α7 Nicotinic acetylcholine receptor-specific antibody induces inflammation and amyloid β42 accumulation in the mouse brain to impair memory.

Nicotinic acetylcholine receptors (nAChRs) expressed in the brain are involved in regulating cognitive functions, as well as inflammatory reactions. Their density is decreased upon Alzheimer disease accompanied by accumulation of β-amyloid (Aβ42), memory deficit and neuroinflammation. Previously we found that α7 nAChR-specific antibody induced pro-inflammatory interleukin-6 production in U373 glioblastoma cells and that such antibodies were present in the blood of humans. We raised a hypothesis that α7 nAChR-specific antibody can cause neuroinflammation when penetrating the brain. To test this, C57Bl/6 mice were either immunized with extracellular domain of α7 nAChR subunit α7(1-208) or injected with bacterial lipopolysaccharide (LPS) for 5 months. We studied their behavior and the presence of α3, α4, α7, β2 and β4 nAChR subunits, Aβ40 and Aβ42 and activated astrocytes in the brain by sandwich ELISA and confocal microscopy. It was found that either LPS injections or immunizations with α7(1-208) resulted in region-specific decrease of α7 and α4β2 and increase of α3β4 nAChRs, accumulation of Aβ42 and activated astrocytes in the brain of mice and worsening of their episodic memory. Intravenously transferred α7 nAChR-specific-antibodies penetrated the brain parenchyma of mice pre-injected with LPS. Our data demonstrate that (1) neuroinflammation is sufficient to provoke the decrease of α7 and α4β2 nAChRs, Aβ42 accumulation and memory impairment in mice and (2) α7(1-208) nAChR-specific antibodies can cause inflammation within the brain resulting in the symptoms typical for Alzheimer disease

The level of α7, β2 or β4 nAChR subunits in the brain sections of experimental mice studied by immunohistochemistry.

<p>Confocal microscopy images of the hippocampus CA1 and striatum of non-treated (Ctrl), α7(1–208)-immunized or LPS-injected mice stained with biotinylated α7-, β2- or β4-specific antibodies and developed with Extravidin-Cy3 (<i>red</i>). Cell nuclei are stained with DAPI (<i>blue</i>). Bar corresponds to 50μm, actual for each fragment of the panel.</p

The GFAP-positive astrocytes (A) and the number of nucleated cells (B) in the brain sections of experimental mice studied by immunohistochemistry.

<p><b>A</b>—Confocal microscopy images of the hippocampus CA1 (Hip), motor/somatosensory cortex (Crtx) or striatum (Str) of non-treated (Ctrl), α7(1–208)-immunized or LPS-injected mice stained with rabbit GFAP-specific antibody and developed with anti-rabbit Alexa 488 (<i>green</i>). Cell nuclei are stained with DAPI (<i>blue</i>). Bar corresponds to 50μm, actual for each fragment of the panel. <b>B</b>—The number of nucleated cells (DAPI-positive) in corresponding brain regions studied in all available sections (12 to 16 for each treatment for each region); *—p<0.05; **—p< 0.005.</p

The level of nAChR-specific antibodies in the blood and of nAChR subtypes in the brain of experimental mice studied by ELISA (A) or Sandwich ELISA (B).

<p><b>A</b>—7(1–208)-specific antibodies in the blood sera (1:50) of mice immunized with 7(1–208) (n = 8) or injected with LPS (n = 5) for 5 months compared to non-treated (Ctrl, n = 9) and adjuvant-“immunized” animals (CFA, n = 5). <b>B</b>—3, 4, 7, β2 and β4 nAChR subunits in the brain detergent lysates of the same groups of mice (4 mice from each group). <b>C</b>—Pearson coefficients (R) of correlation between the levels of nAChR subunits in the brain and those of 7(1–208)-specific antibodies in the blood. The columns correspond to M±SE, *—p<0.05; **—p<0.005; ***—p<0.0005 compared to Ctrl.</p

The levels of different Aβ isoforms in the brain detergent lysates of experimental mice studied by Sandwich ELISA.

<p><b>A</b>—total Aβ₄₀ and Aβ₄₂, <b>B</b>—Aβ₄₀ and Aβ₄₂ bound to α7 nAChR in mice immunized with 7(1–208) (n = 8) or injected with LPS (n = 5) compared to non-treated animals (Ctrl, n = 9) and to those “immunized” with complete Freund’s adjuvant (CFA, n = 5). The columns correspond to M±SE (n = 5); *—p<0.05; **—p<0.005; ***—p<0.0005 compared to Ctrl.</p

Visualization of biotinylated antibody within the brain at different periods after injection.

<p>Confocal microscopy images of the striatum of mice injected with α7(1–208)-specific antibody (A-B) or non-specific IgG (C) in 15 min (A) or 3h (B-C) after injection. Antibodies were developed with Extravidin-Cy3 (<i>red</i>). Bar corresponds to 100μm, actual for each fragment of the panel. D—Antibody-specific ELISA signal of the primary brain supernatants and detergent lysates of mice either pre-treated or not with LPS; the brains were removed in 15 min or 3 h after the antibody injection. Each column corresponds to mean±SE of three repeats in ELISA; *—p<0.05; **—p<0.005 compared to the data of LPS non-treated mice.</p

The level of Aβ₄₀ and Aβ₄₂ in the brain sections of experimental mice studied by immunohistochemistry.

<p>Confocal microscopy images of the hippocampus CA1 or motor/somatosensory cortex of non-treated (Ctrl), α7(1–208)-immunized or LPS-injected mice stained with biotinylated Aβ₄₀- or Aβ₄₂-specific antibodies and developed with Extravidin-Cy3 (<i>red</i>) and with α7-specific antibody developed with anti-rabbit Alexa 488 (<i>green</i>). Bar corresponds to 50μm, actual for each fragment of the panel.</p

Episodic memory of experimental mice studied in the “Novel Object Recognition” test.

<p>Discrimination indexes calculated for mice immunized with 7(1–208) (n = 8) or injected with LPS (n = 5) compared to non-treated animals (n = 9) or those “immunized” with complete Freund’s adjuvant (CFA, n = 5). ***—p<0.0005 compared to non-treated mice (Ctrl).</p