HIV Tat protein and amyloid-β peptide form multifibrillar structures that cause neurotoxicity

Abstract

Deposition of amyloid-β plaques is increased in the brains of HIV-infected individuals, and the HIV transactivator of transcription (Tat) protein affects amyloidogenesis through several indirect mechanisms. Here, we investigated direct interactions between Tat and amyloid-β peptide. Our in vitro studies showed that in the presence of Tat, uniform amyloid fibrils become double twisted fibrils and further form populations of thick unstructured filaments and aggregates. Specifically, Tat binding to the exterior surfaces of the Aβ fibrils increases β-sheet formation and lateral aggregation into thick multifibrillar structures, thus producing fibers with increased rigidity and mechanical resistance. Furthermore, Tat and Aβ aggregates in complex synergistically induced neurotoxicity both in vitro and in animal models. Increased rigidity and mechanical resistance of the amyloid-β-Tat complexes coupled with stronger adhesion due to the presence of Tat in the fibrils may account for increased damage, potentially through pore formation in membranes.

Figure 1. Tat protein increases aggregation and adherence of Aβ fibrils

a) Typical far UV CD spectra shown for 200 µM Aβ (red trace), 200 µM Aβ/0.4 µM Tat (blue trace) and 200 µM Aβ/1.8 µM Tat (black trace) (each an average of 3 traces) indicate that the predominant structure in Aβ fibrils is the β sheet, which increases in the presence of Tat. b) ThyT bulk fluorescence shows increase in aggregation due to Tat and adhesion to surfaces of Aβ–Tat complexes. Data represents mean+ SEM of two independent experiments done with five technical replicates (first experiment) and seven technical replicates (second experiment) and analyzed by unpaired Student’s T test: *p<0.05. c) ThyT labeled Aβ and Aβ-Tat structures (cyan color) adhered to neuronal cells in culture. The Aβ-Tat samples show larger aggregates attached to the cells. The neuronal connections within neurons appear red due to labeling of tubulin fibers with tdTomato. Scale bars represent 30 µm. The exposure times and display range in the color channels of the merged images are the same for both images. The graph shows the fold increase in the size of the aggregates attached to the cells. Data represents mean +SEM of 49 (Aβ with 0.08 µM Tat), 31 (Aβ with 0.4 µM Tat), and 25 (Aβ with 1.8 µM Tat) complexes, representative of three independent experiments. Data was analyzed by ANOVA with Fisher’s means comparison test: *p<0.0001.

Figure 2. Structure of Tat protein

a) AFM topography image of Tat protein as absorbed from a phosphate buffered saline solution at pH 7.4, onto a clean, atomic flat mica surface, shows a large distribution of sizes, ranging from monomers to large oligomers, all presenting globular structure. Scale bar is 50 nm. Insert represents an experimental zoom-in showing a Tat monomer. Scale bar is 20 nm. b) The distribution of sizes for Tat shows that the most frequent structures are monomers, dimers and small oligomers, however 40–50-mers and larger are present in smaller amounts. The data was derived from 150 particles analysed, representative for 3 technical replicates. Insert shows the relative percentage of monomers, dimers and trimers of Tat, from three technical replicates with 139, 150, and 543 particles analysed respectively. c) Far-UV CD spectra of 10 µM Tat in PBS solution at pH 7.4 indicates the presence of α-helical structure. The CD spectra presented is an average of 3 traces and has similar shape with spectra of three independent experiments at 20 µM, 1 µM and 0.1 µM concentrations.

Figure 3. Changes in the Aβ fibril structure induced by Tat protein

Typical AFM topography images of Aβ fibrils in the presence of various concentrations of Tat protein show that the predominant structure observed is a) a typical Aβ fibril, uniform along length, as seen in the graph below, b) a twisted fibrillar structure with 0.08 µM Tat, c) a thick fibril with irregular length and width with 0.4 µM Tat and d) large aggregated patches with 1.8 µM Tat. The graphs represent sections along and across the fibrils shown in the images, at the locations indicated with lines in the corresponding topography image. Scale bars represent 50 nm.

Figure 4. Aβ fibrils untwist and become more mechanically resistant in the presence of Tat

a) An AFM topography image of a typical twisted Aβ fibrillar structure. b) The height at the top of the twist is about twice the height in the groove, and the groove is about the same height as a singular fibril, which shows that two single fibrils are twisted together to give the twisted fibrillar structure. Data represents mean heights + SEM of 21 twists of 7 fibrils and 22 standard fibrils from the Aβ with 0.4 µM Tat sample; *p<1E-6 and there are no significant differences between the height of the grooves and the height of singular fibrils. Similar groove/top and regular fibril height rapports were obtained for the twisted fibrils at all Tat concentrations (not shown). c) A single fibril (arrow) adjacent to a double twisted fibril (arrow head) in the same topography image. d) The distance between twists in these double fibrils increases significantly with Tat concentration, showing that the fibrils untwist due to Tat. Data represents mean + SEM of 18 (Aβ), 50 (Aβ with 0.08 µM Tat), 54 (Aβ with 0.4 µM Tat), and 19 fibrils (Aβ with 1.8 µM Tat) from two independent experiments: *p<0.05 and **p<0.001. e) An AFM topography image of a single Aβ fibril that ruptured under air flow during the drying procedure. f) With increasing Tat concentration, the rupture lengths of single fibrils grow significantly, indicating increased mechanical resistance. Data represents mean + SEM for 21 (Aβ), 30 (Aβ with 0.08 µM Tat), 70 (Aβ with 0.4 µM Tat), 53 rupture lengths of fibrils (Aβ with 1.8 µM Tat) from two independent experiments: *p<0.05 and **p<0.001. All data was analyzed by ANOVA with Fisher’s means comparison test. Scale bars represent 50 nm.

Figure 5. Tat is binding to the external surface of Aβ fibril

a) An Aβ fibril labeled with ThyT. Scale bar is 10 µm. b) The presence of fluorescent Tat in the Aβ fibrils increases in a dose-dependent manner with Tat present at incubation. Data represents mean + SEM for 15 (Aβ with 0.08 µM Tat), 19 (Aβ with 0.4 µM Tat) and 17 fibrils (Aβ with 1.8 µM Tat), from two independent experiments and was analyzed by ANOVA with Fisher’s means comparison test: *p<0.001 and **p<1E-6. (c–d) Computer modeling of Aβ–Tat interaction visualizes the surface binding of Tat to the Aβ fibril: The ab-initio model of Tat B 1–72 generated by I-Tasser using the crystallographic known structure of Tat B 1–48 as template (c) was docked to the Aβ fibril backbone (golden), which was constructed by repeating the “3 hairpin” structure of Aβ fibril, the result being obtained with ClusPro (d). The first three most probable, low energy, independent conformations were superimposed in the same image (d). The view is in cross-section of fibril. The simulation indicates that Tat binds to the external side at the junction between the hairpins terminals and their turns, allowing in this way further interaction for the bound Tat, due to the external residues, not involved in the binding. Hybrid Tat is presented in (c) in cartoon representation and in (d) as Cα trace, colored blue – the first scored solution, red – the second and grey – the third. The side chains of Lys and Arg residues are represented as sticks with the nitrogen atoms colored blue.

Figure 6. Aβ–Tat complexes show synergistic neurotoxicity in cultured neurons

a) Fluorescence images showing rat neuronal cell cultures where tubulin is fluorescently labeled with td-Tomato fusion protein. Cultures were exposed to the Aβ - Tat complexes and images show the changes after 48 hours of exposure. Aβ - Tat complexes were formed at 200 µM Aβ with varying concentrations of Tat (0.08 µM, 0.4 µM and 1.8 µM). The complexes were automatically diluted 10 fold when incubated with the neurons. Final concentrations are presented in the graphs. Damage to neurons is evident by formation of punctae along neurites. Scale bars are 300 µm. b) Tat in the Aβ -Tat complexes, causes neuronal damage in a dose-dependent manner leading to decreased neuronal cell counts and retraction of neurite in the remaining neurons. Data represents mean + SEM for 12 images per media sample and 6 images at each dose and is representative of four independent experiments. Analysis was done by ANOVA with Fisher’s means comparison test: *p<0.01, **p<0.001 and ***p<1E-6.

Figure 7. Formation of Aβ-Tat complexes in vivo

a) Tat injected in mice brains colocalizes with amyloid precursor protein (APP) in APP-PS1 transgenic mice. The brain slices were fixed and immunostained for Tat (red) and APP (green) to show their colocalization. b–c) Tat colocalizes with APP in the brains of APP-Tat double transgenic mice, both inside the neurons, mainly in the granular structures (b) and outside of the neuronal cells (c). Free floating vibratome sections from non-transgenic (n=8), Tat transgenic (n=4), APP transgenic (n=4) and APP/Tat transgenic mice (n=8) age 4–6 months were studied. As shown, in the non-transgenic mice no reactivity was detected, in the Tat transgenic mice immunopositive punctae were detected in association with glial cells, in the APP transgenic mice no Tat reactivity was detected but Aβ plaques were identified in the neocortex and hippocampus. In the APP-Tat transgenic mice plaques displayed co-localization between Tat and Aβ. In (b) and (c) the labeling is with DAPI for cell nuclei, imunostained for Tat (red) and for APP (green). Scale bars are 20 µm for confocal fluorescence images in (a),(b) and (c).

Figure 8. Proposed model of Aβ–Tat interaction and their increased neurotoxicity

a) Tat attaches on the surface of the typical amyloid β fibril. b) At small concentrations, (Tat:Aβ ratio = 1: 2500), the fibrils that randomly come close to each other attach due to Tat and twist around each other to form the double fibril. c) At a 1:500 molar ratio, due to more Tat present, more fibrils, long and small, attach to each other to form the irregular fibrils. d) At a 1:110 molar ratio large patches appear as many fibrils attach to each other. e) These structures are more rigid and have increased adherence due to Tat presence, therefore are binding stronger to the neuronal cell membrane and can induce, only from a mechanical perspective, pore formation. As a minor pathway, being a trans-membrane penetrating molecule, Tat attached to small aggregates is likely to succeed entering the cells with the Aβ “cargo” to induce damage, in the case of the few small Aβ–Tat aggregates present. Scale bars in a-d AFM tridimensional topography images are 50 nm.