Hrd1 Facilitates Hyperphosphorylated Tau Degradation In Neural Cells

Alzheimer's disease is the most common progressive neurodegenerative disease which is pathologically featured by the formation of neurofibrillary tangels mainly consisted of hyperphosphorylated tau. Abnormally hyperphosphorylation of tau plays a significant role in the pathogenesis of neurodegenerative disease, particularly in AD. Some researchers have reported that Hrd1, an Endoplasmic Reticulum membrane ligase, contributing in the degradation of abnormal protein aggregates. Additionally, our previous studies have found that Hrd1 co-localized with tau in cortex and hipocampal neurons. In view of above all, we speculate that Hrd1 maybe enhance the degradation of overphosphorylated tau as well. In present study, we investigated wether up or down-regulating the expresion of Hrd1 could give an effect on the level of exogenous or endogenous hyperphosphorylated tau in neural cells. The experimental results turn out to be as speculated, and we have also observed that Hrd1 knockdown had badly affected the morphological maintenance of primary cultivated emryonic cortic neurons under OA treatment.Objective:To investigate whether ER membrane ubiquitin ligase Hrd1 facilitates the degradation of hyperphosphorylated tau through ER-associated degradation (ERAD) in neural cells, and determine the effect on morphology of primary cultivated embryonic rat cortic neurons after Hrd1 knockdown plus OA treatment.Methods:After identifying the protein-interaction between Hrd1 with tau through co-immunopricipitation and protein complementation assay, followed experiments were performed. First, overexpressing both exogenous Hrd1 and tau in N2A cells, we determined the level of hyperphosphorylated tau induced by OA treament. Second, we checked the quantitative change of endogenous hyperphosphorylated tau in N2A cells after Hrd1 scilencing with validness-approved siRNA. Simultaneously, the relative degradatin rate was also detected via CHX assay. Otherwise, primary cultivation of embryonic rat cortic neurons was performed successfully, then we observed the morphological change caused by Hrd1 knockdown and OA addition.Results:1. Hrd1 interacts with tau1.1 Co-imminopricipitationHEK 293T cells were co-transfected with tau and Hrd1 or Hrd1C1A . Co-transfection of tau and empty vector was used as control. Twenty-four hours after transfection, cells were collected and lysed in Triton X-100 lysing buffer containing protease inhibitors cocktail. After centrifugation, set aside some supernatant for direct loading, the left supernatant were shifted to a new centrifuge tube to mingle with anti-myc and protein-A beads, rotating at 4℃overnight. The immunopriciptates were subjected to IB, and we spotted the band of tau in both Hrd1 and Hrd1C1A lane but vector. These results indicate the interaction between Hrd1 and tau in a RINF-finger domain-independent manner.1.2 Protein Complementation AssayTo further verify the interaction between Hrd1 and tau, we have constructed the splitted luciferase reporter gene plasmids containing Hrd1 or tau cDNA. Twenty-four hours after co-transfection of both constructs in HEK-293T cells, the cells were lysed and centrifuged at 4℃to obtain the supernatant. The relative luciferse activity of determined by Snergy HT Multiskan spectrum reader at 560 nm, remarkably increased in Hrd1/hGluc(1)+hGluc(2)-tau group contrast with the other groups, Zipper/hGluc(1) +Zipper/hGluc(2) was used as a positive control. These results may further confirm the interaction between Hrd1 and tau which contributed to the combination of splited subunit of hGluc.2. Hrd1 facilitates exogenous tau degradationN2A cells were co-transfected with tau and Hrd1, Hrd1C1A or empty vector respectively. Simultaneouly, GSK-3βwas additionally used to hyperphosphorylate the tau protein in a parallel group. The results showed that the level of tau was markedly reduced in Hrd1 lane compared with Hrd1C1A and empty vector lanes. The GSK-3βgroup was the same but generally higher than non- GSK-3β. This action was partially dependent of an intact RING finger domain. 3. Hrd1 reduces the endogenous phosphorylated tau level induced by OA in N2A cellsN2A cells were transfected with Hrd1, Hrd1C1A or empty vector respectively. OA, a widespread used phosphatase inhibitor, was added to induce phosphorylation of endogenous tau. The phosphorylated tau level detecd by AT-8 antibody showed a markedly decrease in Hrd1 lane than Hrd1C1A and vector lanes. Similarly, the action also relies on an intact RING finger domain.4. Hrd1accelerates the degradation rate of exogenous tauIn order to determine wether Hrd1 accelerates the degradation rate of tau, twenty-four hours after transfection in N2A cells, CHX, an inhibitor of protein biosynthesis was added to cultural medium to restrain the continuous synthesis of protein. Cells were collect at three time points (0, 8, 16 hrs) then subjected to IB with tau-5 antibody. In this way, we found Hrd1 group a significantly increase in degradation rate while the level in Hrd1C1A and empty vector groups remained steady.5. Hrd1 knockdown and verificationThree strips of synthetic siRNA-Hrd1 were adopted to knockdown the Hrd1 transcription and further translation.Twenty-four hours after transfetion, both the total RNA and protein proportion were isolated with Trizol reagent.The RT-PCR results showed that the third siRNA-Hrd1 was the most efficient one in the inhibition of the mRNA level, which is consistent with the protein level detected by IB using anti-Syvn1 antibody. Therefore, the third siRNA-Hrd1 was chosen to the subsequent experiments. 6. Hrd1 knockdown stabilizes the level of endogenous phosphorylated tau induced by OATwenty-four hours after the transfection of validness-approved siRNA-Hrd1 into N2A cells, OA and CHX was added to cultural medium orderly, after that, cells were collected at three time points (0, 8, 16 hrs) then subjected to IB with AT-8 antibody. In this way, we found that Hrd1 knockdown had evidently stabilized the phosphorylated tau level and high molecular bands were detected in this group. This finding is much consistent with the notion that hyperphosphorylation promotes formation of neurofibrillary tangles in pathogenesis of Alzheimer's disease.7. Primary embryonic rat cortic neuron cultivation and identification through immunofluorencent stainingHad been digested with 0.05% trypsin in PBS for 10 minutes, embryonic rat cortic tissues were pipeted into single-cell suspension then seeded in 24-well dish. Nerobasal medium containing 10 nM Ara-C was used to maintain the neurons growth and eliminate the glial proportion. At the ninth day, immuocytochemical staining confirmed a successful cultivation of relative high purity of neurons.8. Hrd1 knockdown exacerbates disruption of neuronal axons induced by OA treatmentThe identified neurons were subjected to Hrd1 knockdown and OA treatment. To investigate the effect of Hrd1 knockdown imposed on neuronal morphological change, non-sense shRNA was used as negative control. We observed that Hrd1 knockdown lead to a severe disruption to the neuronal axons morphology with the time. Conclusions:From above experimental results we can draw conclusions that: based on the interaction between them, Hrd1 sufficiently accelerates wether exogenous tau or endogenous tau degradation dependent on an intact RING finger domain. Same like this, Hrd1 facilitates OA-induced hyperphosphorylated tau dagradtion as well. Hrd1 knockdown gives rise to a susceptibility of neuronal axons disruption exposed to OA treatment. Our study maybe shed a little light on seeking potential pharmacologic targets for neurodegenerative disease, especially AD.