Activation of asparaginyl endopeptidase leads to Tau hyperphosphorylation in Alzheimer disease

Abstract

Neurofibrillary pathology of abnormally hyperphosphorylated Tau is a key lesion of Alzheimer disease and other tauopathies, and its density in the brain directly correlates with dementia. The phosphorylation of Tau is regulated by protein phosphatase 2A, which in turn is regulated by inhibitor 2, I2(PP2A). In acidic conditions such as generated by brain ischemia and hypoxia, especially in association with hyperglycemia as in diabetes, I2(PP2A) is cleaved by asparaginyl endopeptidase at Asn-175 into the N-terminal fragment (I2NTF) and the C-terminal fragment (I2CTF). Both I2NTF and I2CTF are known to bind to the catalytic subunit of protein phosphatase 2A and inhibit its activity. Here we show that the level of activated asparaginyl endopeptidase is significantly increased, and this enzyme and I2(PP2A) translocate, respectively, from neuronal lysosomes and nucleus to the cytoplasm where they interact and are associated with hyperphosphorylated Tau in Alzheimer disease brain. Asparaginyl endopeptidase from Alzheimer disease brain could cleave GST-I2(PP2A), except when I2(PP2A) was mutated at the cleavage site Asn-175 to Gln. Finally, an induction of acidosis by treatment with kainic acid or pH 6.0 medium activated asparaginyl endopeptidase and consequently produced the cleavage of I2(PP2A), inhibition of protein phosphatase 2A, and hyperphosphorylation of Tau, and the knockdown of asparaginyl endopeptidase with siRNA abolished this pathway in SH-SY5Y cells. These findings suggest the involvement of brain acidosis in the etiopathogenesis of Alzheimer disease, and asparaginyl endopeptidase-I2(PP2A)-protein phosphatase 2A-Tau hyperphosphorylation pathway as a therapeutic target.

Keywords: Acidosis; Alzheimer Disease; I2PP2A; Legumain; Lysosomes; PP2A; SET; Tau; Tau Hyperphosphorylation.

FIGURE 1.

AEP interacts with I₂^PP2A and in AD brain the levels of activated AEP and I_2NTF are selectively increased. A, AD and control (Ctrl) human brain homogenates were subjected to Western blots developed with antibodies to AEP and I_2NTF. In AD brain, increases in the levels of both activated AEP (36-kDa band) and I₂^PP2A cleavage product I_2NTF were found. B, quantitation of Western blots showed a decrease in the level of pro-AEP and an increase in the activation of AEP and the cleavage of I₂^PP2A into I_2NTF in AD brains. C, immunoprecipitation of AEP with anti-AEP (legumain) showed co-immunoprecipitation of I₂^PP2A from lysates of Cos-7 cells transfected with HA-I₂^PP2A.

FIGURE 2.

I₂^PP2A and AEP are localized mainly in neuronal cytoplasm in AD brain. A, AD and control (Ctrl) hippocampal paraffin sections were subjected to double immunostaining with anti-histone 3 (a nuclear marker) and anti-I₂^PP2A and analyzed by laser scanning confocal microscopy. In AD brains, I₂^PP2A was localized mainly in neuronal cytoplasm, whereas in control cases it was seen in the nucleus. B, immunostaining with anti-LAMP-2 (a lysosomal marker) and anti-AEP showed a diffuse staining of AEP in neuronal cytoplasm in AD and lysosomal localization in control brain neurons. The insets show high magnification of the area indicated by the arrow. Scale bar, 40 μm.

FIGURE 3.

Active AEP is selectively translocated from neuronal lysosomes to the cytoplasm and the nucleus and is associated with abnormally hyperphosphorylated Tau in AD. A, AD and control (Ctrl) human brain samples were subjected to subcellular fractionation, followed by Western blots to determine the distribution of AEP. B, the translocation of active AEP from the neuronal lysosomes to the cytoplasm was found. LAMP-2 was used as specific marker for lysosomal fraction. C, Western blot analysis showed a trend but no significant increase in translocation of active AEP from neuronal lysosomes to the nucleus in AD brain. D, immunohistochemical staining of AEP in AD hippocampal paraffin sections counterstained with hematoxylin showed its localization in neuronal lysosomes (arrowhead), cytoplasm (arrow), nuclei (asterisk), and neurofibrillary tangles (double arrows). E, AD and control hippocampal paraffin sections were subjected to triple immunohistofluorescent staining with anti-AEP, anti-I₂^PP2A, and anti-Ser(P)-262/356 (12E8) Tau and analyzed by laser scanning confocal microscopy. Diffuse cytosolic staining of AEP was observed in Tau pathology-bearing neurons (arrowheads), whereas in cells without hyperphosphorylated Tau AEP was localized in lysosomes (arrow). Scale bar, 10 μm.

FIGURE 4.

GST-I₂^PP2A is cleaved by AEP from the cytosolic fraction of AD brain. A, GST-I₂^PP2A recombinant fusion protein was cleaved by AD brain extract (Ext) in a proteolysis assay, generating GST-I_2NTF in a time-dependent manner. B, GST-I₂^PP2A (W) but not mutated N175Q (M) GST-I₂^PP2A could be cleaved with the brain extract. C, under the same proteolysis conditions GST-I₂^PP2A was cleaved by the IP AEP, but not by the immunodepleted extract (ID). D, the cytosolic fraction isolated from AD brains was markedly more robust than the lysosomal or the nuclear fraction in cleavage of GST-I₂^PP2A into GST-I_2NTF during 0–90 min of incubation. E and F, quantitation of Western blots showed that the level of active AEP was the highest in the cytosolic fraction (E) and that GST-I₂^PP2A was most robustly cleaved by the cytosolic fraction (F). Actin, LAMP-2, and histone 3 were used as specific markers of cytoplasm, lysosomes, and nuclei, respectively.

FIGURE 5.

I₂^PP2A is released from the cell nucleus to the cytoplasm, co-localized with AEP and cleaved into I_2NTF and I_2CTF under acidic conditions. A, Cos-7 cells co-transfected with AEP and HA-I₂^PP2A were subjected to immunocytochemical staining with anti-AEP and anti-HA, respectively. I₂^PP2A was seen mostly localized in the nucleus in Cos-7 cells but translocated to the cytoplasm following incubation with acid pH medium, pH 5.5, for 30 min. Under the acidic condition, AEP was seen diffused into cell cytoplasm and the nucleus. B, protein expressions evaluated by Western blots. Cos-7 cells were either co-transfected or individually transfected with AEP and I₂ ^PP2A-Myc and incubated with neutral pH or acid pH medium for 30 min. At acid pH, the active pro-AEP and active AEP signals were markedly increased. C, in the same conditions as in B, cells were lysed, followed by Western blots developed with anti-I_2NTF (10E7) or anti-I_2CTF (anti-c-Myc). In the presence of acidic conditions, cells co-transfected with AEP and I_2FL showed the generation of both I_2NTF and I_2CTF. NCP, N-terminal cleavage product of I₂^PP2A that neither requires AEP nor acidic pH; NSB, nonspecific band.

FIGURE 6.

Acidic conditions induce AEP activation, cleavage of I₂^PP2A, inhibition of PP2A activity, and Tau hyperphosphorylation. A, rat hippocampal slices after incubation in oxygenated aCSF at three different pH conditions (control, pH 7.4) were subjected to Western blots developed with anti-phospho-Tau and anti-AEP. The blots were analyzed by densitometry. B–E, acidic conditions caused an increase in the level of activated AEP (B), a decrease in the level of full-length I₂^PP2A (C), inhibition of PP2A activity (D), and increase in abnormal hyperphosphorylation of Tau at Ser-262/356, Thr-205, Ser-214, and Thr-212, but not at Ser-199 (E). F and G, Western blots from SH-SY5Y cells either transfected with AEP-siRNA or reagents alone (mock), treated with kainic acid and then lysed. F, knockdown of AEP by siRNA inhibited Tau hyperphosphorylation induced by kainic acid treatment of cells lysed at neutral pH. G, knockdown of AEP by siRNA inhibited hyperphosphorylation of Tau seen in cells lysed at pH 6.0.

FIGURE 7.

Proposed mechanism of I₂^PP2A cleavage and abnormal hyperphosphorylation of Tau by activated AEP in AD. In AD different etiopathogenic mechanisms activate the translocation of active AEP from the neuronal lysosomes to the cytoplasm and the nucleus where it interacts with I₂^PP2A and cleaves it into I_2NTF and I_2CTF, which bind to PP2A and inhibit its activity. The inhibition of PP2A leads to abnormal hyperphosphorylation of Tau.