The Effect Of Human Tau Overexpression On Mitochondrial Dynamics And Cell Degeneration

Background:Intracellular accumulation of microtubule-associated protein tau is the major cause of neurodegeneration in Alzheimer disease (AD) and other tauopathies; however, the mechanism underlying tau-induced neural dysfunction and degeneration is elusive. Since mitochondrial dynamics inblance was discovered in the autopsy neuron of Alzheimer Disease patient. Therefore, the effect of tau overexpression on mitochondrial dynamics and cell degeneration was investigated in this study.Objective:To study tau overexpression on mitochondrial fission/fusion dynamics and cell process degeneration, and the underlying mechanism.Methods:Immunofluorescence staining and live cell time-lapse imaging was used to quantification of mitochondrial size and distribution, Immunoblot was used to study fission/fusion protein level, real time PCR was used to determine whether mRNA level of fusion protein was increased by tau overexpression, Co-immunoprecipitation was used to determine whether tau located in mitochondrial and whether tau overexpression changed the ubiquitination of Mfns.20S proteasome activity assay to determine proteasome activity, and mitochondrial membrane potential assay with JC-1staining, Complex I activity assay and ATP level assay was to determine mitochondrial function. Extract mitochondrial from HEK293cells and tau-1-or htau transgenic mice to detect the level of PINK1. Alkaline and Trypsin treatment of isolated mitochondria was used to determine tau location.Results:In the present study, we found that expression of human full length tau (htau) promoted mitochondria fusion and their perinuclear accumulation in HEK293cells and primary hippocampal neurons. At the molecular level, expression of htau increased the levels of mitofusins Mfn1, Mfn2and OPA1with no effects on fission protein Fisl, and simultaneous downregulation of Mfnl or Mfn2but not OPA1attenuates the htau-induced mitochondrial fusion. We also found that htau was located into the mitochondria, and the mitochondrial allocation of htau increased the membrane potential with reduced levels of PINKl and ubiquitinated Mfnl/Mfn2. Finally, retraction and deprivation of the neuronal processes with decreased ATP level and complex I activity were observed followed by perinuclear mitochondrial accumulation at tau overexpression48h.Conclusion:These data together indicated that htau overexpression enhances mitochondria fusion via cumulating mitofusins without changing mitochondrial function at early stage, while at later stage causes neurodegeneration via impairing mitochondrial functions. It suggests that blocking htau accumulation may protect against neurodegeneration through rescuing mitochondrial functions. Increased inhibitor-2of protein phosphatase-2A (I2PP2A) level has been associated with decreased protein phosphatase-2A (PP2A) activity and tau hyperphosphorylation in Alzheimer’s disease (AD). We recently report that I2PP2A also upregulates glycogen synthase kinase3β (GSK-3β), a crucial tau kinase. Thus, reduction of I2PP2A level should be promising in attenuating tau pathology in AD. To test this, we constructed the shRNA interference plasmids to knockdown I2PP2A in HEK293/tau and N2a/tau cells (pSUPER-siI2PP2A) and in htau transgenic mice (Lenti-siI2PP2A), respectively. We found that knockdown I2PP2A not only restituted PP2A activity, but also suppressed the activity of GSK-3β in vivo and in vitro, demonstrated by the decreased GSK-3β total protein and mRNA levels, and the increased inhibitory phosphorylation of GSK-3β at Ser9. Further studies revealed that activation of protein kinase A (PKA) but not Akt was upstream of the increased GSK-3β phosphorylation by I2PP2A knockdown. Knockdown I2PP2A antagonized tau hyperphosphorylation in HEK293/tau and N2a/tau cells treated with wortmannin or okadaic acid, it also attenuated tau pathologies with ameliorations of cognitive deficit and dendritic complexity in htau transgenic mice. Our data indicate that targeting I2PP2A is a promising strategy in arresting tau-related pathologies by not only restitution of PP2A but also inhibition of GSK-3β.