Abstract
Ubiquitin C-terminal hydrolase L1 (UCH-L1) is critical for protein degradation and free ubiquitin recycling. In Alzheimer’s disease brains, UCH-L1 is negatively related to neurofibrillary tangles whose major component is hyperphosphorylated tau protein, but the direct action of UCH-L1 on tau has not been reported. In the current study, mouse neuroblastoma Neuro2a (N2a) cells were treated by the different concentrations of UCH-L1 inhibitor LDN (2.5, 5 and 10 μM) to inhibit the hydrolase activity of UCH-L1. In addition, we also used UCH-L1 siRNA to treat the HEK293/tau441 cells to decrease the expression of UCH-L1. After LDN and UCH-L1 siRNA treatment, we used immunofluorescence, immunoprecipitation, and tau-microtubule binding assay to measure the microtubule-binding ability and post-translational modifications of tau protein. All the results presented that both inhibition of the activity and expression of UCH-L1 induced the decreased microtubule-binding ability and increased phosphorylation of tau protein. Abnormal aggregation and ubiquitination of tau protein was also observed after UCH-L1 inhibition. The above results suggested that aggregation of tau protein might be devoted to the abnormal post-translational modifications of tau protein. Our study first indicates that dysfunction of UCH-L1 most likely affected normal biological function of tau protein through decreasing degradation of ubiquitinated and hyperphosphorylated tau.
Keywords
INTRODUCTION
Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), also known as protein gene product 9.5 (PGP9.5), is one of the most abundant proteins in the brain, consisting of 223 amino acids and constituted 1–5% of total brain soluble proteins [1]. UCH-L1 is predominantly expressed in neurons and neuroendocrine cells [2] and possesses deubiquitinating enzyme (DUB) activity that catalyzes hydrolysis of C-terminal esters and amides of ubiquitin (Ub) to generate monomeric ubiquitin and this activity depends on its monomeric form [3, 4]. In addition, UCH-L1 also possesses a dimerization-dependent E3 ubiquitin ligase activity [5] and functions as a monoubiquitin (mono-Ub) stabilizer [6]. The stabilizing effect of UCH-L1 is by binding mono-Ub and independent of its enzymatic activity [6]. UCH-L1 contributes to regulate the cellular pool of available Ub and as a DUB, UCH-L1 facilitates Ub recycling; as an Ub-stabilizing factor, UCH-L1 also binds mono-Ub and prevents the degradation by lysosome [7].
UCH-L1 was proved to be critical in cognitive function and its deficiency to be closely related to neurodegenerative disorders [8–11]. UCH-L1 is required for normal synaptic structure and function; pharmacological inhibition of UCH-L1 dramatically alters synaptic protein distribution and spine morphology [8]. In addition, the gracile axonal dystrophy (gad) mouse that lacks functional UCH-L1 expression displays axonal degeneration of the gracile tract and accumulation of ubiquitin and amyloid-β protein deposits [9]. UCH-L1 was associated with Parkinson’s disease (PD); the I93M mutation of UCH-L1 displays increased insolubility and decreased interaction with monoubiquitin and leads to the selective loss of dopaminergic neurons which is a causative mutation of PD [10]. Other significant studies have linked UCH-L1 dysfunction to neurodegeneration of Alzheimer’s disease (AD). Proteomics analysis indicated that the level of UCH-L1 was aberrant in AD hippocampal proteome [12]. Immunohistochemical studies showed UCH-L1 is associated with neurofibrillary tangles (NFTs) and the reduction soluble UCH-L1 was inversely proportional to the number of NFTs in AD brains [13, 14]. Minjarez and colleagues identified the NFTs derived from AD brains contained UCH-L1 component and proved the colocalization of UCH-L1 and hyperphosphorylated tau protein in NTFs [15]. These results implied a close relationship between UCH-L1 abnormality and tau aberration in AD brain.
NFT is one of the typical hallmarks lesions of AD, which is mainly composed of hyperphosphorylated microtubule-associated protein tau protein. In addition to AD, pathological tau protein is observed in tauopathies such as corticobasal degeneration, progressive supranuclear palsy, argyrophilic grain disease, globular glial tauopathies, Pick’s disease, and hereditary frontotemporal dementia associated with mutations in the microtubule-associated protein tau gene (MAPT) [16, 17]. Unlike normal tau protein, the abnormally hyperphosphorylated tau occurs in tauopathies is resistant to proteolysis and subsequently becomes susceptible for polyubiquitinated [18]. It has been reported that oxidative damage and downregulation of soluble UCH-L1 lead to decreased hydrolase activity and increased ligase activity of UCH-L1 that might cause dysfunction of the neuronal ubiquitination/deubiquitination and neuronal degeneration in AD and PD brains [10]. In addition, the association of UCH-L1 with NFTs could also be devoted to the aberrant ubiquitin hydrolase and/or ligase activity of UCH-L1 in AD [10]. Research suggests that the abnormal activity and modification of UCH-L1 should be involved in the formation of NFTs. NFTs are composed of hyperphosphorylated tau, and phosphorylation is essential for the normal biological functions of tau, including microtubule-binding function, signaling pathways modulation, and axonal transport [19, 20]. Tau protein plays a critical role in regulating neuronal microtubules assembly and stabilization [21], while abnormal hyperphosphorylated tau would result in the inhibition of microtubule assembly and disruption of microtubules [20]. Accordingly, we speculated that UCH-L1 dysfunction probably influences the microtubule binding function of tau protein. However, there was no report about the effects of UCH-L1 inhibition on the biological ability of tau protein until now.
The ubiquitination modification is important for the degradation of tau protein by ubiquitin-proteasome system (UPS), therefore, UCH-L1 dysfunction may play critical roles in aberrant tau function and accumulation which observed in tauopathies. In order to investigate the effect of UCH-L1 inhibition on the biological function of tau protein, UCH-L1 inhibitor LDN and UCH-L1 siRNA were used in the present study. We found that the pharmacological suppression of ubiquitin hydrolase activity of UCH-L1 was correlated with a decreased microtubule-binding function and aberrant accumulation of tau protein, and the decreased degradation of tau including hypophosphorylated and ubiquitinated tau might be responsible for tau biological function impairment. Additionally, decreased UCH-L1 expression also induced hyperphosphorylation and dysfunctional microtubule-binding ability of tau protein. Our research implied a new insight to clarify the relationship between UCH-L1 inhibition and tau dysfunction and provided a new strategy to alleviate tau aggregation.
MATERIALS AND METHODS
Reagents and antibodies
UCH-L1 inhibitor (LDN-57444, LDN) was purchased from Calbiochem (San Diego, CA, USA). Thioflavine S was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used to detect aggregates of beta-pleated sheet-rich structures [22]. The dye Hoechst 33258 was used to stain DNA. The following antibodies were used in this study: rabbit anti-ubiquitin antibody (Abcam, New Territories, Hong Kong); rabbit anti-UCH-L1, rabbit anti-β-actin, and rabbit antibodies Tau-5, Thr205, Thr231, and Ser396 against total tau, phosphorylated tau at Thr205, Thr231, and Ser396 site respectively (Bioworld Technology, Louis Park, MN, USA); rabbit anti-α-tubulin (Santa Cruz Biotechnology, CA, USA). AT8 is a monoclonal antibody recognizing phosphorylation sites Ser202 and Thr205 sites of tau proteins [23] (Thermo scientific, Rockford, IL, USA).
Cell culture and drug treatments
Mouse neuroblastoma Neuro2a (N2a) cells were cultured in 45% DMEM and 45% Opti-MEM supplemented with 10% (v/v) fetal bovine serum in a 5% CO2 humidified incubator at 37°C. Different concentrations of UCH-L1 inhibitor LDN (2.5, 5, 10 μM) was used to treat N2a cells for 1 h to inhibit UCH-L1 activity. LDN was dissolved in dimethyl sulfoxide (DMSO). The concentration of DMSO during drug treatments was maintained in 0.1% . HEK293/tau441 cells were cultured in 90% DMEM supplemented with 10% (v/v) fetal bovine serum and 200 μg/mL G418 in a 5% CO2 humidified incubator at 37°C. Different concentrations of UCH-L1 siRNA (20, 40, 80 nM) was used to treat HEK293/tau441 cells for 6 h to inhibit the expression of UCH-L1.
Western blot analysis
The Western blot analysis was performed as described with some modifications [24]. After LDN treatment, whole-cell extracts were prepared for biochemical analyses. The cells were lysed in ice-cold cell lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% [w/v] sodium dodecyl sulfate, 0.1% [v/v] NP-40, 0.5% sodium deoxycholate, 0.02% NaN3, supplemented with protease inhibitors aprotinin [2 μg/ml] and phenylmethysulfonyl fluoride [PMSF, 100 μg/ml]). The protein concentration was determined by BCA (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA). Equal amounts of sample proteins were separated according to their molecular weight on 10% SDS-polyacrylamide gel electrophoresis (PAGE) and then electrically transferred onto nitrocellulose membrane (Millipore, Billerica, MA, USA). Especially, 15% SDS-PAGE were used for detecting the mono-ubiquitin and 10% SDS-PAGE for the poly-ubiquitin. After transfer, membranes were blocked with 5% defatted milk in TBS-T (10 mM Tris-HCl, 150 mM NaCl, 0.1% (v/v) Tween-20, pH 7.5) for 60 min at room temperature. Membranes were probed with the primary antibody diluted with 5% BSA and incubated overnight at 4°C. The following primary antibodies were used: anti-UCH-L1 (1:500), anti-Tau-5 (1:500), anti-phospho-tau (Ser396) (1:500), anti-phospho-tau (Thr205) (1:500), anti-phospho-tau (Thr231) (1:500), anti-β-actin (1:1000), anti-α-tubulin (1:800) and anti-ubiquitin (1:1000). Membranes were rinsed, incubated with IRDye 800CW Conjugated Goat (polyclonal) Anti-Rabbit IgG (LI-COR Biosciences, Nebraska, USA) (1:10000) for 1 h at room temperature and visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences, Nebraska, USA).
Immunofluorescence
Sterilized coverslips were placed in 24-well plates and transferred N2a cells into the plates and cultured in a 5% CO2 humidified incubator at 37°C. When the cells were 40–70% confluent and they were ready for fixation. Rinsed the cells with phosphate buffered saline (PBS) and then fixed with methanol at –20°C for 8 min. Aspirated fixative and then rinsed three times with PBS and subsequently permeabilized with 0.5% Triton X-100 in PBS buffer for 15 min. After rinsing cells three times with 0.2% Triton X-100 in PBS, blocking buffer 5% BSA were added and incubated for 1 h. The immunostaining procedure was performed on the cultured cell lines before the Thioflavine S staining. Primary antibodies mouse anti-AT8 (1:50) was added in blocking buffer and cultures were incubated overnight at 4°C. After three washes with 0.2% Triton X-100 in PBS, cells were incubated in goat anti-rabbit secondary antibodies conjugated to AT8 at room temperature for 1 h. Cells were washed three times with 0.2% Triton X-100 in PBS and added nuclear counterstain Hoechst (1 μg/ml) to incubate for 15 min. The Thioflavine S staining was performed as described with some modifications [25]. Cells were washed three times with 0.2% Triton X-100 in PBS and incubated in 0.01% Thioflavine S for 5 min. Finally, cells were washed three times with ethanol and mounted on slides with buffered glycerol. The images were obtained with fluorescence microscopy on an XDS-1 microscope.
Immunoprecipitation
After harvesting, the cells were washed with ice-cold PBS, drained, and added to ice-cold radio immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.5% [w/v] sodium deoxycholate, 1% [v/v] NP-40, 0.1% [w/v] SDS, 100 μM sodium orthovanadate, 1 mM PMSF and 1 μg/ml aprotinin). The cells were then lysed on ice for 20 min and centrifuged at 12,000 rpm at 4°C for 20 min and then the supernatant was transferred to a fresh tube kept on ice. To reduce the nonspecific binding of proteins, 10 μl protein G agarose beads was added to 100 μl cell lysate and incubated at 4°C for 30–60 min, followed by centrifugation at 12,000 rpm at 4°C for 15 min and removal of the supernatant to a fresh tube. Added primary antibody Tau-5 to this tube and incubated with gentle rocking overnight at 4°C. After that 20 μl protein G agarose beads was added to the tube and incubated with gentle rocking for 1–3 h at 4°C to capture the immune complex. The tube was then centrifuged for 1 min at 12,000 rpm at 4°C, and the pellet was washed three times with ice-cold PBS and drained. The obtained pellet contained the protein G (antibody-binding protein) and was resuspended in SDS sample buffer, heated to 95–100°C for 2–5 min, and then centrifuged for 1 min at 12,000 rpm. The obtained solution was then loaded on SDS-PAGE gel and analyzed by Western blotting.
Tau-microtubule binding assay
The microtubule (MT)-binding assay was performed as described [26]. Briefly, N2a cells were harvested in high-salt reassembly buffer (0.1 M Tris, 0.5 mM MgSO4, 0.75 M NaCl, 1 mM EGTA, 2 mM dithiothreitol, pH 6.8) supplemented with 0.1% Triton X-100, 20 μM taxol, 2 mM GTP, and a mixture of protease inhibitors (2 mM PMSF and 1 μg/ml Aprotinin) at 37°C. Cell lysates were homogenized and then immediately centrifuged for 20 min at 50,000×g at 25°C. The supernatant (S) containing unbound tau was removed, and the protein concentration was determined. The remaining pellet (P) was resuspended in a 2×volume of sample buffer corresponding to the total volume of supernatant after normalizing to total protein. Equal amounts of pellet and supernatant fraction were separated by SDS-PAGE, blotted, and processed for immunodetection with tau-5 and anti-tubulin antibodies. The ratio of tau bound to MTs (P) versus unbound tau (S) was assessed by comparing the tau immunoreactivities in these two fractions.
siRNA interference
For siRNA-mediated knockdown of UCH-L1, the HEK293/tau441 cells were transfected with either the targeting or control siRNA (Santa Cruz Biotechnology, TX, USA). The siRNA pools of three duplexes were used to uch-l1 gene and the sequences of siRNA targeting human UCH-L1 are as follows: (1) sense: 5′-CUUCAGUACUUGUGAAACAtt-3′; antisense: 5′-UGUUUCACAAGUACUGAAGtt-3′; (2) sense: 5′-GUCUUGUAUCCGAUAUCUAtt-3′; antisense: 5′-UAGAUAUCGGAUACAAGACtt-3′; (3) sense: 5′-GGUUUCUGUCUGUAAGUUAtt-3′; antisense: 5′-UAACUUACAGACAGAAACCtt-3′. The siRNA interference procedures were taken according to the manufacturer’s instructions. Briefly, the HEK293/tau441 cells were seeded at in 6-well plates, 2 × 105 cells per well. Medium without antibiotic was added and incubate the cells at 37°C until the cells are 60–80% confluent, when the transfection was conducted. For each transfection, prepared 1 ml transfection mixture including 100 μl siRNA transfection reagent and 900 μl siRNA transfection medium, and the concentrations of UCH-L1 siRNA is 20, 40, and 80 nM. Aspirated the normal growth medium, washed cells with 1 ml transfection medium and added the mixture onto the washed cells, incubated the cells 6 h at 37°C in the CO2 incubator. Aspirated the mixture and replaced with fresh normal growth medium, incubated the cells for 24 h. After assaying the cells, the lysates were collected for Western blot and tau-microtubule binding assay.
Statistic analysis
For the measurement of Western blot observations, data were analyzed with SPSS 13.0 statistical software and expressed as means ± S.D. One-way analysis of variance (ANOVA) was used for multiple groups’ comparison. Differences were considered significant at p < 0.05. Fluorescence intensity of AT8 and Thioflavine S were measured with the Edgefitter NIH ImageJ plug-in (Ghosh Laboratory, University of California, San Diego, La Jolla, CA). Measurements were then automatically logged from NIH ImageJ.
RESULTS
UCH-L1 inhibition drives abnormality of ubiquitin levels
The major function of UCH-L1 is to maintain the level of free monomeric ubiquitin, thus inhibition of UCH-L1 activity can affect ubiquitin pool homeostasis [5, 6]. Firstly, different concentrations of UCH-L1 inhibitor LDN (2.5, 5, and 10 μM) were used to treat N2a cells and we found that UCH-L1 inhibition did not affect cell viability (Supplementary Fig. 1). Secondly, we isolated total protein from LDN treated and non-treated N2a cells to test the level of UCH-L1. Immunoblots of these total lysates confirmed that LDN treatment had no obvious effect on the UCH-L1 expression (Supplementary Fig. 2). Afterwards, to determine the alteration in ubiquitin equilibrium induced by UCH-L1 inhibition, the levels of monomeric ubiquitin and polyubiquitin conjugates in LDN treated N2a cells were tested. We found that treatment of cells with 2.5, 5, and 10 μM LDN for 1 h increased the levels of polyubiquitin conjugates in a dose-dependent manner (Fig. 1A, B), meanwhile, the levels of monomeric ubiquitin decreased significantly after LDN exposure (Fig. 1A, C). The results suggested that LDN affected the ubiquitin hydrolase function of UCH-L1.
Inhibition of UCH-L1 decreases the microtubule-binding function of tau protein
The aberrant ubiquitin hydrolase activity of UCH-L1 associated with hyperphosphorylated tau protein [10], and the abnormal hyperphosphorylation could attenuate the microtubule binding function of tau protein [20]. Therefore, to determine whether UCH-L1 inhibition had an effect on the biological function of tau protein, we carried out the tau-microtubule binding assay. First, we used LDN to treat N2a cells and found that UCH-L1 inhibition did not affect the expression of tubulin (Supplementary Fig. 3). In tau-microtubule binding assay, we used taxol to stabilize the microtubule. Subsequently, the tau protein bound to taxol-stabilized microtubules and the unbound tau were separated by gradient centrifugation, the supernatant containing unbound tau and the pellet containing microtubule-bound tau. After LDN treatment, the levels of tau in the pellet and the supernatant fractions were analyzed respectively. The ratio of immunoreactivities in MTs-bound tau (P) versus unbound tau (S) fractions was represented the changed microtubule-binding function of tau induced by UCH-L1 inhibition. In addition, compared with the control, the level of tau in the supernatant increased while the level of tau in the pellet decreased (Fig. 2A, B), additionally, the ratio of microtubule-bound tau (P) versus unbound tau (S) was decreased induced by UCH-L1 inhibition (Fig. 2C).
Furthermore, we explored whether the microtubule-binding function of tau protein could be disturbed by the UCH-L1 reduction. We used different concentrations of UCH-L1 siRNA to treat HEK293/tau441 cell line which expressed full-length human tau protein. We found that the level of UCH-L1 was decreased obviously by UCH-L1 siRNA at 80 nM (Fig. 2D) and the concentration was used in the consequent test. After MTs were stabilization with taxol and isolated from cells, the amount of tau was determined in the pellet (microtubule-associated fraction) and supernatant (soluble fraction) by Western blot analysis and compared with the immunoreactivity of tubulin in the same fractions. The tau-microtubule binding assay showed that compared to the control, the level of tau in the pellet (P) decreased while the tau protein in the supernatant fractions (S) were increased(Fig. 2E-G). Together, the above results suggested that UCH-L1 deficiency decreased the microtubule-binding ability of tau protein.
UCH-L1 inhibition induces tau aggregation in N2a cells
In AD brain, UCH-L1 was downregulated and associated with NFTs [13], which consisted of abnormal aggregated tau, thus we were interested to determine whether UCH-L1 activity inhibition had any effects on the tau aggregation. After cells were treated with LDN (2.5, 5, and 10 μM), the immunocytochemical distribution of beta-pleated sheet-rich structures aggregates and phosphorylated tau (Ser202/Thr205) were detected by Thioflavine S and AT8, respectively. As shown in Fig. 3, elevated immunoreactivity of AT8 (tau-Ser202/Thr205) revealed significantly hyperphosphorylated tau protein in LDN treated cells; the Thioflavine S staining indicated that the LDN exposure promoted intracellular abnormal aggregation. In addition, the merged results showed the increased co-localization of AT8 and Thioflavin S in a LDN dose-dependent manner. The result implied that UCH-L1 inhibition induced the abnormal aggregation of hyperphosphorylated tau protein in N2a cell.
UCH-L1 inhibition induces hyperphosphorylation of tau protein
UCH-L1 has an effect on promoting UPS-dependent substrates degradation [11] and tau protein is a substrate of UPS [27]. Moreover, hyperphosphorylation of tau protein can result in the dissociation of tau from microtubules which cause abnormal aggregation of tau protein [28]. Since UCH-L1 inhibition weakened the microtubule-binding ability of tau protein (Fig. 2) and led to tau aggregates (Fig. 3), we then examined whether UCH-L1 inhibition had a detectable impact on the degradation and phosphorylation of tau protein. We first treated the N2a cells with LDN and found that both the level of total tau protein (probed by tau-5) and phosphorylated tau at Ser396, Thr205, and Thr231 epitopes were increased in a LDN concentration-dependent manner (Fig. 4A-C). Furthermore, we used UCH-L1 siRNA to inhibit the expression of UCH-L1, as shown in Fig. 4D-F, the levels of phosphorylated tau at Ser396, Thr205, and Thr231 epitopes were increased following UCH-L1 siRNA treatment. These results suggested that the degradation of tau protein, including the phosphorylated tau protein could be disturbed after UCH-L1 inhibition.
Inhibition of UCH-L1 activity increases ubiquitination of tau protein
Since UCH-L1 inhibition increased levels of polyubiquitin conjugates in a LDN dose-dependent manner (Fig. 1) and hyperphosphorylated tau protein was susceptible for ubiquitination modification [29]. We therefore explored the effect of UCH-L1 inhibition on the ubiquitination modification of tau protein by immunoprecipitate. The input is a positive control which used to prove the existence of tau protein and ubiquitin in the samples obtained from LDN treated and untreated cell lysates. After incubating with antibody tau-5, the samples that contained the immune-complex were used for immunoblot analysis. The association of tau protein and ubiquitinated proteins in the immune-complex was validated by tau-5 (probed the total tau protein) and anti-ubiquitin antibody (probed the ubiquitination-conjugated proteins). As the result showed, compared with the untreated cells, the amount of co-immunoprecipitated ubiquitination-conjugated proteins in LDN treated cells was markedly increased. Meanwhile, LDN treatment also increased the amount of co-immunoprecipitated tau protein (Fig. 5). Together, these data point to the existence of ubiquitinated tauprotein and UCH-L1 inhibition may block the degradation of ubiquitinated tau.
DISCUSSION
Deubiquitinating enzyme UCH-L1 appears at high cytoplasmic levels (including neuron) and is critical for cytoplasmic protein degradation, recycling free ubiquitin by catalyzing hydrolysis polyubiquitin gene product and ubiquitin-protein conjugates [30]. Dysfunction and/or impairment of UCH-L1 would influence the protein degradation and affect the normal physiological function of cells. Inhibition of ubiquitin hydrolytic activity of UCH-L1 could result in dramatic alternations in synaptic protein distribution and spine morphology [8]. Additionally, mouse which lacks functional UCH-L1 appears to disturb the reusage of free ubiquitin which results a lowering of free monoubiquitin levels and accumulation of abnormal proteins in brain [31]. These researches demonstrate that the normal function and activity of UCH-L1 are important to the ubiquitin-dependent degradation.
The degradation of tau protein is mainly mediated by ubiquitin-proteasome system [32] and UCH-L1 is involved in the regulation of this process. Research revealed that the dysfunction and abnormal modification of UCH-L1 was positive related to the neurofibrillary tangles which were polymerized by the abnormal hyperphosphorylated and aggregated tau protein [13, 14]. Moreover, the biological activity of tau protein including microtubule-binding function is primarily regulated by the phosphorylation modifications [33]. However, the direct effect and related mechanisms of UCH-L1 inhibition on the microtubule-binding capacity of tau protein are still unknown.
In this study, we used LDN to inhibit UCH-L1 activity and found monoubiquitin level obviously decreased and polyubiquitinated proteins increased in cultured N2a cells. In addition, the alternations of polyubquitin and monoubiquitin indicated the LDN treatment in-duced dysfunction of UCH-L1 and disturbed the ubiquitin-dependent degradation of polyubiquitinated proteins. These results are consistent with the previous reports that impaired or deficient ubiquitin hydrolytic activity of UCH-L1 is capable of affecting the degradation and turnover of proteins [11, 13]. Subsequently, to determine whether UCH-L1 inhibition could affect the interaction of tau and microtubule, the tau-microtubule binding assay was carried out. We found that LDN treatment attenuated the microtubule-binding capacity of tau protein. We also used UCH-L1 siRNA to inhibition the expression of UCH-L1 and found that microtubule-binding capacity of tau protein was attenuated. These results mean that UCH-L1 inhibition promotes the dissociation of tau protein from microtubule. Once tau protein dissociates from microtubule, it is apt to self-aggregate into paired or straight filaments then aggregated in the cytoplasm [28]. Thus, the decreased microtubule binding ability of tau protein triggered us to explore whether UCH-L1 inhibition induced tau aggregation.
Double-staining of AT8 and Thioflavine S showed increased co-localization of hyperphosphorylated tau (Ser202/Thr205) and beta-pleated sheet-rich structures in the LDN treated N2a cells. Antibody AT8 is usually used to detect paired helical filaments (PHF)-tau (Ser202/Thr205), and PHF is a major component of the NFTs. Therefore, we used AT8 to detect the abnormal aggregation containing hyperphosphorylated tau in Fig. 3. The increased immunoreactivity of AT8 and Thioflavine S staining suggested that decreased UCH-L1 activity related to the accumulation of hyperphosphorylated tau (Ser202/Thr205). Hyperphosphorylated tau protein is associated with decreased level and attenuated hydrolase activity of UCH-L1 [10, 11], thus UCH-L1 activity inhibition is likely to affect the ubiquitin-dependent degradation of hyperphosphorylated tau protein and promote the formation of tau aggregates. Polymerization and hyperphosphorylation of tau protein can attenuate the microtubule-binding capacity [21]. The ability to bind and stabilize microtubules is a hallmark of tau and phosphorylation of tau protein at some specific sites plays a critical role in regulating tau-microtubule interactions [16], whereas the hyperphosphorylation modification attenuates or impairs the biological activity of tau protein [34]. Additionally, polymerization of hyperphosphorylated tau inhibits the microtubule assembly and disrupts tau-mediated microtubule dynamics regulation [21]. Thus, we further detected the levels of phosphorylated tau of the sites which related to its microtubule binding function. We observed that both inhibition of UCH-L1 activity and reduction of UCH-L1 expression increased the phosphorylation of tau protein at many epitopes including Thr205, Ser396, and Thr231. Phosphorylation at Thr231 causes a local conformational change that allows the access of kinases to further phosphorylate tau [34] and diminishes the ability of tau protein to bind microtubules in situ [35]. Additionally, phosphorylation at Thr231 and Ser396 enhances self-aggregation of tau into filaments [36]. The phosphorylation of Thr205 in tau has been shown to not only enhance polymerization but also make filaments formation [37]. The hyperphosphorylation of tau protein probably promotes the self-assembly and facilitates aggregation and filaments formation [38]. Therefore, together with the above results, we speculate that UCH-L1 inhibition induced tau hyperphosphorylation impairs the microtubule binding capacity and promotes the abnormal aggregation of tau.
Tau protein is a substrate of UPS and ubiquitination has been found to be a signal of tau protein for proteasome degradation [27]. AD type phosphorylation of tau is the signal for its ubiquitination modification, the CHIP-Hsc70 complex conjugates Ub to hyperphosphorylated tau to degrade through proteasome pathway [39]. In the current study, we found UCH-L1 inhibition induced aberrant hyperphosphorylation and aggregation of tau protein, in addition, the increased ubiquitination of tau protein also observed after LDN treatment. According to the above mentioned, the hyperphosphorylation was likely to be a signal for ubiquitination modification of tau protein in our study. Besides, proteomics analysis of AD hippocampal proteome indicated the expression aberration of UCH-L1 [12]. Minjarez and colleagues identified the NFTs derived from AD brains contained UCH-L1 component and proved the colocalization of UCH-L1 and hyperphosphorylated tau protein in NTFs [15]. These results implied a close relationship between UCH-L1 abnormality and tau aberration in AD brain. Together, these results suggested that dysfunction or aberration of UCH-L1 could decrease the proteasome-dependent degradation of tau via attenuating the hydrolysis of ubiquitin-conjugated tau protein.
In summary, we reported that the inhibition of UCH-L1 increased the content of hyperphosphorylated and ubiquitinated tau protein which further decreased the microtubule-binding function of tau protein and promoted tau abnormal aggregation. Our data suggested that the abnormal modifications and accumulation of tau protein caused by UCH-L1 inhibition probably devoted to the degradation impairment of abnormal tau protein. Accordingly, the current study provides a novel experimental evidence to illustrate the pathological process associated with the decreased biological function and abnormal aggregation of tau protein in tauopathies.
Footnotes
ACKNOWLEDGMENTS
We thank Professor Wang Jianzhi at Tongji Medical College of Huazhong University of Sciences and Technology, Wuhan, China, for providing N2a and HEK293/tau441 cell lines used in these studies. This work was financially supported by grants obtained from the financially supported by self-determined research funds of CCNU from the college’s basic re-search and operation of MOE (CCNU2015A02024 and CCNU15A02029) and National Natural Science Foundation of China (No. 81361120245 and No. 31172102).
