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Monomeric C‐reactive protein induces the cellular pathology of Alzheimer's disease

Abstract Introduction Human study shows that elevated C‐reactive protein (CRP) in blood impacts apolipoprotein E (APOE) ε4, but not APOE ε3 or APOE ε2, genotype to increase the risk of Alzheimer's disease (AD). However, whether CRP is directly involved in cellular AD pathogenesis and in which type of neuronal cells of APOE ε4 carriers are unknown. Methods We aimed to use different primary neuronal cells and investigate if CRP induces cellular AD pathology depending on APOE genotypes. Here the different primary neuronal cells from the different APOE genotype knock‐in mice cortex were isolated and used. Results Monomeric CRP (mCRP) increased amyloid beta production and, in parallel, induced tau phosphorylation in addition to their related proteins in the primary neurons in a pattern of APOE ε4 > APOE ε3 > APOE ε2 in a dose‐ and time‐dependent manner. Consistently, mCRP induced the staining of other neurodegenerative biomarkers, including Fluoro‐Jade B stain (FjB), TUNEL and Cleaved Caspase‐3, in primary neurons in a similar pattern of APOE ε4 > APOE ε3 > APOE ε2. In contrast, pentameric CRP (pCRP) had a tendency to induce cellular AD pathology but did not reach statistical significance. On the other hand, it is intriguing that regardless of APOE genotype, mCRP did not influence the expressions of Iba‐1 and CD68 in primary microglia or the expression of glial fibrillary acidic protein in primary astrocytes, and additionally mCRP did not affect the secretions of interleukin (IL)‐1α, IL‐1β, and tumor necrosis factor α from these cells. Discussion This is the first report to demonstrate that mCRP directly induces cellular AD pathogenesis in neurons in an APOE genotype‐dependent pattern, suggesting that mCRP plays a role as a mediator involved in the APOE ε4‐related pathway for AD during chronic inflammation. Highlights Pentameric C‐reactive protein (pCRP) can be dissociated irreversibly to form free subunits or monomeric CRP (mCRP) during and after the acute phase. mCRP increased amyloid beta production in the primary neurons in a pattern of apolipoprotein E (APOE) ε4 > APOE ε3 > APOE ε2 in a dose‐dependent manner. mCRP induced the expression of phosphorylated tau in the primary neurons in a pattern of APOE ε4 > APOE ε3 > APOE ε2 in a dose‐ and time‐dependent manner. mCRP plays an important mediator role in the APOE ε4‐related pathway of Alzheimer's disease risk.


Highlights
• Pentameric C-reactive protein (pCRP) can be dissociated irreversibly to form free subunits or monomeric CRP (mCRP) during and after the acute phase.
• mCRP increased amyloid beta production in the primary neurons in a pattern of apolipoprotein E (APOE) ε4 > APOE ε3 > APOE ε2 in a dose-dependent manner.
• mCRP induced the expression of phosphorylated tau in the primary neurons in a pattern of APOE ε4 > APOE ε3 > APOE ε2 in a dose-and time-dependent manner.
• mCRP plays an important mediator role in the APOE ε4-related pathway of Alzheimer's disease risk.

BACKGROUND
Apolipoprotein E (APOE) ε4 allele is the major genetic risk factor for late-onset Alzheimer's disease (AD), 1 but not all APOE ε4 carriers develop AD, even among those who reach > 90 years of age. 2 Our study used the data from the Framingham Heart Study (FHS) offspring cohort and found that chronically elevated C-reactive protein (CRP) impacts the risk of AD only in APOE ε4, but not in APOE ε3 and APOE ε2, carriers; 3 another study showed that CRP is associated with the biomarkers of tauopathy in cerebrospinal fluid (CSF) only in APOE ε4/ε4 carriers. 4 However, it is unclear whether CRP is directly involved in cellular AD pathogenesis in the presence of APOE ε4. As APOE ε4 leads to blood-brain barrier (BBB) dysfunction and leakage, 5 it is possible that the leaked peripheral inflammatory factors like CRP directly act on neuronal cells in the brain.
CRP is widely known as the prototypic acute phase reactant, a hepatically produced protein which increases markedly in the blood within hours of any trauma or disease event that causes tissue damage. [6][7][8] As CRP levels increase with age, 9,10 there are two forms of CRP: (1) native CRP (pCRP), a pentameric homoprotein that is produced during active inflammatory disease 11 and (2) during and after the acute phase, pCRP can be dissociated irreversibly to form free subunits or monomeric CRP (mCRP), which has much lower aqueous solubility and damages tissues. 12 mCRP is shown to play a role in the pathogenesis of peripheral chronic diseases including cardiovascular diseases, 13 age-related macular degeneration, 14 and post-stroke inflammation. 15 As the BBB prevents the passage of peripheral neurotoxic molecules into the brain, 16 several studies suggest that the BBB is damaged in the AD brain. 17,18 Thus, it is possible that the damaged BBB provides a mechanism for peripheral CRP proteins, especially mCRP dissociated and released during chronic peripheral inflammation, to enter into the brain during chronic peripheral inflammation and directly are involved in AD pathogenesis in APOE ε4 carriers. One study directly injected mCRP into the hippocampus region of an AD mouse model, 3XTg, and found it increased the AD pathology including tauopathy and amyloid pathology. 19   The area and total intensity after adjusting for the threshold was analyzed by ImageJ software as described previously 24 and the ratio with the number of cell bodies was calculated.

Enzyme-linked immunosorbent assays
Cell media from mCRP or pCRP treated neurons, astrocytes, or microglia was collected and centrifuged at 2800 g for 5 min-

2.6
Western blot assay

Fluoro-Jade B (FjB) labeling
Fluoro-Jade B (FjB) labeling was performed as reported by Gu et al. 26 with some modifications. Cell culture medium containing 0.000025% FjB (Histo-Chem Inc., 1FJB30MG) was freshly prepared each time using a FjB stock solution (10 mg of FjB in 100 mL distilled water). The area and total intensity after adjusting for the threshold was analyzed by ImageJ software.

Statistical analysis
All values are expressed as the mean ± standard error. Statistical analysis was performed using GraphPad Prism 8 (version 8.1.0, GraphPad Software Inc.). One-or two-way analysis of variance (ANOVA) with Tukey's post hoc testing was used for multiple comparisons. For all cell experiments, there were three replications. In all cases, P < .05 was considered significant.

mCRP induces Aβ production of the primary neurons with APOE ε4 genotype
After adding mCRP to the primary neuron cultures, we found that more of mCRP stuck/bound to primary neurons in an APOE genotype- Figure 1A).
Because Aβ42 is an early hallmark of AD pathology, we first investigated if mCRP has any effect on Aβ42 production using the ELISA assay. mCRP induced Aβ42 production in the media of primary neurons in a dose-dependent pattern and APOE genotype-dependent pattern, (Figure 2A). In contrast, pCRP showed a similar trend for inducing Aβ42 production in the primary neurons but didn't reach statistical significance ( Figure 2B). To further investigate, the RNA and protein expressions related to Aβ42 production including amyloid precursor protein (APP) and β-secretase (BACE1) 27 were determined by using RT-qPCR and western blot experiments in these neurons. Figure 2C-2E revealed that mCRP increased the RNA and protein levels of APP and BACE1 in a dose-dependent manner and the intensity pattern of APOE ε4 > APOE ε3 > APOE ε2 in primary cortical neurons. Interestingly, the effect of mCRP on the protein levels F I G U R E 1 The binding of monomeric C-reactive proteins (mCRP) on primary cortical neurons. Postnatal day 0-2 cortical neurons from mice expressing different apolipoprotein E (APOE) genotypes were isolated and cultured for 14 days. The neurons were treated with medium only versus adding different concentrations of mCRP for 16 hours. A, The neurons were fixed, incubated with human CRP antibodies to detect the binding of mCRP to the cells. The representative images were shown (scale bar: 50 μm). B, The immunofluorescence staining of human CRP antibodies was normalized by 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) and quantitated. Values were the mean ± standard error from all experiments. Compared to the untreated APOE ε2 neurons, differences with statistical significance were shown with * P < .05, * * P < .01, * * * P < .001, and * * * * P < .0001. Compared to the same concentration of mCRP treated neurons, the differences were shown with statistical significance # P < .05, ## P < .01, ### P < .001, and #### P < .0001 of APP and BACE1 were more pronounced in APOE ε2 neurons than APOE ε4 neurons ( Figure S1A and 1B in supporting information). In addition, the brain RNA levels of APP and BACE1 were also increased in mCRP-treated APOE ε4mice ( Figure S2A, S2B in supporting information). There was no significant change for the expression of presenilin 1 (PS1) in these brain samples ( Figure S2C).

mCRP induces cellular tauopathy of the primary neurons with APOE ε4 genotype
To test the effect of mCRP on cellular tauopathy, we added different concentrations of mCRP to the primary cortical neurons derived from WT, APOE ε2, APOE ε3, and APOE ε4 mice ( Figure 3A). mCRP induced cellular tau phosphorylation as detected by PHF1 antibody (Ser396/Ser404) immunostaining in a dose-dependent (from 1.25 to 5 μg/mL; Figure 3A,B) and time-dependent (5 μg/mL, 0-80 minutes) manner, with staining intensity patterns of APOE ε4 > APOE ε3 > APOE ε2 ( Figure 3C). In contrast, pCRP showed similar trends and pattern for inducing cellular tauopathy but did not reach statistical significance ( Figure 3D,E).
To characterize if mCRP influences the key enzymes to phosphorylate tau protein, RNA and protein were extracted and the expressions of tau phosphorylation-related RNA and proteins, including cyclindependent kinase 5 (CDK5) and glycogen synthase kinase 3 beta (GSK3β) 27 and Phospho-GSK3β (Ser9), were determined by using RT-qPCR and western blot experiments. Figure 3F-3H revealed that mCRP increased the RNA and protein levels of CDK5 and GSK3β in a dose-dependent manner, while decreasing the protein levels of GSK3β. Figure S3 in supporting information also shows that the RNA levels of CDK5 and GSK3β were increased in the brain of mCRP-treated APOE ε4 mice (Figure S3A, S3B).

F I G U R E 2
The effects of monomeric C-reactive proteins (mCRP) on the amyloid beta (Aβ)42 secretion in primary cortical neurons. Postnatal day 0 to 2 cortical neurons from mice expressing different apolipoprotein E (APOE) genotype were isolated and cultured for 14 days. The neurons were treated with medium only or adding different concentrations of mCRP or pentameric CRP (pCRP) for 16 hours. A, Primary neurons from different APOE mice were treated with control medium versus different concentrations of mCRP. Cell culture media were collected and the expression levels of Aβ42 were detected by enzyme-linkedimmunosorbent assay (ELISA). Values were the mean ± standard error (SE) from all experiments by using ANOVA with with Tukey's post hoc testing. Within each genotype, compared to the untreated neurons, differences of Aβ42 level with each concentration of mCRP are shown with statistical significance * P < .05, * * P < .01, * * * P < .001, and * * * * P < .0001. Within each concentration of mCRP, the comparisons of different APOE genotypes were conducted by using analysis of variance (ANOVA) with Tukey's post hoc testing. Statistical significance is shown with # P < .05, ## P < .01, ### P < .001, and #### P < .0001. B, Primary neurons from different APOE mice were treated with control medium versus different concentrations of pCRP. Cell culture media were collected and the expression levels of Aβ42 were detected by ELISA. Values were the mean ± SE from all experiments by using ANOVA with with Tukey's post hoc testing. C, Real-time polymerase chain reaction (PCR) assays were conducted to reveal the expressions of amyloid precursor protein (APP) in neurons. Values are expressed relative to untreated condition in each genotype, which were set as 1. Values were the mean ± SE from all experiments. Within each genotype, compared to the untreated neurons, differences with statistical significance were shown with * P < .05 and * * P < .01. D, Real-time PCR assays were conducted to reveal the expressions of β-secretase (BACE1) in neurons. Values are expressed relative to untreated condition in each genotype, which were set as 1. Values were the mean ± SE from all experiments. Within each genotype, compared to the untreated neurons, differences with statistical significance were shown with * P < .05. E, Western blot assays for the expressions of APP and BACE1 in different primary APOE neurons treated with mCRP were performed and quantified after normalization against β-actin. The representative photographs are shown. Values were the mean ± SE. Within each genotype, compared to the untreated neurons, differences with statistical significance were shown with * * * P < .001

F I G U R E 3
The effects of monomeric C-reactive proteins (mCRP) and pentameric CRP (pCRP) on the phosphorylated tau (p-tau) expression in primary cortical neurons. Postnatal day 0 to 2 cortical neurons from mice expressing different apolipoprotein E (APOE) genotype were isolated and cultured for 14 days. The neurons were treated with medium only or adding different concentrations of mCRP for different time. A, Neurons from wild-type (WT) mice or mice expressing different APOE genotypes were treated with control medium versus different concentrations of mCRP. The neurons were fixed, incubated with the p-tau antibody, PHF1, to detect cellular tauopathy. The representative images were shown (scale bar: 50 μm). B, The level of tau phosphorylation was normalized by NeuN and quantitated. Values were the mean ± standard error (SE) from all

Dose-dependent effects of mCRP on other biomarkers of neurodegeneration in primary neurons based on APOE genotypes
Caspase-3, one of the members of caspase family, is responsible for the majority of proteolysis during apoptosis. Therefore, the detection of cleaved caspase-3 is considered a reliable marker for cellular apoptosis, 28 and we applied it to our experimental system. To determine whether mCRP also induce apoptosis of neurons, we performed cleaved caspase-3 staining by applying increasing concentration of mCRP to primary cortical neurons derived from APOE ε2, APOE ε3, and APOE ε4. Figure 4A  We further investigated if mCRP has effects on neuron synapses using immunocytochemistry on the expressions of synaptophysin (SYP) and PSD95. In cortical neurons, we found that mCRP did not influence the expression of SYP regardless of the APOE genotype ( Figure 4E). While in the absence of mCRP APOE ε2 neurons had the highest expression of PSD95 compared to APOE ε3 and APOE ε4 neurons, only APOE ε2 neurons seemed to have tendency of a dosedependent decrease of PSD95 expression responding to mCRP, but again did not reach significance ( Figure 4F). These data indicate that mCRP probably did not have significant effects on neuronal synapses in our experimental system.

The effects of mCRP on primary microglia
Microglia were isolated from mouse cortex and the primary cultures were incubated with mCRP with increasing concentrations (1.25 μg/mL to 5 μg/mL). The expressions of CD68 and Iba-1 in primary microglia were examined by immunostaining ( Figure 5A-C). Surprisingly, despite that these concentrations of mCRP showed a dose-dependent influence on primary neurons described above, mCRP had no or minimum

The effects of mCRP on primary astrocytes
Consistent with the microglia results, the expressions of GFAP were stained in primary astrocytes and we found that there were no differences after adding different concentrations of mCRP ( Figure 6A,B).
experiments and compared using analysis of variance (ANOVA) with Tukey's post hoc testing. Within each genotype, differences with different concentrations of mCRP for each genotype were shown with statistical significance * P < .05, * * P < .01, and * * * P < .001. Within the same concentration of mCRP treated neurons, the differences among different APOE neurons were compared with ANOVA with Tukey's post hoc testing. Statistical significance is shown #P < .05. C, The neurons were treated with control medium or 5 μg/mL mCRP for different incubation time. The level of tau phosphorylation was normalized by NeuN and quantitated. Values were the mean ± SE from all experiments by using ANOVA with Tukey's post hoc testing. Within each genotype, differences with statistical significance for each genotype were shown with * P < .05, * * P < .01, and * * * P < .001. D, The neurons were treated with control medium versus different concentrations of pCRP. The level of tau phosphorylation was normalized by NeuN and quantitated. Values were the mean ± SE from all experiments. E, The neurons were treated with control medium versus 5 μg/mL pCRP for different incubation time. The level of tau phosphorylation was normalized by NeuN and quantitated. Values were the mean ± SE from all experiments. F, Real-time polymerase chain reaction (PCR) assay was conducted to reveal the expressions of CDK5 in neurons. Values are expressed relative to untreated APOE ε2 mouse primary neurons, which were set as 1. Values were the mean ± SE from all experiments by using ANOVA with Tukey's post hoc testing. Within each genotype, differences with statistical significance were shown with * P < .05. G, Real-time PCR assay was conducted to reveal the expressions of GSK3β in neurons. Values are expressed relative to untreated APOE ε2 mouse primary neurons, which were set as 1. Values were the mean ± SE from all experiments by using ANOVA with Tukey's post hoc testing. Within each genotype, differences with statistical significance were shown with * P < .05 and * * P < .01. H, Western blot assays for the expressions of GSK3β, pGSK3β, and CDK5 in neurons were performed and quantified after normalization against β-actin. The representative photographs are shown. Values were the mean ± SE. Within each genotype, compared to the untreated neurons, differences with statistical significance were shown with * P < .05 and * * P < .01

F I G U R E 4
The impact of monomeric C-reactive proteins (mCRP) on the biomarkers of neurodegeneration in primary cortical neurons. Postnatal day 0 to 2 cortical neurons from mice expressing different apolipoprotein E (APOE) genotype were isolated and cultured for 14 days. The neurons were treated with medium only or adding different concentrations of mCRP for different time followed by immunostaining with different antibodies. The representative images were shown (scale bar: 50 μm). A, The neurons were fixed, incubated with the pre-caspase 3 antibodies to detect cellular apoptosis. B, The immunofluorescence staining of pre-caspase 3 antibodies was normalized by 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) and quantitated. Values were the mean ± standard error (SE) from all experiments by using analysis of variance (ANOVA) with Tukey's post hoc testing. Within each genotype, differences with statistical significance for each genotype were shown with * P < .05 and * * * * P < .0001. Compared to the same concentration of mCRP treated neurons, the differences were shown with statistical significance #P < .05. C, The neurons were fixed and stained with Fluoro-Jade B (FjB) to detect neuronal degeneration. The representative images were shown. D, The immunofluorescence staining of FjB was normalized by DAPI and quantitated. Values were the mean ± SE from all experiments by using ANOVA with Tukey's post hoc testing. Within each genotype, differences with statistical significance were shown with * P < .05 and * * P < .01. Compared to the same concentration of mCRP treated neurons, the differences were shown with statistical significance ## P < .01. E, The neurons were fixed and stained with synaptophysin (SYP) antibodies to detect synapse loss. The immunofluorescence staining of synaptophysin antibodies was normalized by DAPI and quantitated. Values were the mean ± SE from all experiments. F, The neurons were fixed and stained with PSD95 antibodies to detect synapse loss. The immunofluorescence staining of PSD95 antibodies was normalized by DAPI and quantitated. Values were the mean ± SE from all experiments.

DISCUSSION
This study extends our previous research 3 These cellular pathologies are consistent and parallel with the interactive effects of APOE ε4 and CRP for AD risk 3 and AD brain biomarkers in humans. 4 Our study used in vitro primary neurons to demonstrate that APOE ε4 neurons are more vulnerable than APOE ε3 and APOE ε2 neurons to be exposed to mCRP and develop cellular AD pathology including Aβ and tau phosphorylation (Figures 2, 3). This process may be through mCRP increasing the expressions of APP and BACE1 for Aβ production, CDK5 and GSK3β for p-tau generation in the neurons.
Paradoxically, the effect of mCRP on the protein levels of APP and BACE1 were more pronounced in APOE ε2 genotype than APOE ε4 genotype ( Figure S1), but the effect of mCRP on the production of Aβ was more pronounced in APOE ε4 than APOE ε2 (Figure 2A). It is likely and consistent with other studies that the APOE isoform dependent-Aβ interaction after Aβ production mainly regulates Aβ level, aggregation, and clearance. 31 While many studies demonstrate that amyloids and tauopathy induce neuroinflammation, 32 our experiments demonstrate that inflammatory factor, mCRP, can cause production of Aβ and ptau in neurons depending on APOE genotypes. The data suggest that typical AD pathology, for example, Aβ or tau protein, and neuroinflammation enhance the formation of each other in AD pathogenesis. Most importantly, neurovascular dysfunction is shown in the AD brain. 33 Several studies have shown that APOE ε4 leads to BBB disruption, 5 which is shown to be a key step in late-onset AD pathogenesis, 5,34 and could cause leakage of peripheral inflammatory factors like mCRP into the brain and become toxic to neurons. Clinical study shows that an increased CSF/serum ratio for albumin and immunoglobulin G in a subgroup of AD patients, 35 suggesting that peripheral inflammatory factors leaked into the AD brain due to some cerebrovascular pathology. 36 It is intriguing that unlike in neurons, mCRP had no direct effects on astrocytes (Figure 6), although astrocytes are shown to be closely associated/connected with the brain endothelia in BBB; the "default" condition, like peripheral inflammation, for endothelia are more leaky inducing influences on associated cells especially astrocytes. 37,38,39 In addition, microglia cells also have interaction with the BBB to regulate neuroinflammation in the brain, 40 but also were not influenced by mCRP ( Figure 5). It is probable that during peripheral inflammation, blood mCRP could not directly influence astrocytes and microglia, but induced cerebrovascular inflammation of endothelia or pericytes to release some other proinflammatory factors, which will further influence astrocytes and microglia. Indeed, our recent research shows that peripheral intraperitoneal injection of mCRP into APOE knock-in mice induced significant neuroinflammation of astrocytes and microglia in the brain of APOE ε4 mice. 41 On the other hand, the receptor(s) for mCRP's activities in neurons is unknown. However, it is shown that CRP can bind to phosphocholine in cell membrane and bind to FcgammaRI and FcgammaRIIa in immune cells. 42 Because neurons have enriched membrane phospholipids and do not have FcgammaRI and Fcgam-maRIIa receptors, it is likely that mCRP binds to phosphocholine or some unidentified receptor of neuron cell membrane to induce AD cellular pathology. On the contrary, microglia cells express Fcgam-maRI and FcgammaRIIa but do not respond to added mCRP for the activation.
Although our study did not show the correlation between mCRP and synaptic changes in the neurons ( Figure 4E,F), we argue that during chronic inflammation mCRP induces Aβ and p-tau production, as well as causes neurodegenerative changes in APOE ε4, but not in APOE ε2 and minimally in APOE ε3, neurons (Figures 2-4) is an early and accumulative step before synapsis impairment in AD pathogenesis. Our findings in this study are consistent and relevant with the findings in humans. After following for 20 years, we observed that chronically elevated CRP levels predicted accelerated global cognitive decline and AD incidence in APOE ε4 carriers, but no such effect was observed among participants who were APOE ε4 non-carriers. 3 Using an Alzheimer's Disease Neuroimaging Initiative study, we additionally observed a positive association between baseline plasma CRP and trajectory levels of CSF total tau and p-tau biomarkers among those who had two APOE ε4 alleles. 4 Interestingly, this effect was reversed among those without an APOE ε4 allele.

CONCLUSION
Our results suggest that CRP may play an important mediator role in the APOE ε4-related pathway for AD risk. Treatment of chronic lowgrade inflammation to reduce CRP levels and block the dissociation of mCRP from pCRP, especially in APOE ε4 carriers, may delay AD onset.

ACKNOWLEDGMENTS
We want to express our thanks to the FHS participants for their decades of dedication and to the FHS staff for their hard work in collecting and preparing the data. This work was supported by the National Heart, Lung, and Blood Institute contract (N01-HC-25195) and by grants from the National Institute of Neurological Disorders and Stroke, NS-17950 and from the National Institute on Aging AG-008122, AG-16495, AG-022476. The sponsor institutes did not play any role in design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. Qiushan Tao and Ting Fang Alvin Ang had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.