Hrd1 Facilitates Tau Degradation By Ubiquitin-proteasome Pathway

Aberrant aggregates of tau have been documented a common feature of many neurodegenerative diseases, collectively called tauopathy, including Alzheimer's disease, progressive supranuclear palsy and frontotemporal dementia with Parkinsonism linked to chromosome 17. Our previous studies found that Hrd1 and tau co-localized in cortex and hippocampal neurons of AD. There is an inverse relationship between Hrd1 and aggregated tau. We found that the expression of Hrd1 can decrease the total level of tau in cell models. On the basic of these studies, we investigated the mechanism of Hrd1 decreased the level of tau. Thus it would be helpful not only to further elucidate the pathological mechanism of tau, but also to offer theoretic basis for drug screening and therapy for patients with tauopathies based on Hrd1 target.OBJECTIVE:The purposes of this study were to investigate whether Hrd1 can enhance tau degradation through ubiquitin-proteasome pathway. METHODS:The plasmid encoding human tau was constructed and transfected to 293T cells along with wt-Hrd1 or Hrd1C1A (a mutant of Hrd1 with loss of E3 activity) using Lipofactamine 2000. The plasmid encoding GSK-3βwas transfected to cells to induce tau phosphorylation. Fluorescent microscopy and cofocal microscopy were used to observe cell morphology; Western blotting was used to determine the levels of proteins; Immunoprecipatation and immunofluorescent staining were used to observe the interaction and coexpression of tau and Hrd1.RESULTS:1. Hrd1 decreases the total level of tau in 293T cellsTo test whether Hrd1 can decrease the level of tau, we transfected tau or tau plus Hrd1 to 293T cells. It was found that the fluorescent intensity of GFP-tau in the cells was attenuated when co-transfected with Hrd1, compared with that transfected with tau alone. As the control, Hrd1 did not affect the fluorescent intensity of GFP vector. Meanwhile, Western blotting showed that cellular level of tau was decreased by cotransfected with wild type Hrd1, but not Hrd1C1A, a type Hrd1 with the loss of E3 activity. This indicates that Hrd1 decreased the total level of tau.2. Hrd1 facilitates tau degradationTo address whether the suppression on the total level of tau is correlated with the degradation of tau, cycloheximide (CHX) chase analysis was used to examine its rate of disappearance after protein synthesis was blocked. We chose CHX chase analysis since it has been widely used in determining ER-anchored E3s-mediated degradation of proteins in both yeast and mammalian cells. Cells were transfected either with tau alone or with tau plus Hrd1. 24 h after transfection, cells were treated with CHX and were chased for 4, 8, and 16 h. Levels of tau were determined by IB. The results showed that Hrd1 promoted tau elimination. These data indicate that Hrd1 removes tau by facilitating its degradation.3. Hrd1 facilitates phosphorylated tau degradationHyperphosphorylation of tau is crucial in the age-related formation of NFTs which is correlated well with neurotoxicity. The previous studies demonstrated that tau phosphorylation is a recognition requirement for the addition of ubiquitin by the ubiquitin ligase CHIP. We wondered whether ubiquitin ligase Hrd1 targets phosphorylated tau for proteasomal degradation. To verify this question, we cotransfected the plasmids encoding tau and GSK-3βto 293T cells to induce tau phosphorylation, based on the report that GSK-3β-induced hyperphosphorylation leads to the formation of paired helical filament (PHF)-like tau. We found that the total level of tau elevated after phosphorylation. This may be due to the increase of tau stability after phosphorylation. Hrd1 reduced the total amount of GSK-3β-induced phosphorylation of tau when it was cotransfected. This suggests that Hrd1 facilitated the degradation of phosphorylated tau.4. Proteasome is involved in Hrd1-mediated tau degradationTau can be degraded by proteasome. Hrd1 should target tau for proteasomal degradation in terms of the requirement of an intact RING finger of Hrd1 in tau degradation. To address this issue, we determined the levels of tau in 293T cells cotransfected with tau plus Hrd1 after treatment with the proteasome inhibitor MG132 and lysosome inhibitor ammonium chloride. The result shows that inhibition of proteasome activity by MG132 stabilized tau in the lysate either expressing Hrd1 or not, while ammonium chloride did not stabilize the levels of tau. When deubiquitination was inhibited by 5mM N-ethylmaleimide (NEM, Sigma), we observed high molecular weight tau accumulation stained with tau-5 antibody, suggesting proteasome involves in Hrd1-mediated tau degradation. 5. Hrd1 interacts with tau and enhances tau ubiquitinationIn our previous report, we found the cytosolic protein polyglutamine-containing huntingtin is a substrate for Hrd1. We wondered whether Hrd1 was able to interact with and ubiquitinate tau, although Hrd1 is an ER membrane protein and tau is the cytosolic protein. To test this, we first conducted co-immunoprecipitation experiments. Myc-tagged Hrd1 was cotransfected into 293T cells with tau and then immunoprecipitation was performed with the monoclonal tau-5 antibody. Detection of co-immunoprecipitating Hrd1 was performed by western blotting with monoclonal anti-myc antibody. Hrd1 was found to co-immunoprecipitate with tau, which did not depend on its E3 activity.To further ascertain whether Hrd1 ubiquitinates tau, 293T cells were cotransfected with myc-tagged Hrd1, His-tagged tau, and myc-tagged ubiquitin. High molecular weight tau was used as a potential indicator of ubiquitination. Proteasome inhibitor MG132 was used to block possibly ubiquitinated tau degradation. We found that Hrd1 overexpression increased high molecular weight forms of tau. We obtained the same result when His-tau was replaced with GFP-tau. Immunoprecipitated tau showed prominent anti-ubiquitin immunoreactivity in Hrd1-expressing cells. This suggests that the formation of high molecular weight forms of tau correlates with the increased ubiquitination. To characterize the effect of phosphorylation on Hrd1-mediated tau ubiquitination, the plasmid encoding GSK-3βwas cotransfected into the cells. the amount of high molecular weight tau increased dramatically in the cells expressing GSK-3β, suggesting that GSK-3β-induced phosphorylation can stabilize ubiquitinated tau, which may have potential functional implications for the role of ubiquitination in tau biology.CONCLUSIONS: These results suggest that Hrd1 facilitates tau degradation, which may be at least partially associated with ubiquitin-proteosome pathway.