Proteasome inhibition stabilizes tau inclusions in oligodendroglial cells that occur after treatment with okadaic acid

Abstract

Tau-positive inclusions in oligodendrocytes are consistent neuropathological features of corticobasal degeneration, progressive supranuclear palsy, and frontotemporal dementias with Parkinsonism linked to chromosome 17. Here we show by immunohistochemistry that tau-positive oligodendroglial inclusion bodies also contain the small heat-shock protein (HSP) alphaB-crystallin but not HSP70. To study the molecular mechanisms underlying inclusion body formation, we engineered an oligodendroglia cell line (OLN-t40) to overexpress the longest human tau isoform. Treatment of OLN-t40 cells with okadaic acid (OA), an inhibitor of protein phosphatase 2A, caused tau hyperphosphorylation and a decrease in the binding of tau to microtubules. Simultaneously, tau-positive aggregates that also stained with the amyloid-binding dye thioflavin-S as well as with antibodies to tau and alphaB-crystallin were detected. However, they were only transiently expressed and were degraded within 24 hr. When the proteasomal apparatus was inhibited by carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG-132) after OA treatment, the aggregates were stabilized and were still detectable after 18 hr in the absence of OA. Incubation with MG-132 alone inhibited tau proteolysis and led to the induction of HSPs, including alphaB-crystallin and to its translocation to the perinuclear region, but did not induce the formation of thioflavin-S-positive aggregates. Hence, although tau hyperphosphorylation induced by protein phosphatase inhibition contributes to pathological aggregate formation, only hyperphosporylation of tau followed by proteasome inhibition leads to stable fibrillary deposits of tau similar to those observed in neurodegenerative diseases.

Figure 1.

Colocalization of tau and αB-crystallin in oligodendrocytes from patients with tauopathies. A-L, Immunohistochemistry on sections from the globus pallidus of CBD (A, E, I), PSP (B, F, J), FTDP-17 (C, G, K), and schizophrenia (D, H, L) brains were stained with antibodies to PHF-1 (A-D), αB-crystallin (E-H), and HSP70 (I-L). Insets show high-power magnification of tau pathology (A-C) and αB-crystallin staining (E-G) in oligodendrocytes obtained from adjacent sections. Note the abundant tau pathology in the brains of patients with CBD, PSP, FTDP-17 (A-C), and αB-crystallin immunoreactivity (E-G), respectively, which was not observable in sections of schizophrenia brains (D, H). Only baseline levels of the inducible HSP70 could be detected (I-L). Double-immunofluorescence staining of the globus pallidus from a CBD brain shows the individual labeling of tau (M) and αB-crystallin (N) as well as the colocalization of both (O, arrowheads). Staining was performed with polyclonal rabbit anti-tau antibody N-tau (green) and MAb anti-αB-crystallin antibody (red). Scale bars: A-H, M-O, 50 μm; I-L, 100 μm.

Figure 2.

Effect of okadaic acid on tau phosphorylation and heat-shock protein expression. OLN-t40 cells were treated with 20 nm OA for the indicated times. Lysates of control (Co) and OA-treated cells were prepared and subjected to immunoblot analysis using phosphorylation-independent antibody 17026 reacting with total tau, and phosphorylation-dependent antibodies 12E8, Tau-1, and PHF-1, recognizing nonphosphorylated and phosphorylated tau proteins, respectively, and MAb anti-α-tubulin antibodies. Heat-shock proteins were identified by antibodies reacting with HSP/HSC70, HSP32, and αB-crystallin, respectively.

Figure 3.

Okadaic acid decreases the binding of tau to microtubules in OLN-t40 cells. Cells were treated with OA or LiCl as indicated for 24 hr. Cell lysates were separated into cytoskeletal (P) and soluble (S) fractions after MT assembly and subjected to immunoblot analysis using polyclonal tau 17026 and MAb anti-α-tubulin antibodies. OA leads to the detachment of tau from the MTs, as indicated by an increase of tau in the soluble fraction and a decrease in the cytoskeletal fraction. LiCl increases the binding affinity of tau to MTs, as indicated by an increase of tau in the cytoskeletal fraction.

Figure 4.

Okadaic acid leads to thioflavin-S-positive aggregates in OLN-t40 cells. Control (a-c) cells and cells treated with 20 nm OA for 3 hr (d-f) or 6 hr (g-i) were subjected to indirect immunofluorescence staining using polyclonal anti-tau 17026 antibodies, followed by staining with thioflavin-S. For nuclear staining (blue), DAPI was included in the mounting medium. Scale bar, 20 μm.

Figure 5.

Inhibition of proteasome activity by MG-132 occurs without decreasing the levels of proteasomal subunits. a, OLN-t40 cells were incubated with increasing concentrations of MG-132 as indicated for 4 hr, and postglutamyl-peptidase-hydrolase and chymotrypsin-like proteasome activity were determined using fluorogenic substrate S2 and S3, respectively (see Materials and Methods for details). The cleavage of both substrates is inhibited by MG-132 in a concentration-dependent manner. Data are expressed as a percentage of the untreated control and represent the means of three independent experiments. b, Immunoblot analysis of proteasome subunits. OLN-t40 cells were incubated with increasing concentrations of MG-132 as indicated for 18 hr. Cell lysates were prepared and immunoblot analysis was performed using antibodies against the proteasome subunit 20Sα and 20Sβ, respectively. No decrease in the individual subunits was observed. Numbers on the right indicate the molecular weight in kilodaltons.

Figure 6.

a, b, Induction of heat-shock proteins by MG-132. OLN-t40 cells were treated with MG-132 at various concentrations for 18 hr (a) or with 10 μm MG-132 for 1-24 hr (b). Cell lysates were prepared and subjected to immunoblot analysis using a panel of antibodies against the following after-heat-shock proteins: HSP90, HSP70, HSP60, HSP40, HSP32, and αB-crystallin. In comparison, the immunoreactivity of α-tubulin was determined using MAb anti-α-tubulin. Co, Untreated control.

Figure 7.

MG-132 inhibits tau proteolysis. OLN-t40 cells were incubated with 10 μm MG-132 for 1-24 hr, and cell lysates were prepared. Immunoblot analysis was performed using phosphorylation-independent tau 17026 antibody reacting with total tau and phosphorylation-dependent antibodies tau-1 and PHF-1, recognizing nonphosphorylated and phosphorylated tau proteins, respectively, and MAb anti-α-tubulin antibodies. MG-132 inhibits the formation of proteolytic tau fragments. Co, Untreated control.

Figure 8.

Hyperphosphorylated tau is stabilized in OLN-t40 cells by MG-132. OLN-t40 cells were treated with MG-132 alone (MG; 0.5 μm; 18 hr), with OA (20 nm; 6 hr) followed by MG-132 (0.5 μm; 18 hr) in the absence of OA (OA-MG), or with OA (20 nm; 6 hr) followed by a recovery period of 18 hr in the absence of OA (OA). Co, Untreated control. Cell lysates were analyzed by immunoblot procedure using a panel of phosphorylation-dependent and phosphorylation-independent tau antibodies, antibodies against HSPs, and antibodies against tubulin, as described in Figure 2.

Figure 9.

Accumulation of cytoplasmic inclusions by proteasome inhibition after OA treatment. OLN-t40 cells were subjected to 0.5 μm MG-132 for 24 hr (d-f), to 20 nm OA for 24 hr (g-i), or to 20 nm OA for 6 hr followed by 0.5 μm MG-132 for 18 hr in the absence of OA (j-l). Indirect immunofluorescence was performed using MAb anti-αB-crystallin (red) followed by thioflavin-S staining (green). For nuclear staining (blue), DAPI was included in the mounting medium. a-c, Untreated control. a, d, g, j,αB-crystallin (red). b, e, h, k, Thioflavin-S (green). c, f, i, l, Overlay with DAPI. Scale bar, 20 μm.

Figure 10.

Cytoplasmic inclusions contained tau and αB-crystallin. OLN-t40 cells were treated with 20 nm OA for 6 hr, followed by 0.5 μm MG-132 for 18 hr in the absence of OA. Cells were subjected to indirect immunofluorescence using polyclonal tau antibody 17026 (a-c), MAb PHF-1 (d-f), or MAb αB-crystallin (g-i), followed by thioflavin-S staining (green). To demonstrate tau and αB-crystallin colocalization, double labeling was performed using polyclonal tau antibody 17026 (red) and αB-crystallin (green) (j-l). For nuclear staining (blue), DAPI was included in the mounting medium. Scale bar, 10 μm.

Figure 11.

Cytoplasmic inclusions in OLN-t40 cells are composed of filamentous aggregated tau. Immuno-EM was performed on OLN-t40 cells treated with OA followed by MG-132. Cells were stained with rabbit anti-recombinant tau (17026) as the primary antibody visualized with nanogold-conjugated secondary antibody. Squares in a mark the areas in the inclusion body magnified in b and c. Arrows indicate tau-positive filaments. Scale bars: a, 2 μm; b, c, 100 nm.