1. Introduction
As aging is a biological phenomenon that can lead to a decline in the physiological functions of the brain, an increase in the number of elderly people in the world termed the “silver tsunami” highlights the present need to understand the processes that characterize early or potentially pathological aging. With increasing age, changes in brain morphology as well as levels of neurotransmitters and hormones have been observed [
1,
2]. These, in large part, correlate with cognitive decline, memory loss and a reduction in executive function. Aging is accompanied by expression changes in multiple genes and particularly in inflammatory genes, which are the most upregulated in the aging brain within several key regions [
3,
4,
5]. Pro-inflammatory cytokines levels have been found to be greater in the brains of old-aged mice (22–24 months) as compared with adults (3–4 months) [
6]. Additionally, peripheral markers of inflammation are elevated in aging people, and appear to act as a risk factor for the development or progression of age-related neurodegenerative disease [
7]. Albeit that aging is the strongest risk factor for neurodegenerative disorders, and whereas aging is not considered a disease, neuroinflammation is one of the key factors that drives neurodegeneration in several brain diseases, as epitomized by Alzheimer’s disease (AD). Neuroinflammation compromises the maintenance of homeostasis as well as neuronal function in the aging brain [
8] and likely drives cognitive and behavioral alterations by multiple mechanisms, among which are the regulation of gene expression, alterations in neuronal function and reduced neurogenesis [
9,
10,
11]. In this light, a vicious circle is established, in which aging may amplify the effects of neuroinflammation on cognition, and inflammation can exacerbate the effects of aging on cognitive decline.
There is established evidence indicating deterioration of cholinergic function during brain aging [
12,
13,
14,
15] and a marked surge in neuroinflammation with senescence. According to the cholinergic hypothesis of AD, a correlation between changes in cholinergic function and cognitive decline has been proposed in dementia and aging. Support for this hypothesis is based on pharmacological research and on histological analysis of brain pathology in AD patients [
16,
17,
18]. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) by inactivating acetylcholine (ACh) can potentially modulate inflammation [
19,
20,
21] as evident in various clinical conditions [
22,
23,
24]. Inhibition of the neuronal AChE enzyme augments ACh levels at the cholinergic synapses, partly counteracts the cholinergic loss and can ameliorate AD cognitive symptoms [
25]. Shytle et al. proposed the presence of a “cholinergic anti-inflammatory pathway” (CAP) within the central nervous system (CNS) mediated by the activation of nicotinic acetylcholine receptors (nAChR) α7, whose dysfunction would tip the balance towards greater inflammation [
26]. Thus, hallmarks of aging and neurodegenerative disorders can be represented by and associated with neuroinflammation and cholinergic dysfunction. The brain is organized into multiple areas with relatively specialized functions, and multiple inter-areal interactions generally underlie its various behaviors and cognitive functions. Function alterations can precede histopathological degeneration in aging brain, suggesting that analysis of brain gene expression may offer unique insights into the molecular mechanisms underlying age-related changes [
27]. Thus, a breakdown in inter-regional coordination is likely an important pathophysiological mechanism that underpins neurodegenerative disorders such as Parkinson’s disease (PD) and AD [
28,
29].
In the hippocampus, aging is associated with neuronal deterioration, evidenced by a reduction of neuronal size and count [
30], decrease of synaptic connections [
31], intracellular pathology [
32] and complexity, and plasticity [
33], suggesting that the hippocampus is particularly exposed to the effects of aging. Although the mechanisms are not yet fully understood, increased neuroinflammation appears to have a key role in these age-related declines and reduced cholinergic function has been related to such increases in neuroinflammation and progressive memory deficits in both normal and accelerated aging, corresponding to deficits in hippocampal neuronal plasticity [
13,
34,
35,
36,
37,
38,
39].
As indicated above, many age-related changes of inflammatory and cholinergic markers have been evaluated, but without highlighting a relationship between them. To understand whether there is an aging-associated inter-relationship between cholinergic and inflammatory changes within the hippocampus, we evaluated this in the spontaneous senescence-accelerated mouse prone-8 (SAMP8) model as it displays a phenotype of accelerated aging with memory, cognitive and behavioral impairments. This murine model is accompanied by molecular features typical of AD and is a valuable facsimile for obtaining more in-depth information on potential age-related neurodegenerative processes [
15,
40,
41,
42]. We additionally used the senescence-accelerated-resistant mouse (SAMR1), which has a similar genetic background and is extensively used as a control model as it shows normal aging characteristics [
43].
In this study, we collected brain and hippocampus samples, as the latter is a particularly important brain area related to learning and memory. This allowed us to perform hippocampal and brain proteomics to define their protein profiling in both SAMP8 and SAMR1 animals, as well as to compare the hippocampus as a brain region involved in cognition and implicated in AD with a composite brain sample that combined multiple other brain regions. Additionally, to better understand alterations in hippocampal processes underlying aging, we integrated proteomic evaluation with gene expression profiles by quantitative Real-Time PCR (qPCR) carried out to evaluate the relationship between the expressions of nAChR cholinergic receptor subtypes with those of the cholinergic esterase enzymes and inflammatory cytokines, interleukin (IL)-1β and tumor necrosis factor (TNF)α, and to investigate the way in which gene expression levels change concurrently with aging.
3. Discussion
The hippocampus is a brain area fundamental to cognition, which possesses a key role in learning and memory, and previous studies have revealed that hippocampal aging is largely similar in humans and in animal models [
44,
45]. During aging, a cholinergic hypofunction, related to progressive memory impairment, has been detected. Normal aging is accompanied by a gradual loss of cholinergic function caused by dendritic, synaptic, and axonal loss as well as a decrease in trophic support, whereas pathological aging has been characterized by neuronal cell loss. Age-related losses of hippocampal-dependent memory may be due to altered synaptic plasticity mechanisms, including long-term potentiation (LTP) that can lead to the dysregulation of inflammatory pathways, neurogenesis, and ultimately to apoptosis [
46,
47,
48]. To date, interactome mapping and network-based examinations of gene-specific targets, with functional significance in hippocampal aging, have not yet been fully explored.
Based on proteomic evaluation, we have characterized the molecular function, biological process and cellular distribution of hippocampal proteins that may be involved in early or pathological aging. We compared these to proteins deriving from a composite (i.e., combined) brain sample containing multiple brain regions, but not hippocampus, to highlight potential key differentially expressed proteins, and evaluated SAMP8 and SAMR1 mice on an alike genetic background as the former displays a phenotype of accelerated aging with ensuing memory, cognitive and behavioral impairments by 6 to 8 months of age [
40,
41,
43].
The Protein Atlas database confirmed a strong expression of proteins was involved in metabolic and cellular processes in neuronal and glial cells of the hippocampus. Functional analysis of our data with the PANTHER database revealed that most of the differentially expressed proteins are involved in metabolic processes important for energy generation, apoptosis, cellular transport, organization of the cytoskeleton and cellular response, thereby highlighting the various molecular mechanisms involved in learning and memory capacity under the influence of the brain aging process in accordance with other studies that highlighted the role of metabolic alterations during aging [
49,
50,
51]. In this regard, in hippocampus of SAMP8 and SAMR1 proteoforms, we found different regulations of proteins involved in the cytoskeleton and cell motility, metabolism, protein biosynthesis and cell response, signal transmission and cellular transport, suggesting a characteristic aging onset.
Sustained release of inflammatory cytokines may promote cognitive aging and diminished memory. Increased IL-1α levels in the hippocampus may be a trigger for impairments in LTP in age- and stress-induced rats [
52]. The heightened secretion of TNFα which can alter astrocyte-neuron signaling and the excitability of hippocampal synapses and promote an increased possibility of engulfment of synapses by activated microglia [
53] could result from a suppressed cholinergic anti-inflammatory pathway.
The nAChRs together with cholinesterase activity, the status of the cholinergic anti-inflammatory pathway, and inflammatory cytokines are important factors in the development of neurodegenerative disorders such as AD, and hence their dysfunction has been extensively studied across several animal models [
54,
55]. It is known that memory deficits in both normal and accelerated aging are characterized by a reduction of the intrinsic neuronal plasticity of the hippocampus, and it has been hypothesized that changes in the expression of ion channels and/or receptors that perform a key role in the regulation of neuronal excitability and plasticity may provide a basis for the onset of memory deficits [
56]. In this context, nAChRs have key roles in development and synaptic plasticity, and alterations in nicotinic mechanisms that participate in learning, memory and attention contribute to a variety of neurological disorders and diseases [
57,
58]. Several lines of evidence have led to the idea that there is a relationship between the activation of nAChRs and the maintenance of cognitive function in aging [
59,
60]. Aberrant alterations in muscarinic and nicotinic receptor expression may hence contribute to cholinergic dysfunction [
61]. Among neuronal nAChRs, receptor proteins that form ligand-gated ion channels in the plasma membranes of neurons, α4-β2 and α7 are broadly expressed in the human brain and are involved in cognitive functions such as attention, learning, and memory [
62]. In this regard, the hippocampus is a region with a high density of nAChRs as well as microglia and astrocytes [
63], and the cholinergic stimulation of the hippocampus not only has direct neuronal effects, but also affects the microglia and astrocytes that may modulate neuronal function. Cognitive dysfunction caused by an interruption of the cholinergic pathway can potentially be ameliorated by activation of nAChRs present within the hippocampus, and since ACh lowers the release of inflammatory cytokines, an inhibition of cholinesterase activity modulates ACh hydrolysis, augmenting its level and thereby implementing its impact on the cholinergic blockade of inflammatory processes [
64]. The activation of α7 is known to alter the phenotype of both macrophages and microglia from a pro-inflammatory macrophage (M)1-like to a more quiescent M2-like phenotype [
65,
66]. Consequently, any dysfunction in nAChRsα7 and related signaling processes could tip the balance towards more inflammation. However, ion channels and receptors are under-represented in many proteomic studies due to the heterogeneous and sparse nature of these proteins, especially when compared to highly abundant mitochondrial and cytosolic proteins [
67]. To overcome this bias, we performed a qPCR, which allowed us to highlight a decreasing trend of β2 nAChR subtype expression in brain of both SAMR1 and SAMP8 mice, with a more significant variation in SAMP8 mice. The nAChR α4 expression was higher in brain tissue of 4-month-aged SAMP8 and not significantly different between SAMP8 and SAMR1 brains at 8 months; however, it spiked in the hippocampus of 8-month SAMP8 mice. Our results agree with the study of Tohgi et al., which showed an unaltered expression of α4 subunit mRNA and a significantly decreased level of β2 subunit mRNA expression in cortex and hippocampus in normal human aging [
68].
The α7 subunits are likely expressed as homomeric assemblies that, when combined with heteromeric α4β2 assemblies, constitute the two major nAChR subtypes expressed in brain. We report the time-dependent up-regulation of the expression of the nAChRα7 subunit mRNA in hippocampus of both SAMR1 and SAMP8 mice with a higher expression in SAMP8 mice. Furthermore, significant differences were detected between hippocampus compared with composite brain, with higher nAChRα7 expression in 4-month-aged and higher nAChRb2 in 8-month-aged SAMP8 mice hippocampus. This suggests that, in SAMP8 mice, an increased nAChRα7 expression in hippocampus could stimulate auto receptor-mediated ACh release to augment the declining cholinergic signal as well as to maintain a stable presence of α7 in the hippocampus and compensate for its decrease in other brain areas. Our data are in accordance with Counts et al., who reported that in hippocampus there was little or no decrease in high-affinity nicotine binding during aging, whereas in the entorhinal cortex and in the presubiculum, a major loss of high-affinity nicotine binding was detected [
69]. In human, a statistically significant association has been found between increasing nAChRα7 mRNA levels and a higher likelihood of AD by the National Institute on Aging and the Reagan Institute Working Group criteria [
69]. The nAChRα7 subtype is widely considered to participate in several mechanisms of neuroprotection [
70,
71], such as in nicotine-mediated neuroprotection against either glutamate excitotoxicity or Aβ peptide challenge [
72,
73], and by blocking the release of pro-inflammatory cytokines [
74].
The greater availability of cholinergic receptors is correlated with an increase in available binding sites and to greater receptor activation. Activation of cholinergic receptors triggers multiple pathways that cause post-translational modifications (PTMs) in multiple proteins to ultimately bring about changes within the nervous system. PTMs mediated by cholinergic receptors, in part, substantially influence the biosynthesis, proteolysis, degradation, and expression of many proteins. Our results showed a similar time-dependent increasing trend for IL-1β and TNFα expression, in line with a study that demonstrated that pro-inflammatory cytokines could influence the outcome of nicotinic receptor assembly and that cytokines modulating the assembly of receptors can influence the response of key neurotransmitter receptors [
75].
Previous studies of the cholinergic system in aging SAMP8 and SAMR1 mice have produced inconsistent results with increases, decreases or no changes reported in pre- and post-synaptic cholinergic parameters [
76,
77]. A decreased choline acetyltransferase (ChAT) activity was reported in two subregions of the hippocampus of SAMP8, but not in the SAMR1 mice, by Strong et al., 2003, and during aging the spontaneous release of [3H]ACh was not significantly different in SAMP8 and SAMR1 mice [
14,
78]. Keeping this in mind, we focused our attention on the expression of pro-inflammatory cytokines and several key cholinergic system components. The CNS is a privileged immunological site, and the inflammatory mediators are relatively low in abundance, and thus, to guarantee high sensitivity, we carefully established and optimized our qPCR settings and were able to show that expression levels of all the evaluated inflammatory cytokines are increased with age.
Other characteristics shared by our SAMP8 mice are the non-significant differences of AChE and BuChE expression in the brain samples between 4 and 8-month-aged SAMP8 and SAMR1 mice. In the hippocampus of 4- and 8-month-aged SAMP8 mice, a significantly higher expression of BuChE was detected with respect to aged-matched SAMR1 mice. These results are partially in accordance with results of Fernández-Gómez et al. that suggested a senescence-induced up-regulation of BuChE activity and invariable expression levels in SAMP8 brains [
15].
A recently proposed role for BuChE as a compensatory mechanism for AChE activity, should it become insufficient, might explain the significant increases of BuChE in contrast to no variation of AChE in hippocampus of SAMP8 with respect to SAMR1 mice, as BuChE has been proposed as a co-regulator of cholinergic transmission in neurons when strongly stimulated and exposed to high amounts of ACh [
25,
79] or in a pathological setting as occurs in AD [
80,
81].
In the brain and hippocampus of SAMP8 mice, the mRNA expression of cholinergic markers was down-regulated over time; to the contrary, a higher expression was detected in the hippocampus of both 4- and 8-month-aged SAMP8 with respect to SAMR1 mice. Proteomic analysis did not highlight the presence of translated protein of cholinergic markers. This absence of correlation between mRNA expression and protein levels did not surprise us. Indeed, several studies have shown a poor or absent correlation between expression levels of protein and mRNA in mammals, and multiple factors such as post-transcriptional modifications of mRNA or protein stability that are not mutually exclusive could explain such poor correlations [
82,
83,
84].
Among numerous factors, translational efficiency for mRNA may be modified by methylation, and actions of miRNA and post-transcriptional modifications of mRNA may be the reasons for low protein with respect to the mRNA level in a tissue [
85]. Protein stability is another factor, as proteins may differ substantially in their in vivo half-lives. Whereas some proteins have a long half-life, others must be immediately destroyed or modified to support their time-sensitive function. Protein damage/degradation by reactive oxygen species (ROS) may contribute more in relation to the reverse result, and, in the brain of animal models, the evaluation of protein expression is complicated by the relatively low and often very heterogeneous expression within tissues of mixed cellular composition.
The complexity of the human genome is enhanced by alternative splicing. This certainly applies to AChE mRNA that is susceptible to alternative splicing [
86], and this leads to three post-transcriptional species that derive from the same gene [
87]. In human brain and leukocytes, six mRNA splice variants of the α7 gene have been identified, although it remains unclear whether several of these transcripts are processed to functional protein [
88]. Indeed, not all splice variants can be translated, and others cannot be readily quantified as they are of a different size, but analysis of large-scale mRNA and protein isoforms expression is beyond the aim of this study and can potentially be performed by proteogenomic assays.
Our results provide evidence that SAMP8 mice have changes in their cholinergic system from 4 to 8 months of age and an increase in pro-inflammatory cytokines expression, in line with reported declines in their cognitive capacity at 4 to 8 months of age [
40,
41,
43].
More detailed future studies of the way in which each of these sets of molecules affects the local network of the hippocampus could potentially augment our understanding of the onset of neurodegenerative diseases and the way these differ from normal aging.