Protective Effects And The Mechanisms Of Long-term ?-linolenic Acid Supplement On Age-associated Cognitive Impairment And Alzheimer's Disease-like Neuropathology In Natural Aging Rats

Part 1 Effect of long-term a-linolenic acid supplement on learning and memory ability in natural aging ratsObjectives:Aging is an inevitable process that is normal but complicated. Cognitive dysfunction is one of the most prominent features, a-linolenic acid (ALA) is an essential n-3 polyunsaturated fatty acid (PUFA), which exerts a role in neuroprotection and anti-aging. The major purpose of this section was to investigate the effect of long-term ALA intake on learning and memory ability in natural aging rats.Methods:A total of 160 specific-pathogen free (SPF) female Sprague-Dawley rats aged 6 months (initial weight,250-300 g) were randomly divided into four groups, namely the aged control group (Aged), high-fat control group (HF), low- and high-dose ALA group (L-ALA and H-ALA), which were chronically treated as long as 12 months with regular diet (AIN-93M),10%(w/w) lard diet,5%(w/w) lard+5%(w/w) flaxseed oil, and 10%(w/w) flaxseed oil, respectively. Another 30 rats (4-month-old) from the same source and genetic background served as young controls, and were fed on a regular diet for one month. Daily food intake and weekly body weight of rats in each group were monitored, so as to calculate the food efficacy ratio (FER). Spatial learning and memory abilities were determined by evaluating animals'performance in the Morris water maze tests (MWM). After sacrificed, the major organs were weighted. Subsequently, HE and Nissl staining were performed to evaluate the pathological changes in rat brain. Total lipids were extracted and analyzed by a gas chromatography from hippocampus and cortex.Results:12-month dynamic monitoring results among the 4 groups of old rats showed that, HF rats had the most weight gain and food intake, resulting in the highest FER (all P< 0.05), whereas the rats with ALA diets presented less weight gain and food intake, as well as reduced FER. Compared with the Young rats, Aged and HF rats showed memory deficits in MWM, as evidenced by increased latency time and total distance swam in the place navigation test (all P< 0.05), as well as decreased time/distance travelled in the platform quadrant compared to the total time/distance traveled in the spatial probe test (all P< 0.05). However, ALA dose-dependently shortened the latency, and strengthened the locating capability in the target quadrant (all P< 0.05). Pathological examination showed that the rates of neuronal apoptosis and degeneration were significantly increased in Aged and HF rat brains, while markedly ameliorated in ALA treated rats despite aging and high fat diet. Fatty acids analysis displayed that compared to the non-ALA treated rats (Young, Aged and HF), ALA increased the contents of both pe se and its metabolites in cortex and hippocampus (all P< 0.05). Moreover, ALA also elevated n-3 polyunsaturated fatty acid (PUFA) but declined total n-6 PUFA, leading to a significantly lower (n-6)/(n-3) ratio and higher unsaturated coefficient (P< 0.05).Conclusions:Long-term dietary ALA supplement regulated the fatty acid composition of brain, protected neurons and improved learning and memory function in natural aging rats.Part 2 The mechanisms of long-term ALA supplement alleviates cognitive impairment in natural aging rats:a role of hippocampal CREB signalingObjectives:It has been documented that the dominant trend of synaptic plasticity has been to interpret age-related cognitive changes. Of note, cAMP-response element-binding protein (CREB) plays a key role in neuronal survival, memory consolidation, as well as synaptic modification. The major purpose of this section was to investigate whether the improvement on age-related cognitive impairment by ALA associated with CREB signaling and to determine the mechanisms involved.Methods:Rats that had undergone the MWM tests were decapitated and the brains were immediately removed. Hippocampus and cortex were dissected as well. Transmission electron microscopy was applied to estimate synaptic morphometry of the CA1 regions of hippocampus. The expression of functional proteins and genes, including the CREB signaling and its upstream regulator, such as extracellular signal-regulated kinase (ERK), protein kinase B (PKB/Akt), protein kinase A (PKA) and calcium calmodulin kinase IV (CaMKIV) in the cortex and hippocampus were determined by western blotting analysis and real time RT-PCR.Results:Alterations from hippocampal synaptic structure showed that Aged and HF rats displayed significantly reduced synaptic number (all P< 0.05) and obvious lipofuscin aggradations, accompanied with postsynaptic membrane density decreasing (all P< 0.05) while the width of synaptic cleft increasing (all P< 0.05), as compared to the Young rats. However, ALA supplement notably reversed the alterations induced by aging and high fat diet. Functional genes analysis indicated that aging and high lard diet (Aged and HF) down-regulated the protein expression of phosphorylated CREB (CREB-P) and its downstream brain-derived neurotrophic factor (BDNF), as well as the high affinity receptor tyrosine kinase B (TrkB) phosphorylation levels in the hippocampus (all P.< 0.05). ALA dramatically enhanced the expression of CREB-BDNF-TrkB pathway (P< 0.05). Further study identified that hippocampal ERK and Akt rather than CaMKIV and PKA, the upstream signaling of CREB, were also activated by ALA supplement. Additionally, gene and proteins in the cortex were not response to ALA (all P> 0.05).Conclusions:These results suggest that aging and high fat diet down-regulated hippocampus CREB signaling and impaired the learning and memory ability in rats. Long-term dietary ALA intake enhanced CREB-BDNF-TrkB pathway through the activation of ERK and Akt signalings in a hippocampal manner, which contributes to its ameliorative effects on cognitive deficits in natural aging.Part 3 Long-term ALA treatment alleviates age-associated Alzheimer's disease-like neuropathology in natural aging rats:a role of PERK/eIF2aObjectives:The classic pathological features of Alzheimer's disease (AD) consist of extracellular plaques of the amyloid-? (A?) peptide and intracellular neurofibrillary tangles of hyperphosphorylated aggregates tau protein, also occurred in natural aging process. Recent studies have found that endoplasmic reticulum (ER) stress has a protective effect in the early stages of AD lesions. This study aimed to verify whether there is AD-like pathology during aging, and explore the mechanism mediated by ER stress underlying ALA alleviates AD-like pathology.Methods:A total of 160 SPF female Sprague-Dawley rats aged 6 months (initial weight,250-300 g) were randomly divided into four groups, namely the aged control group (Aged), high-fat control group (HF), low-and high-dose ALA group (L-ALA and H-ALA), which were chronically treated as long as 12 months with regular diet (AIN-93M),10% (w/w) lard diet,5%(w/w) lard+5%(w/w) flaxseed oil, and 10%(w/w) flaxseed oil, respectively. Another 30 rats (4-month-old) from the same source and genetic background served as young controls, and were fed on a regular diet for one month. In an attempt to verify the AD-like pathology, after the rats were sacrificed, brain sections were prepared and stained with Congo red and thioflavin-T to assay A? plaques in the parenchyma and vascular. Cortical and hippocampal A?42 and A?40 levels were measured using the rat ELISA kits. In addition, immunohistochemical technique to detect phosphorylated Tau protein (pS396-Tau) levels. All protein extracted from the cortex and hippocampus were subjected to western blotting analysis to investigate the alterations of ?-site APP-cleaving enzyme 1 (BACE1) processing the amyloid-P precursor protein (APP), and the activation of glycogen synthase kinase 3? (GSK-3?), an enzyme well known for its role in the phosphorylation of Tau. Meanwhile, the expression of mRNA and proteins related to one signaling branch of ER stress and the unfolded protein response, the protein kinase RNA-like endoplasmic reticulum kinase/eukaryotic initiation factor 2a (PERK/eIF2a), which is a key pathway regulating the activation of BACE1 and GSK-3? were also determined.Results:Compared with those in Young rats, Aged and HF rats had remarkably raised senile plaque burdens in the parenchyma and vascular in both hippocampus and cortex of were (all P< 0.05), as evidenced by prominent A? plaque and cerebral amyloid angiopathy. The area fractions of Tau-phospho-Ser396 positive neurons in the subregions of hippocampus of both the Aged and HF groups were also significantly increased (all P< 0.05). Treatment with ALA obviously reversed high senile plaque formation and Tau pathology in the Aged and HF rats (all P< 0.05). Further study found that the suppression of the PERK/eIF2a signaling induced by ALA may be responsible for neuroprotection mechanisms, including inhibition of A? production by suppressing BACE1 expression and suppression of Tau phosphorylation by inhibiting GSK-3? activation.Conclusions:All results suggested that AD-like pathology occurred in the brain of natural aging rats. Long-term supplement of ALA suppressed ER stress and the PERK/eIF2a branch of UPR signaling, which mediated the protective effect by ALA in aging-associated AD-like neuropathology.