S100A9-Driven Amyloid-Neuroinflammatory Cascade in Traumatic Brain Injury as a Precursor State for Alzheimer’s Disease

Pro-inflammatory and amyloidogenic S100A9 protein is an important contributor to Alzheimer’s disease (AD) pathology. Traumatic brain injury (TBI) is viewed as a precursor state for AD. Here we have shown that S100A9-driven amyloid-neuroinflammatory cascade was initiated in TBI and may serve as a mechanistic link between TBI and AD. By analyzing the TBI and AD human brain tissues, we demonstrated that in post-TBI tissues S100A9, produced by neurons and microglia, becomes drastically abundant compared to Aβ and contributes to both precursor-plaque formation and intracellular amyloid oligomerization. Conditions implicated in TBI, such as elevated S100A9 concentration, acidification and fever, provide strong positive feedback for S100A9 nucleation-dependent amyloid formation and delay in its proteinase clearance. Consequently, both intracellular and extracellular S100A9 oligomerization correlated with TBI secondary neuronal loss. Common morphology of TBI and AD plaques indicated their similar initiation around multiple aggregation centers. Importantly, in AD and TBI we found S100A9 plaques without Aβ. S100A9 and Aβ plaque pathology was significantly advanced in AD cases with TBI history at earlier age, signifying TBI as a risk factor. These new findings highlight the detrimental consequences of prolonged post-TBI neuroinflammation, which can sustain S100A9-driven amyloid-neurodegenerative cascade as a specific mechanism leading to AD development.

. S100A9 and Aβ in Human TBI Hippocampi. Sequential immunohistochemistry of a representative proteinaceous precursor-plaque with (A) Aβ, (B) amyloid oligomer specific A11 and (C) fibril specific OC antibodies, respectively (ca. 1 day post-TBI time). Sequential immunohistochemistry of neuronal cells with (D) Aβ, (E) amyloid oligomer specific A11 and (F) fibril specific OC antibodies, respectively (ca. 1 day post-TBI time). Pair of sequential immunohistochemistry images with (G) S100A9 and (H) A11 antibodies of TBI tissues (ca. 1 day post-TBI time) indicating that the precursor-plaques were reactive with S100A9 but not with A11 antibodies, while S100A9 immunopositive cells were stained also with A11 antibodies. Pair of sequential immunohistochemistry images with (I) S100A9 and (J) fibril specific OC antibodies of TBI tissues (ca. 1 day post-TBI time), indicating that the S100A9 immunopositive precursor-plaques were not reactive with OC antibodies. Pairs of sequential immunohistochemistry images of blood vessel in TBI tissues (ca. 1 day post-TBI time) with (K) S100A9 ̶ (L) A11 antibodies and (M) S100A9 ̶ (N) fibril specific OC antibodies, respectively. (O) Part of sequential immunohistochemistry procedure with S100A9 antibodies complementary to the image of Aβ immunopositive precursor-plaques in Figure 1B, indicating the lack of immunoreactivity of these plaques with S100A9 antibodies. Pair of sequential immunohistochemistry images of precursor-plaques in TBI brain tissues (ca. 1 day post-TBI) with (P) S100A9 and (Q) Aβ antibodies, respectively, indicating that the S100A9 positive precursorplaques were not reactive with Aβ antibodies. Scale bars are 20 μm (A-F and O) and 50 μm in (G-N, P and Q). Supplementary Fig. S2. Intracellular S100A9/Aβ aggregation and apoptosis in human TBI hippocampi.

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Parts of sequential immunohistochemistry of neuronal cells in the TBI hippocampus with (A) S100A9 and (B) A11 antibodies, respectively, which are complementary to the Aβ-positive immunostaining ( Figure 2K) (C) Representative image of double immunohistochemistry with S100A9 and Aβ antibodies of the hippocampus tissues in control non-demented patient, showing lack of immunostaining. Parts of sequential immunohistochemistry of neuronal cells in the TBI hippocampus with (D) S100A9 and (E) A11 antibodies, S5 respectively, which are complementary to the Bax-positive immunostaining ( Figure 2L). (F and G) Parts of sequential immunohistochemistry of neuronal cells with (F) S100A9 and (G) A11 antibodies, respectively, which are complementary to the immunostaining with activated caspase-3 antibodies ( Figure   2M). Scale bars are 50 µm (A, B, D-G) and 500 µm (C). S6 S7 Supplementary Fig. S3. Effect of age on TBI amyloid, neuroinflammatory and apoptotic responses. n=13 TBI patients. Bubble charts showing counts of (A) the precursor-plaques reactive with S100A9 and Aβ antibodies, (B) neuronal and microglial cells reactive with S100A9 and Aβ antibodies and (C) neuronal cells reactive with A11, Bax and activated caspase-3 antibodies, respectively, depending on the subject age.
Each bubble corresponds to individual subject. Supplementary Fig. S4. S100A9 and Aβ plaques in human brain tissues with Alzheimer's disease. Senile plaques stained with sequence of (A) S100A9, (B) Aβ and (C) OC antibodies. S100A9 plaques stained with sequence of (D) S100A9, (E) A11 and (F) Aβ antibodies. S100A9 plaques stained with sequence of (G) S100A9 and (H) OC antibodies. Scale bars are 50 µm in (A-H). Supplementary Fig. S5. In vitro S100A9 amyloid formation and proteinase K digestion. (A and B) Normalized kinetics of S100A9 amyloid formation monitored by h-FTAA fluorescence at (A) 42 °C and (B) 37 °C under shaking with glass beads. S100A9 amyloid kinetics in 10 mM PBS, pH 7.4, 2 mg/ml is shown in red and at 5 mg/mlin green; amyloid kinetics in 20 mM sodium acetate, pH 4.5, 2 mg/ml is shown in blue and at 5 mg/mlin magenta. (C) AFM height images of S100A9 amyloids (5 mg/ml) subjected to 72 h treatment with proteinase K at pH 7.4, 37 °C and (D)at pH 4.5, 42 °C. Scale bars are 2 µm in (C) and 200 nm in (D). S10 Supplementary Fig. S6. Effect of S100A9 fibrillar sample on Aβ42 amyloid formation kinetics monitored by thioflavin-T fluorescence assay. Concentrations of Aβ42 and S100A9 samples are indicated in caption.  Supplementary Fig. S7. Representative confocal microscopy image of wild-type mouse neurons expressing S100A9 (shown by red immunofluorescence) induced by addition of Aβ42 oligomers. DAPI nuclei staining shown in blue. S100A9-specific immunofluorescence signal per cell was quantified by using an Imaris (Bitplane) software. Two-dimensional cell images obtained by confocal microscopy were reconstructed by an Imaris into three-dimensional volumetric data sets and the volumetric data, reflecting S100A9, rendered iso-surfaces used for quantification. Scale bar is 10 µm.

Preparation of S100A9 amyloid fibrils
Freeze-dried S100A9 protein was dissolved in PBS buffer at pH 7.4. The solution was filtered through a spin 0.2 µm membrane filter to remove any aggregated species. Fibrils were prepared by incubating S100A9 solution (final concentration of 400 µM) in PBS buffer, pH 7.4, at 50 °C in 500 µl reaction volume on a rotating shaker (300 rpm) during 3 days. Fibril morphology was confirmed by AFM.

Thioflavin-T kinetics assays
Aβ42 (Tocris Bioscience) was freshly dissolved and filtered through a spin 0.2 µm membrane filter to remove aggregated species. 30 µM Aβ42 in PBS buffer, pH 7.4, was incubated alone and in the presence of S100A9 fibrillar samples taken at 6 and 30 µM concentrations in a 96-well plate mixed with 20 µM thioflavin-T at 42°C. The resulting fluorescence was measured each 10 minutes during 13 hours using a fluorescence plate reader (Infinite F200 pro, TECAN) in triplicate samples. Excitation and emission wavelengths were set at 430 nm and 485 nm, respectively.