Ubiquitin Specific Protease 13 Regulates Tau Accumulation and Clearance in Models of Alzheimer’s Disease 1

Abstract

Ubiquitin Specific Protease-13 (USP13) is a de-ubiquinating enzyme that regulates protein ubiquitination and clearance. The role of USP13 is largely unknown in neurodegeneration. In this study we aim to demonstrate whether tau accumulation and/or clearance depends on ubiquitination/de-ubiquitination via USP-13. We used transgenic animal models of human amyloid precursor protein (APP) or P301L tau mutations and genetically knocked-down USP13 expression via shRNA to determine USP13 effects on tau ubiquitination and levels. We found a two-fold increase of USP13 levels in postmortem Alzheimer’s disease (AD) brains. USP13 knockdown significantly increased the activity of the 20S proteasome and reduced the levels of hyper-phosphorylated tau (p-tau) in primary cortical neurons. USP13 knockdown also reduced the levels of amyloid and increased p-tau ubiquitination and clearance in transgenic animal models that overexpress murine tau as a result of the expression of familial APP mutations (TgAPP) and the human mutant P301L tau (rTg4510), respectively. Clearance of p-tau appears to be mediated by autophagy in these animal models. Taken together, these data suggest that USP13 knockdown reduces p-tau accumulation via regulation of ubiquitination/de-ubiquitination and mediates its clearance via autophagy and/or the proteasome. These results suggest that USP13 inhibition may be a therapeutic strategy to reduce accumulation of plaques and toxic p-tau in AD and human tauopathies.

Keywords

Alzheimer’s disease amyloid tau ubiquitin USP13

INTRODUCTION

Ubiquitin specific protease (USP)-13 is a de-ubiquitinase member of the cysteine-dependent protease superfamily [1]. Several findings implicate de-ubiquitinases in neurodegeneration [2 –7]. In melanoma cells, USP13 regulates the degradation of several proteins primarily via ubiquitination and de-ubiquitination [8, 9]. However, the significance of USP13 in regulating protein clearance in Alzheimer’s disease (AD) and the tauopathies is largely unknown. USP13 regulates stress granules containing lysine48- and lysine63-linked ubiquitin chains, through deubiquitylating protein-conjugated ubiquitin [10]. USP13 knockdown increases alpha-synuclein ubiquitination and clearance in several cell and animal models of neurodegeneration [11]. Ubiquitination leads to degradation of neurotoxic proteins via the proteasome [12 –14] or lysosome [15 –19]; while de-ubiquitination may reduce degradation, leading to protein accumulation.

Tau hyperphosphorylation and accumulation in AD may be present with plaque deposits containing amyloid-β (Aβ) peptides [20, 21]. At autopsy, several neurotoxic proteins (tau, Aβ, and alpha-synuclein) may co-exist in postmortem AD brains, as well as other tauopathies. Both autophagy and the proteasome may play important role in tau degradation and clearance [22]. Evidence suggest that autophagy is impaired in neurodegeneration [23 –30]. Autophagy may prevent protein accumulation in several models of neurodegeneration [31 –34].

In this study we aim to demonstrate whether tau accumulation and/or clearance depends on ubiquitination/de-ubiquitination via USP-13. To study this hypothesis, we used animal models that overexpress human P301L mutant tau (rTg4510) or carry the Swedish, Dutch, and Iowa mutations of human amyloid precursor protein (TgAPP) to mimic AD pathology. We utilized gene transfer strategies to manipulate USP13 expression using lentiviral shRNA or cDNA models. We found USP13 to be elevated in postmortem autopsies of AD. Primary cortical culture studies demonstrate that USP13 knockdown via shRNA increases proteasome activity and hyperphosphorylated tau (p-tau) clearance. Animal data also demonstrate that USP13 knockdown increases p-tau ubiquitination and clearance and reduces plaque levels in AD models. These data suggest that USP13 inhibition may be a therapeutic strategy to increase p-tau ubiquitination, thus facilitating protein degradation and clearance.

MATERIAL AND METHODS

Transgenic mice and stereotaxic surgeries

Stereotaxic surgeries were performed to inject 1×10⁹ moi lentiviral USP13 cDNA or USP13 shRNA [11] into the right hippocampus of 6-8-month-old TgAPP or 4-6-month-old rTg4510 male and female mice as we previously described [31 –33]. Transgenic TgAPP mice express neuronally derived human APP gene, 770 isoform, containing the Swedish K670N/M671L, Dutch E693Q, and Iowa D694N mutations (TgAPP) under the control of the mouse thymus cell antigen 1, theta, Thy1, promoter [35]. rTg4510 mice express human P301L Tau [36] and have the Tet-responsive element (TRE or tetO) and mouse prion protein promoter sequences (PrP or Prnp) directing expression of the P301L mutant variant of human four-repeat microtubule-associated protein tau (4R0NTau P301L) [37]. All animals were sacrificed 3 weeks post-surgery. To verify lentiviral expression in vivo, brain sections were immunostained with anti-V5 or GFP antibodies that is used as tag for lentiviral expression. Animal procedures strictly followed the Institutional Animal Care and Use Committee guidelines approved protocols at Georgetown University Medical Center (IACUC 2016-1194).

Primary cortical neurons

Fetal wild type (C57BL/6) mouse primary cortical neurons were dissected and gently triturated with a fire-polished Pasteur pipette, and the resulting pool of dissociated cells were plated in 24 well dishes pre-coated with polyethyleneimine (1 mg/ml) containing 0.5 ml basal growth medium, which was supplemented with 5% horse serum and 0.5% fetal calf serum and replaced with fresh medium every three days. DIV 14 or when the density of cells reached 3×10⁴ cells, neurons were infected with 1μl of lentiviral plasmids (total multiplicity of infection (moi) is 1×10⁴) containing USP13 cDNA (overexpression) or USP13 shRNA or mock for 24 h.

Western blot analysis

To extract the soluble proteins from postmortem human tissues or mouse brain lysates, tissues were isolated and homogenized in 1x STEN buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.2% NP-40, 0.2% BSA, 20 mM PMSF and protease cocktail inhibitor), centrifuged at 10,000× g for 20 min at 4°C and the supernatant containing the soluble protein fraction was collected. Extracts were analyzed by western blot (WB) on 4– 12% SDS NuPAGE Bis-Tris gel (Invitrogen, NP0301BOX). Beta-actin (β-actin) was probed (1:3000) with monoclonal antibody (Emdmillipore, MAB1501R). P-tau was probed (1:1000) with monoclonal AT180 antibody (Thermofisher, MN1040) or monoclonal AT8 antibody (Thermofisher, MN1020). Tau was probed (1:1000) with monoclonal antibody Tau-5 (NeoMarkers, Mab MS247-P1ABX). Mouse monoclonal (6E10) antibody (Biolegend, 803001) (1:1000) was used to detect Aβ. Other probes used were antibodies for Beclin-1 (1:1000) (Cell Signaling, 3495), Atg5 (1:1000) (Cell Signaling, 12994), LC3 (1:1000) (Invitrogen, PA1-16931), USP13 (1:1000) (ThermoFisher, PA5-12014), and ubiquitin (1:5000) (ThermoFisher, PA3-16717). Blots were visualized using Super Signal™ West Dura Extended Duration Substrate (Cat 37071, ThermoFisher) on the Amersham™ Imager 600 (GE Healthcare Life Sciences). WBs were quantified by densitometry using Quantity One 4.6.3 software (Bio Rad) and Image J.

Tau immunoprecipitation

Mouse brain tissues were homogenized in 1XSTEN buffer and the soluble fraction was isolated as indicated above. The lysates were pre-cleaned with immobilized recombinant protein A/G agarose (Santa Crutz, sc-2003), and centrifuged at 2500× g for 1 min at 4°C. The supernatant was recovered and quantified by protein assay and a total of 300 μg protein was incubated overnight at 4°C with primary anti-Tau-5 (1:100) (NeoMarkers, Mab MS247-P1ABX) antibodies in the presence of sepharose G and an IgG control. The immunoprecipitates were collected by centrifugation at 2500× g for 3 min at 4°C, washed 5× in PBS, with spins of 3 min, 2500× g using detergent-free buffer for the last washing step and the proteins was eluted according to Pierce instructions (Pierce #20365). After IP, the samples were size-fractionated on 4– 12% SDS-NuPAGE and transferred onto 0.45 μm nitrocellulose membranes. The primary antibodies used for WB analysis of Tau-5, p-tau, and ubiquitin were the same as those used for above WB analysis. WB detection was then performed using HRP conjugated secondary antibodies.

Immunohistology and proximity ligation assay (PLA)

Animals were deeply anesthetized with a mixture of Xylazine and Ketamine (1:8), washed with normal saline for 1 min and then perfused with 4% paraformaldehyde (PFA) for 15– 20 min. Brains were quickly dissected out and immediately stored in 4% PFA for 24 h at 4°C, and then transferred to 30% sucrose at 4°C for 48 h. Brains were cut using a cryostat microtome into 20μm thick coronal sections and stored at – 20°C. We optimized the concentration of p-tau and ubiquitin antibodies prior to PLA. Primary mouse anti-p-tau (AT180) antibodies (1:200, Thermofisher, MN1040) or anti-p-tau (ptau181) antibodies (1:200, Thermofisher, MN1050) and rabbit anti-ubiquitin antibodies (1:200, ThermoFisher, PA3-16717) were applied to 20 μm thick sections of mouse brains overnight at 4°C. Species-specific secondary antibodies or PLA probes used were Duolink In Situ PLA^® probes anti-mouse Minus (Duo92004-30RXN) and Duolink In Situ PLA^® probes anti-rabbit Plus (Duo92002-30RXN), each with a unique short DNA strand attached to it (Sigma-Aldrich) as described in manufacturer’s protocol. When the PLA probes are in close proximity, the DNA strands interact through a subsequent addition of two other circle-forming DNA oligonucleotides. After joining of the two added oligonucleotides by enzymatic ligation, they are amplified via rolling circle amplification using a polymerase to highlight the interaction. Fluorescence in each single-molecule amplification product is easily visible as a distinct bright spot when viewed with a fluorescence microscope.

Immunohistochemistry was also performed for tau-5, p-tau, ubiquitin, Aβ and plaques using the same antibodies above on the 20-μm-thick sections. Nuclear staining with 4^′,6-diamidino-2-phenylindole (DAPI) was performed according to the manufacturer’s protocol (Life Technologies, Miami, FL, USA).

Fluorescence and WB image analysis

Fluorescence images were acquired with Fluorescence microscope EVOS^®fl Cell Imaging System (Thermofisher, USA). Alexa-488 (p-tau or GFP) was visualized in the green channel (EX 495 nm, EM 519 nm) and Alexa-594 (ubiquitin or PLA product) in the red channel (EX 590 nm, EM 617 nm). The same equipment parametric settings for treatment groups were applied equally to control groups. The WB images were visualized and acquired using Super Signal™ West Dura Extended Duration Substrate (Cat 37071, ThermoFisher) on the Amersham™ Imager 600 (GE Healthcare Life Sciences). Quantitative analysis of fluorescence images and WBs were carried out using Quantity One 4.6.3 software (Bio Rad) and ImageJ software (National Institutes of Health).

Enzyme-Linked Immunosorbent Assay (ELISA)

The ELISA for total tau (Millipore, HNABTMAG68K), specific p-Tau ser396 (Invitrogen, KHB7031), human Tau thr181 (Invitrogen, KHO0631), and Aβ1– 42 (Invitrogen, KHB3442) were performed according to manufacturer’s protocol on tissue soluble extracts from midbrain lysates in 1XSTEN buffer (see above). We also measured Aβ₄₂ levels on tissue insoluble extracts from midbrain lysates. To extract insoluble portion, the pellet isolated from 1XSTEN buffer was re-suspended in 70% formic acid for 30 min at room temperature followed by centrifuge at 28,000 g at 4°C for 1 h. The supernatant was collected and neutralized with 1M Tris-base (1:20) immediately before use.

Quantification of plaque load

Quantification of plaque load or counting plaque number was performed by a blind investigator using ImageJ by drawing a line around individual plaques within 1 mm² radius of six randomly selected hippocampal and cortical regions in 6E10 + DAB stained slides. Aβ plaques were defined as intensely stained brown deposits with a clear boundary compared to background. The number of plaques was counted and averaged per mm².

20S proteasome activity assay

Primary cortical neurons were co-infected with 1μl of lentiviral plasmids (total multiplicity of infection (moi) is 1×10⁴) containing USP13 cDNA (overexpression) or USP13 shRNA or mock for 24 h. As a control 20μM of proteasome inhibitor MG132 was applied for 6 h prior to the beginning of proteasome activity assays. Cell extracts (100μg) were incubated with 250μM of the fluorescent 20 S proteasome specific substrate Succinyl-LLVY-AMC (BML-P802-0005, Enzo Life Sciences) at 37°C for 2 h. The medium was discarded and proteasome activity was measured in tissue homogenates.

Stereological methods

A blinded investigator performed unbiased stereology analysis (Stereologer, Systems Planning and Analysis, Chester, MD) to determine the total positive cell counts randomly and systematically from each animal. We counted 20 random fields from 3-4 slides cut at different levels of interested region from each animal and used 6 different animals in each treatment group to obtain a total of 400– 500 fields. The AT180+ neurons in each treatment group were averaged.

Caspase-3 activity assay

It was performed according to manufacturer’s protocols as we previously described [32, 33]. To measure caspase-3 activity in the animal models, we used EnzChek^® caspase-3 assay kit #1 (Invitrogen) on hippocampal extracts and Z-DEVD-AMC substrate and the absorbance was read according to manufacturer’s protocol.

Statistical analysis

All statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad software, Inc, San Diego, CA). Two-tailed student’s t-test was used in WB analysis for comparison of USP13 levels between control and AD postmortem brains. In all other experiments, one-way analysis of variance (ANOVA) followed by Newman-Keuls or Dunnett comparison post-hoc tests were used. Asterisks denote actual p-value significances (*<0.05), and N is the number of animals or the number of independent experiments (cell culture) per group. Unless otherwise indicated, data are expressed as Mean±SD.

RESULTS

USP13 levels are increased in postmortem AD brains

Postmortem autopsies of human AD hippocampus were collected 2– 4 h from 12 AD [19] and 9 age-matched control subjects [38]. The level of USP13 was significantly increased (>2-fold) in the hippocampus (Fig. 1A, B) of AD brains. Based on this increase of USP13 in postmortem AD brains, we performed USP13 knockdown via shRNA to demonstrate whether alteration of USP13 levels changes tau in primary cortical neurons. We used lentiviral infection of primary cortical neurons to express human wild type 4R tau together with USP13 cDNA (overexpression) or knockdown via shRNA. Wild type (C57BL/6) mouse primary cortical neurons were grown in 24-well dishes (Falcon^®) as previously described [19] and infected at DIV 14 with 1μl of lentiviral plasmids (total multiplicity of infection (moi) is 1×10⁴) for 24 h. WB analysis shows that lentiviral USP13 cDNA significantly increased USP13 levels (Fig. 1C&D) but shRNA significantly reduced USP13 levels relative to actin. Quantification of tau protein via ELISA shows that lentiviral infection resulted in a significant increase in total tau (tau-5 antibody) compared to mock infected cells (Fig. 1E). The increase in total tau also resulted in a significant 2-fold increase in ptau181 (Threonine 181) and 3-fold increase in ptau396 (Serine 396). Importantly, USP13 knockdown significantly reduced the level of total tau, ptau181, and ptau396 compared to tau expression alone or with USP13 over-expression.

Fig. 1

USP13 expression is elevated in postmortem AD brain. A) Western blot (WB) of human postmortem hippocampus showing USP13 level relative to the neuronal marker Map-2 on 10% NuPAGE SDS gel, and B) Densitometry of WB. * indicates significantly different to control. N = 9 Control and 12 AD hippocampus, p < 0.01, two tailed t-Test. C) Mouse primary cortical neurons were grown until day 14 and infected with human 4R wild type lentivirus tau together with either USP13 cDNA (overexpression) or shRNA (knockdown) for 24 h and cells were extracted in 1X STEN buffer and protein levels were measured. D) Densitometry of WB of the same samples shows a significant 2-fold increase in USP13 overexpression (cDNA) compared to mock (shRNA that does not affect USP13 levels). E) ELISA results show reduction of p-tau181, p-tau396 and total tau levels following co-expression of lentiviral human 4R tau with USP13 shRNA using primary cortical neurons. One Way ANOVA analysis * indicates p < 0.05. N = 4 per group. F) Mouse primary cortical neurons were grown until day 14 and infected with 4R wild type lentiviral tau together with either USP13 cDNA (overexpression) or shRNA (knockdown) for 24 h and cells were extracted in 1X STEN buffer to measure proteasome activity. Mock infected cells were treated with a lentivirus shRNA that does not affect USP13 levels. 20S Proteasome activity was significantly reduced when tau was expressed or alone with USP13 in primary neurons but USP13 shRNA increased proteasome activity even higher than baseline levels in mock infected cells. One Way ANOVA analysis * indicates p < 0.05. N = 6 per group.

USP13 knockdown with shRNA significantly increases proteasome activity in primary neurons expressing tau

We also measured 20S proteasome activity in cells co-expressing tau and USP13. Wild type (C57BL/6) mouse primary cortical neurons were grown in 24-well dishes and infected at DIV 14 with 1μl of lentiviral (total moi = 1×10⁴) human 4R wild type tau together with USP13 cDNA or USP13 shRNA (or mock, which is shRNA that does not knockdown USP13) for 24 h (Fig. 1F, n = 6). Proteasome activity was significantly reduced when tau was expressed compared to control mock (Fig. 1F), and this activity remained low when USP13 was overexpressed, but USP13 shRNA significantly increased proteasome activity above control level. These data show that USP13 knockdown increases 20S proteasome activity and reduces p-tau levels.

USP13 knockdown via shRNA reduces amyloid plaques and tau levels in transgenic TgAPP mice

To determine whether USP13 affects protein accumulation in animal models in vivo, we performed stereotaxic surgery to inject lentiviral USP13 cDNA or shRNA (1×10⁹ moi) into the right CA1 hippocampus of 6-8-month-old male and female mice that express neuronally derived human APP gene, 770 isoform, containing the Swedish K670N/M671L, Dutch E693Q, and Iowa D694N mutations (TgAPP) under the control of the mouse thymus cell antigen 1, theta, Thy1, promoter [35]. Staining with anti-amyloid 6E10 antibody with DAB (3,3-diaminobenzidine) of 20μM thick brain sections 3 weeks post-injection shows no difference in Aβ levels between mock injected left hemisphere (Fig. 2A) and the USP13 cDNA injected right hemisphere (Fig. 2B) of the same animal. However, the level of Aβ was noticeably reduced in the USP13 shRNA injected (ipsilateral) brains (Fig. 2D) compared to mock injected left (contralateral) hemisphere (Fig. 2C). Quantitative measurement of Aβ₄₂ using ELISA shows that 1xSTEN buffer extracted (soluble) and 30% formic acid extracted (insoluble) Aβ₄₂ were significantly reduced with shRNA injection (Fig. 2E) compared to mock or USP13 overexpression in hippocampal extracts. Further quantification of plaque load showed no difference in plaques between mock (Fig. 2F) and USP13 overexpression (Fig. 2G). However, USP13 shRNA resulted in significant reduction of plaque load (Fig. 2I, J) compared to mock (Fig. 2H, J). There was no difference in the level of cell death between treatments as indicated by caspase-3 activity assays (Fig. 2K).

Fig. 2

USP13 knockdown with shRNA significantly reduces Aβ levels and plaque load in TgAPP mice. Male and female TgAPP mice (6– 8 months old) were injected with lentiviral USP13 cDNA or shRNA in the right hippocampus, and USP13 mock (shRNA that does not knockdown USP13) in the left hippocampus for 3 weeks. Immunohistochemistry shows no difference in Aβ levels between A) USP13 mock and B) USP13 cDNA injected hemispheres, but D) Lentiviral shRNA injection reduces Aβ levels compared to C) USP13 mock as shown via quantitative E) Aβ₄₂ ELISA. Immunohistochemistry shows no difference in plaque load between F) USP13 mock and G) USP13 cDNA injected hemispheres, but I) Lentiviral shRNA injection reduces plaque levels compared to H) USP13 mock as shown via J) quantitative analysis of plaque load. K) No differences were observed in capsase-3 activity between treatment groups. One Way ANOVA analysis and * indicates p < 0.05. N = 4 per group. Aβ was extracted in 1X STEN buffer (soluble) and 30% formic acid (insoluble) from hippocampal lysates.

We previously demonstrated that TgAPP mice develop progressive amyloid and tau pathology as early as 4 months of age [39]. Therefore, we examined tau changes in this 6-8-month cohort and found that, similar to Aβ, there is no change in tau hyperphosphorylation at Threonine 231 (AT180) between mock (Fig. 3A, E) and USP13 overexpression (Fig. 3B, E), but USP13 knockdown significantly reduced AT180 staining (Fig. 3D, E) compared to mock (Fig. 3C, E). WB analysis of p-tau at Serine202/Threonine205 (AT8) in hippocampus extracts also shows that UPS13 shRNA significantly reduces p-tau levels relative to actin compared to control or USP13 overexpression (Fig. 3F, G). Taken together, these data suggest that USP13 knockdown may simultaneously reduce Aβ and tau accumulation in TgAPP mice.

Fig. 3

USP13 knockdown with shRNA significantly reduces p-tau levels in TgAPP mice. Male and female TgAPP mice (6– 8 months old) were injected with lentiviral USP13 cDNA or shRNA in the right hippocampus, and USP13 mock (shRNA that does not knockdown USP13) in the left hippocampus for 3 weeks. Immunohistochemistry shows no difference in AT180 levels between A) USP13 mock and B) USP13 cDNA injected hemispheres, but D) Lentiviral shRNA significantly reduces AT180 levels compared to C) USP13 mock. E) The level of AT180 staining was quantified via stereological methods. Figures show right and left hemispheres in the same mouse. One Way ANOVA analysis and * indicates p < 0.05. N = 4 per group. WB of hippocampal lysates showing F) the levels of tau and p-tau (AT8) relative to actin on 4– 12% SDS-NuPAGE gel in above TgAPP mice, and G) the densitometry. Asterisks indicate statistic significant difference *p<0.05. One Way ANOVA with Newman Keuls was used for analysis. N = 5-6. All values presented as Mean±SD.

USP13 knockdown increases p-tau ubiquitination and reduces p-tau level in TgAPP mice

To further determine whether USP13 affects tau/p-tau localization with ubiquitin, we performed immunostaining in the ipsilateral hippocampus of TgAPP mice using p-tau181 (Threonine 181) antibody in mock (Fig. 4A) compared to USP13 cDNA (Fig. 4B) and USP13 shRNA (Fig. 4C). Ubiquitin staining in mock injected (vehicle) hippocampus (Fig. 4D) shows the level of ubiquitin compared to mice injected with USP13 cDNA (Fig. 4E) and USP13 shRNA (Fig. 4F). Merged p-tau and ubiquitin staining in vehicle (Fig. 4G), compared to USP13 cDNA (Fig. 4H) and USP13 shRNA (Fig. 4I) shows that p-tau co-localizes with ubiquitin in mice injected with shRNA USP13, but USP13 overexpression reduces this co-localization. Optic density of co-localization of p-tau-ubiquitin shows a significant increase (86%) in co-localization in the hippocampus when mice were injected with shRNA compared to mock control (Fig. 4M), while UPS13 overexpressing animals showed a trend toward decreased localization (N = 5-6 animals per treatment). Further analysis with Proximity Ligation Assay (PLA) shows interaction between p-tau and ubiquitin (Fig. 4J, N) in mock animals, but USP13 overexpression (Fig. 4K, N) reduces this interaction, while shRNA knockdown (Fig. 4L, N) increases interaction between p-tau and ubiquitin. Immunoprecipitation of tau protein from hippocampal extracts (Fig. 4O) followed by WB shows that USP13 knockdown decreases p-tau (Ser396) levels (top blot) and increases ubiquitination (third blot) in the ipsilateral brain compared to contralateral side, while USP13 overexpression reduces p-tau ubiquitination.

Fig. 4

(p. 434) USP13 knockdown increases p-tau ubiquitination and reduces p-tau level in TgAPP mice. Immunostaining of 20μm thick hippocampal sections of TgAPP mice injected unilaterally with lentiviral plasmids shows p-tau (ptau181) staining in A) mock injected (vehicle), B) USP13 cDNA overexpression and C) USP13 shRNA knockdown. Ubiquitin staining in D) mock injected (vehicle), E) USP13 cDNA overexpression, and F) USP13 shRNA knockdown. Merged staining in G) vehicle, H) USP13 cDNA overexpression, and I) USP13 shRNA knockdown shows that p-tau co-localizes with ubiquitin but USP13 overexpression reduces this co-localization, while USP13 knockdown increases p-tau/ubiquitin co-localization. N = 5-6 animals per treatment. Proximity ligation assay (PLA) shows interaction between p-tau and ubiquitin in J) vehicle (mock) animals, but K) USP13 overexpression reduces this interaction, while shRNA knockdown L) increases interaction between p-tau and ubiquitin, verified by quantitation of p-tau/ubiquitin co-localization in M) and PLA quantitation optic density of staining in N). N = 4 animals per treatment. O) Immunoprecipitation of tau protein from hippocampal extracts followed by WB shows that USP13 knockdown decreases p-tau (Ser396) levels (top blot) and increases ubiquitination (third blot) in the ipsilateral brain compared to contralateral, while USP13 overexpression reduces p-tau ubiquitination. Asterisks indicate statistic significant difference ^*p < 0.05. One Way ANOVA with Newman Keuls was used for analysis. N = 5-6 animals per treatment. All values presented as Mean±SD.

USP13 knockdown alters autophagy in TgAPP mice

We previously showed that autophagy impairment in TgAPP mice results in accumulation of light chain protein (LC3)-II, which is indicative of reduced autophagosome clearance [32]. LC3-I lipidation and conversion to LC3-II is a marker of autophagosome formation, while LC3-II reduction may be indicative of autophagosome clearance. WB analysis of hippocampal brain lysates demonstrates that USP13 overexpression via cDNA does not affect LC3-I and LC3-II protein levels compared to mock (vehicle) injected mice (Fig. 5A, B). However, USP13 shRNA significantly reduces the ratio of LC3-I relative to LC3-II, suggesting clearance of autophagosome. Further WB analysis also indicates that the autophagy protein markers Beclin-1 and Atg-5 are both increased in USP13 shRNA (Fig. 5C, D) compared to mock or USP13 cDNA injected mice. These data suggest increased autophagic activity as a result of USP13 knockdown.

Fig. 5

USP13 knockdown via shRNA increases autophagy in TgAPP mice. TgAPP mice were injected unilaterally with either mock (vehicle), lentiviral USP13 cDNA or USP13 shRNA into the right hippocampus. WB of hippocampus lysates showing A) the levels of LC3-I and LC3-II relative to actin, and the ratio of LC3-II to LC3-I levels, and B) the corresponding densitometry of WB. C) The levels of beclin-1 and Atg-5 relative to actin on 4– 12% SDS-NuPAGE gel and D) the corresponding densitometry. Asterisks indicates statistically significant, p < 0.05. One Way ANOVA with Newman Keuls was used for analysis. N = 5-6 animals per treatment. All values presented as Mean±SD.

USP13 knockdown increases p-tau ubiquitination and reduces p-tau level in rTg4510 mice

To further determine whether USP13 affects tau/p-tau ubiquitin localization in animal models that express mutant tau alone, we performed stereotaxic surgery to inject 1×10⁹ moi lentiviral USP13 cDNA or shRNA into the right CA1 hippocampus (ipsialateral) of 6-month-old male and female rTg4510 mice. Immunostaining of 20μm thick hippocampal sections of rTg4510 injected unilaterally with lentiviral plasmids shows p-tau (p-tau181, Threonine 181) staining in mock injected (vehicle) (Fig. 6A) compared to USP13 cDNA (Fig. 6B) and USP13 shRNA (Fig. 6C) injected brain, and ubiquitin staining in mock injected (vehicle) (Fig. 6D) compared to USP13 cDNA injected (Fig. 6E) and USP13 shRNA injected brain (Fig. 6F). Merged p-tau and ubiquitin staining in vehicle (Fig. 6G), compared to USP13 cDNA (Fig. 6H) and USP13 shRNA (Fig. 6I) shows that p-tau co-localizes with ubiquitin but USP13 overexpression reduces this co-localization, while USP13 knockdown increases p-tau/ubiquitin co-localization. N = 5-6 animals per treatment. Proximity ligation assay (PLA) shows interaction between p-tau and ubiquitin in vehicle-treated animals (Fig. 6J, M), but USP13 reduces this interaction (Fig. 6K, M), while shRNA (Fig. 6L, M) increases interaction between p-tau and ubiquitin. N = 5-7 animals per treatment. Immunoprecipitation of tau protein from hippocampal extracts (Fig. 6N) followed by WB shows that USP13 knockdown decreases p-tau (Ser396) levels (top blot) and increases ubiquitination (third blot) in the ipsilateral brain compared to contralateral side, while USP13 overexpression reduces p-tau ubiquitination. ELISA measurement of p-tau in the same extracts (Fig. 6O) demonstrates that USP13 knockdown reduces p-tau levels.

Fig. 6

(p. 436) USP13 knockdown increases p-tau ubiquitination and reduces p-tau level in rTg4510 mice. Immunostaining of 20μm thick hippocampal sections of male and female 6-month-old rTg4510 injected unilaterally with lentiviral plasmids shows p-tau (ptau181) staining in A) mock injected (vehicle), B) USP13 cDNA overexpression and C) USP13 shRNA knockdown, and ubiquitin staining in D) mock injected (vehicle), E) USP13 cDNA overexpression and F) USP13 shRNA knockdown. Merged staining in G) mock (vehicle), H) USP13 cDNA overexpression and I) USP13 shRNA knockdown shows that p-tau co-localizes with ubiquitin but USP13 overexpression reduces this co-localization, while USP13 knockdown increases p-tau/ubiquitin co-localization. N = 5-6 animals per treatment. Proximity ligation assay (PLA) shows interaction between p-tau and ubiquitin in J) vehicle animals, but K) USP13 overexpression reduces this interaction, while shRNA knockdown L) increases interaction between p-tau and ubiquitin, verified by quantitation of PLA M) Optic density of PLA staining, N = 5-7 animals per treatment. Immunoprecipitation of tau protein from hippocampal extracts N) followed by WB shows that USP13 knockdown decreases p-tau (Ser396) levels (top blot) and increases ubiquitination (third blot) in the ipsilateral brain compared to contralateral, while USP13 overexpression reduces p-tau ubiquitination. ELISA measurements of p-tau in the same extracts O) demonstrates that USP13 knockdown reduces p-tau levels. Asterisk indicate statistic significant difference ^*p < 0.05. One Way ANOVA with Newman Keuls was used for analysis. N = 5-6 animals per treatment. All values presented as Mean±SD.

Verification of USP13 expression and p-Tau interaction in rTg4510 mice

To verify lentiviral expression in vivo, brain sections were immunostained with anti-GFP antibodies and showed that stereotaxic injection of GFP-tagged lentiviral USP13 mock (Fig. 7A), USP13 cDNA (Fig. 7B) or USP13 shRNA (Fig. 7C) results in lentiviral expression. Proximity ligation assay to determine p-tau and USP13 interaction showed that USP13 shRNA results in reduction of interaction of p-tau and USP13 (Fig. 7F, J) compared to USP13 overexpression (Fig. 7E, J) and mock (Fig. 7D, J), suggesting that USP13 knockdown may directly affect interaction with p-tau. Further p-tau (AT8) staining in serial cortical sections also shows that USP13 knockdown significantly reduces p-tau levels (Fig. 7I, K) compared to USP13 overexpression (Fig. 7H, K) and mock (Fig. 7G, K).

Fig. 7

USP13 alters p-tau-USP13 interaction and p-Tau levels in 6-month-old rTg4510 cortex. DAPI staining and IHC for GFP of 20μm thick cortical sections of rTg4510 mouse brains injected with 1×10⁹ m.o.i of GFP-tagged lentiviral clones driving the expression of A) mock, B) USP13 cDNA, and C) USP13 shRNA, showing expression of all lentiviral clones. Proximity ligation assays (PLA) in serial cortical sections from rTg4510 showing interaction between p-tau (AT8) and USP13 in D) mock, E) USP13 overexpression, and F) USP13 knockdown with shRNA, counterstained with DAPI. Immunostaining with the p-tau antibody (AT8) and counterstaining with DAB show the level of p-tau in the cortex of 6-month-old rTg4510 injected with lentiviral G) mock, H) USP13 cDNA, and I) USP13 shRNA. J) Optic density of p-tau-USP13 staining. K) Stereological counting of p-tau staining in rTg4510 cortex. Asterisks indicate significant difference, p < 0.05. One Way ANOVA with Newman Keuls was used for analysis. N = 5-6 animals per treatment. All values presented as Mean±SD.

DISCUSSION

Our results demonstrate for the first time a two-fold increase of USP13 levels in postmortem AD brains. To our knowledge, there is no prior reports that describe the role of USP13 in AD or in the regulation of tau accumulation and/or clearance in the tauopathies. USP13 is a ubiquitin-specific enzyme that cleaves ubiquitin off protein substrates to reverse ubiquitin-mediated protein degradation [40]. Several other USPs have been described to play a role in neurodegeneration [2 –7]. An increase in the level of USP13 in postmortem AD brains may be indicative of an active role of USP13 in reducing ubiquitination of hyperphosphorylated tau species at several epitopes (e.g., Serine 396, Threonine 181, Threonine 231, Serine202/Threonine205), which would result in reduced accumulation and increased p-tau clearance. Ubiquitin targets proteins to major degradation pathways, including the proteasome and the lysosome [40]. In melanoma cells, USP13 regulates the degradation of several proteins primarily via ubiquitination and de-ubiquitination [8, 9]. In models of neurodegeneration, the proteasome and the lysosomes are involved in the degradation of p-tau [22 , 41– 44], and these findings are consistent with our results showing that p-tau may be reduced via the proteasome and/or autophagy when USP13 level is decreased. It is possible that hyperphosphorylation of tau results in a post-translational modification that would favor the clearance of p-tau over basally phosphorylated tau. For example, prior data show that tau acetylation prevents tau clearance [45] and our data suggest that ubiquitination of post-translationally modified tau via hyperphosphorylation increases p-tau ubiquitination as a result of lowering USP13 level. It is logical to hypothesize here that under normal conditions basal phosphorylation of the microtubule associated protein tau may lead to p-tau ubiquitination and recycling to maintain homeostasis. However, under pathological conditions USP13 levels are increased and may lead to de-ubiquitination and reduced clearance of p-tau. Taking together, our data indicate that USP13 may control tau ubiquitination/de-ubiquitination and facilitates its degradation via autophagy and/or the proteasome (13– 17).

USP13 knockdown also reduced the levels of amyloid plaques and increased tau ubiquitination and clearance in TgAPP mice that develop progressive amyloid and murine tau pathology as early as 4 months of age [39]. There is no evidence that USP13 directly interacts with APP or other amyloid fragments. However, we previously demonstrated that USP13 affects the ubiquitination and activity of the E3 ubiquitin ligase parkin [46], which appears to ubiquitinate Aβ₄₂ [33] and contribute to tau and Aβ₄₂ clearance [33 , 48]. The reduction of plaques, Aβ₄₂ and p-tau when USP13 expression is lowered suggest interaction between tau and amyloid pathology. TgAPP mice show accumulation of murine p-tau as a result of APP mutations, reminiscent of AD. Tau accumulation may exacerbate Aβ pathology [49], and tau deletion attenuates Aβ toxicity [22, 50]. These findings suggest that normal tau function may promote Aβ clearance and reduce plaque accumulation [22], consistent with our results showing that p-tau clearance is associated with Aβ reduction. Conversely, USP13 knockdown in rTg4510 mice that express P301L tau, independent of APP mutations and plaque formation, also demonstrates increased p-tau ubiquitination and clearance, further suggesting that USP13 expression may hinder p-tau clearance via de-ubiquitination. Taken together, our results in TgAPP and rTg4510 mice suggest that USP13 knockdown may have a dual effect separately on Aβ and p-tau, resulting in independent ubiquitination and clearance of these proteins.

De-ubiquitinases, including USPs, have been described in several neurodegenerative processes [2 –7]. USP13 level is increased in postmortem Parkinson’s disease brains and its knockdown enhances clearance of alpha-synuclein [11]. USP8 was also shown to regulate alpha-synuclein (de)ubiq-uitination and proteasome clearance [51]. USP13 also control the ubiquitination/de-ubiquitination cycle of stress granules in models of motor neuron disease [10]. Tau (de)ubiquitination and proteasome clearance may also be mediated by USP14 [52] and Ovarian Tumor (OTU, Ubiquitin Aldehyde Binding-1) OTUB-1 [53]. Therefore, clearance of tau and alpha-synuclein may depend on the balance between ubiquitination [22 , 54– 63] and de-ubiquitination [53 , 64– 66] to control their degradation. At autopsy, neurotoxic proteins including p-tau, Aβ, and alpha-synuclein co-exist in AD, Parkinson’s disease, and dementia with Lewy bodies [20 , 67].

Collectively, these data indicate that USP13 may regulate proteostasis via protein (de)ubiquitination and autophagy/proteasome clearance. Furthermore, these studies suggest that USP13 is a therapeutic target to lower tau and other neurotoxic proteins in neurodegenerative diseases, including the tauopathies and AD.

Footnotes

ACKNOWLEDGMENTS

These studies were supported by Emerald Foundation, Inc. grants to Charbel E-H Moussa.

Authors’ disclosures available online ().

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