Mechanism of Tau Hyperphosphorylation Involving Lysosomal Enzyme Asparagine Endopeptidase in a Mouse Model of Brain Ischemia

Abstract

Dementias including Alzheimer’s disease (AD) are multifactorial disorders that involve several different etiopathogenic mechanisms. Cerebral ischemia has been suspected in the altered regulation of protein kinases and phosphatases that leads to hyperphosphorylation of tau and further neurofibrillary pathology, a key hallmark of AD and related neurodegenerative diseases. However, the deregulation of these enzymes and their relationship with ischemia and AD remain unclear. Previously, we reported a mechanism by which the lysosomal enzyme asparagine endopeptidase (AEP) is associated with brain acidosis and AD. In this study, we subjected mice to middle cerebral artery occlusion and found that compared with wild type mice, the ischemia-induced brain injury and motor deficit in AEP-knockout mice are reduced, probably because ischemia activates AEP. AEP cleaves inhibitor 2 of protein phosphatase 2A (I₂PP2A), which translocates from the neuronal nucleus to the cytoplasm and produces hyperphosphorylation of tau through inhibition of PP2A. These findings suggest a possible mechanism of tau pathology associated with ischemia.

Keywords

Alzheimer’s disease asparagine endopeptidase ischemia tau

INTRODUCTION

Brain ischemia is believed to be one of the etiopathogenic aspects involved in Alzheimer’s disease (AD) [1 –4]. Cognitive impairment induced by vascular damage in individuals with AD and in aged individuals is common, and several vascular risk factors for AD are linked to hypoxia. Vascular pathology coexists in at least 33% of AD cases, suggesting that vascular factors might play a role in the pathogenesis of AD [5 –8]. However, how stroke leads to the onset of AD remains obscure.

Neurofibrillary tangles are the hallmark of AD and are composed mainly of the microtubule-associated protein (MAP) tau, which is seen in an abnormally hyperphosphorylated state in these lesions [9, 10]. The hyperphosphorylation of tau negatively regulates its binding to microtubules; the hyperphosphorylated tau sequesters normal tau and other MAPs, causing aggregation, breakdown of the microtubule network, and eventually cell death [11 –15]. It is well known that an imbalanced regulation in tau protein kinases and phosphatases can directly cause tau hyperphosphorylation [16]. An increasing number of studies has demonstrated that glycogen synthase kinase 3β (GSK-3β) is one of the major kinases responsible for the abnormal phosphorylation of tau in AD [15 , 18]. Protein phosphatase 2A (PP2A) is the main regulator of tau phosphorylation, accounting for 70% of the adult human brain’s phospho-serine/threonine tau protein phosphatase activity [19, 20]. PP2A is highly regulated by inhibitor 2 of PP2A (I₂PP2A) [21, 22]. We previously reported that I₂PP2A is cleaved at Asn-175 in the neuronal cytoplasm by the lysosomal enzyme asparagine endopeptidase (AEP), also named legumain. PP2A activity is compromised by the interaction of two I₂PP2A cleavage products (N- and C-terminal fragments) with the PP2A catalytic subunit (PP2Ac) and leads to tau hyperphosphorylation in AD brain [22 –25]. AEP also truncates tau by cleaving at Asn-255 and 368 [26] and mediates stroke-provoked I₂PP2A cleavage and cell death in the brain [27]. Thus, AEP is one of the proteinases activated by acidosis triggering neuronal injury during ischemia. Pro AEP is a 56-kDa protein that is autocatalytically processed into a 36-kDa active AEP under acidic conditions [28]. Several studies suggest that vascular lesions promote tau phosphorylation at different sites [3 , 29] and that the decrease in PP2A activity plays an important role in this pathogenesis. However, the nature of this mechanism is not fully understood. In the present study, employing cerebral ischemic model in wild type (WT) and AEP-knock out (AEP-KO) adult mice, we showed that AEP is involved in the infarct size and motor deficit, activation of I₂PP2A, and consequent tau hyperphosphorylation.

MATERIALS AND METHODS

Animals and tissue processing

Adult female mice (3 months old, body weight 20–22 g) WT and AEP-KO [30] with C57BL/6J background [31] were housed and bred according to the United States Public Health Service Policy on Human Care and Use of Laboratory Animals, with 2–3 animals per cage, a 12:12 h light-dark cycle, and ad libitum access to food and water. Studies on animals were carried out according to a protocol approved by the Animal Welfare Committee of the New York State Institute for Basic Research. A total of 36 mice were subjected to middle cerebral artery occlusion (MCAO) for different experiments as follows: for biochemical analysis, seven WT and six AEP-KO and another two sham-operated mice per group were used as controls. The left (contralateral) and right (ipsilateral) cerebral hemispheres were dissected separately into hippocampus, cerebral cortex, striatum, and subcortical structures and kept at –80°C till used. Another three mice per group were used for immunohistochemical staining. The brains of the mice from each group were formalin-fixed, paraffin-embedded, and sectioned into 5-μm-thick sections for immunohistochemistry. The last group of eight WT and nine AEP-KO mice were used to analyze ischemic infarct size by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Middle cerebral artery occlusion

The MCAO surgery was performed as described previously [32]. Briefly, mice were anesthetized with intraperitoneal injection of 2.5% Avertin. Through a ventral midline incision, the right common carotid artery, internal carotid artery, and external carotid artery were surgically exposed. A 6-0 nylon suture with silicon coating (Doccol Corporation, Redlands, CA) was inserted into the internal carotid artery through the external carotid artery stump and was gently advanced to occlude the middle cerebral artery. For the infarct volume and motor deficit analysis, after 1 h of MCAO, the suture was carefully removed to restore blood flow (reperfusion), the neck incision was closed, and the mice were allowed to recover. For the rest of the studies, the MCAO was carried out for 24 h. The sham-operated mice underwent identical surgery, but the suture was not inserted. Animals were killed by cervical dislocation.

Evaluation of brain infarct volume and motor deficit

After the mice were subjected to MCAO and reperfusion, their brains were extracted from skulls, cut into five 1.5-mm-thick coronal sections, and stained with 2% TTC for 30 min and then fixed with 4% paraformaldehyde. Damaged areas not stained red with TTC were analyzed quantitatively with Sigma Scan Pro 5. Infarct volume was calculated through slice thickness and damaged area, expressed as a percentage of total hemisphere [32]. The motor deficits of mice subjected to MCAO were evaluated by an examiner without knowing the experimental conditions by using the scales described by Longa et al. in 1989 [33]: 0 point, mouse behaves normally; 1 point, mouse cannot fully stretch its left front legs; 2 points, mouse turns around into a circle; 3 points, mouse falls down to the left side; 4 points, mouse cannot move voluntarily, losing consciousness.

Immunohistochemistry and TUNEL apoptosis staining

Double or triple staining with different antibodies combined with TUNEL staining was used to study the spatial relationship between the assessed proteins and the apoptotic cells. This assay also allowed us to identify specifically the infarct area. Apoptotic cells were detected by Dead End Fluorometric TUNEL System (Promega, Madison, WI, USA); the protocol was performed according to the manufacturer’s instructions. Briefly, brain paraffin sections from WT and AEP-KO mice subjected to 24 h MCAO were rehydrated and incubated in 20 μg/ml proteinase K for 10 min, followed by washes with 0.1% Triton X-100 in 0.1 M PBS and blocking with 2% normal goat serum and 1% bovine serum albumin. The sections were then incubated overnight at 4°C with the following primary antibodies: mouse mAb anti-I₂PP2A N-term (10E7; 1:200 [22]), rabbit anti-I₂PP2A N-term (1483; 1:200 [22]), anti-pT-Ser262/356 (12E8; 1:200 [34]), anti-pT-Ser396/404 (PHF-1; 1:200, a gift from Dr. Peter Davies, Albert Einstein College of Medicine, New York, NY, USA), sheep anti-mouse legumain (1:50; R&D Systems, Minneapolis, MN, USA), anti-GSK-3β (1:200; Cell Signaling, Danvers, MA, USA), anti-βIII tubulin (1:100, Covance Inc. Emeryville, CA, USA), GFAP (1:500; Thermo, Rockford, IL, USA), and Iba 1 (1:200; Wako, Osaka, Japan). Thereafter, secondary antibodies were added to the TUNEL reaction mix (equilibration buffer, nucleotide mix, rTdT enzyme) and incubated for 60 min at 37°C in a humidified chamber in dark. The secondary antibodies used at 1:500 dilution were Alexa 555-conjugated donkey anti-sheep IgG (H+L), Alexa 555-conjugated goat anti-mouse IgG (H+L), Alexa 488-conjugated goat anti-mouse IgG (H+L), CY5 goat anti-mouse IgG (H+L), Alexa 555-conjugated goat anti-rabbit IgG (H+L), and Alexa 488-conjugated goat anti-rabbit IgG (H+L). Nuclei were counter-stained with TOPRO3 (1:1000; Invitrogen, Carlsbad, CA, USA). Finally, sections were analyzed using confocal microscope Nikon Eclipse 90i, the sections were scanned through the z axis, and horizontal z sections were generated and analyzed individually for colocalization and projected as superimposed stacks (merge). The quantitation of the cell density–positive staining was done in the striatum from three mice per group by using ImageJ National Institutes of Health.

Western blots

The tissue from brain striatum was homogenized to 10% (wt/vol) final concentration in cold buffer containing 50 mM Tris-HCl (pH 7.4), 8.5% sucrose, 2 mM EDTA, 2 mM EGTA, 10 mM β-mercaptoethanol, 5 mM benzamidine, 0.5 mM AEBSF, 4 μg/L pepstatin A, 10 μg/L each of aprotinin and leupeptin, 20 mM β-glycerophosphate, 100 mM sodium fluoride, 1 mM sodium vanadate, and 100 nM okadaic acid (OA). Tissue homogenates were heated in Laemmli buffer, and protein concentrations were determined by 660 nm Protein Assay (Thermo Scientific) according to the manufacturer’s instructions, and subjected to sodium dodecyl polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidine difluoride membrane of 0.45-μm pore size, and membranes were blocked with 5% nonfat dry milk. The following primary antibodies were used: 12E8 (1:500), sheep anti-mouse legumain (1:2000), 134D (1:3,000), and anti-GAPDH (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoblots were probed with the corresponding anti-mouse, anti-rabbit horseradish or anti-sheep horseradish peroxidase secondary antibodies (1:5,000; Jackson ImmunoResearch, West Grove, PA, USA) and were detected using enhanced chemiluminescence reagents (Thermo Scientific). Multi-Gauge V3 software (Fuji Photo Film, Tokyo, Japan) was used to quantify the density of the protein bands in western blots. The quantified values were analyzed statistically with the non-parametric t-test.

RESULTS

Infarct size and motor deficit are reduced after ischemia injury in AEP-KO mice

To study whether AEP is involved in the pathophysiological process of brain ischemia, WT and AEP-KO mice were subjected to brain ischemia by MCAO, followed by reperfusion, and the brain infarct size was assessed by TTC staining (Fig. 1A). We found that AEP-KO mice had smaller infarct volume than WT mice (Fig. 1B). In addition, we scored the animals for motor deficit by using scales as described by Longa et al. [33]. Consistent with the brain lesion size, AEP-KO mice showed reduced neurological deficits compared to WT mice (Fig. 1C). Western blots of the affected area showed that AEP was activated in WT mice; as expected, AEP could not be detected by either immunofluorescence or western blots in AEP-KO mice (Fig. 1D). These results suggest that AEP is involved in the regulation of ischemia-induced brain injury, and the lack of AEP diminished the pathology and the motor deficit.

Fig.1

Reduced infarct size and motor deficit after ischemia-reperfusion injury in AEP-KO mice. A) TTC staining of mouse brain slices 24 h after MCAO. B) Quantification of the infarct sizes detected by TTC staining. C) Scores of motor deficits from WT (n = 8) and AEP-KO (n = 9) mice. D) Active AEP was identified by western blots from WT (n = 5) but not AEP-KO (n = 4) mice.

AEP is activated and translocated from neuronal lysosomes to the cytoplasm and the nucleus after ischemia

We previously reported that under acidic conditions, AEP can translocate to outside of the lysosomes and promote the hyperphosphorylation of tau [25]. To determine the AEP localization after ischemia, we immunostained and analyzed by laser confocal microscopy paraffin sections from MCAO WT and AEP-KO mice. We found that in the infarct core (IC) of the ipsilateral side, AEP was mainly colocalized with TUNEL staining in the neuronal nucleus (Fig. 2A and B), and in the peri-infarct (PI) area, it was diffusely distributed in the cytoplasm in WT mice (Fig. 2B and C), whereas in the MCAO WT-contralateral side, AEP showed as granular staining, suggesting lysosomal localization (Fig. 2D). These data indicated that ischemia promotes both activation and translocation of AEP from the neuronal lysosomes to both the cytoplasm and the nucleus.

Fig.2

Ischemia induces AEP activation. Ipsilateral paraffin sections from 24 h-MCAO WT mice were subjected to double staining with TUNEL and anti-AEP, followed by laser scanning confocal microscopy analysis. A–C) AEP was mainly distributed in both the neuronal nuclei of infarct core (IC) colocalizing with TUNEL assay (A) and neuronal cytoplasm of the periinfarct area (PI) (B). D) AEP cytoplasmic granular staining was shown in the contralateral side section. Scale bar 50 μm.

Absence of AEP prevents I₂PP2A translocation from neuronal nucleus to cytoplasm

I₂PP2A cleavage and translocation from the neuronal nucleus to the cytoplasm is a key mechanism for tau hyperphosphorylation mediated by AEP in AD, and it is known that I₂PP2A is cleaved in WT but not in AEP-KO mice during ischemia [22 , 27]. However, it was not known whether tau hyperphosphorylation occurs through AEP activation during ischemia. To determine the involvement of AEP, we studied the distribution of I₂PP2A by immunostaining paraffin sections from the ipsilateral ischemic area of WT and AEP-KO mice. We found translocation of I₂PP2A from the neuronal nucleus to the cytoplasm in only the ipsilateral brains of WT (Fig. 3A) and not in AEP-KO ipsilateral area, WT-contralateral area and WT-sham mice (Fig. 3B-D); the I₂PP2A translocation was mainly located in the infarct area, colocalizing with AEP (Fig. 3E), suggesting that AEP is required for I₂PP2A translocation.

Fig.3

AEP-KO mice avoid the cleavage and cytoplasmic translocation of I₂PP2A caused by ischemia. WT and AEP-KO mice paraffin sections from 24 h-MCAO were subjected to double staining with anti-I₂PP2A and TUNEL or TO-PRO then analyzed by laser scanning confocal microscopy. A) Cytoplasmic I₂PP2A distribution was mainly found in ipsilateral-WT compared to the nuclear staining of AEP-KO-ipsilateral (B), WT-contralateral (C) and WT-sham (D) mouse brain hemispheres. E) In ipsilateral-WT mice sections, I₂PP2A colocalizes with AEP in the neuronal cytoplasm compared to AEP-KO mice (F). Scale bar 20 μm.

AEP is involved in phosphorylation of tau induced by ischemia

It was previously reported that truncation of I₂PP2A by AEP leads to hyperphosphorylation of tau in AD [25]. Here we investigated by immunohistochemistry whether this pathway is involved during ischemia. We found that ischemia induced in the ipsilateral side of WT mice hyperphosphorylation of tau at Ser262/356 site in the striatum (Fig. 4A), the most affected area after ischemic injury, and the positive staining was markedly less in the cortex (Fig. 4B) and hippocampus (Fig. 4C) of these animals. It is known that glial cells play a role in the regulation of neuron repair after injury. We therefore carried out double immunofluorescence with anti-pT-Ser262/356 and anti-GFAP (Fig. 4D) or Iba1 (Fig. 4E) and found that hyperphosphorylated tau-positive staining corresponds to neurons and not glial cells. While we found that hyperphosphorylated tau did not colocalize with either astrocytes (GFAP) or microglia (Iba1), we observed colocalization between hyperphosphorylated tau and the neuronal marker βIII tubulin, and between hyperphosphorylated tau and TUNEL staining that labels apoptotic cells, by triple immunofluorescence staining (Fig. 4F).

Fig.4

Apoptotic neuronal tau hyperphosphorylation is induced by ischemia in the striatum. Ipsilateral paraffin sections from WT mice subjected to 24 h MCAO were stained with anti-pT-Ser262/356 and contrastained with TUNEL assay and/or TO-PRO. A–C) Tau phosphorylation was mainly distributed in the striatum (A) compared to the cortex (B) and the hippocampus (C). D–F) Phosphorylation of tau at Ser262/356 does not colocalize with either GFAP (D) or Iba1 (E), but colocalizes with the neuronal marker βIII tubulin, shown in purple, merged image in F. Scale bar 50 μm.

To study the role of AEP in the phosphorylation of tau after ischemia, we compared the phosphorylation of tau at Ser262/356 in the ipsilateral side of ischemic brains from WT and AEP-KO mice. We found that neurons bearing hyperphosphorylated tau were decreased in AEP-KO compared to WT mice (Fig. 5A). We carried out quantitation of Tau anti-pT-Ser262/356–positive cells as well as western blots from the same area and found a significant decrease in the hyperphosphorylation of tau-positive cells in AEP-KO compared with WT mice (Fig. 5B, C). Similar results were found when phosphorylation of tau at Ser396/404 was assessed (data not shown). These data suggested that AEP is involved in the hyperphosphorylation of tau.

Fig.5

Tau hyperphosphorylation induced by ischemia is reduced in AEP-KO mice. A, B) Ipsilateral paraffin sections from WT and AEP-KO mice subjected to 24 h MCAO were stained with anti-pT-Ser262/356, βIII tubulin. Less hyperphosphorylation of tau was found in AEP-KO mice compared to WT mice, in the neuronal population located in the penumbra; the quantitation of pT-Ser262/356–positive staining density is shown in panel B. C) Western blots of ipsilateral striatum from WT (n = 5) and AEP-KO (n = 4) mice developed with R134d (anti-pan-tau), anti-pT-Ser262/356, and anti-GAPDH. The levels of total tau (R134d/GAPDH) and phosphorylated tau (pT-S262/356/R134d) are shown as mean±SEM. Scale bar 40 μm.

Different pathways induce the hyperphosphorylation of tau in ischemic injury

To determine the association between I₂PP2A translocation from the neuronal nucleus to the cytoplasm and the hyperphosphorylation of tau, we analyzed by immunofluorescence Tau Ser(P)-262/356 and I₂PP2A and found that hyperphosphorylated tau coexisted with the translocation of I₂PP2A (Fig. 6A arrows upper panel). However, some cells did not follow this trend when they were stained with Tau Ser(P)-396/404 (Fig. 6A, lower panel). In addition, we found that GSK3β and Tau Ser(P)-396/404 staining coexisted in some affected cells (Fig. 6B, arrowheads), suggesting the involvement of GSK-3β in ischemia-induced hyperphosphorylation of tau.

Fig.6

I₂PP2A and GSK-3β are associated with hyperphosphorylated tau after ischemia. Ipsilateral paraffin sections from 24 h-MCAO WT mice were subjected to triple staining and analyzed by laser scanning confocal microscopy. A) Cytoplasmic I₂PP2A is highly associated with phosphorylated tau at Ser262/356 (arrow) but less at Ser396/404 (arrowheads). B) GSK-3β showed colocalization with phosphorylated tau at Ser396/404. Scale bar 50 μm.

DISCUSSION

Ischemic damage is known to trigger various pathways that lead to cell death and neural diseases [35]. Dementias develop progressively, and cerebral stroke increases the risk of their development [36]. AD is the most prevalent dementia and shares common neuropathology features such as Aβ and tau pathologies with stroke or as a consequence of it [37 –39]. Some of these features are related to the leaking of lysosomal enzymes into the cytoplasm after ischemia. We previously reported that the lysosomal enzyme AEP is involved in AD pathogenesis [25]. The present study shows the involvement of AEP in ischemia and its association with tau hyperphosphorylation. We found that the deficiency of AEP reduces both infarct size and motor deficit after ischemia induced by MCAO. However, contrary to our results, Ishizaki et al. reported that in aged mice, the AEP deficiency did not affect the infarct volume and animal behavior compared with the respective WT animals [40]. Neuronal degeneration and post-stroke inflammation contributing to stroke recovery are known to be significantly altered in the aged rodent brain [41]. Consistent with our data, Liu et al. (2008) showed that neuronal cell death is markedly blocked in AEP-KO mice after MCAO (24–48 h occlusion) [27], suggesting that AEP might be the major proteinase mediating this process. Another possible explanation of the role of AEP in ischemia resides in the fact that the deficiency of AEP increases reactive hematopoiesis [42] and elevates erythropoietin level, which induces protection against cerebral ischemia [43].

Previously it was shown that acidic pH activates AEP during ischemia [27, 40]. In the present study, we analyzed the activation and distribution of AEP in WT mice after MCAO to determine its role during this injury. We found AEP in the neuronal nucleus in the IC of the ischemic area and distributed in the neuronal cytoplasm in the PI area. Apparently, during ischemia, the translocation of AEP to specific compartments corresponds to the level of cell damage, because the nuclear location of AEP is observed in apoptotic cells, and it is in the cytoplasm in non-apoptotic cells. The different subcellular distribution of AEP has been associated with different pathologies such as cancer, multiple sclerosis, and AD [44 –49]. Collectively, these data suggest that AEP is activated and translocated probably as a part of the cell death mechanism due to ischemia. Interestingly, the non-lysosomal localization of AEP is believed to trigger different pathological mechanisms, targeting proteins such as TDP-43 [50], tau [26], amyloid-β precursor protein [51], and I₂PP2A [25] involved in cognitive impairment and ischemia.

We postulate that AEP promotes the onset of tau pathology following ischemia. We base our hypothesis on two findings: 1) I₂PP2A is cleaved at Asn-175 by AEP after 24 h MCAO, but not in AEP-KO mice under the same conditions [27], and 2) I₂PP2A is translocated from the neuronal nucleus to the cytoplasm due to its cleavage by AEP, leading to tau hyperphosphorylation in AD [25]. In the present study, we found that after MCAO, I₂PP2A is translocated from the neuronal nucleus to the cytoplasm in the ipsilateral hemisphere in WT mice compared to the ipsilateral hemisphere in AEP-KO mice. Supported by our previous data in AD [25], under acidic conditions induced by ischemia, AEP is actively released in the cytoplasm, where it cleaves I₂PP2A at Asn 175 into N-terminal and C-terminal fragments, which translocate between the nucleus and the cytoplasm during normal and pathological conditions [52, 53], inducing the inhibition of PP2A activity. Another possible mechanism is that AEP translocates to the nucleus, where it cleaves I₂PP2A, which on release into the cytoplasm interacts with PP2A and inhibits it, causing hyperphosphorylation of tau.

Brain ischemia is known to produce acidosis of the tissue and cleavage of I₂PP2A into N-terminal and C-terminal fragments by AEP [27]. The cleavage of I₂PP2A by AEP leads to its translocation from the neuronal nucleus to the cytoplasm, where the N- and the C-terminal fragments of I₂PP2A, which are potent inhibitors of PP2A, the major regulator of tau phosphorylation, can inhibit the phosphatase and lead to hyperphosphorylation of tau [22 , 54]. In AD brain, AEP is translocated from neuronal lysosomes to the cytoplasm and the nucleus, and AEP at acidic pH cleaves I₂PP2A as described previously [25]. Herein, we found that the hyperphosphorylated tau is distributed in the striatum and thalamic-neurons of the ischemic brains. A previous study reported that MCAO increases the total levels of tau in glial cells; however, the study did not show specific markers of glial cells, and the cells located in the ischemic area were identified only by their morphological appearance [55]. After analyzing with different cell markers, we found that cells located in this region correspond to neurons and not to glial cells; consistent with previous reports, these neurons undergo hyperphosphorylation of tau and apoptosis after cerebral ischemia [4]. We observed higher phosphorylation of tau during MCAO in WT than AEP-KO. This issue has been contradictory, because some labs reported that the phosphorylation decreases during MCAO and is restored to normal levels compared to sham mice during reperfusion [39, 56]. Other labs reported that the hyperphosphorylation of tau increases during MCAO [4, 29]. However, in these studies, the level of hyperphosphorylated tau was compared to that in sham mice, and in none of these studies was the ratio of phospho-tau/total-tau analyzed in the ischemic area. We found that after MCAO, the level of total tau was markedly different in sham mice from ischemic mice (data not shown). The loss of tau is due to the cell death and protein loss caused by ischemia [57, 58]. Herein, we observed that the distribution and increase in hyperphosphorylated tau staining was consistent with a previous report but not comparable to its biochemical data that compared tau hyperphosphorylation in the ischemic area with the contralateral hemisphere using western blots [4]. This discrepancy is probably because in the present study we observed by immunohistochemistry that the hyperphosphorylated tau protein was highly localized in neurons in specific areas of the damaged tissue. However, overall, the total protein level in non-ischemic tissue was high, but was evenly distributed in the neurons and was difficult to quantify by microscopy. Likewise, consistent with other studies [59], we found higher levels of total tau, phosphorylated tau, and constitutive cell proteins in both sham mice (WT and AEP-KO sham) and contralateral hemisphere than the ischemic area in WT MCAO mice. The staining and quantitation revealed that under the same ischemic conditions, the deficiency of AEP in AEP-KO mice caused less hyperphosphorylation of tau than in WT mice, specifically in the striatum of the ischemic hemisphere, suggesting that tau pathology is restricted to a specific region after MCAO.

The hyperphosphorylation of tau during ischemia is highly attributed to the increase in GSK-3β activity and decrease in PP2A activity [39 , 61]. PP2A is known to upregulate the activity of GSK-3β by dephosphorylating it at Ser 9 [62]. In the present study, we found that the deficiency of AEP reduces the effect of ischemia and down-regulates this pathway of tau hyperphosphorylation. Supporting this hypothesis, we found a high colocalization between cytoplasmic I₂PP2A and tau hyperphosphorylation at Ser262/356 that is regulated by PP2A, and we observed a high colocalization between GSK-3β and phosphorylated tau at Ser396/404, suggesting that hyperphosphorylation of tau results from the involvement of both PP2A and GSK-3β through activation and translocation of AEP from neuronal lysosomes to the cytoplasm and the nucleus during ischemia.

Footnotes

ACKNOWLEDGMENTS

This work was supported in part by the New York State Office for People With Developmental Disabilities, NIH (Grant #: AG019158); Convocatoria Institucional de Investigación, University of Guanajuato; the Jiangsu Postdoctoral Science Foundation; the China Postdoctoral Science Foundation (2016M601866); and the U.S. Alzheimer’s Association (Grant #: DSAD-15-363172).

Authors’ disclosures available online ().

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Mechanism of Tau Hyperphosphorylation Involving Lysosomal Enzyme Asparagine Endopeptidase in a Mouse Model of Brain Ischemia

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Animals and tissue processing

Middle cerebral artery occlusion

Evaluation of brain infarct volume and motor deficit

Immunohistochemistry and TUNEL apoptosis staining

Western blots

RESULTS

Infarct size and motor deficit are reduced after ischemia injury in AEP-KO mice

AEP is activated and translocated from neuronal lysosomes to the cytoplasm and the nucleus after ischemia

Absence of AEP prevents I2PP2A translocation from neuronal nucleus to cytoplasm

AEP is involved in phosphorylation of tau induced by ischemia

Different pathways induce the hyperphosphorylation of tau in ischemic injury

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Absence of AEP prevents I₂PP2A translocation from neuronal nucleus to cytoplasm