Expression Pattern of Scavenger Receptors and Amyloid-β Phagocytosis of Astrocytes and Microglia in Culture are Modified by Acidosis: Implications for Alzheimer’s Disease

Abstract

The pathological hallmarks of Alzheimer’s disease (AD) are amyloid-β (Aβ) plaques, neurofibrillary tangles, and glia activation. The pathology also includes vascular amyloidosis and cerebrovascular disease. Vascular compromise can result in hypoperfusion, local tissue hypoxia, and acidosis. Activated microglia and astrocytes can phagocytose Aβ through membrane receptors that include scavenger receptors. Changes in glial cells induced by extracellular acidosis could play a role in the development of AD. Here, we assess whether extracellular acidosis changes glial cell properties relevant for Aβ clearance capacity. Incubation of glial cells on acidified culture medium (pH 6.9 or 6.5) for 24–48 h resulted in decreased cell diameter, with thinner branches in astrocytes, slight reduction in cell body size in microglia, a transient decrease in astrocyte adhesion to substrates, and a persistent decrease in microglia adhesion compared with control media (pH 7.4). Astrocyte Aβ phagocytosis decreased at pH 6.9 and 6.5, whereas microglia phagocytosis only transiently decreased in acidified media. Scavenger receptors class B member I (SR-BI) increased and scavenger receptors-macrophage receptors with collagenous structures (SR-MARCO) decreased in astrocytes cultured at pH 6.5. In contrast, in microglia exposed to pH 6.5, expression of SR-BI and SR-MARCO increased and fatty acid translocase (CD-36) decreased. In conclusion, the acidic environment changed the adhesiveness and morphology of both microglia and astrocytes, but only astrocytes showed a persistent decrease in Aβ clearance activity. Expression of scavenger receptors was affected differentially in microglia and astrocytes by acidosis. These changes in scavenger receptor patterns can affect the activation of glia and their contribution to neurodegeneration.

Keywords

Acidosis amyloid-β peptide glial activation phagocytosis scavenger receptors

INTRODUCTION

Alzheimer’s disease (AD) is the degenerative disorder that most commonly causes dementia [1]. Risk factors for AD include aging, the ɛ4 allele of apolipoprotein E, atherosclerosis, diabetes, hyperlipidemia, high blood pressure, and neurovascular and cardiovascular diseases [2, 3]. Several of these risk factors can be associated with reduced cerebral perfusion leading to a hypoxic and acidic microenvironment, which in turn can result in oxidative stress, brain tissue damage, and AD progression [4 –8]. Brain tissue acidification can be observed in normal processes like aging and in pathological conditions like ischemia and inflammation. It has been hypothesized that aging and ischemia increase cellular metabolism, accumulation of acidic metabolites, and inactivation of genes responsible for controlling pH, leading to acidification [9]. Acidification may induce cell death and promote development of AD or vascular dementia. In fact, enzymes associated with amyloid-β (Aβ) generation [10], like β-secretase and γ-secretase [11 –13], and changes in amyloid-β protein precursor processing are induced by hypoxia-related increase in lactic acid [14] and aggregation of Aβ [15, 16]. Furthermore, Aβ degradation by insulin-degrading enzymes (IDE) could further decrease under acidic conditions given that the effect of low pH on enzyme formation, assembly, and stability may inactivate IDE [17].

A characteristic feature of inflammation is local acidosis. It has been suggested that acidic microenvironments inhibit immune function in certain respiratory conditions [18] and during neoplastic growth and invasion [19, 20]. So far, no pH data have been reported for chronic neuroinflammatory conditions of the central nervous system, but there are reports indicating that the brains of AD patients have decreased tissue pH in the frontal cortex and caudate nuclei [21, 22], in part due to a greater accumulation of lactate [21]. Acidosis also appears to induce changes in amyloidogenesis and contribute to neuronal death [10, 23]. Increased amyloidogenesis could worsen AD neurodegeneration because Aβ produces cytotoxic activation of microglia, which in turn produces oxygen radicals and cytokines [24]. Nevertheless, as we have shown previously, acidification has a neuroprotective effect for hippocampal neurons exposed to Aβ [25].

Local extracellular pH also fluctuates during normal brain functioning as a consequence of neuronal activity. Neuronal activity increases carbohydrate metabolism, increasing lactic acid, CO₂, and the release of synaptic vesicles containing protons that acidify the synaptic space. In addition, astrocyte and microglia pH regulation involves the contributions of carbonic anhydrase, the Na⁺-HCO₃^– cotransporter, the Na⁺/H⁺ exchanger, and the Na⁺-dependent or Na⁺-independent Cl^-/HCO₃^– antiporters [26 –30]. Acid-sensing or CO₂-sensing receptors are found in neurons and glia. TRP channels endow astrocytes with the capacity to sense CO₂ [31]. Astrocytes in the retrotrapezoid nucleus are capable of sensing increases in CO₂ and H+ and in response increase the cytoplasmic calcium concentration and releasing ATP [32]. Several mechanisms can account for the H+ and CO₂-sensitivity of astrocytes and microglia. Microglia express the voltage-sensitive proton channel Hv1, which under physiological conditions regulates intracellular pH and facilitates NADPH oxidase-dependent generation of reactive oxygen species [33]. Astrocytes and microglia can be depolarized by inhibition of inwardly rectifying potassium (Kir) channels, which contribute to extracellular potassium regulation [28 , 35]. Cx32 has been detected along with other connexions in activated microglia [28], and Cx26, Cx30, and Cx32 are expressed in astrocytes. These connexins have a carbamylation motif, a binding site for CO₂, the activation of which opens Cx hemichannels [36].

Microglia and astrocytes are the major players in neuroinflammation [37, 38]. In AD brains, microglia and astrocytes are activated at the Aβ deposition site closely associated with plaques. When activated, they increase their expression of inducible nitric oxide synthase (iNOS) and they secrete inflammatory molecules like interleukin-1β, MCP-1, and RANTES, among others [39 –43], reviewed in [44]. Glial cell activation can be both deleterious and protective in neurodegenerative diseases, depending on the co-stimulants and temporal context [44 –50]. Activated microglia and astrocytes can phagocytose Aβ in vitro [50, 51]. Aβ phagocytosis appears to be impaired in aged animals [52, 53]. Reduction of microglial cells results in an increased accumulation of Aβ in a murine AD model [47] and impairment of glia function by inflammatory conditions appears to contribute to AD pathogenesis by decreasing Aβ clearance [37 , 54–56] and increasing neurotoxicity [57]. Scavenger receptors (SR) are among the receptors mediating Aβ clearance by glial cells as well as glial cell activation [47 , 59]. Glial cells express scavenger receptor class A (SR-A) [60] and SR-MARCO [61], class B member I (SR-BI) [62], and fatty acid translocase (CD36) [63], the receptor for advanced glycation end products [64], low density lipoprotein receptor-related protein [65], and mannose receptor [66]. Glial cell expression of scavenger receptors is modified by several factors, some depending on acute changes in cerebral homeostasis and on chronic changes like those produced by hypoperfusion, aging, and chronic inflammation [37 , 68].

Changes in SR levels secondary to extracellular acidification could be especially relevant for AD, because of potential changes on both Aβ clearance and their participation in the activation of glial cells. Several pattern recognition receptors including Toll-like receptors (TLR) [69] and SR [58, 70] appear to trigger the activation of specific inflammatory pathways according to the ligand they bind. To evaluate the effect of reduced extracellular pH on glial activity, we determined the expression of SR on astrocyte and microglial cell cultures maintained at pH 6.9 and 6.5, and assessed the effect of pH in Aβ clearance activity. First we assessed whether reduced pH had different effects in the binding of various substrates, indicating that it was affecting specific recognition and not general processes. Next we performed competition experiments in Aβ binding with SR ligands to reveal if SR were indeed mediating interaction with Aβ. Finally, we assessed whether extracellular acidification differentially affected astrocyte and microglia activation, glial cell adhesion, and Aβ phagocytic activity.

MATERIALS AND METHODS

Chemicals and reagents

Poly-L-lysine, fucoidan, polyinosinic acid (Poly-I) and polycytidylic acid (Poly-C), bovine serum albumin (BSA), lidocaine, and trypsin EDTA were purchased from Sigma. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were obtained from Invitrogen. Tissue culture dishes and plastic ware were from Falcon (Franklin Lakes, NJ) and Nalgene Nunc (Rochester, NY). The cytotoxicity detection kit (LDH) was from Roche Applied Science. Other reagents were purchased from Sigma or Merck. The Aβ_1–42 was a generous gift from Dr. Heinz Döbeli (Hoffmann-La Roche, Switzerland). All animal experimentation was performed according to the protocol submitted to and approved by the Animal Research Ethics Committee of the Pontificia Universidad Católica de Chile, and in compliance with the current Chilean law.

Primary glial cell culture

Astrocytes and microglial cell cultures were prepared from the brains of 1–2-day-old neonatal rats [71]. Cortices were minced and incubated with 0.25% trypsin-EDTA in Hanks’ solution (0.4 g/l KCl, 0.06 g/l KH₂PO₄, 0.048 g/l Na₂HPO₄, 8 g/l NaCl, 1 g/l D-glucose, and 3.5 g/l NaHCO₃) for 15 min and mechanically dissociated. Cells (one brain per flask) were seeded in 75-cm² culture flasks coated with poly-L-lysine (Sigma) in complete medium (DMEM/F-12, 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin) and incubated in a water-saturated atmosphere with 5% CO₂ at 37°C. After 14–28 days of culture, flasks were treated with 12 mM lidocaine and shaken at 37°C for 10 min to detach microglia. Astrocytes were obtained by trypsinization. Cells were pre-plated for 1 h; cells that were not adherent were recovered, counted, and seeded for experimental use. This procedure yielded highly enriched astrocyte cultures (95% or more) and microglial cell cultures over 99% pure. Cell typing was done by labeling fixed cultures with fluorescein isothiocyanate (FITC)-conjugated lectin Griffonia simplicifolia (1 : 200; Sigma), which recognizes microglia and an antibody against glial fibrillary acidic protein (GFAP; Dako, Denmark) to identify astrocytes.

Cell adhesion assay at various values of pH

The 96-well tissue culture plate was left uncoated (plastic) or coated with one of the following substrates: 50 μg/ml of poly-L-lysine, 1 μg/ml BSA, 50 μg/ml collagen IV or 10% gelatin, or 25 μg/ml melibiose-BSA (mel-BSA) for 1 h, in Dulbecco’s modified Eagle’s medium and nutrient mixture F-12 (DMEM-F12), with 10% of fetal bovine serum, and then washed with distilled water and air-dried under sterile conditions. For the adhesion assay, 3×10⁴ cells/well were seeded in a 96-well plate with 100 μl of DMEM-F12 equilibrated at various values of pH (7.4; 6.9; 6.5) and placed in an incubator at 37°C for 24 h. To evaluate viability of detached cells, floating cell were recovered and plated at pH 7.4 for an additional 24 h to evaluate their adhesion. The number of cells that were adherent was determined by quantifying the LDH released by the cells after exposure to non-ionic detergent by the Cytotoxicity LDH assay^TM (Roche Molecular Biochemicals) according to the manufacturer’s instructions. A standard curve was generated by plating 0.5–30×10³ cells, which were processed in parallel with each LDH assay. For quantification, adhesion in various matrices at different pH levels was compared with the adhesion of astrocytes and microglia to poly-L-lysine at pH 7.4.

For the SR-A ligand competition assay, glial cells were preincubated in suspension in DMEM-F12+1 mg/ml BSA with the various ligands for scavenger receptors: 200 μg/ml fucoidan, mel-BSA, or poly-I, or 200 μg/ml of the control ligand poly-C, with gentle agitation at 37°C for 1 h. After preincubation, the cells were plated on Aβ-coated wells as previously described.

Aβ phagocytosis

Glass coverslips were placed in 24-well plates and coated with 500 μl of 50 μg/ml poly-L-lysine at 37°C for 24 h, and washed three times with distilled water. Then, 10 μl of 10 μg/ml Cy3-Aβ was placed on the coverslips, and air-dried under sterile conditions. Purified astrocytes and microglia were plated at a density of 3 × 10⁴ cells/well. The phagocytosis assay was run in DMEM-F-12 at varied pH (7.4; 6.9 or 6.5) for 5 h, 24 h or 5 days for microglia, and 24 h, 48 h, or 6 days for astrocytes. After the phagocytosis assay, cells were immunolabeled for cell identity markers or endosomal markers (EEA1; early endosome antigen 1) and Cy3-Aβ (Red) to assess phagocytosis. For quantification, 8 fields per cell culture of 5 independent experiments were photographed and the cells that took up Aβ were quantified with the program Image J (NIH).

Immunofluorescence

After the Aβ phagocytosis assay, astrocytes and microglia were washed with PBS with 1 mM Ca^2 +, and fixed in 2% p-formaldehyde at 20°C for 15 min. Cells were permeabilized with 0.2% Triton X-100 in PBS for 15 min, blocked with 10% goat serum in PBS and incubated at 4°C overnight with rabbit anti-GFAP (1 : 200; Dako), rabbit human anti iNOS (1 : 200; Santa Cruz), monoclonal human anti-EEA1 (1 : 200; kindly provided by Dr. Alfonso Gonzalez, PUC School of Medicine), or Alexa-488-conjugated lectin from Griffonnia simplicifolia (1 : 200; Molecular Probes, Inc., Eugene, OR). Except for samples labeled with lectin, coverslips were washed in PBS and incubated with the corresponding secondary antibody, either anti-rabbit Alexa 488 (1 : 100; Molecular Probes) or mouse anti-human Alexa 546 (1 : 200; Origen) in blocking solution at room temperature for 2.5 h. Nuclei were stained with 0.1 μg/ml Hoechst 33258 (B2883; Sigma). Coverslips were washed in PBS and water and mounted in low fluorescence mounting medium (Dako).

Immunoblot analysis

After cells were exposed to the experimental conditions, they were harvested and lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and protease and phosphatase inhibitors). Protein concentration was determined by the BCA method. The same amount of protein for each sample was added to sample buffer (80 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 2% 2-mercapthoethanol, 0.02% Coomassie Blue G250) and heated for 5 min. SDS-PAGE was performed in 10 and 12% (w/v) polyacrylamide gels and transferred to a PVDF membrane (Thermo Scientific). The membrane was incubated with the primary antibody in 5% milk-PBST buffer: anti-SR-A antibody (1 : 1,000, R&D), anti-SR-B antibody (1 : 2,000; Novus Biologicals), anti-SR-MARCO antibodies (1 : 1,000, Serotec) or anti-CD36 antibody (1 : 1,000; Santa Cruz Biotechnology) overnight. Primary antibodies were rinsed and membranes incubated with the appropriate secondary antibody conjugated with horseradish peroxidase (1 : 5,000 in blocking buffer) for 2 h. Signals were detected by enhanced chemiluminescence in accordance with the manufacturer’s instructions. Positive controls were obtained from adrenal gland (SR-B), liver (SR-A and SR-MARCO), and heart (CD36). Quantification of bands from independent immunoblotting groups were subjected to a densitometry analysis using the Image J software (NIH).

Statistical analysis

Values are expressed as mean ± SD or SEM as indicated. Statistical analysis was performed with an ANOVA, followed by the Newmann–Keul test. Statistical significance was established for p < 0.05.

RESULTS

Astrocytes exposed to an acidic environment showed differential adhesion to various substrates

We evaluated the effect of an acidic environment on the adhesion of astrocytes to various substrates. Changes in astrocyte pH-dependent adhesion were different depending on the type of adhesion surface. Adhesion was maximal on poly-L-lysine at pH 7.4 (Fig. 1A). Adhesion was moderate on plastic, gelatin, and mel-BSA (50%) and poor for BSA (<10%). Adhesion to BSA and plastic did not change significantly at acidic pH. Astrocytes adhesion to poly-L-lysine, gelatin, and mel-BSA was sensitive to pH changes. However, whereas adhesion to poly-L-lysine and gelatin decreased to 80% and 10%, respectively (p < 0.001) with acidification, adhesion to mel-BSA increased by almost 200% when cells were cultured in an acidic environment (p < 0.001; Fig. 1B). These results show that acidic environments have different effects on adhesion to various substrates. For the experiments related to adhesion, the adhesion on poly-L-lysine was considered to represent 100% adhesion. The substrates BSA and collagen IV were non-adherent for astrocytes and microglia, respectively [50].

Extracellular acidosis decreased glial cell adhesion but not cell viability

Glial cells presented morphological changes and decreased cell adhesion when exposed to an acidic environment. Astrocytes at physiological pH (pH 7.4-7.4) had extensive cytoplasm and robust extensions (Fig. 2). However, when changed to an acidic environment for a further 24 h (pH 7.4-6.5), they showed thinner extensions with decreased cell diameter compared with the control condition, compatible with the morphology of activated astrocytes. In contrast, astrocytes that were initially exposed to an acidic environment for 48 h and then transferred to a physiological pH environment for 24 h (pH 6.5-7-4) reverted to their normal morphology. iNOS immunodetection to evaluate glia inflammatory activation revealed that iNOS immunolabeling was slightly higher in astrocytes cultured in acidosis than in those cultured at pH 7.4.

Microglia showed very mild morphological differences when exposed to acidic pH. At physiological pH (7.4-7.4), microglia were round shaped, and after exposure to an acidic environment (7.4-6.5), they were smaller than those under the control condition. When the culture medium was changed to physiological pH for 24 h (6.5-7-4), microglia morphology returned to that under control conditions. Acidosis was not effective as an inflammatory activator of microglia, since iNOS labeling was similar at the different pH levels (Fig. 2).

The adhesion assays showed that astrocyte adhesiveness decreased by 25% in an acidic environment compared with controls (p < 0.001). This loss in adhesiveness was transiently observed at 24 h of culture (Fig. 3A), whereas the adhesion assessed at 48 h was similar at all pH conditions. In contrast, microglial cell adhesion decreased by 40% after 24 h under acidic conditions (p < 0.001) and was further reduced by 70% after 48 h of culture in an acidic environment (p < 0.001; Fig. 3A).

To evaluate if reduced adhesiveness at low pH depended on changes in the adhesion mechanism of glia or on reduced cell viability, detached glial cells from cultures at acidic pH were recovered and cultured at physiological conditions (pH 7.4) for 24 h. These glial cells recovered adhesiveness at pH 7.4, presenting similar viability to that of cells maintained at pH 7.4 (Fig. 3B).

Adhesion of astrocytes and microglia to surfaces coated with Aβ was mediated by SRs

Microglia and astrocytes adhesion to Aβ was concentration dependent. Glial cell adhesion to non-fibrillary Aβ (nfAβ) and fibrillary Aβ (fAβ) was similar up to 1 μg/ml Aβ, whereas at 2.5 μg/ml Aβ, adhesion to fAβ decreased by 30% compared with nfAβ (Fig. 4A,B). Adhesion of astrocytes was 104±3.7% for nfAβ and 77±4% for fAβ (Fig. 4A). Adhesion of microglia reached a maximum of 94±4.3% for nfAβ and 63±3% for fAβ. Decreased adhesion of glial cells to fAβ could depend on the cytotoxicity of fAβ, or on the inflammatory activation of glial cells.

We assessed the ability of different SR ligands to inhibit the adhesion of glial cells to 1 μg/ml Aβ, including fucoidan, a polysaccharide that inhibits SR class A, Poly-I and mel-BSA (chemically modified protein), which binds all SR. The three ligands were capable of inhibiting astrocyte (Fig. 4C) and microglia adhesion (Fig. 4D) to fAβ. Fucoidan inhibited astrocyte adhesion to fAβ by 75±5% and microglial adhesion by 90% ±3. Mel-BSA and Poly-I inhibited astrocyte adhesion by 85% and microglia adhesion by more than 90% (p < 0.001). Poly-C (negative control) did not inhibit astrocytes or microglia adhesion to fAβ. Our results indicate that the adhesion of glial cells to an otherwise non-adhesive substrate containing Aβ is mediated by SR.

Aβ phagocytosis by glial cells decreased in an acidic environment

Differences in adhesion at different pH values may affect Aβ uptake, which in turn probably affect the capacity of glial cells to clear Aβ. The effect of acidosis on phagocytosis was assessed for various durations (from 5 h to 6 days). Astrocyte Aβ uptake was 30 to 40% lower at pH 6.9 and 6.5 than at pH 7.4 (p < 0.001) at 24–48 h in phagocytosis assays. Aβ uptake was still significantly lower in the 6-day assays at pH 6.5 (Fig. 5A). Aβ uptake by microglia was 25 and 35% lower at pH 6.9 and 6.5 (p < 0.01) respectively, in the 5 h assay. In the 24 h assay, Aβ uptake at pH 6.9 was similar to the phagocytosis observed at pH 7.4, but a 10% decrease persisted at pH 6.5 (p < 0.05). However, Aβ phagocytosis was not affected by pH in the 5-day phagocytosis assays (Fig. 5B), indicating that acidic environments have only transient effects on Aβ uptake by microglia.

Acidification changed the expression pattern of scavenger receptors depending on glial cell type

The protein levels of some scavenger receptors in astrocytes were modified by an acidic environment (Fig. 6). There were no significant changes in SR-A (Fig. 6A) or CD-36 (Fig. 6D) expression by astrocytes at acidic conditions. SR-MARCO expression was unaffected at pH 6.9, but decreased by 25% at pH 6.5 (p < 0.05; Fig. 6B). SR-BI expression was 50% higher at pH 6.9 and pH 6.5 than under the control condition (p < 0.05; Fig. 6C).

The presence of scavenger receptors in microglia was also modified by an acidic environment (Fig. 7). Whereas no significant changes were observed in SR-A levels under acidic conditions (Fig. 7A), SR-MARCO increased by 55% at pH 6.9 and by 50% at pH 6.5 (p < 0.01; Fig. 7B), and SR-BI increased by up to 30% at pH 6.5 compared with the control condition (p < 0.001; Fig. 7C). In contrast, CD-36 expression decreased by 20 to 30% after incubation of microglia under acidic conditions (p < 0.01; Fig. 7D).

Aβ phagocytosed by glial cells was observed in the early endosome compartments

In astrocytes and microglia, Cy3-Aβ and early endosomes labeled with EEA1 antibody shared the same plane localization in confocal analysis. The localization was similar at pH 6.5 and pH 7.4 (Fig. 8). Z-scan images taken to verify Aβ uptake revealed that the Cy3-Aβ was internalized by glial cells and not bound to the cell surface (Fig. 8).

DISCUSSION

Deposition of Aβ as senile plaques is a well-described pathological hallmark of AD and neuroinflammation appears to have a major participation in the disease progress [72]. Microglia can phagocytose Aβ [51, 73] and have both a protective and a deleterious effect. The neuroinflammatory response mediated by activated microglia and astrocytes results in increased concentrations of cytokines, chemokines, nitric oxide, and reactive oxidative species [74 –76], all of which are found to be increased in the brain of AD patients [77]. If noxious stimuli fail to be resolved, and a persistent inflammatory condition develops, microglia acquire a cytotoxic activation that could favor the progression of neurodegeneration [57].

We have previously observed that SR are present and participate in the interaction of glial cells with Aβ [50, 59], and regulate inflammatory activation [70]. Here, we show that under acidic conditions like those observed under conditions of impaired perfusion and brain ischemia [4 , 78], the properties of phagocytosis, adhesion, and expression of scavenger receptors of glial cells change, leading to impairment of Aβ clearance. Transient acidification also occurs in response to acute sub-lethal ischemic injury, a condition that can be observed in age-dependent pathophysiological changes. In this sense, acidification could constitute a preconditioning factor for other potentially noxious stimuli or inflammation and Aβ.

Diabetes has been associated as a risk factor for AD [79]. The proposed mechanisms for the association include augmented oxidative stress [80], changes in the inflammatory response [81], cerebrovascular pathology mediated damage [82], and also insulin resistance as a process that impairs brain function [83]. Acidosis may also be a diabetes-associated mechanism favoring AD onset and progression. It has been described that diabetic ketoacidosis leads to glial activation in the brain [84], and reports on a murine model indicate that it could result in decreased pH in the hippocampus [85].

Acidosis at both tissue and cell levels has been detected in the nervous system during diseases such as stroke, traumatic brain injury, epilepsy, Parkinson’s disease, and AD [86, 87]. In ischemia, acidosis is an important component of pathological events leading to brain damage [4, 5]. Moderate ischemia can result in a fall of parenchymal pH to around 6.6 without clear evidence of irreversible cell damage [88], although there are reports that cholinergic neurons die when pH is lowered to 6.8 [23]. In severe ischemia, anaerobic glycolysis leads to accumulation of acids, causing pH to decrease to around 6.0 [88], a condition associated with cell death and irreversible damage. Both ischemia lesions and brain acidosis have been reported in AD [21, 23]. In AD brains, the lysosomal enzyme asparaginyl endopeptidase, is selectively activated and translocated from neuronal lysosomes to the cytoplasm. Asparaginyl endopeptidase cleaves and inhibits key phosphatases, such as the protein phosphatase 2A, which appears to be involved in the abnormal hyperphosphorylation of tau [89]. Prolonged acidosis may in fact contribute to the dysregulation of Aβ and subsequent plaque deposition and cell death of cholinergic neurons. Under acidic conditions (pH 6.0), cholinergic neurons degenerate in brain slices, an effect that is accompanied by aggregation of Aβ peptides [90]. Furthermore, acidosis enhances iron-catalyzed production of reactive oxygen species [91], which in turn favors Aβ aggregation and neurodegenerative changes.

In AD, acidosis also increases potentially amyloidogenic enzymes such as β- and γ-secretase [9, 10], whereas acidosis reduces IDE activity, which is involved in the clearance of Aβ [92, 93]. These changes as a whole could lead to an overproduction of Aβ and promote Aβ accumulation induced by acidosis, facilitating the development of AD.

We observed that decreased adhesion and morphological changes induced by low pH were reversible, returning to basal levels when cells were again plated at physiological pH. In astrocytes, reduction of adhesiveness was transient. In contrast, microglial cell adhesion appeared to be more sensitive to acidification than that of astrocytes. Considering that microglia are naturally moving cells, decreased adhesion could be associated with greater mobility toward injured regions. However, this could also result in impaired recognition of environmental signals. Our in vitro approach has the advantage of allowing us to expose cells to well defined stimuli, but on the other hand, the complex regulatory signaling that is present in the normal in vivo situation is not available.

Changes in glial cell adhesion to substrates associated with acidification were also matrix dependent. The best adhesion was obtained with poly-L-lysine, a cationic polymer associated by epsilon C-bond rather a normal peptide bond (α-C). On other matrices, like gelatin, which is 90% collagen, adhesiveness worsened in an acidic environment, which could depend on protonation of proteins. In contrast, for mel-BSA matrix, which is a BSA modified by negative charges, adhesion increased in an acidic environment. Neutral matrices like BSA were not changed under acidified conditions.

The effect of acidic environments on Aβ phagocytosis by glial cells is especially interesting. Acidification permanently reduced the capacity of astrocytes for Aβ phagocytosis. In contrast, the phagocytic capacity of microglia is only transiently affected by acidification. In the context of an injury, microglia are activated earlier and move more easily to the lesion site than do astrocytes [94]. To orchestrate their response, glial cells have a large battery of membrane receptors, including SR and TLRs [94]. Although astrocytes express receptors similar to those of microglia, microglia appear to be more efficient at Aβ clearance. The difference can be due to the mobility of microglia in reaching Aβ plaques or to the fact that astrocytes express lower levels of TLRs and SR [95]. Low levels or the absence of certain receptors could explain differences in Aβ phagocytosis by glial cells, as well as its modulation by acidic environments. However, given the elevated number of astrocytes, their participation in phagocytosis could be highly relevant for the homeostasis of the brain.

Although our results clearly indicate the negative impact of acidic environments on binding to and phagocytosis of Aβ, we did not establish a clear correlation between the changes in the SR expression in astrocytes and microglia and the functional effect. We propose that the pattern of expression of the various SR is relevant for overall cell activation. We have previously shown that the absence of SR-A results in the dysregulation of inflammatory signaling and the response of microglia [58 , 96]. Furthermore, it has recently been shown that macrophages are modulated by environmental conditions depending on the expression of surface receptors, including SR [97]. The absence of SR-A, SR-B, and CD36 has been associated with increased levels of inflammatory cytokines [98 –100] in various tissues and injury models, suggesting a role for SR in the production and regulation of diverse cytokines and other inflammatory mediators [101 –105]. The absence of SR significantly increases induced inflammation, contributing to inflammation-induced tissue injury [99, 100]. In peritoneal macrophages and macrophage cell lines, the induction of TNFα, IL1β, and NO production by SR ligands appears to depend on the activation of mitogen-activated protein kinases (MAPKs) and NFκB signaling pathways [106 –108].

It is particular important to study the expression of specific receptors that can be involved in Aβ phagocytosis and microglia activation because brain pH is lower in AD patients than in healthy individuals [109]. Acidosis also occurs after ischemia and is associated with neuronal injury [110, 111]. It is unknown if acidosis, in addition to affecting the clearance properties of glial cells, induces other phenotypic cellular changes that enhance neurodegeneration.

SR activation can result in ligand internalization and production of extracellular superoxide by microglia [62]. The class B scavenger receptor CD36 mediates free radical production and tissue injury in cerebral ischemia [112]. Both SR-A [113] and SR-BI [114] mediate adhesion and endocytosis of fibrillary Aβ by microglia. Interestingly, SR-A, SR-BI, and CD36 [59, 63] participate in the production of radical species by microglia in response to Aβ fibrils and other SR ligands [96]. SR-MARCO, an inducible member of the class A scavenger receptor family, has also been implicated in the adhesion of microglia and astrocytes to Aβ [50] and in the mediation of cytoskeleton rearrangements in fibroblast and microglia [115, 116].

There were no significant differences in the relative abundance of SR-A or SR-CD36 in astrocytes. However, SR-MARCO decreased and SR-Bs increased, both significantly, in response to acidic environments, which could explain the persistent decrease in Aβ phagocytosis by astrocytes cultured in acidic environments. In contrast, despite the notable change in cell adhesion when exposed to acidic media, microglia increased relative abundance of SR-BI and SR-MARCO and a slight decrease in CD36. Expression of SR-A in microglia is upregulated in the brains of patients with AD [117], and microglia also upregulate scavenger receptors in response to injury and to cytokines [118]. However, we did not find a significant increase in SR-A in response to acidification for 48 h. It is possible that repeated or longer lasting exposure to acidic environments is required for the modification of SR-A expression.

In conclusion, we have shown here that when glial cells are exposed to acidic extracellular conditions, they modify SR expression, inducing several phenotypic changes, including morphological changes to a more rounded shape, reduced adhesion to substrates, and impaired Aβ phagocytic capability. Modification of SR was a complex response, whereas SR-BI expression increased in glial cells under acidic conditions; SR-MARCO expression increased in microglia but decreased in astrocytes. Changes in SR expression patterns could explain key phenotypic changes observed in acidosis: reduced Aβ phagocytosis and reduced adhesion by astrocytes and microglia (Fig. 9), leading to decreased uptake of Aβ; thus favoring its accumulation. Since several age-related pathologies, including ischemia, trauma, and cardiorespiratory pathology induce tissue acidosis, the study of the changes induced by acidosis in glial cells provides a better understanding of the immune behavior of glial cells associated with an injury. In addition, given the role played by SR in neurodegenerative diseases such as AD, understanding how the environment modifies SR expression and function in glial cells can provide key data in the search for better therapies based on SR function. Authors’ disclosures available online (http://j-alz.com/manuscript-disclosures/16-0083r2).

Footnotes

ACKNOWLEDGMENTS

Supported by Grants Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) 1130874 (JE) and 1131025 (RvB), and the Comisión Nacional de Ciencia y Tecnología (CONICYT) Fellowship 21120013 (FC). We thank Dr. Heinz Döbeli (Hoffmann-La Roche, Basel, Switzerland) for providing the Aβ and Dr. Alfonso Gonzalez (PUC School of Medicine, Santiago, Chile) for kindly providing the human monoclonal anti-EA1.

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