An in situ and in vitro investigation of cytoplasmic TDP-43 inclusions reveals the absence of a clear amyloid signature

Figures & data

Figure 1. Overexpression of full length TDP-43 leads to nuclear depletion of endogenous TDP-43 and formation of TDP-43 cytoplasmic inclusions. (A) Representative STED microscopy images of NSC-34 cells (n = 3) transfected with 20 µg of pCI-neo plasmid expressing human TDP-43 and analyzed immediately after and 40 h after transfection. Red fluorescence: total TDP-43 (endogenous and exogenous). Higher magnifications show round (left) and elongated (right) TDP-43 inclusions. (B) 3D reconstruction of the z-stack analysis (5-μm-thick slices) of the specimens shown in (A). A NSC-34 cell was virtually dissected on the zy plane to show more clearly the intracellular TDP-43 inclusions. Red and green fluorescence: cell membrane (WGA) and exogenous (human) TDP-43, respectively. Yellow and white arrows: round and elongated inclusions, respectively.

Figure 2. Cytoplasmic inclusions of TDP-43 do not stain with amyloid-diagnostic dyes able to cross biological membranes. Representative confocal microscopy images of NSC-34 cells (n = 3) transfected with vehicle, 0.15 μM BSA, 20 µg of pCI-neo plasmid expressing human TDP-43, or preformed Aβ₄₂ fibrils at 2 µM monomer equivalents. Cells were then stained with Amytracker 630 (upper panels), Compound 3 (middle panels), and compound 4j (lower panels). Green and blue fluorescence: amyloid-bound diagnostic dyes and DAPI-stained nuclei, respectively.

Figure 3. Cytoplasmic inclusions of TDP-43 do not have an enrichment of β-sheet structure. (A,B) Raman spectra (n = 15) of control cells (black) and TDP-43 expressing cells (red) in the 500–1200 cm⁻¹ wavenumber range with excitation at 532 nm (A) and in the 1200–1800 cm⁻¹ wavenumber range with excitation at 785 nm (B). (C,D) Magnification of the 1500–1800 cm⁻¹ region (n = 15) containing the amide I band for control cells (C) and TDP-43 expressing cells (D), showing the experimental spectrum (black), the best fitted spectrum using a sum of five Lorentzian functions (red) and individual fitted components (indicated colors). (E) Quantification of secondary structure components for both control cells and TDP-43 expressing cells. The last column reports the data of the mammalian proteome [49,50]. (F) Difference spectrum obtained by subtracting the control cell spectrum from that of TDP-43 expressing cells: experimental (black), smoothed (grey), best fitted (red) spectrum, and individual fitted components (indicated colors). (G) Quantification of secondary structure components of the difference spectrum.

Figure 4. Cytoplasmic inclusions of TDP-43 imaged with TEM accumulate in lysosomes and show a non-fibrillar morphology. (A) Representative electron micrograph showing lysosomal accumulation (arrowheads) in NSC-34 cells expressing TDP-43 after 40 h from transfection. Note the round shaped electron-dense inclusions within lysosomes (arrows). Plasma membrane (PM) and nucleus are also indicated. (B) Higher magnification image showing lysosomes (arrowheads) engulfed with electron-dense material and putative TDP-43 inclusions (arrows). (C) Further enlargement of an image showing a lysosome with non-fibrillar dense inclusions (arrows). (D) Positive control with a high magnification image of a lysosome from a primary neuron of PS19 mice expressing P301S mutant human tau (gene MAPT), showing filamentous inclusions (red arrowheads).

Figure 5. Tinctorial, structural and morphological analysis of TDP-43 aggregates formed in vitro from purified full-length TDP-43 show a non-amyloid nature. (A) ThT fluorescence spectrum in the presence of TDP-43 preincubated at a concentration of 5 µM, in 20 mM acetate buffer, 150 mM NaCl, 5% (w/v) PEG8000, 2 mM TCEP, pH 5.0 (Final pH 6.0), 25 °C, under agitation at 560 rpm for 4 days (solid line). The ThT-independent baseline deriving from aggregate-generated light scattering is shown (dotted line). The ThT fluorescence spectrum in the absence of protein (blank) is also shown (dashed line). (B) Far-UV CD spectrum of TDP-43 pre-incubated as described above. (C) Left: representative confocal microscopy images showing TMR-TDP-43 protein pre-incubated as described above. Center: higher magnifications of the aggregates shown from the boxed areas. Right: depth coding profiles along the xyz-axes of the 3D reconstruction of the same aggregates (different colors represent different planes along the xyz axes).

Figure 6. Scheme of TDP-43 inclusion formation. Self-assembly of full-length TDP-43 (blue) begins with the nucleus-to-cytoplasm mislocalization of the protein. In the cytoplasm it forms non-amyloid inclusions of different sizes. Later on, filaments distinct from the classical amyloid fibrils may or may not form. In the first case, they can either form directly from a structural reorganization of the initial inclusions, or from nuclei derived slowly, rarely, and independently of them. In the latter scenario, filaments would then rapidly grow from TDP-43 reservoirs provided by the initial non-filamentous inclusions. The process also involves phase separation within stress granules and TMEM106B CTD amyloid fibril formation, which have been neglected in the figure for simplicity (Created with BioRender.com).

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Data availability statement

The data that support the findings of this study are available on request from the corresponding author.