Figure 1.
High molecular weight TDP-43 band on SDD-AGE gels found in FTLD-TDP but not control brain homogenates.
(A) Immunoblot of brain homogenates separated on an analytic SDD-AGE gel and probed with polyclonal anti-TDP-43 serum. Twenty-five µL of a 10% brain homogenate from the frontal or anterior temporal cortex was loaded in each lane. A slow-migrating band (upper bracket) is present only in the FTLD-TDP cases. Arrowheads indicate the position of molecular weight markers. The arrowhead labeled “titan” marks the position of the ∼ 3000-kDa titan band on a Coomassie-stained gel of chicken muscle extract on an SDD-AGE gel run under identical conditions. (B) Ratio of upper to lower band greater in FTLD than in controls. The quantities of TDP-43 in the range of the upper bracket and the lower bracket (Fig. 1A) were determined as described in the Methods, and then the ratio of the upper to the lower band calculated. This ratio is greater in every FTLD case than in any control and the difference between the two groups is significant (P = 0.013, one-way t-test). The plots depict the range (thin horizontal bars), median (white line) and 25–75 percentile range (box) of values. (C) TDP-43 in upper band correlates with FTLD diagnosis, not with total quantity of TDP in sample. Quantity of TDP-43 in the lower band plotted against that in the upper band. Filled circles are FTLD cases, open circles are control cases. While there appears to be a correlation of TDP-43 quantity in the upper region with that in the lower for FTLD cases, no such correlation exists for controls. Quantities in the lower region for FTLD cases and controls largely overlap.
Figure 2.
TDP-43 is non-covalently bound in a high molecular weight aggregate in FTLD-TDP.
(A) Independently derived antibodies to TDP-43 bind to high-molecular weight band. Immunoblots of 25 µL of brain homogenate from a case of FTLD-TDP, probed with two distinct primary and secondary antibody combinations (p+s), or the secondary antibody alone (s): either polyclonal (poly) anti-TDP-43 serum and an HRP-anti-rabbit IgG conjugate, or a murine monoclonal antibody (mono) directed against TDP-43 and HRP-anti-mouse IgG. Both primary antibodies recognize the same high molecular weight band, while neither secondary antibody gives significant signal in this region when used alone. (B) Denaturation eliminates the high molecular weight band. Immunoblots of semi-denaturing agarose gels probed with TDP-43 antiserum. For urea denaturation, homogenates were incubated in the indicated concentrations of urea for 1 hour then before loading on the gel. For the boiling study, samples were mixed with loading buffer then either incubated at room temperature for 10 minutes or heated to 100°C for 5 minutes. Boiling in 1% SDS/12.5 mM DTT or incubating with 6 M urea eliminates most of the high molecular weight TDP-43 band.
Figure 3.
The method for analyzing components of aggregates isolated on SDD-AGE gels.
(A) An unstained large format SDD-AGE gel after a typical preparative electrophoretic run. The dashed boxes depict the regions that were excised for concentration and analysis of proteins. The upper box labeled “A” circumscribes the “aggregate region” in which the major portion of aggregated TDP-43 associated with FTLD-TDP is found. The lower box labeled M contains the presumably monomeric TDP-43 forms found in both controls and FTLD cases (“monomer region”). The wide dark band at the bottom of the gel is bromphenol blue. Positions of pre-stained protein mass standards are indicated on the right. (B) Schematic depiction of the methods employed to analyze components of the TDP-43 aggregate region. The figure demonstrates the most commonly used method. Variations, when employed, are described in the text. (1) Brain homogenate (400 μg total protein) subject to electrophoresis on an SDD-AGE gel. The black dots represent TDP-43 and other proteins separated by the method. (2) The region in which the aggregate predominantly migrates is excised and transferred to a 15 mL conical tube. SDS is added to 1% and DTT to 12.5 mM. (3) Excised region of gel boiled and the volume brought to 4 mL with ultrapure water. (4) Molten agarose is transferred to glass tube with a 40% polyacrylamide plug at bottom. (5) Proteins are electrophoretically concentrated to bottom of agarose column. (6) The ∼1.5 mm (∼120 µL) section of agarose immediately adjacent to the polyacrylamide plug is excised. (7) Laemmli buffer (4×) added and the concentrated proteins are separated on a 10% polyacrylamide SDS-PAGE gel (typical volume loaded 35 µL for immunoblotting). SDS-PAGE gels further analyzed by silver staining or immunoblotting (not depicted).
Figure 4.
Protein components of the aggregate and monomer regions of SDD-AGE gels.
(A) Proteins concentrated from aggregate or monomer regions of an FTLD case compared. Silver stained SDS-PAGE gel with equal volumes of a 40-fold dilution of the monomer region concentrate (M) and undiluted aggregate region concentrate (A) are loaded on the gel. These fractions comprise different proteins. Semi-quantitative analysis indicates the total protein content of the aggregate fraction is approximately 1% that of the monomer fraction. Bars on the left indicate the migration of molecular weight protein standards of the depicted masses (kDa). (B) Comparison of proteins concentrated from the aggregate region of SDD-AGE gels for two FTLD cases and a control (the volume loaded is ∼22% of the concentrate from a region such as in figure 3a). The lane labeled W contains approximately 1 µg (total protein) from a whole brain homogenate for comparison. The aggregate regions of either FTLD cases or controls comprise a complex mixture of proteins. No consistent difference in the band pattern is seen between cases and controls. The TDP-43 bands seen in immunoblots of proteins from the aggregate region of FTLD cases are not distinctly seen in these silver stained gels.
Figure 5.
Aggregates include a 43-kDa form of TDP-43 without apparent covalent modifications.
(A) SDS-PAGE gels loaded with concentrates of aggregate region from FTLD (F) and control (C) cases. A band of 43-kDa (filled arrowhead) is seen in FTLD brain homogenates, but is absent or barely visible in most controls. Several other forms of TDP-43 are seen only in FTLD cases: a pair bands near 25-kDa (lower bracket); a pair of broad bands near 37-kDa (middle bracket); a 45-kDa band (open arrowhead); an ∼60-kDa band, and a broad band of high molecular weight species (upper bracket). In each gel a lane loaded with 0.5 µg (total protein) of 10% brain homogenate (H) is included both to indicate the apparent molecular weight of the 43-kDa species of TDP-43 that predominates in brain, and as an indication of the relative quantity of TDP-43 isolated from the aggregate. The 3 panels represent the results of 3 independent preparations from brain tissue specimens. T-4077 is represented in the first and the second panel of the figure because it was part of two preparations. The positions of protein mass standards are indicated with horizontal bars. (B) Quantity of 43-kDa and 45-kDa bands in concentrates of the SDD-AGE aggregate region. Quantities were determined as described in the Methods and are expressed as equivalents, expressed as µg total protein loaded in the well, of the 43-kDa band from unfractionated control brain homogenate. The quantities of both the 45-kDa band (P = 0.0003, one-tailed T-test) and 43-kDa band (P = 0.021, one-tailed T-test) are significantly greater in FTLD than in control concentrates. The box-whisker plots depict the range (thin line), median (white line) and 25–75 percentile range (box) of values.
Figure 6.
The 43-kDa TDP-43 band is not phosphorylated at S409/410 and not derived from 45-kDa band during processing.
(A) Immunoblot prepared exactly as those in Fig. 5A, then stripped of bound antibody and re-probed with an antibody against pS409/410 TDP-43. Phosphorylated species correspond to most of the FTLD-specific bands seen in A, including the approximately 45 kDa band (open arrowhead), but the 43-kDa TDP-43 band (closed arrowhead) is not phosphorylated at the S 409/410 site. (B) Brain homogenates were subject to conditions designed to minimize or maximize phosphatase activity (see Methods), then proteins were concentrated from the aggregate region. The quantity of the 45-kDa and 43-kDa bands were determined as described in the methods, and the ratio of the 43-kDa to 45-kDa band calculated. The box-whisker plots depict the results (range, 25–75 percentile, median) of at least 5 measurements of the ratio. The figure depicts the ratio from homogenates of 3 FTLD cases either prepared with both protease and phosphatase inhibitor stored on ice (PPI), prepared with both inhibitors and heated (PPH), or prepared with only protease inhibitor and heated (PH). An increase in the ratio of the 43-kDa to 45-kDa species in the PH condition, relative to either the PPI or PPH condition would suggest that significant endogenous phosphatase activity present in sample processing might generate the 43-kDa species from the phosphorylated 45-kDa species. No consistent pattern is seen.
Table 1.
Clinical information on sources of cerebral cortex samples used.