Effect Of L-Carnitine On Alzheimer-like Tau Hyperphosphorylation And Spatial Memory Retention Deficits

Alzheimer's disease (AD) is the most common neurodegenerative disease in the aged population. With the aged tendency of population, the morbidity of AD increases year after year. NFTs is the known neuropathological feature in AD, they are intracellular fibrillar structures composed of aggregations of PHFs, which are made up of abnormally phosphorylated tau. Hyperphosphorylated tau is believed to be an early pathological event of AD and the amount of NFTs is closely correlated with the clinical dementia degree of AD patients. Thus target the underlying pathogenic mechanisms of tau hyperphosphorylation in AD might be a potential therapeutic strategy for prevention or treatment of the diseae.L-Carnitine is an L-lysine derivative, which was first discovered in beef by scientists from Russia in 1905. Its primary role appears to be facilitating the transport of long chain fatty acids into mitochondria in order to enter theβ-oxidation cycle. In our present study, we have investigated the pharmacological effect of L-Carnitine on WT/GFX induced AD-like pathological models and its involved mechanism of the effection. Each rat was fed orally with 50 mg/ day of L-Carnitine in drinking water or nomal drinking water as control for two weeks. At the day 8, the rats meanwhile were trained in Morris water maze everyday for 7 days. At the day15, wortmannin (WT, a specific PI3K inhibitor) and GF-109203X (GFX, a specific PKC inhibitor) (100Μm of each, total volume of 10μl) were co-injected into the left ventricle of the rats and the spatial memory retention was tested at 24h after the brain injection. The samples were prepared for western blotting and immunocytochemistry research after the spatial memory retention test.1.L-Carnitine improves WT/GFX-induced spatial memory retention deficitsThe results are shown as follows: after the co-injection of WT/GFX, the latency of finding the hidden platform increased remarkably and swimming path was tortuous. Pre-treatment with L-Carnitine significantly shortened the WT/GFX-induced increase of the latency and improved the searching strategy of the rats. These results suggest that L-Carnitine can effectively prevent the rats from WT/GFX-induced spatial memory deficits.2.L-Carnitine attenuates WT/GFX-induced tau hyperphosphorylation in rat hippocampusTo detect the effect of L-Carnitine on WT/GFX-induced tau phosphorylation, the level of total tau using antibody Tau-5, no significant difference was observed. We carried out western blot by using phosphorylation-dependent Tau-1 (reacts with non-phosphorylated tau at Ser 199/202), PT205, PS396 and PS404. We found that the immunoreactivity of Tau-1 was remarkably weaker, and of PT205, PS396 and PS404 was obviously stronger after the co-injection of WT/GFX than in control rats. Pre-administration with L-Carnitine attenuates the WT/GFX-induced tau hyperphosphorylation at PT205 and Tau-1 epitopes, but not at PS396 and PS404 epitopes. The level of phosphorylation at PS214, no significant difference was observedImmunohistochemistry was used to detect the distribution of the phosphorylated tau. It was shown that L-Carnitine prevented WT/GFX-induced hyperphosphorylation of tau at site of Thr205 and Ser199/202. Co-injection of WT/GFX caused a dramatically increasing PT205 staining of the hippocampus and a decreasing Tau-1 staining in neural fibers of the CA3 regions. L-Carnitine markedly reversed the hyperphosphorylation of tau at these regions. 3. L-Carnitine decreases WT/GFX-induced overactivation of GSK-3βwith Akt-independent mannerwe measured the total level and the activity-dependent Ser9-phosphorylated level of GSK-3βin the hippocampal extracts by western blotting. No obvious change was seen in total level of GSK-3βin four groups. However, the level of pSer9 -GSK-3β(representing the inactivated form of the kinase) was significantly decreased in WT/GFX-injected group, L-Carnitine could increase the level of the pSer9-GSK-3β, which suggested that L-Carnitine could prevent GSK-3βfrom WT/GFX-induced overactivation.To study whether the protective effect of L-Carnitine on GSK-3βand tau phosphorylation involves protein kinase B (Akt), an upstream effector of GSK-3β. We tested the activity-dependent Ser473-phosphorylated level of Akt and total AKT. WT/GFX treatment induced suppression of Akt demonstrated by a dramatic decline of the pAkt at Ser473. L-Carnitine cannot increase ratio of the pSer473Akt to total Akt. The results showed that L-Carnitine could attenuate WT/GFX-induced GSK-3βactivation with Akt-independent manner.4. L-Carnitine enhances the expression of c-Fos independent of PKA/CREBIn order to investigate the molecular mechanism of memory protection of L-Carnitine, We used immunohistochemisty to detect the c-Fos expression and distribution in rat hippocampus, L-Carnitine could dramaticly increased the level of c-Fos expression in the hippocampus which decreased by WT/GFX. The expression of c-Fos is significantly increased in long-term memory. L-Carnitine improve learning and memory through targeting c-Fos .In order to investigate the upstream mechanism, we measured PKA catalytic subunit , regulatory subunit and transcription factor CREB phosphorylation at Ser133. PKA catalytic subunit was no significance of four groups. Injection with WT/GFX induced the increasing of PKA regulatory subunit, L-Carnitine did not reverse WT/GFX- induced the increasing. The colour of pSer133-CREB- positive staining neurons was no significantly of four groups. It is suggested that L-Carnitine enhances the expression of c-Fos independent of PKA/CREB .5. L-Carnitine attenuates phosphorylation of PP-2Ac at Tyr307We also tested total PP-2AC and Tyr307-phosphorylated PP-2AC (the inactive form of PP-2A). It was shown that total PP-2AC was no significant difference among four groups and the level of Tyr307-phosphorylated PP-2A catalytic subunit did not change after the injection of WT/GFX, however, pre-administered with L-Carnitine can attenuates phosphorylation of PP-2Ac at Tyr307 as control group. The results suggested that L-Carnitine decreased the lever of tau hyperphosphorylation and improve spatial memory retention deficits partly because of upregulating PP-2A activity.