MSR1 and NEP Are Correlated with Alzheimer’s Disease Amyloid Pathology and Apolipoprotein Alterations

Abstract

Background:

In mouse models of amyloidosis, macrophage receptor 1 (MSR1) and neprilysin (NEP) have been shown to interact to reduce amyloid burden in the brain.

Objective:

The purpose of this study is to analyze these two gene products in combination with apolipoproteins and Aβ_1-42 in the cerebrospinal fluid (CSF) and plasma of individuals at different stages of Alzheimer’s disease (AD), as well as in autopsied brain samples from ROSMAP (Religious Orders Study and Memory and Aging Project).

Methods:

CSF/plasma levels of MSR1 and NEP were measured using the sensitive primer extension assay technology. CSF Aβ_1-42 was assessed with ELISA, while CSF ApoE and ApoJ were measured with the Luminex’s multiplex technology. Brain MSR1, APOE, and CLU (APOJ) mRNA levels were measured with RNA-Seq and contrasted to amyloid plaques pathology using CERAD staging.

Results:

While plasma and CSF MSR1 levels are significantly correlated, this correlation was not observed for NEP. In addition to be highly correlated to one another, CSF levels of both MSR1 and NEP are strongly correlated with AD status and CSF Aβ_1-42, ApoE, and ApoJ levels. In the cortical tissues of subjects from ROSMAP, MSR1 mRNA levels are correlated with CLU mRNA levels and the CERAD scores but not with APOE mRNA levels.

Conclusion:

The discrepancies observed between CSF/plasma levels of MSR1 and NEP with CSF Aβ_1-42 and ApoE concentrations can be explained by many factors, such as the disease stage or the involvement of the blood-brain barrier breakdown that leads to the infiltration of peripheral monocytes or macrophages.

Keywords

Alzheimer’s disease amyloid biomarkers inflammation MSR1 NEP

INTRODUCTION

In addition to extracellular amyloid-β (Aβ) plaques [1], neuronal and synaptic loss [2, 3], intra-neuronal neurofibrillary tangles [4], and cortical thinning in the temporal, orbitofrontal, and parietal regions [5], Alzheimer’s disease (AD) is characterized by chronic inflammation [6] as well as an altered cholesterol metabolism [7, 8]. According to multiple studies, scavenger receptors are directly implicated in the neuropathological features that characterize AD. In addition to mediating the uptake and the degradation of acetylated and oxidized low-density lipoproteins (LDLs), which both lead to massive cholesterol deposition in the neuropil [9], these pattern recognition receptors are highly expressed by amyloid deposit-associated microglia [10]. Moreover, the scavenger receptors have been implicated in the binding and uptake of oligomeric and fibrillar Aβ by microglia [11 –13]. In opposition to the familial form of AD, which is caused by the overproduction of Aβ₄₂ [14], the sporadic form of AD, which is more common, is mostly characterized by a reduced Aβ₄₂ clearance [15], a process that is mediated in part by the microglial uptake of this peptide.

Macrophage scavenger receptor 1 (MSR1) is one of the main receptors implicated in Aβ uptake by immune cells. In humans, MSR1 is predominantly found on macrophages [16] and monocytes in the periphery [11], and microglia in the CNS [13]. A diverse array of ligands is recognized by MSR1: Aβ [17], heat shock proteins [18], modified (i.e., acetylated or oxidized) LDL [9], as well as surface molecules from hepatitis C virus [19] and bacteria [20]. The majority of endogenous MSR1 ligands are connected to age-related degenerative diseases; oxidized lipoproteins being the driving force behind atherosclerosis, advanced-glycosylated end products resulting from diabetic glucose overload, and Aβ fibrils representing the central component of amyloid plaques in AD [21]. This receptor has also been shown to modulate the TLR4-mediated inflammation, which has been well documented in AD [17 , 22–28].

In recent years, multiple human studies have identified a correlation between MSR1 and AD. Indeed, a study using the Lothian Birth Cohort 1936 have shown that a significant proportion (36%) of the negative correlation between plasma MSR1 levels and cognitive ability was mediated by the total brain volume [29]. Moreover, a GWAS performed on nearly 4,000 European AD cases identified a variant in the 3’-UTR region that displays a suggestively significant correlation with the rate of cognitive decline [30]. In autopsied cases, elevated microglial MSR1 expression was correlated with a higher risk of having a diagnosis of AD and was correlated, in AD subjects, with reduced pre-mortem cognitive performances as well as higher densities of neuritic/diffuse plaques and neurofibrillary tangles [31]. Single-cell RNA sequencing also revealed that MSR1, along with APOE, are highly expressed in microglia from AD subjects (Single-cell atlas of the Entorhinal Cortex in Human Alzheimer’s Disease; ddnetbio.com) [32].

MSR1 has been implicated in the pathological processes of multiple mouse models of amyloidosis, tauopathy, neuroinflammation, and cerebral ischemia. RgTg4510 tau transgenic mice, a model of tauopathy [33], showed increased microglial Msr1 mRNA levels in contrast to wild-type mice [34]. Eight and fourteen-month-old APP/PS1 mice showed elevated microglial Msr1 mRNA levels compared to age-matched wild-type littermates [35]. Furthermore, APP/PS1 mice deficient for Msr1 (Msr1 –/–) displayed increased mortality rates over time, enhanced Aβ deposition, and a decreased gene expression for neprilysin (NEP) and insulin [17], two key Aβ-degrading enzymes [36, 37]. Moreover, glatiramer acetate, which is known to reduce spatial learning or memory deficits and to decrease the number of hippocampal Aβ plaques in APP/PS1 mice [38], increased MSR1 immunoreactivity one hour and 24 h after treatment on cultured monocyte-derived macrophages extracted from WT mice [39].

NEP is an integral membrane zinc metallopeptidase that is produced by mononuclear phagocytes [35], whose active site is present in the extracellular space [40]. NEP-deficient mice showed a significantly reduced but not completely abolished Aβ degradation, suggesting that, although being the most potent Aβ-degrading enzyme [36, 41], NEP degrades Aβ₄₂ along with other enzymes [42]. Intronic variants in the NEP gene were also correlated with sporadic AD [43]. A recent meta-analysis showed that AD subjects displayed lower levels of NEP gene expression in the frontal cortex [44]. Cerebrospinal fluid (CSF) NEP levels were also reduced in mild cognitive impairment (MCI) and AD subjects and were correlated, in these patients, with lower Mini-Mental State Examination scores as well as higher CSF total tau (t-tau) levels [45].

The purpose of the present study is to examine the relationship between MSR1, NEP, and neuropathological traits of AD in both the pre-symptomatic and symptomatic phases of the disease. Therefore, we systematically examined the relationships between plasma/CSF MSR1 and NEP levels versus disease progression and CSF Aβ_1-42, ApoE, and ApoJ concentrations in a living cohort of asymptomatic individuals with a parental history of AD (the Presymptomatic Evaluation of Novel or Experimental Treatments for Alzheimer’s Disease [PREVENT-AD]) [46] as well as in MCI/AD subjects from the Canadian Consortium on Neurodegeneration in Aging (CCNA) [47]. Data from autopsied brain samples from the Religious Orders Study and Memory and Aging Project (ROSMAP) [48] were used to examine cortical MSR1 gene expression in relation to APOE and CLU mRNA levels as well as CERAD scores [49].

METHODS

PREVENT-AD

Data used in the preparation of this article were obtained from data release 5.0 (November 30, 2017). Each participant and study partner provided written informed consent. All procedures were approved by the McGill University Faculty of Medicine Institutional Review Board. All research complied with ethical principles of the Declaration of Helsinki [46, 50].

Subjects

Pre-symptomatic participants enrolled in PREVENT-AD are cognitively normal volunteers with a parental or multiple-sibling history of sporadic AD. Most were 60 years of age or older, but persons aged 55–59 years were eligible if their age was within 15 years of their youngest-affected relative’s onset. To confirm their cognitive integrity throughout the longitudinal study, two cognitive screening tests were used: the Montreal Cognitive Assessment (MoCA) [51] and the Clinical Dementia Rating (CDR) [52]. When cognitive status was in doubt at any follow-up, (MoCA < 26/30 or CDR > 0), a complete evaluation was obtained from a neuropsychologist. A ∼30-min MRI session was also run to rule out structural brain disease.

DNA extraction and APOE genotyping

Automated DNA extraction from buffy coat samples was performed using the QIAsymphony DNA mini kit (Qiagen, Hilden, Germany). APOE genotype was determined using the PyroMark Q96 pyrosequencer (Qiagen, Hilden, Germany). DNA was amplified using polymerase chain reaction with the following primers: rs429358 forward 5^′-ACGGCTGTCCAAGGAGCTG-3^′, rs429358 reverse biotinylated 5^′-CACCTCGCCGCGGTACTG-3^′, rs429358 sequencing 5^′-CGGACATGGAGGACG-3^′, rs7412 forward 5^′-CTCCGCGATGCCGATGAC-3^′, rs7412 reverse biotinylated 5^′-CCCCGGCCTGGTACACTG-3^′, and rs7412 sequencing 5^′-CGATGACCTGCAGAAG-3^′.

Plasma and CSF extraction

Following an overnight fast, two samples of 13 mL of blood were withdrawn from patients and placed into EDTA tubes. Tubes were centrifuged for 10 min at 4°C and 3,000 rpm. Plasma was then aliquoted into 500μL samples and stored at –80°C. On the same day, lumbar punctures were performed with a Sprotte 24-gauge atraumatic needle to withdraw 20–30 mL of CSF samples (PAJUNK, Geisingen, Germany). After centrifugation, CSF was aliquoted into 500μL propylene cryotubes and stored at –80°C.

Measurement of MSR1 and NEP using the primer extension assay technology

CSF/plasma MSR1 and NEP levels were measured at the McGill Genome Centre with more than 90 other neurology-related protein biomarkers by using the Olink® Neurology panel, which uses the Proximity Extension Assay technology (Olink Proteomics, Uppsala, Sweden) [53]. The data were pre-processed using the NPX Manager software. Standardized NPX values that were below the limit of detection and samples that were marked as failed were removed.

CSF measurement of Aβ_1-42, ApoE, and ApoJ

CSF concentrations of Aβ_1-42 were assessed using the Innotest enzyme-linked immunosorbent assay kits (Fujirebio, Ghent, Belgium) as described before [54]. ApoE and ApoJ concentrations were assessed with the help of the Bio-Plex Pro Human Apolipoprotein Assay Panel, 10-plex (Bio-Rad, Hercules, CA, USA) as per manufacturer’s instructions.

CCNA

The Comprehensive Assessment of Neurodegeneration and Dementia (COMPASS-ND) Study is enrolling 1,650 memory-impaired/concerned subjects from 31 centres across Canada. Participants typically undergo a comprehensive baseline evaluation, including clinical and neuropsychological assessments, biospecimen collections, polymorphisms mapping, and MRI neuroimaging [47]. Data are made available to investigators from the Canadian Consortium on Neurodegeneration in Aging (CCNA) through the Longitudinal Online Research and Imaging System (LORIS) database (https://ccna-ccnv.ca/national-platforms/). CSF collection and measurement of Aβ_1-42 were performed as described for PREVENT-AD. A subset of CSF samples from CCNA (40) were analyzed along with CSF samples from PREVENT-AD using the Olink® Neurology panel, as described above. However, no plasma sample was analyzed with the Olink® technology, and no APOE ɛ4 genotyping was performed.

ROSMAP

ROSMAP consists of two longitudinal clinical–pathologic cohort studies of aging and AD from the Rush Alzheimer’s Disease Center in the US (http://www.radc.rush.edu/, accessed on 28 May 2019; GEO platform accession GPL18461) [48]. In this study, a subset containing individuals with RNA sequencing and pathology data was used. Briefly, 638 individuals both passed quality control for RNA sequencing and had phenotype data. Of these, 13 individuals were removed because they had other dementias [55]. Mixed MCI (defined as MCI and another cause of cognitive impairment) and mixed dementia (caused by AD and another dementia) were included respectively in the MCI and AD cases.

Statistical analysis

All analyses were performed in SPSS. In analyses combining PREVENT-AD and CCNA samples, Pearson correlations adjusted for age and sex (for Figs. 2B, 3, 4B-D, 5C-D, and 6C-D) were used. Analyses performed only on PREVENT-AD subjects (Figs. 1, 2A, 4A-C, 5A-B, and 6A-B, as well as Supplementary Figure 1) used Pearson correlations adjusted for age, sex, and APOE ɛ4 status. In ROSMAP, because mRNA levels were not distributed normally, analyzes were performed using Spearman correlations adjusted for age groups (65–79/80–89/90+), sex, postmortem interval, and APOE ɛ4 status (Figs. 7 and 8). In ROSMAP, MSR1, CLU and APOE mRNA levels that were 3 interquartile ranges above the median were removed.

Fig. 1

Correlation between plasma and CSF MSR1 A) and NEP B) levels in asymptomatic subjects from PREVENT-AD. Each dot represents an individual subject. CSF, cerebrospinal fluid; MSR1, macrophage scavenger receptor 1; NEP, neprilysin; NPX, normalized protein expression; PREVENT-AD, Presymptomatic Evaluation of Novel or Experimental Treatments for Alzheimer’s Disease.

Fig. 2

Correlation between plasma (A) or CSF (B) NEP and MSR1 levels in asymptomatic individuals from PREVENT-AD and MCI/AD subjects from CCNA. Each dot represents an individual subject. AD, Alzheimer’s disease; CCNA, Canadian Consortium on Neurodegeneration in Aging; CSF, cerebrospinal fluid; MCI, mild cognitive impairment; MSR1, macrophage scavenger receptor 1; NEP, neprilysin; NPX, normalized protein expression; PREVENT-AD, Presymptomatic Evaluation of Novel or Experimental Treatments for Alzheimer’s Disease.

Fig. 3

CSF MSR1 A) and NEP B) levels in PREVENT-AD and CCNA according to diagnosis. Bars represent mean NPX values±SEM. AD, Alzheimer’s disease; CCNA, Canadian Consortium on Neurodegeneration in Aging; CSF, cerebrospinal fluid; MCI, mild cognitive impairment; MSR1, macrophage scavenger receptor 1; NEP, neprilysin; NPX, normalized protein expression; PREVENT-AD, Presymptomatic Evaluation of Novel or Experimental Treatments for Alzheimer’s Disease; SEM, standard error of the mean.

Fig. 4

Relationship between plasma (A, C) or CSF (B, D) MSR1 (A-B) or NEP (C-D) and CSF Aβ_1-42 levels in asymptomatic individuals from PREVENT-AD and MCI/AD subjects from CCNA. Each dot represents an individual subject. Aβ_1-42, amyloid-beta 1-42; AD, Alzheimer’s disease; CSF, cerebrospinal fluid; CCNA, Canadian Consortium on Neurodegeneration in Aging; MCI, mild cognitive impairment; MSR1, macrophage scavenger receptor 1; NPX, normalized protein expression; PREVENT-AD, Presymptomatic Evaluation of Novel or Experimental Treatments for Alzheimer’s Disease.

Fig. 5

Correlation between plasma (A-B) or CSF (C-D) MSR1 levels and CSF ApoE (A,C) or ApoJ (B, D) levels in asymptomatic subjects from PREVENT-AD. Each dot represents an individual subject. ApoE, apolipoprotein E; ApoJ, apolipoprotein J; CSF, cerebrospinal fluid; MSR1, macrophage scavenger receptor 1; NPX, normalized protein expression; PREVENT-AD, Presymptomatic Evaluation of Novel or Experimental Treatments for Alzheimer’s Disease.

Fig. 6

Correlation between plasma (A-B) or CSF (C-D) NEP levels and CSF ApoE (A,C) or ApoJ (B, D) levels in asymptomatic subjects from PREVENT-AD. Each dot represents an individual subject. ApoE, apolipoprotein E; ApoJ, apolipoprotein J; CSF, cerebrospinal fluid; NEP, neprilysin; NPX, normalized protein expression; PREVENT-AD, Presymptomatic Evaluation of Novel or Experimental Treatments for Alzheimer’s Disease.

Fig. 7

Correlation between brain MSR1 mRNA levels and CERAD scores in ROSMAP. Bars represent mean MSR1 FPKM values±SEM. AD, Alzheimer’s disease; CERAD, Consortium to Establish a Registry for Alzheimer’s Disease; FPKM, Fragments Per Kilobase of transcript per Million mapped reads; mRNA, messenger ribonucleic acid; MSR1, macrophage scavenger receptor 1; ROSMAP, Religious Orders Study and Memory and Aging Project; SEM, standard error of the mean.

Fig. 8

Relationship between brain expression of MSR1 and APOE/CLU in ROSMAP. Each dot represents an individual subject. AD, Alzheimer’s disease; APOE, apolipoprotein E; CTL, control, CLU, clusterin (APOJ); FPKM, Fragments Per Kilobase of transcript per Million mapped reads; MCI, mild cognitive impairment; mRNA, messenger ribonucleic acid; MSR1, macrophage scavenger receptor 1; ROSMAP, Religious Orders Study and Memory and Aging Project.

RESULTS

Demographic characteristics of PREVENT-AD and CCNA

Along the disease continuum, participants are significantly older: asymptomatic individuals had a mean age of 63.5 years old, while MCI and AD subjects were 71.3 and 76.4 years old respectively (R² = 0.353, p < 0.001; Table 1). The proportion of women was also significantly higher in asymptomatic individuals (69.1 %) compared to the MCI (50 %) and AD (41.7 %) group (χ ²[2, N = 150] = 6.140, p = 0.046; Table 1). Along the disease continuum, participants with cognitive impairments (MCI and AD) displayed significantly lower CSF Aβ₄₂ levels relatively to asymptomatic subjects (R² = 0.407, p < 0.001; Table 1; 1129 pg/mL for asymptomatic individuals, 702 pg/mL for MCI subjects, and 469 pg/mL for AD cases).

Table 1

Demographic characteristics of the PREVENT-AD and CCNA participants with available plasma or CSF levels of MSR1 and NEP

	PREVENT-AD	CCNA
	Asymptomatic	MCI (n = 28)	AD (n = 12)	p
	individuals
	(n = 110)
Age±SEM	63.5±0.5	71.3±1.3	76.4±1.6	< 0.001
Women - n (%)	76 (69.1)	14 (50.0)	5 (41.7)	0.046
APOE ɛ4+ - n (%)	45 (40.9)	–	–	–
CSF Aβ_1-42 levels (pg/ml±SEM)	1,129±28	702±53	469±50	< 0.001

% refers to the percentage within each diagnosis group. Aβ_1-42, amyloid-beta 1-42; AD, Alzheimer’s disease; APOE ɛ4+, carriers of the apolipoprotein E ɛ4 genotype; CCNA, Canadian Consortium on Neurodegeneration in Aging; CSF, cerebrospinal fluid; MCI, mild cognitive impairment; PREVENT-AD, Presymptomatic Evaluation of Novel or Experimental Treatments for Alzheimer’s Disease; SEM, standard error of the mean.

Demographic characteristics of ROSMAP

In ROSMAP, AD subjects were significantly older (χ ²[4, N = 625] = 44.062, p < 0.001; Table 2) and showed a higher proportion of APOE ɛ4 positivity (χ ²[2, N = 624] = 31.391, p < 0.001; Table 2). Moreover, according to the CERAD scores (χ ²[6, N = 625] = 103.826, p < 0.001; Table 2), their brains displayed an increased density of neocortical amyloid plaques. However, there was no significant difference in postmortem intervals (R² = 0.002, p = 0.291), the proportion of women (χ ²[2, N = 625] = 2.150, p = 0.341), and APOE and CLU mRNA levels (R² = 0.001, p = 0.386 for APOE; R² < 0.001, p = 0.854 for CLU) among control, MCI, and AD subjects.

Table 2

Demographic characteristics of the ROSMAP cohort

	Cognitively	MCI	AD	p
	unaffected	(n = 168)	(n = 256)
	(n = 201)
Age				< 0.001
n (%) < 80	38 (18.9)	13 (7.7)	13 (5.1)
n (%) 80 s	107 (53.2)	82 (48.8)	105 (41.0)
n (%) ≥90	56 (27.9)	73 (43.5)	138 (53.9)
Women – n (%)	78 (38.8)	62 (36.9)	83 (32.4)	0.341
APOE ɛ4+ – n (%)	33 (16.4)	32 (19.2)	96 (37.5)	< 0.001
PMI (h±SEM)	7.4±0.3	7.7±0.4	6.9±0.3	0.291
CERAD score (%)				< 0.001
No AD	39.3	31.5	10.5
Possible AD	14.9	12.5	5.9
Probable AD	33.8	33.9	35.5
Definite AD	11.9	22.0	48.0
APOE mRNA levels (FPKM±SEM)	575±16	596±21	557±13	0.386
CLU mRNA levels (FPKM±SEM)	2,370±54	2420±61	2,361±44	0.854

% refers to the percentage within each diagnosis group. AD, Alzheimer’s disease; APOE, apolipoprotein E; APOE ɛ4+, carriers of the apolipoprotein E ɛ4 genotype; CERAD, Consortium to Establish a Registry for Alzheimer’s Disease; CLU, clusterin; FPKM, Fragments Per Kilobase of transcript per Million mapped reads; MCI, mild cognitive impairment; mRNA, messenger ribonucleic acid; PMI, postmortem interval; SEM, standard error of the mean.

Relationships between plasma/CSF MSR1 and NEP\\ levels, CSF AD markers, and CSF apolipoproteins

While plasma and CSF MSR1 levels are strongly correlated (R² = 0.141, p < 0.001; Fig. 1A), plasma and CSF NEP levels do not correlate (R² = 0.001, p = 0.700; Fig. 1B). Both CSF MSR1 and NEP levels are not correlated with CSF/plasma ratios of albumin and of immunoglobulin G (IgG) (MSR1: R² < 0.001, p = 0.995 for albumin; R² = 0.002, p = 0.778 for IgG; Supplementary Figure 1A, B; NEP: R² < 0.001, p = 0.917 for albumin; R² < 0.001, p = 0.916 for IgG; Supplementary Figure 1C, D). While CSF MSR1 and NEP levels are highly correlated with one another (R² = 0.417, p < 0.001; Fig. 2B) and are both reduced as a function of AD status (MSR1: R² = 0.114, p < 0.001, Fig. 3A; NEP: R² = 0.058, p = 0.004, Fig. 3B), plasma MSR1 and NEP levels are not correlated with one another (R² = 0.015, p = 0.207; Fig. 2A). In contrast to plasma MSR1 levels, CSF MSR1 concentrations are significantly correlated with CSF Aβ_1-42 levels (plasma: R² =0.008, p = 0.370; CSF: R² = 0.113, p < 0.001; Fig. 4A, B). Likewise, plasma concentrations of NEP are not correlated with CSF Aβ_1-42 levels (plasma: R² = 0.021, p = 0.156), but CSF NEP levels are (CSF: R² = 0.032, p = 0.038; Fig. 4C, D). In contrast to plasma concentrations, CSF MSR1 levels are significantly correlated with CSF ApoE and ApoJ concentrations (plasma: R² = 0.020, p = 0.160 for ApoE; R² = 0.016, p = 0.209 for ApoJ; Fig. 5A, B; CSF: R² = 0.095, p = 0.002 for ApoE; R² = 0.082, p = 0.004 for ApoJ; Fig. 5C, D). While plasma NEP levels are only correlated with CSF concentrations of ApoE (ApoE: R² = 0.066, p = 0.010; ApoJ: R² = 0.027, p = 0.104; Fig. 6A, B), CSF NEP levels are significantly correlated with both CSF concentrations of ApoE and ApoJ (ApoE: R² = 0.056, p = 0.018; ApoJ: R² = 0.061, p = 0.014; Fig. 6C, D).

Correlations between MSR1 mRNA levels, amyloid pathology, as well as APOE and CLU mRNA levels in the brain tissues of ROSMAP subjects

Higher brain MSR1 mRNA levels are correlated with a definite diagnosis of AD on the CERAD scale (r_s = 0.097, p = 0.017; Fig. 7). While mRNA levels from CLU (i.e., the gene encoding ApoJ) are correlated with brain MSR1 expression, APOE mRNA levels are not (r_s = 0.031, p = 0.942 for APOE; r_s = 0.273, p < 0.001 for CLU; Fig. 8A, B). NEP mRNA levels could not be analyzed due to the low expression of this gene in the brain (data not shown).

DISCUSSION

Plasma and CSF MSR1 levels are strongly correlated in asymptomatic subjects from PREVENT-AD (Fig. 1). This observation suggests that a large proportion of MSR1 originates from peripheral macrophages [16] and monocytes [11] that infiltrated the brain. Since CSF/plasma ratios of albumin and IgG, which are indicators of blood-brain barrier permeability [56], and CSF MSR1 levels are not correlated in this asymptomatic cohort (Supplementary Figure 1A, B), this infiltration likely occurs via active transport in the brain and not through a leakage in the blood-brain barrier [57]. Once in the brain, cell-surface MSR1, like for proteins such as TREM2, is released from the microglial plasma membrane upon the action of specific sheddases such as ADAM10 or ADAM17 [58] and can therefore be detected in the CSF. On the other hand, since 1) CSF NEP levels do not correlate with CSF/plasma ratios of albumin and IgG (Supplementary Figure 1C, D) and 2) there is nearly no NEP gene expression in the CNS (data not shown), this suggests that most of the NEP that is detected in the CSF originates from the active infiltration of NEP-expressing peripheral monocytes/macrophages in the CNS. However, the lack of correlation between plasma and CSF NEP levels also suggests that some signals unique to the brain environment might influence CNS NEP protein levels.

As expected from Frenkel’s original observations in APP mice [17], CSF levels of MSR1 and NEP are highly correlated in pre-symptomatic subjects, sharing almost half of their variance (Fig. 2B). Plasma MSR1 and NEP levels are not correlated (Fig. 2A), which is consistent with a CNS-specific process. CSF MSR1 and NEP levels are also inversely correlated with clinical disease progression, with lower levels found in MCI and AD subjects (Fig. 3). Results for NEP are consistent with the literature [45], while those for MSR1 are consistent with our hypothesis that CSF levels of the latter protein reflect the proportion that is released from the surrounding environment of amyloid plaques. Therefore, a reduction in the CSF concentrations of MSR1 could be viewed as an informative marker of higher levels of this protein in amyloid plaque-associated brain tissues.

Relationships between levels of MSR1 and CSF Aβ_1-42 are also quite different depending on the origin of MSR1. In PREVENT-AD, while CSF MSR1 concentrations are positively correlated with CSF levels of Aβ_1-42, plasma MSR1 levels are not (Fig. 4A, B), consistent again with a CNS-specific process with little or no contribution from the periphery. As the disease progresses toward a higher density of neocortical amyloid plaques (i.e., a definite diagnosis of AD according to the CERAD classification), higher MSR1 gene expression is observed in cortical areas (Fig. 7), presumably as a compensatory response to control for the increased amyloid deposition in brain tissues [11]. Although there have been some discrepancies in the ability of peripheral NEP to reduce brain Aβ [59, 60], our results tend to show that plasma NEP levels have no impact on CSF Aβ_1-42 levels (Fig. 4C). In the CSF, NEP levels correlate positively with Aβ_1-42 levels (Fig. 4D), which implies that lower CSF NEP levels are correlated with higher brain amyloid deposition. This observation is quite consistent with a recent study performed by Grimmer et al. on AD patients [61].

In the early 1990s, MSR1 was identified as one of the receptors that mediate the uptake of both acetyl-LDL and oxidized LDL by foam cells (i.e., cholesterol-loaded macrophages) [62 –64]. Ligand detection for MSR1 using purified lipoprotein complexes identified ApoAI, ApoAII, and ApoE as selective ligands that compete with known MSR1 ligands, such as acetylated or oxidized LDL, for their binding to the receptor [65]. Furthermore, MSR1-mediated adhesion of apolipoproteins to macrophages/microglia may represent a mechanism by which MSR1 contributes to the trapping of these cells at sites of pathological apolipoprotein accumulation or deposition, such as amyloid plaques and tangles [65].

These findings prompted us to investigate the possible interactions between MSR1 and the two major HDL-associated apolipoproteins found in the CSF, which are also widely believed to be implicated in AD [66]: ApoE and ApoJ. MSR1 levels are significantly correlated with CSF concentrations of ApoE and ApoJ, which is again consistent with a CNS-specific process (Fig. 5). In the injured or diseased brain, ApoE and ApoJ are known to work in tandem to coordinate the lipid transport and mobilization associated with synaptic remodeling [67 –69]. The involvement of MSR1 in the early phase of synaptic damage is certainly consistent with its known function during apoptosis after neuronal injury [70]. In cognitively unaffected individuals from ROSMAP, brain MSR1 mRNA levels correlate with the cortical expression of CLU (i.e., the gene encoding ApoJ), but not APOE (Fig. 8). In the CSF of cognitively unaffected individuals, NEP concentrations are however correlated with both ApoE and ApoJ levels (Fig. 6C, D). It is conceivable that it is the APOE genotype, which significantly modulates ApoE protein levels independently from its gene expression in the CNS, that serves as the driving force behind the correlation between CSF MSR1 and ApoE concentrations, as seen in Fig. 5C [66 , 71–73] These observations are consistent with evidence that suggests that lipidated ApoE might be implicated in the cleavage of Aβ by neprilysin within microglia [74]. Plasma NEP levels are inversely correlated with CSF ApoE levels (Fig. 6A), which might be explained by the implication of CNS ApoE in the reallocation of peripheral NEP-expressing mononuclear phagocytes to the CNS. Plasma NEP levels are however not correlated with CSF ApoJ levels (Fig. 6B).

Although we believe it might unravel a portion of the mechanisms by which MSR1 and NEP collaborate to clear amyloid plaques, our study contains a few limitations. First, CCNA’s COMPASS-ND cohort lacked a few important data, such as APOE ɛ4 genotypes and CSF/plasma ratios of albumin and IgG, which would have allowed us to perform comparisons on the whole AD spectrum. Although CSF collection and measurements were performed in the same way between the PREVENT-AD and the COMPASS-ND studies, important differences, such as those pertaining to the eligibility criteria, might have also introduced biases when comparing data from these cohorts. Furthermore, CSF samples were centrifuged, which would remove all the microglia cell surface content from our analyses. Therefore, we can only assume that the soluble portion of the proteins that we measured constitutes a proxy of the microglial-bound content. Another potential confounding issue in this article is the fact that the discrepancies observed between PREVENT-AD/CCNA and ROSMAP can be explained by factors other than differences related to mRNA versus protein levels, such as age, sample sizes, and soluble versus membrane-bound protein regulations. Finally, while we have shown multiple significant correlations throughout this article, these do not necessarily imply direct causality, and future research is needed to better understand the mechanisms in place.

Overall, these results suggest that, in addition to be highly correlated to one another, CSF MSR1 and NEP levels are both correlated with the disease status, as well as CSF Aβ_1-42, ApoE, and ApoJ concentrations. Additionally, both CSF MSR1 and NEP levels are not correlated with CSF/plasma ratios of albumin and IgG. However, while plasma NEP concentrations are correlated with CSF ApoE levels, plasma MSR1 concentrations are not. Moreover, although CSF MSR1 levels are correlated with plasma MSR1 concentrations, CSF NEP levels are not correlated with plasma NEP concentrations. Furthermore, while brain MSR1 mRNA levels are correlated with the CERAD scores and CLU mRNA levels, parenchymal NEP expression is nearly inexistant. The multiple discrepancies described in this paragraph could be partly explained by the differential gene/protein expression of NEP and MSR1 by peripheral monocytes/macrophages before and after their infiltration in the CNS. Additional research is needed to investigate the possible use of MSR1 and NEP as potential plasma or CSF biomarkers of the response to Alzheimer’s disease pathology.

Footnotes

ACKNOWLEDGMENTS

This study was supported by the Canadian Institutes of Health Research (grant N° PJT 153287), the Natural Sciences and Engineering Research Council of Canada (grant N° RGPIN-2020-04702), the Fonds Québécois de la Recherche en Santé (grant FRQ-264732), the J.-Louis Lévesque Foundation, the Lemaire Family Foundation, and the ICAO Charity Drive.

Data used in preparation of this article were obtained from the PREVENT-AD program (), data release 5.0 (November 30, 2017). A complete listing of PREVENT-AD Research Group can be found in the PREVENT-AD database: https://preventad.loris.ca/acknowledgements/acknowledgements.php?date=[2021-09-27.

The investigators of the PREVENT-AD program contributed to the design and implementation of PREVENT-AD and/or provided data but did not participate in the analysis or writing of this report.

We also thank Rush University, the ROSMAP project, and their participants for the RNA-sequencing data. The ROSMAP project was supported by funding from the National Institute on Aging (AG034504 and AG041232).

We would also like to acknowledge CCNA, which is supported by a grant from the Canadian Institutes of Health Research and funding from several partners, including the Saskatchewan Health Research Foundation, the Centre for Aging and Brain Health, and the Alzheimer Society of Canada (ASC).

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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MSR1 and NEP Are Correlated with Alzheimer’s Disease Amyloid Pathology and Apolipoprotein Alterations

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

METHODS

PREVENT-AD

Subjects

DNA extraction and APOE genotyping

Plasma and CSF extraction

Measurement of MSR1 and NEP using the primer extension assay technology

CSF measurement of Aβ1-42, ApoE, and ApoJ

CCNA

ROSMAP

Statistical analysis

RESULTS

Demographic characteristics of PREVENT-AD and CCNA

Demographic characteristics of ROSMAP

Relationships between plasma/CSF MSR1 and NEP\\ levels, CSF AD markers, and CSF apolipoproteins

Correlations between MSR1 mRNA levels, amyloid pathology, as well as APOE and CLU mRNA levels in the brain tissues of ROSMAP subjects

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

CSF measurement of Aβ_1-42, ApoE, and ApoJ