Distinct modulation of microglial amyloid β phagocytosis and migration by neuropeptidesⁱ

Besides the well described pathological hallmarks, the deposition of the amyloid β peptide (Aβ) in fibrillar plaques and neurofibrillar tangles of tau protein, neuroinflammation has a prominent role during Alzheimer disease (AD) [1]. This is expressed by activated microglia and reactive astrocytes localized to amyloid plaques, and the release of biochemical markers, like cytokines, chemokines and nitric oxide, that are found to be increased in the brains of patients with AD[2]. Microglia, the main immune effector of the brain, are able to migrate to sites of Aβ deposition and to eliminate Aβ by phagocytosis upon activation by multiple receptors amongst the toll-like receptors (TLR2, TLR4), scavenger receptor A and CD36.

It is known that certain neuropeptides decline during aging before the onset of AD [3, 4]. Neuropeptides belong to a family of signalling molecules released by neurons acting on cell surface receptors. Their role in inflammation has been widely studied in peripheral tissues [5, 6], but their particular function on glial cells has not been studied in detail so far. There is growing evidence that neuropeptides are able to alter neurodegenerative processes during AD [7‐10]. Strikingly, brain regions like the hippocampus and the cortex that are affected early during AD, are also the regions where neuropeptides like bradykinin and somatostatin, as well as their receptors, are prominently present [11‐13].

Somatostains are cyclic peptides that are widely expressed throughout the brain where they act as neuromodulators [14]. Five G protein-coupled somatostatin receptors have been described (SSTR1-5), which are expressed in distinct yet overlapping patterns within the brain [15]. Of note, two of them, SSTR2 and SSTR4, are expressed in the hippocampus and cortex [16]. In AD, elimination of SST from the cortex and hippocampus is correlated to reduced cognitive function and memory impairment [10, 17]. In addition, SSTRs show marked depletion in the AD brain [18‐20].

Bradykinin (BK) is a short-lived nonapeptide generated from the kininogen precursors that is upregulated after lesions in the CNS [21]. Because of its attenuating effect on the secretion of pro-inflammatory cytokines from microglia BK has anti-inflammatory and thereby neuroprotective properties [21]. Like somatostatin, bradykinin binds to G protein-coupled receptor at the cell surface. Two subtypes of bradykinin receptor, B1 and B2, have been characterized so far. The expression of B2 bradykinin receptors has been confirmed in the brain stem, basal nuclei and cerebral cortex, whereas B1 bradykinin receptors are present in the entorhinal cortex, dentate gyrus, and pyramidal neurons of the hippocampus. Besides neurons and astrocytes, primary microglia have been shown to express BK receptors [22].

Another prominent neuropeptide system in the brain is the endothelin (ET) system. Endothelins are 21 amino acid long cyclic peptides. Currently there are three described isoforms (endothelin 1-3), that derive from precursor proteins by proteolytic processing [23]. They signal through two major receptor subtypes known as ETA and ETB receptors belonging to the superfamily of G-protein coupled receptor [23]. ET-1, ET-3 as well as ET receptors are expressed in glial and neuronal cells [23]. During AD it has been observed that endothelin-1 levels are decreased in the cerebrospinal fluid of AD patients [9] and its expression has been found to be elevated in frontal and occipital cortex in AD [7].

Since there are multiple indications that suggest an important role for neuropeptides in inflammation in general and in particular during the chronic inflammatory processes of AD, we investigated the impact of these signaling molecules on inflammatory core functions of microglial cells.

In order to assess whether neuropeptides are involved in the regulation of microglial functions, we first assessed the presence of the neuropeptide receptors on the cells lines and murine primary cells utilized in the following experiments. BV2, N9 and primary murine microglia expressed the bradykinin receptors 1 and 2, the endothelin receptor A and B as well as the somatostatin receptors 2,3 and 4 (Figure 1). These immunocytochemical experiments document that indeed all the above described neuropeptide receptors can be detected on the microglial cell surface and thus may be involved in mediating the modulating effects on Aβ_1-42 phagocytosis and migration described below.

Microglial Aβ_1-42 phagocytosis was assessed in two microglial cell lines, BV2 and N9 cells as well as primary rat microglia which were incubated with either bradykinin, endothelin or somatostatin for 15 h and subsequently exposed to aggregated and FAM-labelled Aβ_1-42. While concentrations of 10^-9-10^-7 M of bradykinin and somatostatin increased Aβ_1-42 phagocytosis concentration-dependently, endothelin showed the opposite effect (Figure 2). There was no principle difference in quantity of quality regarding the observed effects in microglial cell lines compared to primary rat microglia, suggesting that neuropeptide effects can be studied reliably in the two immortalized cell lines which were subsequently used.

Principally, the observed effects of neuropeptides on microglial Aβ_1-42 uptake after 4 h could also have resulted from an impairment of Aβ_1-42 degradation within microglia instead of an increase of phagocytosis. We therefore tested whether neuropeptides would influence the Aβ_1-42 content of microglia over time after an initial short exposure of cells to the peptide for only 30 min. Importantly, microglial cells did phagocyte Aβ_1-42 within the first 30 min. However, neither bradykinin, endothelin nor somatostatin affected the microglial Aβ content which showed a progressive decline over 4 h, consistent with the hypothesis of an unaffected intracellular degradation (Figure 3). None of the neuropeptides accelerated the decomposition of Aβ_1-42 within microglia. Together, these experiments validate that neuropeptides positively modulate the phagocytosis of Aβ but do not affect its intracellular degradation. Next it was analyzed whether the observed modulation of Aβ_1-42 phagocytosis was preserved when cells were simultaneously pre-exposed to an immune challenge. Therefore, BV2 and N9 cells were incubated for 15 h either with lipopolysaccharide (LPS) alone (0.1 μg/ml) or together with bradykinin, endothelin or somatostatin. LPS stimulation alone increased the phagocytosis of Aβ_1-42 (Figure 4). In contrast to the previously observed stimulatory effect of bradykinin, costimulation of microglial cells with LPS lead to a concentration-dependent decrease of bradykinin stimulated Aβ_1-42 phagocytosis (Figure 4A). LPS also abolished the attenuating effect of endothelin or the enhancing modulation of somatostatin that were observed under non-inflammatory conditions. However, all neuropeptides impaired the LPS-induced increase of Aβ_1-42 phagocytosis in microglial cells.

Nevertheless, the possible effect of neuropeptides on the expression of two major Aβ degrading activities, namely, the insulin degrading enzyme (IDE) and neprylisin (NEP), was investigated under control conditions and in the presence of Aβ_1-42 (Figure 5). While IDE was not found to be influenced by any of the conditions investigated, bradykinin increased the NEP under control conditions (Figure 5B). This effect, however, was abolished when cells were coincubated with Aβ_1-42.

While phagocytosis of Aβ was differentially regulated by neuropeptides, all neuropeptides investigated increased the capacity of microglia to migrate in a more or less concentration-dependent fashion (Figure 6), reaching indeed very strong effects already at the lowest concentrations.

To assess whether Aβ_1-42-induced inflammatory gene transcription is being influenced by neuropeptides Aβ_1-42-stimulated microglial BV2 and N9 cells were incubated with either bradykinin, somatostatin or endothelin. While exposure of cells to Aβ_1-42 alone constantly decreased the expression of cyclooxygenase-1, all other targets investigated including cyclooxygenase-2, tumor necrosis factor-α, interleukin-1β, interleukin-6, inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 and macrophage inflammatory protein-1α were robustly increased (Figure 7). Of the latter, only a few targets were negatively regulated by neuropeptides. Thus, bradykinin reduced iNOS gene transcription in N9 cells (Figure 7A), whereas this phenomenon did not reach the level of statistical significance in BV2 cells. In BV2 cells however, endothelin decreased IL-6 and MCP-1 gene transcription (Figure 7B), a results which was not reproduced in N9 microglial cells. Somatostatin did not affect inflammatory gene transcription of the above targets. In summary this analysis may indicate that the analyzed neuropeptides are no major regulators of inflammatory gene transcription and hence the observed effects on Aβ_1-42 phagocytosis and migration are not influenced by inflammatory mediators.

Microglial cells represent the brain's innate immune system and are actively involved in maintaining parenchymal homeostasis through active tissue scanning and removal of debris by phagocytosis [25]. In neurodegenerative diseases, activation of microglia leads to an alteration of these physiological functions, which in turn may contribute to disease progression and neuronal death. Next to cytokines and chemokines, neurotransmitters including norepinephrine, glutamate and acetylcholine can modulate the inflammatory action of microglia and influence their functions [26, 27]. Thus, neuronal activity and release of these factors may determine the activation status and finally the conditions whether microglia turn towards a beneficial or detrimental phenotype. In AD, various neurotransmitter systems are compromised and may thereby impair microglial functions in response to Aβ deposition including phagocytosis and migration [28]. Impaired microglial phagocytosis of Aβ, however, may directly contribute to disease progression and neuronal dysfunction.

The presence of neuropeptide receptors on microglia suggests that these factors may be actively involved in the regulation of microglial functions [22, 29, 30]. After confirming the expression of bradykinin, endothelin and somatostatin receptors on primary murine microglia as well as on the microglial cell lines, the capacity of microglia to phagocyte fibrillar Aβ after preincubation with increasing concentrations of either one of the respective neuropeptides was studied. Of note, the overall levels of the peptides are low in the normal rodent and human brain. In particular, the basal levels of endothelins have been found to be 0.37 × 10^-9 M in human cerebrospinal fluid [31], but undergo a massive upregulation upon brain injury [32, 33]. Further, the release of somatostatin from incubated rat hypothalamus has been determined to be 3 × 10^-9 M [34]. Finally, the exact levels of brain bradykinin are unknown. Nevertheless, the literature is unanimous in that bradykinin, used at 10^-7 M and higher, stimulates glial inflammation [35]. We therefore used these peptides in the range from 10^-9 to 10^-7 M, corresponding to physiological as well as 10 to 100 fold increased concentrations.

While bradykinin and somatostatin increased microglial Aβ phagocytosis in a concentration-dependent manner, endothelin showed the opposite action, suggesting a distinct regulation of Aβ uptake by neuropeptides. Importantly, none of the investigated peptides altered the intracellular degradation of Aβ thus confirming that the observed differences are solely due to increased uptake by microglia. While there is no previous information on the modulation of microglial phagocytosis by these substances, in general a positive regulation of phagocytosis by bradykinin had been reported in peripheral leucocytes after intravenous application of this neuropeptide in rabbits [36]. In contrast, somatostatin had been found to suppress the phagocytic capacity of macrophages for Leishmania major parasites [37]. Together, these data suggest that the neuropeptides do not generally up- or downregulate phagocytosis, but the later action strongly involves target-specific mechanisms.

Since somatostatin is reduced in AD brain and in CSF of AD patients [38, 39], one can speculate that this reduction contributes to Aβ deposition in the AD brain through impaired microglial phagocytosis along with the previously described negative influence on Aβ degradation by neprilysin [40]. Likewise, the increase of endothelin expression observed in AD frontal and occipital cortex could compromise the effective clearance of the brain from Aβ by reducing microglial phagocytosis [7].

Interestingly, immunostimulation of microglia with bacterial lipopolysaccharide (LPS) almost completely abolished the regulatory effects of neuropeptides on microglial Aβ phagocytosis. Systemic bacterial infection has been reported to aggravate the clinical symptoms of AD patients [41], a phenomenon which may be explained by several mechanisms. However, extrapolating the findings of this study, it can be hypothesized that an additional immune challenge, in this case by LPS, leads to the reduction of beneficial microglial functions evoked neuropeptides such as bradykinin and somatostatin -even if those are decreased in AD brains as stated above, since the increased Aβ uptake observed in response to these neuropeptides is compromised by pre-exposure to LPS.

Since in the CNS migration of microglial towards inflammatory lesion sites plays an important role in resolving neuroinflammation and restoring local homeostasis, the effect of the three neuropeptides on migration were investigated. In contrast to the divergent effects on phagocytosis, all three neuropeptides collectively increased microglial migration. Bradykinin has been shown before to stimulate microglial migration in a concentration- and time-dependent manner and seems to involve the bradykinin 1 receptor [42], however no positive regulation on migration in general, but inhibition of Leishmania major promastigote-induced macrophage migration had been reported previously for somatostatin [43].

Once phagocyted, microglial cells are able to degrade Aβ fibrils [44]. Several enzymatic systems have been described to be involved in this process, but the detailed mechanisms underlying phagolysosomal decomposition have not been elucidated. Both neprilysin (NEP) and insulin degrading enzyme (IDE) play the pivotal role for Aβ degradation in the brain [45]. While NEP is presumably acting within a cell, IDE is also being secreted and involved in extracellular Aβ degradation [46]. To test whether neuropeptides are also influencing the generation of these systems, in the absence or presence of Aβ, microglial expression of these enzymes was detected. In the case of Bradykinin there was a trend to increased levels of IDE, however this effect did not reach the level of statistical significance. Of note, the same neuropeptide increased microglial NEP levels, an effect that disappeared when cells were challenged with Aβ. The latter phenomenon suggests that Aβ has a negative regulatory function on NEP levels. The two other neuropeptides tested did not influence either NEP or IDE levels suggesting that this effect is specific for bradykinin. Since previous studies suggested an anti-inflammatory role of neuropeptides, in particular of bradykinin [47], we analyzed the regulation of inflammatory gene mRNA levels in response to Aβ stimulation and neuropeptide incubation. Under all conditions, incubation of microglial cells with Aβ strongly upregulated inflammatory gene transcription including tumor necrosis factor α, interleukin-1β, interleukin-6, inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, cyclooxygenase-2, while cycloxygenase-1 was downregulated by the identical treatment. However, a significant decrease of inflammatory mRNA levels was only observed in N9 cells, where bradykinin decreased iNOS and in the BV2 cell line, where IL-6 and MCP-1 were negatively controlled by endothelin. It seems possible that the chosen time point have not allowed for the detection of stronger regulatory effects, however, it still suggests that the observed neuropeptide regulation of microglia function is a direct effect and not secondary to chemo- or cytokine regulation.

Together our data suggest that neuropeptides may be critically involved in the modulation and guidance of microglial functions relevant for AD. Since these neuropeptides are generated and secreted from neurons, the latter may be intimately involved in immune cell regulation and modification of the neuroinflammatory component of AD. Future studies are needed to unravel the intracellular signaling pathways involved and to verify these data in appropriate animal models.