| Tau, a neuronal microtubule-associated protein, is known to stabilize and promote the formation of microtubules. Aberrant aggregates of tau have been documented a common feature of many neurodegenerative diseases, collectively called tauopathy. The dysfunction of tau degradation under some conditions may be the main cause of tau aggregation. Therefore, reducing intracellular abnormal tau should be an effective method to protect cells from tau-induced toxicity. Human hrd1 (Hrd1) is a newly identified endoplasmic reticulum (ER) membrane-spanning E3 with its catalytically active RING finger facing the cytosol, mediating protein ubiquitination and degradation. Our previous studies found that there is an inverse relationship between Hrd1 and NFTs in cortical and hippocampal neurons of AD. This implies that Hrd1 may be protective against tau-mediated toxicity. In this study we investigated the toxic effect of tau on neuro-derived or non-neuronal cells. 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 the toxic effect of tau on neuro-derived or non-neuronal cells and the protective effect of Hrd1 against it.METHODS:The plasmid encoding human tau was constructed and transfected to 293T and SH-SY5Y 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; MTT assay was used to determine cell viability; Western blotting was used to determine the levels of proteins; Immunofluorescent staining was used to observe the coexpression of tau and Hrd1.RESULTS:1. The toxic effect of tau on SH-SY5Y and 293T cells To investigate the direct toxicity of GFP-tau to cells, we transfected tau or blank vector to SH-SY5Y and 293T cells. For tau phosphorylation, tau was cotransfected with GSK-3β. It was found that many of the cells transfected with tau were deattached and some of them became round 24 h after transfection. Transient transfection with tau easily leads to axons or pseudopods disappearance and cell death. Meanwhile, multinucleared cells and large size cells can be seen after tau transfection. The long-term toxicity of tau developing over a period of days in culture is associated with cell apoptosis. Most evidently, the cell nucleus were disassembled into small particles of condensed chromatin, which represent degraded nuclear DNA fragments. Moreover, plasma membrane blebbing was also observed. These results suggest that overexpression of tau is directly toxic to cells.The fluorescent intensity of GFP-tau in the cells was enhanced and the morphology of cells became worse after cotransfection of tau with GSK-3βor co-incubation with OA. This indicates that tau toxicity increased after phosphorylation.2. Hrd1 protects cells from tau-mediated toxicityTo test the potentially protective effects of Hrd1, we cotransfected human tau with Hrd1 into 293T cells. Tau and Hrd1 expressions were observed under cofocal microscope followed by double-labelled staining of immunofluorescence. It was found that most of the cells either expressing Hrd1 alone or coexpressing Hrd1 plus tau attached well with pseudopods 24 h after transfection, but not that transfected tau alone. In the investigation of single cell, we found that the cell expressing small amount of tau and large amount of Hrd1 presents normal morphology with long pseudopods. Hrd1 became less and the morphology of cells got worse along with the increase of tau expression.Up to fifth day after transfection, we observed typically apoptotic characteristics in those cells transfected with tau alone. Interestingly, if the cells were cotransfected stably with tau plus Hrd1, the cells in a large proportion still survived with almost normal morphology. This indicates that Hrd1 can antagonize tau toxicity and rescue cells from apoptosis.3. Hrd1 E3 activity is required for the protection from tau toxicityTo test if lacking of E3 activity abolishes the effect of Hrd1 on tau toxicity, we used the construct of pcDNA3.1-Hrd1C1A (Hrd1 mutant with loss of E3 activity). Wild type Hrd1 was used as a positive control. It was found that the morphology of cells was abnormal after cotransfection tau with Hrd1C1A, while wild type Hrd1 maintained cells in almost normal shapes. These data suggest that Hrd1 E3 activity is involved in maintenance of normal cell morphology.Additionally, not like wild type Hrd1, Hrd1C1A did not enhance cell survive when cotransfected with tau. On the contrary, Hrd1C1A deteriorated the morphology of cells. 4. Hrd1 reduces the total level of tau in 293T cells and E3 activity is required for Hrd1-mediated removal of tauDuring exploring the mechanisms by which Hrd1 protect cells from tau toxicity, we notice an interesting phenomenon that the fluorescence of GFP-tau in the cells cotransfected with wild type Hrd1 became weak, compared with that in the cells transfected with tau alone. More interestingly, if we focus on single cell, we found the local tau accumulation was removed by wild type Hrd1 observed under cofocal microscope, while Hrd1C1A did not remove the local tau where it expressed. Western blot showed that Hrd1 reduced the total level of tau, compared with tau alone, but not Hrd1C1A. This suggests that Hrd1 E3 activity is involved in, at least partially, Hrd1-mediated tau reduction.5. Hrd1 decreases the level of phosphorylated tau in 293T cellswe phosphorylated tau by using GSK-3β. In current study, we found GSK-3βphosphorylated tau in a dose-dependent manner when cotransfected it with tau. Meanwhile, GSK-3βincreased the total level of tau. If we cotransfected tau, GSK-3β, and Hrd1 to 293T cells, Hrd1 decreased the total level of tau either treated or untreated with GSK-3β. This suggests that Hrd1 not only decreased native tau, but also decreased phosphorylated tau.CONCLUSIONS:The protective effects of Hrd1 against tau-mediated toxicity may be related to removal of tau from cells, which may be, at part associated with ubiquitin-proteosome pathway. |
