Regulation Of β-amyloid Production By C-reactive Protein And Its Implications In The Pathogenesis Of Alzheimer’s Disease

Objective:Alzheimer’s disease (AD), a central neurodegenerative disorder with progressive cognitive deficit, is pathologically characterized by the presence of extracellular senile plaques (SPs), intracellular neurofibrillary tangles (NFTs) and severe loss of neurons in AD brain. The key component of SPs is β-amyloid protein (Aβ), which is a key factor in AD pathogenesis. CRP, a member of the pentraxin family, is a trace serum protein that markedly increases during an inflammatory reaction. CRP, a member of the pentraxin family, is a trace serum protein that markedly increases during an inflammatory reaction. Incidence studies demonstrated that the elevated CRP in serum is associated with cognitive impairment and dementia. CRP was introduced into rats by intracerebroventricular (i.c.v.) injection in our previous study, the CRP infusion, similar with Aβ, was found to cause learning and memory impairments, suggesting that CRP may trigger AD-like dementia. Using PC12cells to mimic neurons,1,6-Bis(phosphocholine)-hexane (bis(PC)-H), a specific small molecule CRP inhibitor, was utilized to initially investigate the regulatory relationship of CRP and Aβ in the present study, especially to verify the regulatory relationship of CRP with amyloid precursor protein (APP), beta-site APP cleaving enzyme (BACE-1), and presenilins (PS-1and PS-2), from which the supporting for CRP as a novel target in AD pathogenesis can be established. Meanwhile, the toxicity antagonistic test between bis(PC)-H and Aβ was carried out to identify whether CRP is the potential upstream regulator of Aβ. APP/PS1transgenic (Tg) mice were utilized to simulate the early stages of AD, the relevance of CRP and Aβ as well as potential mechanisms were studied by examining the cerebral levels of CRP and Aβ, and the mRNA levels of serum amyloid P component (SAP), complement component1q (C1q), component3(C3), and tumor necrosis factor-a (TNF-a). Moreover,4months Tg mice were observed in behavioral tests and cerebral Aβ plaque to point out the effect of CRP on the learning and memory of early-stage AD and the related mechanisms.Methods:(1) Using PC12cells as the research object, we firstly established the range of non-toxic concentrations of bis(PC)-H. The control and model groups were then incubated with DMEM, bis(PC)-H group with bis(PC)-H, the supernatants were aspirated after3h of the incubation, the inside dishes were washed with phosphate buffer solution (PBS) before CRP or DMEM treatments. Toxicity inhibition was evaluated using MTT reduction assay and LDH activity assay as well. In addition, PC12cells cultured in a10cm-diameter Petri dish were incubated with a sub-toxic concentration of CRP (0.2μM) or DMEM for48h after3h incubation with bis(PC)-H (20,40,80μM) or DMEM. The supernatants were collected for ELISA analyse, and the cells were collected for analyzing the expreesions of Aβ and the related factors by PCR and Western blot. For all experiments, the treatment with DMEM containing1%FBS for48h was used as the control.(2) PC12cells were incubated with various concentrations of Aβ25-35(1.25,2.5,5.0,10,20,30μM) and Ap1-42(0.25,0.5,1.0,2.5,5.0, and10μM) for48h, then the cells and the supernatants were analyzed using MTT assay and LDH activity assay, respectively. After establishing the cytotoxicities and optimal cytotoxic concentrations of Aβ25-35and Ap1-42, bis(PC)-H was dissolved into the culture medium to incubate the cells for3h, Aβ25-35and Aβ1-42treatments were carried out after the supernatants were aspirated, respectively. Cytotoxicity was evaluated using MTT reduction assay and LDH activity assay.(3)1,2, and3-month-old Tg and wild type (WT) mice were housed a week for habituation. Genotype identification of APP/PS1transgenic mice was conducted by PCR. The mice were decapitated under a deep anesthesia with an overdose of chloral hydrate. Their hemibrains, excluding the cerebellum, were then dissected and homogenated by extract solution. The levels of CRP and Aβ1-42were examined by ELISA, followed by the relevance analysis.40mg brain tissues per mouse were isolated on the ice, the total RNA was extracted from the hemibrains using the TRIzol reagent. Reverse transcription of the isolated RNA was performed after finishing the RNA concentration examination. The mRNA levels of CRP and the related factors SAP, Clq, C3, and TNF-α were measured by real-time PCR using a Stratagene Mx3000P QPCR system.(4)3-month-old Tg and wild type (WT) mice were housed for a month. Genotype identification was conducted by PCR. The animals were divided into5groups:WT vehicle group (WT, aCSF). APP/PS1vehicle group (APP/PS1, aCSF), APP/PS1bis(PC)-H0.2group (APP/PS1,0.2nmol/mouse), APP/PS1bis(PC)-H1.0group (APP/PS1,1.0nmol/mouse) and APP/PS1CRP group(APP/PS1,0.02nmol/mouse). The experimental mice were fixed to the stereotaxis instrument under anesthesia. The locating of dexter cerebri ventriculus was carried out under the exposure of their anterior fontanelle, and fixed up the cannula with a inner. Bis(PC)-H (0.2or1.0nmol/mouse) or CRP (0.02nmol/mouse) was infused q.o.d. into ventricle8day after implanting the cannula into right ventricle, and aCSF was applied to the control and model groups.60days after administration, behavioral tests were carried out in accordance with the order of Object recognition test (ORT), Morris water-maze (MWM), and Step-down passive avoidance (SDPA). Animals were sacrified24h after the behavioral tests, the brains were collected for paraffin section, Aβ immunohistochemistry was measured.Results:(1)30～3000μM bis(PC)-H were verified to be non-toxic for PC12cells. MTT results revealed that the viability of PC12cells treated with1μM CRP for48h decreased compared with the control,12.5～400μM bis(PC)-H reversed the CRP-induced cell viability reduction, the significant reductions were induced by25,50,100,200,400μM bis(PC)-H. LDH results revealed that the cytosolic LDH leakage of PC12cells treated with1μM CRP for48h significantly increase compared with the control,12.5～400μM bis(PC)-H reversed the CRP-induced LDH leakage increase, the significant increases were induced by25,50,100,200,400μM bis(PC)-H.We then investigated the neurochemical mechanisms of CRP cytotoxicity with sub-toxic concentration of CRP. Real-time PCR analysis show that the mRNA levels of APP, BACE-1, PS-1, PS-2, and endogenous CRP increased after the48h incubation with0.2μM CRP,80μM bis(PC)-H markedly suppressed the CRP-induced mRNA upregulations of APP, BACE-1, PS-1, PS-2, and endogenous CRP. Western blotting analysis show that the48h incubation with0.2μM CRP significantly increased the expressions of APP, BACE-1, and PS-1of PC12cells,40μM bis(PC)-H significantly suppressed the CRP-induced upregulations of BACE-1and PS-1,80μM bis(PC)-H markedly suppressed the CRP-induced mRNA upregulations of APP, BACE-1, and PS-1, PS-2. ELISA result shows that the0.2μM CRP-induced Aβ1-42elevation was significantly suppressed by40and80μM bis(PC)-H.(2) The cytotoxicity of Aβ325-35on PC12cells was determined using MTT reduction assay, and shown that the viability significantly decreased after incubation with10,20,30μM Aβ25-35.30μM was chosen as the final cytotoxic concentration of Aβ25-35to display observable cytotoxicity. To verify whether bis(PC)-H decreased the cytotoxicity of Aβ25-35, PC12cells were pretreated with30～3000μM bis(PC)-H. A48h incubation with30μM Aβ25-35followed. We found that30～3000μM bis(PC)-H did not reversed Aβ25-35-induced viability decrease and LDH leakage.We then replaced AP25-35by Aβ1-42to have the next study. The cytotoxicity of Aβ1-42on PC12cells was determined using MTT reduction assay, and shown that the viability significantly decreased after incubation with1.0,2.5,5.0,10μM Ap1-42.5.0μM was chosen as the final cytotoxic concentration of Aβ1-42to display observable cytotoxicity. To verify whether bis(PC)-H decreased the cytotoxicity of Aβ1-42, PC12cells were pretreated with30～3000μM bis(PC)-H. A48h incubation with5.0μM Aβ1-42followed. We found that30～3000μM bis(PC)-H did not reversed Aβ1-42-induced viability decrease and LDH leakage.(3) To verify the relevance of CRP and Aβ in the early stages of AD,1-,2-, and3-month-old APP/PS1Tg mice were used to determined brain Aβ1-42and CRP levels. ELISA results show that Aβ1-42in2-and3-month-old APP/PS1Tg mice significantly increased compared with that in littermate WT mice of the same age. Similarly, the CRP level in the APP/PS1Tg mice increased with aging. CRP and its mRNA in3-month-old Tg mice clearly increased compared with those in WT mice. We also analyzed the relationship between CRP and Aβ1-42in APP/PS1Tg mice and WT mice using linear regression analysis, and found that the brain levels of Aβ1-42were positively correlated with CRP (r=0.818).To investigate the potential molecular participants of the relevance of CRP and Aβ in the early stages of AD, the mRNA levels of CRP and the related factors (SAP, Clq, C3, and TNF-a) were measured. The mRNA level of SAP and TNF-a in3-month-old APP/PS1Tg mice significantly increased compared with WT mice of the same age. CRP and C1q mRNA of both2-and3-month-old APP/PS1Tg mice increased. No significant difference was observed for C3mRNA in1-,2-, and3-month-old APP/PS1Tg mice.(4) In the ORT, we found that different genotypes and treatments did not influence the animal recognition of the identical object. The novel-object recognition index of APP/PS1Tg mice was in the decline trend, indicating some impairment of their learning and memory. The impairment of learning and memory in APP/PS1Tg mice was reversed after received bis(PC)-H, indicating blockage of CRP can lessen the memory impairment. The memory of APP/PS1Tg mice was destroyed obviously after received CRP, indicating CRP can aggravate their memory impairment.In the acquisition trial of the MWM, the latancy of WT mice to reach the platform was shorten gradually, indicating that daily training can enhance the learning and memory, but APP/PS1Tg mice need longer time to find a hidden platform, suggesting a certain degree of impairment of spatial memory, and these impairment can be reversed by blocking CRP with bis(PC)-H. The latancy to reach the platform revealed a significant prolong in APP/PS1Tg mice received CRP, indicating that CRP can aggravate the learning and memory function of APP/PS1Tg mice. There is no difference in average swimming speed, indicating different genotypes and treatments did not affect the ability of movement in the mice. In the probe trial of the MWM, the duration in the target quadrant of APP/PS1Tg mice decreased compared to WT mice, suggesting a certain degree of impairment of spatial memory, and the impairment can be reversed by blocking CRP with bis(PC)-H. The duration revealed a significant decrease in APP/PS1Tg mice received CRP, indicating that CRP can aggravate the learning and memory function of APP/PS1Tg mice. Meanwhile, the entries in the target quadrant of APP/PS1Tg mice decreased, suggesting a certain degree of memory impairment, and the impairment can be reversed by blocking CRP with bis(PC)-H. The entries revealed a significant decrease in APP/PS1Tg mice received CRP, indicating that CRP can aggravate the learning and memory function of APP/PS1Tg mice. Moreover, there is no difference in average swimming speed in the probe trial, indicating different genotypes and treatments did not affect the ability of movement in the mice.In the SDPA, WT mice remembered electrical stimulation after accepting4learning trials, but APP/PS1Tg mice need5learning trials, indicating their memory in passive stimulation got a certain degree of impairment, and these impairment can be reversed by blocking CRP with bis(PC)-H. APP/PS1Tg mice received CRP remembered electrical stimulation after accepting6learning trials, indicating that CRP can aggravate the memory impairment in passive stimulation of APP/Ps1Tg mice. After24h, the retention of SDPA test in APP/PS1Tg mice decreased compared to WT mice, suggesting a certain degree of memory impairment in passive stimulation, and the impairment can be reversed by blocking CRP with bis(PC)-H. The retention revealed a significant decrease in APP/PS1Tg mice received CRP, indicating that CRP can aggravate the memory impairment in APP/PS1Tg mice.Immunohistochemistry results shown that wild type mice received aCSF by i.c.v. have little of Aβ staining, suggesting no changes of Aβ plaque formation in the treatments. APP/PS1Tg mice appeared typical Aβ plaques, APP/PS1Tg mice received bis(PC)-H shown a decrease of Aβ plaque burden, demonstrating that blocking CRP can inhibit the plaque formation of APP/PS1Tg mice. In addition, APP/PS1Tg mice received CRP revealed a significant increase of Aβ plaque burden compared with APP/PS1Tg mice received aCSF, indicating that CRP can induce the plaque formation of APP/PS1Tg mice. These results suggest that the differences of learning and memory of the mice in different genotypies and treatments related to the cortical plaque formation.Conclusion:CRP exerts cytotoxic injury on PC12cells; bis(PC)-H reversed the CRP-induced cell viability reduction and LDH leakage by antagonizing the CRP activity. A sub-toxic concentration of CRP can induce the mRNA upregulations of Aβ-related factors and endogenous CRP of PC12cells, and finally upregulated the expressions of Aβ-related factors and Aβ1-42. Bis(PC)-H markedly reversed the CRP-induced mRNA upregulations of APP, BACE-1, PS-1, PS-2, and endogenous CRP, meanwhile, significantly reversed the CRP-induced upregulations of APP, BACE-1, PS-1, and Aβ1-42. Aβ25-35and Ap1-42exert cytotoxic injury on PC12cells, but bis (PC)-H can not antagonize the Aβ25-35and Aβ1-42cytotoxic injury. The in vitro experiments show that CRP can regulate Aβ generation, may be involved in the pathogenesis of AD as an upstream factor of Aβ. The results of animal experiments show that CRP and Aβ levels increased with aging in APP/PS1Tg mice, revealing a positive correlation. Combined with in vitro experiments, we presumed that CRP may involve in the early-stage pathogenesis of AD by regulating Aβ1-42generation, this process may involve in SAP, Clq and TNF-a. All of these suggest that CRP is a new target for early intervention of AD development. After the two-month early administration in APP/PS1Tg mice, we found that CRP affected their learning and memory and cerebral Aβ plaque formation. Antagonizing the CRP can reverse the mild memory impairment and the increased Aβ plaques formation in APP/PS1Tg mice, giving the CRP can aggravate the memory impairment and the Aβ plaques formation in APP/PS1transgenic mice, proofing that CRP involves in the pathogenesis of AD Tg mice from the positive and negative perspectives.