The Effects And Mechanisms Of TRPV4 Activation On Tau Phosphorylation

BackgroundAlzheimer’s disease(AD)is the most common dementia that mainly occurs in the elderly.The onset of AD is often insidious and it is mainly manifested as progressive memory and cognitive dysfunction,drastically interfering with the patients’ daily life and social sustainable development.Neurofibrillary tangles,which consist of abnormal aggregation of intraneuronal phosphorylated tau,represent one of the classical neuropathological hallmarks of AD.Tau hyperphosphorylation is believed to appear early in the course of AD,which is positively correlated with the dementia severity in AD patients.In recent years,the role of transient receptor potential vanilloid 4(TRPV4)in the central nervous system has gradually been recognized.TRPV4 expression was significantly increased in the cortex and hippocampus of aged rats,which suggested that the abnormal function of TRPV4 receptor was closely related to cognitive impairment.However,whether TRPV4 activation affects the phosphorylation of tau protein and its potential mechanism have not yet been elucidated.It has been reported that the disorder of brain cholesterol metabolism promotes the abnormal phosphorylation of tau protein in neurons,but the relationship between the cholesterol unbalance and TRPV4 activation and whether cholesterol unbalance participates in the regulation of tau phosphorylation by TRPV4 remains unclear.Part 1 The effects of TRPV4 activation on cognitive function and tau phosphorylation ObjectiveTo explore the regulatory effects of TRPV4 activation on cognitive function and tau phosphorylation in P301 S mice.MethodsFive-month-old tau transgenic P301 S mice and C57BL/6 wild-type mice were used and TRPV4 si RNA or TRPV4 agonist GSK1016790 A was injected into the lateral ventricle of P301 S mice by stereotactic technique.The experiment was divided into four groups: C57BL/6 group(negative control group,given PBS solvent control treatment),P301S-Control si RNA group(P301S mice were given control si RNA treatment by stereotactic injection),P301S-TRPV4 si RNA group(P301S mice were given TRPV4 si RNA treatment by stereotactic injection),and P301S-GSK1016790 A group(P301S mice were given GSK1016790 A treatment by stereotactic injection).Morris water maze was used to evaluate the spatial learning and memory ability of the mice in each group,and the escape latency,movement trajectory,and platform crossing times were recorded.The phosphorylation levels of tau protein in the cerebral cortex and hippocampus of the mice in each group were detected by immunofluorescence staining and Western Blot.ResultsMorris water maze results showed that the escape latency of P301S-GSK1016790 A group was significantly longer than that of P301S-Control si RNA group,while the escape latency of P301S-TRPV4 si RNA group was decreased.In the probe test,P301SGSK1016790 A group mice crossed the platform location less than P301S-Control si RNA group mice,while P301S-TRPV4 si RNA group crossed the platform location more than P301S-Control si RNA group mice.Immunofluorescence staining and Western Blot results showed that compared with P301S-Control si RNA group,the levels of phosphorylated tau protein in the cortex and hippocampus of P301S-GSK1016790 A group was significantly increased,while TRPV4 si RNA treatment could reduce the expression of phosphorylated tau protein in the cortex and hippocampus of P301 S mice.The above results indicated that TRPV4 activation aggravated the cognitive dysfunction of P301 S mice,and increased the tau phosphorylation level in the cerebral cortex and hippocampus of the mice.ConclusionTRPV4 activation aggravates the learning and memory deficits by inducing the hyperphosphorylation of tau protein in the cortex and hippocampus.Part 2 TRPV4 activation induces tau hyperphosphorylation via intraneuronal cholesterol accumulation ObjectiveTo explore the regulatory effects of TRPV4 activation on tau phosphorylation and its relationship with cholesterol unbalance in primary neurons.MethodsPrimary rat cortical neurons were transiently transfected with TRPV4 si RNA or treated with GSK1016790 A.The experiment was divided into four groups: Blank group(given PBS treatment),Control si RNA group(given control si RNA treatment),TRPV4 si RNA group(given TRPV4 si RNA group treatment),and GSK1016790 A group(given GSK1016790 A treatment).The phosphorylation levels of tau protein in primary cortical neurons were detected by Western Blot,and the cholesterol contents in neurons were detected by Filipin staining and a cholesterol quantitative kit.Besides,we upregulated the intraneuronal cholesterol level by silencing the cholesterol outflow regulator ABCA1(ATP binding cassette transporter A1),and detected the levels of tau phosphorylation in neurons by Western Blot.ResultsWestern blot analysis showed that compared with Blank group,the expression of phosphorylated tau protein was increased in primary cortical neurons treated with GSK1016790 A,while compared with Control si RNA group,silencing TRPV4 receptor could reduce the expression of phosphorylated tau protein in neurons.Filipin staining and quantitative data showed that GSK1016790 A treatment could increase the intraneuronal cholesterol content compared with Blank group,while TRPV4 si RNA treatment reduced the cholesterol content compared with Control si RNA group.In addition,Western Blot analysis showed that compared with control si RNA group,ABCA1 si RNA treatment increased the cholesterol content and the level of phosphorylated tau protein in neurons.TRPV4 silencing could partially reduce the increase of phosphorylated tau protein in neurons caused by ABCA1 si RNA treatment.These results suggested that intraneuronal cholesterol accumulation was involved in tau hyperphosphorylation mediated by TRPV4 activation.ConclusionTRPV4 activation can induce intracellular cholesterol accumulation and promote tau hyperphosphorylation.