Study On The Role And Mechanism Of Hyperphosphorylated Tau In Neuronal Death

Alzheimer’s disease(AD)is a fatal progressive neurodegenerative disease,and it is also the most common neurodegenerative disease affecting older people today.As of2018,there are 50 million dementia patients worldwide,and it is estimated that the number of patients will increase to an astonishing 152 million by 2050.There are currently around 10 million people living with AD in China and it is already the 5th leading cause of death.However,at present,the cause of AD has not yet been elucidated and there is no effective treatment that can prevent or block the progress of the disease.The main pathological features of AD are extracellular senile plaques made of amyloid-β(Aβ)and neurofibrillary tangles(NFTs)made of hyperphosphorylated tau(p Tau),and widespread death of neurons.The progressive loss of function and death of neurons in response to various mechanisms of damage is the most important cause of memory and cognitive decline.However,the mechanisms of neuronal death in AD remain uncertain.Therefore,further elucidation of the mechanisms regulating neuronal death in AD is of great importance in the search for effective targets and the development of therapeutic drugs.Necroptosis is a form of regulatory cell death that can be caused by the activation of death receptors,including the tumour necrosis factor receptor.It has been shown that necroptosis is involved in the pathogenesis of AD,with upregulated levels of RIPK1 and significantly increased levels of phosphorylation and oligomerisation of MLKL in the brains of AD patients.Furthermore,RIPK1 and MLKL were positively correlated with Braak stage,indicating that activation of necroptosis may be associated with p Tau.To date,however,the key factors and associated mechanisms that induce necroptosis in neurons of AD patients have not been reported.In addition,neuroinflammation also plays an important role in the pathological progression of AD.Several experimental evidences suggest that microglia and astrocytes are involved in the pathological progression of Aβ and Tau,and that their activation and subsequent release of proinflammatory factors can contribute to neuronal damage.Meanwhile,necroptosis has been found to be involved in regulating neuroinflammation in a variety of neurodegenerative diseases,including AD,and activated RIPK1 has also been shown to promote neuroinflammation.Thus,these findings suggest that necroptosis may be a key event in the regulation of cell death and inflammation in neurodegenerative diseases.However,the role of necroptosis in mediating neuroinflammation during AD and its underlying mechanisms have not been fully elucidated.In order to further unravel the key factors,molecular mechanisms and downstream events involved in the regulation of necroptosis in AD,this thesis explores the regulatory role and molecular mechanisms of p Tau in neuronal necroptosis at the cellular and animal model levels,we first determined whether p Tau induces necroptosis in neuronal cells lines by transient transfection.We found that p Tau can induce neuronal cell death,which can be inhibited by the necroptosis inhibitor Nec-1 but not by caspase inhibitors,and flow cytometry analysis further indicates that this form of death is necrosis,while p Tau can upregulate RIPK1-RIPK3-MLKL and promote necrosome formation,suggesting that p Tau can induce p necroptosis in neuronal cells.The altered gene expression levels during p Tau-induced necroptosis were then analysed by transcriptome sequencing which showed that p Tau mainly upregulated multiple immune-related passages including cytokine-cytokine receptor interaction pathways.Subsequent validation by q PCR,Western bolt and flow cytometry confirmed that p Tauinduced necroptosis was accompanied by high expression of multiple inflammatory factors and upregulation of ROS levels,and that the upregulated inflammatory factors could further promote microglia chemotaxis.Next,the signalling pathways mediating the upregulation of inflammatory factors were analysed using CRISPR-Cas9 knockdown technology and related pathway inhibitors,and the results showed that p Tau can activate NF-κB signaling pathway,while inhibitors of the NF-κB signalling pathway or knockdown of RIPK1-RIPK3-MLKL could inhibit inflammatory factor upregulation,and thus this process was mainly regulated by the RIPK1-RIPK3-MLKL axis-dependent NF-κB signalling pathway.Finally,the occurrence of necroptosis and neuroinflammation in the brain was examined in Tau P301 S transgenic AD model mice,and the therapeutic effects of necroptosis inhibitor Nec-1s on neuronal death,neuroinflammation,cognitive impairment and behavioural deficits in AD mice were explored.The results showed that p Tau also induced necroptosis and neuroinflammation in Tau P301 S mice,while treatment with Nec-1s reduced neuronal damage and improved neuroinflammation by inhibiting the NF-κB signaling pathway and microglia hyperactivation,and ultimately improved cognitive and behavioral deficits and significantly prolonged survival in AD model mice.In summary,this thesis demonstrates for the first time the key role of p Tau in inducing necroptosis in neurons,revealing the molecular mechanism by which p Tau synergistically induces necroptosis and neuroinflammation through activation of the RIPK1-RIPK3-MLKL axis and NF-κB signaling pathway,leading to neuronal injury and neuronal death.It also confirmed the therapeutic effect of targeted necroptosis on neuronal death and cognitive impairment in p Tau-induced AD model mice.This study not only has theoretical implications for understanding the molecular mechanisms of neuronal degeneration in AD patients,but also has important implications for clinical diagnosis and drug development in AD.