C-reactive Protein Induces Cognitive Deficits And Molecular Biological Alteration In Rat: Implications For Alzheimer's Disease

Background:Alzheimer's disease (AD) is a progressively neurodegenerative disorder in older. Its pathologycal features include intracellular neurofibrillary tangles(NFT), and senile plaques (SP), which primarily consist of extracellularβ-amyloid peptides (Aβ). Aβpeptides are generated by sequential cleavages of APP byβ-andγ-secretases. Although the pathogenesis of AD is unclear, Ap is considered to be a common factor and key link of AD. C-reactive protein (CRP), a prototypic acute-phase protein, is a sensitive marker of inflammation and tissue damage. There is much evidence showing that CRP may be implicated in the pathogenesis of AD and contribute to the cognitive problem in AD. However, there is no direct evidence showing that CRP impairs memory and causes neurochemical changes as those observed in AD.Objective:To investigate the effect of CRP, associated with Alzheimer's disease (AD), on cognitive deficits and molecular biologial alteration in rat, we examined the effects of CRP on memory performance and levels of inflammatory cytokines (IL-1βp, IL-6, and TNF-α), CRP, and markers of the endogenous production of Aβ(APP, PS-1, PS-2 and BACE) in rats, which are associated with AD. In addition, in order to further study the mechanism of CRP impair momory, we determined the cytotoxicity of CRP and the concentration and time effects of CRP using PC12 cells.Methods:(1) Forty Male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into four groups with ten animals each:control (naive animals) group, vehicle group, CRP group, and Aβ25-35 group (positive control). Animals were infused with same voleme of sterile distilled water (vehicle), aggregated Aβ25～35 (10μg/side), or CRP (12.8μg/side) into each cerebral lateral ventricle. Two weeks after operation, the place navigation test and spatial probe test were performed to evaluate both reference and spatial memory in the Morris water-maze. From the 20th to the 21th day, long-term memory was measured by using the passive avoidance test. The mRNA levels of inflammatory cytokines (IL-1β, IL-6, TNF-α), endogenous CRP, APP, PS-1and PS-2 in the hippocampus and cerebral cortex were measured by real time RT-PCR. The protein levels of inflammatory cytokines (IL-1β, IL-6, TNF-α), endogenous APP and BACE in the hippocampus and cerebral cortex were measured by Western blot. (2) In order to investigate the cytotoxicity of CRP, the methyl thiazolyl tetrazolium (MTT) assay was applied to evaluate the cell viability and biochemical method was used to determine the lactate dehydrogenase (LDH) activity in PC 12 cells. (3) In order to investigate the concentration effects of CRP, the mRNA expression levels of PS-1, PS-2, BACE-1 and APP were determined by Real-time PCR and ELISA was applied to evaluate the content of Aβ1-42. (4) In order to investigate the time effects of CRP, the mRNA expression levels of PS-1, PS-2, BACE-1 and APP were determined by Real-time PCR and ELISA was applied to evaluate the content of Aβ1-42.RESULTS:(1) In the place navigation test, behavioral tracking results revealed that, although most rats showed surrounding searches at the beginning of training, the naive and vehicle-treated rats learned more quickly than the drug-treated rats to swim away from the side walls to find the platform in the target quadrant, leading to shorter escape latency and swimming path length. The latency to reach the platform and the swimming distances during the first training trial were not changed in rats treated with Aβ25-35 or CRP, compared to the vehicle controls. During days 2-5, Aβ25-35-or CRP-treated rats displayed increases in the latency to reach the platform and the swimming distances, relative to vehicle-treated or naive rats (P<0.01, P<0.05.) However, these were no significance between vehicle-treated and naive rats(P>0.05). In the spatial probe trial, the distances traveled in all the four quadrants were not different among groups. Compared to the vehicle control, Aβ25-35 decreased both the duration and swimming distance (P<0.05, P<0.01) in the target quadrant. Similarly, CRP also decreased both indices (P<0.01 for duration and P<0.05 for distance) relative to the vehicle control, suggesting impairment of spatial memory. In addition, all the rats took approximately 23 s before entering the dark compartment, regardless of the treatment during the training; there was no difference among groups (P>0.05). In contrast,24 h after initial training (21d post-infusion), both naive and vehicle-treated rats displayed significantly increases in retention, suggesting that animals remembered the association of the aversive stimulus with the dark compartment. In contrast, the retention was significantly decreased in rats treated with either Aβ25-35 or CRP (P<0.01 and P<0.001, respectively), compared to the vehicle control, suggesting impaired long-term memory. Real-time RT-PCR revealed that both CRP and Aβ25-35 increased the mRNA levels of APP, IL-1β, IL-6, TNF-α, and CRP in the cerebral cortex and hippocampus. Immunoblotting analysis revealed that the protein levels of total APP, IL-1β, and IL-6 were deferentially changed by CRP in the cerebral cortex and hippocampus. The expression of APP, IL-1β, and IL-6 was increased, but that of BACE and TNF-αwas unaltered in the cerebral cortex; in the hippocampus, the expression of all the five proteins except IL-6 was increased. (2) In the cytotoxicity test, the results demonstrated that the concentration-dependent effects of CRP on the viability of PC 12 cells as well as on the LDH leakage of PC 12 cells. The viability significantly decreased and the LDH leakage significantly increased when the PC12 cells treated with CRP at the concentration between 12.5 and 100mg/L for 48h (P<0.01, P<0.05). (3) The concentration-dependent upregulated effects of CRP on the mRNA expression levels of PS-1, PS-2, BACE-1 and APP as well as on the content of Aβ1-42. The content of Aβ1-42 and the mRNA expression levels of PS-1, PS-2, BACE-1 and APP increased significantly while PC12 cells treated with CRP at the subtoxic concentration of 5 mg/L for 48h (P<0.01, P<0.05). (4) The time-dependent upregulated effects of CRP on the mRNA expression levels of PS-1, PS-2, BACE-1 and APP as well as on the content of Aβ1-42. The content of 2 and the mRNA expression levels of PS-1, PS-2, BACE-1 and APP increased significantly while PC 12 cells treated with CRP at the subtoxic concentration of 5 mg/L for 48 h (P<0.01, P<0.05). Particularly, the mRNA expression levels of BACE-1 also significantly increased for 24h (P<0.01)CONCLUSION:CRP contributes to memory loss and early phase of pathogenesis of AD. CRP can be a novel target for therapeutic intervention in AD, in particular in the memory loss associated with AD.