5-HT receptors and adrenergic receptors contain multiple subtypes, while alpha (1A)-adrenergic receptors (ADRA1A) and serotonin 1A receptor (5-HT1AR) have been reported to be more closely associated with depression. Inhibition of alpha (1)-adrenergic receptor (α1-AR) has been reported to induce significant depressive-like behavior in mice, whereas mice expressing constitutively active mutant α1A-ARs (CAM α1A-AR) exhibit antidepressant-like behavior (Doze et al. 2009). Consistent with this, decreased 5-HT1A receptor density has been observed in major depressive disorder (MDD) in positron emission tomographic (PET) and postmortem studies, while 5-HT1AR deficiency attenuates depression- and anxiety-like behavior in mice (Gorinski et al. 2019). Regardless of the ADRA1A, the 5-HT1A receptor was downregulated in the hippocampus of 3×Tg-AD mice with depression-like behavior, indicating that the regulation of neurotransmitter receptors by pathological changes of AD is not consistent.
Studies have suggested that the imbalance between major excitatory and inhibitory neurotransmitters glutamate and GABA, respectively, plays a role in depression (Alaiyed et al. 2020; Godfrey et al. 2018). However, changes in the two neurotransmitters may vary depending on different studies, some of which show increases (Sanacora et al.2004), while others show decreases (Croarkin et al. 2011; Hasler et al. 2007) or no changes (Truong et al.2021). In elderly female 3×Tg-AD mice, decreased L-glutamic acid and increased GABA levels were observed, but there was no statistical significance; in contrast, the glutamine levels were significantly up-regulated. Furthermore, we also observed that pGluA1(Ser831) was also increased evidently. Notably, glutamate, adenosine triphosphate, and ammonia can be used to synthesize glutamine in astrocytes under the catalysis of glutamine synthetase (GS) (Castegna and Menga 2018). Glutamate A1 receptor (GluA1) is not only one of the-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptors, but also one of the three glutamate ionotropic receptors in presynaptic and postsynaptic membranes. GluA1 is widely expressed in the CNS and plays a unique role in various nervous disorders, especially depression (Zhang and Abdullah 2013). Studies have demonstrated that increased phosphorylation of Ser831-GluA1 alleviates depression by production and maintenance of long-term potentiation (LTP) and long-term depression (LTD), similar to many antidepressants, such as ketamine, lamotrigine and fluoxetine (Rame et al. 2017; Svenningsson et al. 2002). GABAergic dysregulation has been linked to depression in preclinical and clinical studies (Koester et al. 2013; Fatemi et al.2013), and GABAergic effects are mainly mediated via ionotropic GABA(A) receptors, but the specific subtype involved remains unknown. We demonstrated that 3×Tg AD mice displayed downregulation of the GABRA2 receptor in the hippocampus while exhibited depression-like behavior. This is supported by recent findings that global knockout of the GABAAR α2 subunit gene (α2KO) produces anxiety- and depressive-like phenotypes, supporting that α2-containing GABAARs (GABRA2) may be related to depression-like behavior (Vollenweider et al. 2011; Engin et al. 2012; Benham et al. 2017). Our data and these findings suggest that AD-induced depression may be involved by glutamatergic and GABAergic receptors, instead of the corresponding neurotransmitters.
The neuronal histaminergic system is also involved in symptoms of depression. Histamine is a neurotransmitter and is synthesized from the essential amino acid histidine in a reaction catalyzed by histidine decarboxylase (Hdc). Previous studies have shown that chronic brain histamine depletion in adult mice induced depression-like behavior (Yamada et al. 2020). In addition, clinical studies have proved that deficiency of L-histidine is associated with moderate depression in elderly women, while histidine intake improves depression and mental task performance (Solís-Ortiz et al. 2021). In this study, we also found that 3×Tg AD mice display significant increases in histidine, while histamine is not changed, which seems to be inconsistent with the conclusions of previous studies (Yamada et al. 2020; Solís-Ortiz et al. 2021). The feedback regulation of brain neurotransmitters may account for this, but further studies are needed to clarify it. The present study focused on the H3 receptor, as it plays a significant role in the pathogenesis of depression (Pérez-García et al. 1999). Activation of H3 receptors, the autoreceptors in presynaptic membranes of histaminergic neurons, inhibits histamine release. Moreover, some H3 receptor antagonists attenuate depression-like behavior in mice (Iida et al. 2017; Boccella et al. 2021). Give that H3 receptors were significantly overexpressed in 3×Tg-AD mice, we speculate that AD may have little effect on the histamine levels; AD-related depression may be contributed by up-regulating H3 receptors and reducing histamine release from pre-synapses.
Central cholinergic transmission has long been known to be involved in both depression and AD. Cholinergic signaling in the brain is dysregulated in AD (Bekdash 2021). Human and animal studies have demonstrated that hyperactive ACh signaling in the hippocampus contributes to depressive symptoms (Higley and Picciotto 2014). Changes in ACh levels of AD accompanied by depression are rarely reported. Our results showed that ACh levels were decreased significantly in 3×Tg-AD mice with depression-like behavior. This could be induced by Aβ and Tau accumulation as evidence suggests that Aβ and Tau disorder can hijack normal cholinergic mechanisms and degrade the brain cholinergic system (Majdi et al. 2020). This indicates that the abnormal transmission of cholinergic signals may not be the main cause of depression in AD, or there may be a more complex mechanism which needs further studies. Coincidentally, some systematic reviews of randomized control trials do not support the use of cholinesterase inhibitors to treat depressive symptoms in dementia (Ford and Almeida 2017).
We also found that 3×Tg-AD mice displayed high levels of L-arginine in the hippocampus. As far as we know, this has not been investigated. L-Arginine is considered an essential amino acid in early life; it is the precursor of the synthesis of the signaling molecule nitric oxide (NO), which is involved in the pathogenesis of depression and depression-related disorders (Ergün Y and Ergün UG 2007; Dong et al. 2020). L-arginine produces depression-like behavior at high doses such as 1000 mg/kg, but attenuates chronic stress-induced depression-like behavior at low doses such as 10 and 20 mg/kg in rats (Maia et al. 2009). Therefore, depression-like behavior observed in the AD mice may be attributed to the high levels of L-arginine in the hippocampus.
The imbalance of neurotransmitters is undoubtedly important in the pathogenesis of depression. Recent studies have identified molecules and cellular pathways relevant to synaptic plasticity, such as the transcription factor CREB (Blendy 2006). Despite conflicting results, most studies indicate that CREB is upregulated by chronic antidepressant treatment, and increasing CREB levels in the brain of rodents produces antidepressant-like behavior (Keshavarz et al. 2019). The present study showed no significant changes in the expression of CREB, while pCREB was significantly decreased in 3×TgAD mice with depression-like behavior. This seems to be supported by the findings that pCREB, the active form of CREB, is downregulated in patients and animal models of depression (Sulser 2002).