Distinct FTDP-17 missense mutations in tau produce tau aggregates and other pathological phenotypes in transfected CHO cells

Abstract

Multiple tau gene mutations are pathogenic for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), with filamentous tau aggregates as the major lesions in the CNS of these patients. Recent studies have shown that bacterially expressed recombinant tau proteins with FTDP-17 missense mutations cause functional impairments, i.e., a reduced ability of mutant tau to bind to or promote the assembly of microtubules. To investigate the biological consequences of FTDP-17 tau mutants and assess their ability to form filamentous aggregates, we engineered Chinese hamster ovary cell lines to stably express tau harboring one or several different FTDP-17 mutations and showed that different tau mutants produced distinct pathological phenotypes. For example, delta K, but not several other single tau mutants (e.g., V337 M, P301L, R406W), developed insoluble amorphous and fibrillar aggregates, whereas a triple tau mutant (VPR) containing V337M, P301L, and R406W substitutions also formed similar aggregates. Furthermore, the aggregates increased in size over time in culture. Significantly, the formation of aggregated delta K and VPR tau protein correlated with reduced affinity of these mutants to bind microtubules. Reduced phosphorylation and altered proteolysis was also observed in R406W and delta K tau mutants. Thus, distinct pathological phenotypes, including the formation of insoluble filamentous tau aggregates, result from the expression of different FTDP-17 tau mutants in transfected Chinese hamster ovary cells and implies that these missense mutations cause diverse neurodegenerative FTDP-17 syndromes by multiple mechanisms.

Figure 1

Topography of FTDP-17 missense mutations in tau- and epitope-specific anti-tau antibodies. Schematic representation of the longest tau isoform, designated Tau40, containing 441 amino acids. The locations of six FTDP-17 missense mutations studied here are identified in the schematic diagram of tau. The two 29-amino-acid-long inserts are defined by white boxes at the amino-terminal region and the MT binding repeats appear as four black blocks near the carboxy terminal of tau. Each MT repeat is numbered 1–4. The defined epitopes recognized by each of the anti-tau antibodies used in this study are shown below the tau schematic with their corresponding amino acid length and position as well as the code name of the specific antibody that recognizes each epitope. The exact site(s) of phosphorylation detected by the phosphorylation-dependent anti-tau MAbs are shown here below the code names.

Figure 2

SDS-PAGE analysis of the electrophoretic mobility of Wt and mutant tau expressed in CHO cells. Western blot analysis of cell lysates from CHO cells stably expressing either the Wt or mutant tau proteins and probed with a cocktail of MAbs T14 and T46 (T14/46; A). Tau proteins harboring the RW and the triple mutation (VPR) migrate rapidly and nearly in parallel with Wt recombinant tau (R_τ). Extracted Wt and mutant tau proteins were either untreated (−) or treated (+) with alkaline phosphatase (Alk Phos) and probed with T14/46 (B). Note that after treatment with Alk Phos all tau proteins migrate as one strong band with R_τ.

Figure 3

RW and VPR tau mutants exhibit reduced phosphorylation at Ser396 and Ser404. The phosphorylation of Wt and mutant tau at multiple sites was determined by immunoblot analysis with a panel of anti-tau antibodies. Note that the phosphorylation of the RW and VPR tau mutants expressed in CHO cells is reduced at the Ser 396/404 site (as detected by PHF1 and T3P antibodies), but not at Thr181 (AT270), Ser262 (12E8), or Thr231 (PHF6).

Figure 4

Turnover of Wt and mutant tau in CHO cells is similar. Wt and mutant tau proteins were immunoprecipitated at various times after pulse labeling with [³⁵S]methionine. No significant difference in the turnover rate of mutant versus Wt tau was apparent in these cells. However, although Wt tau and PL tau mutants migrated more slowly over the chase period, an indication of increase phosphorylation, the slower migrating tau isoforms are not detected in cells harboring the RW and VPR mutations.

Figure 5

Alteration in proteolytic processing by FTDP-17 tau mutations. Immunoblot analysis of tau extracted from CHO cells expressing Wt and FTDP-17 mutant tau proteins showing the intact, full-length species of each tau isoform and the profile of breakdown products generated, as visualized by the T46 MAb (A) and the T1 MAb (B). The banding pattern is altered for the ΔK, RW, and VPR tau mutants relative to Wt tau, suggesting that these mutations induce a conformational change that modifies the proteolysis of these proteins.

Figure 6

FTDP-17 missense mutations induce tau aggregation and disrupt MT bundling. CHO cells expressing Wt and mutant tau were plated onto glass coverslips and allowed to settle overnight. After fixation in 0.3% glutaraldehyde, cells were permeabilized with Triton X-100 and immunostained with recombinant tau (17026) (A–D and I–L) and α-tubulin (E–H and I–L). Colocalization and bundling of tau and tubulin are readily apparent in cells with Wt (A, E, and I) and RW (B, F, and J) tau. This pattern is completely disrupted with the expression of the ΔK and VPR tau mutants. Note that in CHO cells expressing these tau mutants, tau and tubulin no longer colocalize (K and L), the tubulin network is not bundled (G and H), and aggregates of tau are apparent (C and D). Bar, 30 μm at 20×.

Figure 7

FTDP-17 missense mutations decrease the binding of tau to MT. Cell lysates from CHO cells stably expressing Wt or mutant tau were separated into cytoskeletal (P) and soluble fractions (S) after MT assembly. The amount of tau and α-tubulin present in each fraction was determine by immunoblot analysis with I¹²⁵-conjugated secondary antibody for quantification (A and C). (B and D) Quantitation of the percentage of tau in the soluble fraction relative to the total amount of tau present. Note that the ΔK and VPR mutations significantly reduce the binding of tau to MT, leaving more of these mutant proteins free in the soluble fraction. (n = 4, *p < 0.01), and that the dramatic loss of MT binding was evident in three different CHO cell clones that expressed the VPR tau mutants (C and D).

Figure 8

Aggregates of FTDP-17 tau mutants grow larger with time. CHO cells stably expressing Wt tau and mutant tau protein were plated at low density on coverslips and grown for either 1 or 3 d. (A–D) Double-labeled immunofluorescence images of CHO cells expressing the VPR tau mutants immunostained with rabbit antirecombinant tau (17026; A and C) and a MAb to α-tubulin (B and D) in cells grown for 1 d (A and B) or 3 d (C and D). (E–H) Immuno-EM detection of tau by using 17026 as the primary antibody visualized either with secondary antibody and silver-enhanced DAB (E and F) or nanogold conjugated secondary antibody (G and H). Bar, 10 μm at 60× for immunofluorescence photomicrographs; bar, 100 nm for immuno-EM.

Figure 9

Aggregates with fibrillar structures are detected in CHO cells expressing the VPR tau mutants. Transmission EM of tau aggregates at low (A and C) and corresponding high (B and D) magnifications. (C and D) Images of aggregates that were treated with formic acid. The image in D is a higher magnification of the lower left portion of the inclusion seen in C. Arrowheads in C correspond to the same region that is marked by arrowheads at higher magnification in D. Arrows in B and D highlight fibril-like structures within the aggregates. Bar, 100 nm.

Figure 10

FTDP-17 tau mutant become increasingly insoluble in CHO cells. Soluble and insoluble fractions of tau from CHO cells expressing Wt and FTDP-17 mutant tau protein were isolated and evaluated by immunoblot analysis with Tau14/46 (A). Quantitative analysis (n = 3) of the percentage of insoluble tau relative to the soluble fraction in Wt-, ΔK-, and VPR-expressing CHO cells (B). There is an increase in the amount of insoluble tau present in cells expressing the ΔK and VPR tau mutants relative to Wt tau.