Bangladesh J Pharmacol. 2016; 11: S92-S97

Available Online: 6 March 2016

DOI: 10.3329/bjp.v11iS1.26412

Amyloid precursor protein in peripheral granulocytes as a potential biomarker for Alzheimer’s disease

Abstract

The aim of this study was to assess the potential of amyloid precursor protein in peripheral granulocytes as a diagnostic biomarker for early detection of Alzheimer’s disease. Immunohistochemistry and flow cytometry were used to evaluate amyloid precursor protein expression levels and subcellular localization in Alzheimer’s disease. Much higher amyloid precursor protein expression was observed in some leukocytes from Alzheimer’s disease patients, compared with samples from non-Alzheimer’s disease controls. In addition, flow cytometry data indicated significantly higher amyloid precursor protein expression in granulocytes from Alzheimer’s disease patients compared with control values. No statistically significant differences in amyloid precursor protein expression were obtained in lymphocytes or monocytes between the patient groups. In conclusion, amyloid precursor protein expression level in peripheral blood granulocyte is a potential biomarker for early diagnosis of Alzheimer’s disease.

Introduction

Peripheral blood samples are more convenient for diagnosis compared with other tissues such as brain tissues or CSF. Routine diagnosis of Alzheimer's disease and mixed forms of dementia from CSF has several draw-backs: Lumbar puncture and CSF collection is an invasive method with potential adverse effects; screening of patients is often difficult, and follow-up analysis over several years is problematic. Consequently, identifying useful Alzheimer's disease biomarkers from peripheral blood to provide insights on Alzheimer's disease diagnosis has gained increasing attention (Li et al., 1999). Regarding APP in peripheral blood as a potential biomarker for the diagnosis of Alzheimer's disease, some interesting data have been reported (Borroni et al., 2002; Mukaetova-Ladinska et al., 2012). Other researchers investigated APP-N expression in platelets, and found that APP-N levels were negatively correlated with cognitive scores (Mangia-lasche et al., 2013) and MCI with nearly 90% accuracy.

Our preliminary data showed that APP expression in peripheral blood cells was increased in Alzheimer's disease patients compared to non-Alzheimer's disease controls (data not shown). The APP protein plays an important role in the pathogenesis of Alzheimer's disease, and APP changes appear very early within and outside the central nervous system in patients with Alzheimer's disease (Shad et al., 2013). To further explore the effectiveness of peripheral blood cell APP as an Alzheimer's disease biomarker, we assessed the expression levels and sub-cellular localization of APP in peripheral blood cells by immunochemistry/immunofluorescence and flow cytometry techniques. Our findings indicate that APP expression in peripheral granulocytes is a potential biomarker for Alzheimer’s disease, providing a basis for Alzheimer's disease prevention and cure.

Materials and Methods

Cases

Samples were obtained from a total of 18 individuals. All specimens were collected with informed consent and under IRB-approved protocols.

A total of 9 newly diagnosed Alzheimer's disease patients (4 males and 5 females; 53-87 years old) from Jiangbin Hospital (ZhenJiang City, Jiangsu Province) were randomly selected. Patients had no previous drug treatment and fulfilled the American NINCDS- ADRDA Alzheimer’s criteria. Each patient underwent the minimum mental state examination which was scored. Combined with electroencephalography, CT, MRI or SPECT, patients with other types of dementia were excluded.

The control group comprised randomly selected healthy people (males 4; females 5) at the Jiangbin Hospital in ZhenJiang. They were 52-85 years old, without Alzheimer's disease or other diseases diagnosed by physical examination.

Methods

Immunohistochemistry

Blood was collected into tubes containing sodium citrate. Leukocytes were then isolated by lysing RBC with BD Pharm LyseTM lysing buffer (BD Biosciences, USA), followed by centrifugation at 2,000 rpm for 5 min. After a series of washes in PBS, cells were re-suspended in PBS. Cells were then applied onto poly-L-lysine coated glass slides, which were air dried and stored at room temperature until use. For IHC slides were washed with PBS and fixed for 15 min with 3.7% paraformaldehyde in PBS. After sequential rinsing with PBS and PBS + 0.5% Triton X–100, and blocking with BSA blocking buffer, slides were incubated overnight in a humid chamber at 40°C with rabbit polyclonal primary antibodies targeted at the N-terminus of APP (1:100, Abgent, USA). After washing, bound primary antibodies were detected either by FITC-conjugated (Sigma) or AP-conjugated (Jackson) secondary antibodies. The BCIP/NBT kit was used in alkaline phosphatase (AP)-based immunoblotting detection. Images were acquired on an Olympus IX71 microscope.

For statistical analysis, t-test was used to compare the expression of APP in different cell types between AD patients and non-dementia controls.

Flow cytometry

APP protein expression levels in peripheral blood cells were analyzed precisely using flow cytometry. 100 µL of peripheral blood from Alzheimer's disease and control patients, respectively, were assessed. After red blood cell lysis with BD Pharm LyseTM lysing buffer (BD Biosciences, USA), leukocytes were treated with 2% paraformaldehyde, and permeabilized with 90% cold methanol. Primary antibody specific to an APP amino-terminal epitope (Abgent, USA) and FITC-conjugated secondary antibodies (Sigma) were used. Antibody-bound cells were finally re-suspended in 1 mL PBS. The fluorescence of 10,000 cells was measured using FACS Calibur (BD Bioscience, USA). FITC green fluorescence (APP signal) was analyzed within the context of a forward scatter (FSC) and side scatter (SSC) gate established to include granulocytes, monocytes and lymphocytes. Fluorescence intensity data were express-ed as geometrical mean (GEO mean) for each cell type.

Results

Higher APP expression in some peripheral blood leukocytes from Alzheimer's disease patients

Leukocyte APP protein expression in samples from Alzheimer's disease patients and controls was assessed by fluorescence inverted microscope after immunohistochemical staining (Figure 1, 400x magnification). Figure 1 A and B show immunofluorescence data, while Figure 1C and D depict alkaline phosphatase staining results. Although basal expression of the APP protein was found in the control group, the intensity of APP signals was higher in some cell types from Alzheimer's disease patient samples. Interestingly, APP was mainly expressed in the cell membrane and/or cytoplasm for most positive cells, with the cellular expression often not evenly distributed.

Granulocytes from Alzheimer's disease patients express significantly higher amounts of APP compared with control values

Flow cytometry was used to assess APP expression in peripheral blood leukocytes. Blood cells from AD patients and healthy individuals (n = 9) were assessed by flow cytometry after fluorescence labeling. Figure 2A shows the clustering of various types of peripheral blood cells. APP expression levels in AD patients and controls were detected and compared for lymphocytes, monocytes, and granulocytes. Interestingly, APP expression levels in lymphocytes, monocytes, and neutrophils from Alzheimer's disease patients were all higher compared with control values (Figure 2B and C).

Independent sample t-test (Table I) was conducted to compare the Alzheimer's disease and control groups, with 95% confidence intervals determined. T values of 1.045 (p = 0.312) and 1.264 (p = 0.224) were obtained for lymphocytes and monocytes, suggesting a higher APP expression in lymphocytes and monocytes from patient.

For statistical analysis, t-test was used to compare the expression of APP in different cell types between AD patients and non-dementia controls.

Flow cytometry

APP protein expression levels in peripheral blood cells were analyzed precisely using flow cytometry. 100 µL of peripheral blood from Alzheimer's disease and control patients, respectively, were assessed. After red blood cell lysis with BD Pharm LyseTM lysing buffer (BD Biosciences, USA), leukocytes were treated with 2% paraformaldehyde, and permeabilized with 90% cold methanol. Primary antibody specific to an APP amino-terminal epitope (Abgent, USA) and FITC-conjugated secondary antibodies (Sigma) were used. Antibody-bound cells were finally re-suspended in 1 mL PBS. The fluorescence of 10,000 cells was measured using FACS Calibur (BD Bioscience, USA). FITC green fluorescence (APP signal) was analyzed within the context of a forward scatter (FSC) and side scatter (SSC) gate established to include granulocytes, monocytes and lymphocytes. Fluorescence intensity data were expressed as geometrical mean (GEO mean) for each cell type.

Discussion

Current clinical diagnosis of Alzheimer's disease requires intensive examination that includes neuropsychological testing and costly brain imaging techniques, and a definitive diagnosis can only be made upon post-mortem neuropathological evaluation. Additionally, ante-mortem clinical Alzheimer's disease diagnosis is typically administered following onset of cognitive and behavioral symptoms. As these symptoms emerge relatively late in the disease course, therapeutic intervention generally occurs after significant neurodegeneration, with limited efficacy. Therefore, identification of noninvasive diagnostic biomarkers of Alzheimer's disease is becoming increasingly important; they would provide diagnosis tools to clinics with limited access to neuropsychological testing or state of the art brain imaging, reduce clinical diagnosis cost, constitute biological parameters to track the course of therapeutic interventions, and most importantly, allow for earlier diagnosis possibly even during the prodromal phase, with hopes of therapeutic intervention prior to appreciable neurodegeneration.

In this study, APP expression levels in different peripheral blood leukocyte types were assessed by flow cytometry, a highly sensitive method. The results obtained by flow cytometry and immunohistochemistry were consistent. By analyzing APP expression in peripheral blood leukocytes, including lymphocytes, monocytes, and granulocytes, high APP levels were found in some cells, especially in the membrane and/or cytoplasm. Statistical analysis suggested that APP expression in granulocytes from Alzheimer's disease patients is significantly higher compared with values obtained in healthy individuals; however, no statistically significant differences were observed for lymphocytes and mononuclear cells.

Magaki et al. (2008) reported increased APP expression on lymphocyte or monocyte surface in MCI subjects compared with controls, with no differences observed in granulocytes (Magaki et al., 2008). Here, surface as well as intracellular APP levels in lymphocytes, mono-cytes, and granulocytes were measured. The increased APP expression in granulocytes is probably due to the higher intracellular APP production. The changes of methylation patterns and expression of multiple genes in Alzheimer's disease patient leukocytes (Hou et al., 2013) might explain the change of APP expression in leukocytes.

Platelets, considered a major source of peripheral APP, can generate the different APP forms via cleavage by α- and β- and other secretases, followed by storage in platelet α granules. When platelets are activated, various APP isomers and Aβ are released, some of which penetrate through the compromised blood brain barrier and are deposited in the extracellular space in the brain, leading to senile plaque generation (Di Luca et al., 2000; Li et al., 1998). As shown above, granulocytes, lymphocytes and monocytes from peripheral blood cells all express the APP protein, and its expression levels are significantly higher in samples from Alzheimer's disease patients than those from control subjects. It has been suggested that Alzheimer's disease is associated with granulocyte density (Jaremo et al., 2013). In addition, ineffective phagocytosis of amyloid-beta by macrophages/monocytes in Alzheimer’s disease patients was reported (Fiala et al., 2005). Furthermore, the phagocytic activity of peripheral neutrophils is altered in Alzheimer's disease patients. Neutrophils were shown to retain the ability to engulf microbes, but their digestive activity decreased at the early stage of Alzheimer’s disease (Davydova et al., 2003). We speculate that APP might be engulfed and digested by peripheral neutrophils/PMN leukocytes, when circulating at high levels under disease state. Therefore, decreased digestive activity of APP in PMN leukocytes might lead to the elevated APP levels observed in granulocytes from Alzheimer's disease patients. Our results indicate a potential close correlation between the APP accumulation in peripheral PMN leukocytes and Alzheimer's disease pathogenesis, which may help further uncover the physiological and biochemical mechanism of Alzheimer's disease; this merits further study.

Conclusion

APP levels in peripheral blood granulocytes may be used as attractive Alzheimer's disease biomarker candidate for early diagnosis, as peripheral blood cells can be obtained in a minimally invasive manner and are easily analyzed by widely available cytometry techniques.

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