Loss of muscarinic M1 receptor exacerbates Alzheimer's disease-like pathology and cognitive decline

Abstract

Alzheimer's disease (AD) is pathologically characterized by tau-laden neurofibrillary tangles and β-amyloid deposits. Dysregulation of cholinergic neurotransmission has been implicated in AD pathogenesis, contributing to the associated memory impairments; yet, the exact mechanisms remain to be defined. Activating the muscarinic acetylcholine M(1) receptors (M(1)Rs) reduces AD-like pathological features and enhances cognition in AD transgenic models. To elucidate the molecular mechanisms by which M(1)Rs affect AD pathophysiological features, we crossed the 3xTgAD and transgenic mice expressing human Swedish, Dutch, and Iowa triple-mutant amyloid precursor protein (Tg-SwDI), two widely used animal models, with the M(1)R(-/-) mice. Our data show that M(1)R deletion in the 3xTgAD and Tg-SwDI mice exacerbates the cognitive impairment through mechanisms dependent on the transcriptional dysregulation of genes required for memory and through acceleration of AD-related synaptotoxicity. Ablating the M(1)R increased plaque and tangle levels in the brains of 3xTgAD mice and elevated cerebrovascular deposition of fibrillar Aβ in Tg-SwDI mice. Notably, tau hyperphosphorylation and potentiation of amyloidogenic processing in the mice with AD lacking M(1)R were attributed to changes in the glycogen synthase kinase 3β and protein kinase C activities. Finally, deleting the M(1)R increased the astrocytic and microglial response associated with Aβ plaques. Our data highlight the significant role that disrupting the M(1)R plays in exacerbating AD-related cognitive decline and pathological features and provide critical preclinical evidence to justify further development and evaluation of selective M(1)R agonists for treating AD.

Figure 1

Genetic ablation of M₁R disrupts cognitive function. Eighteen-month-old 3xTgAD mice were tested on a spatial reference version of the Morris water maze (A and B), object recognition (C), social recognition (D), contextual fear conditioning (E), and open field (F) tasks. The cognitive performance of M₁R^−/− and 3xTgAD-M₁R^−/− mice was significantly impaired when compared with their respective nTg and 3xTgAD littermates. The values represent the mean ± SEM (N = 12 to 15). *P < 0.05 and **P < 0.01 compared with nTg mice; ^††P < 0.01 compared with 3xTg-AD mice. TQ indicates target quadrant.

Figure 2

M₁R genetic ablation dysregulates memory-related intracellular signaling but not expression of synaptic-related protein. A: Total c-Fos and phosphorylated CREB levels were reduced in the brains of 18-month-old M₁R^−/− and 3xTgAD-M₁R^−/− mice when compared with their respective littermates. B: Similarly, these mice presented disrupted PKA activity. Although 3xTgAD mice show reduced expression of synaptic-associated proteins when compared with nTg mice, no changes were found in the levels of PSD-95 (A) and synaptophysin (C) in mice with an M₁R deletion. D: Quantification of c-Fos, phosphorylated CREB, PSD-95, and synaptophysin levels. The values represent the mean ± SEM (N = 4 to 6). *P < 0.05 and **P < 0.01 compared with nTg mice; ^†P < 0.05 and ^††P < 0.01 compared with 3xTgAD mice. Scale bar = 25 μm (C). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PSD-95, postsynaptic density protein 95.

Figure 3

Deletion of M₁R accelerates the parenchymal and vascular Aβ deposition through up-regulation of amyloidogenic APP processing and down-regulation of PKC activity in transgenic mouse models of AD. Eighteen-month-old 3xTgAD-M₁R^−/− mice have higher Aβ₄₀ and Aβ₄₂ levels (A), 6E10 immunoreactivity (B), and thioflavin S–positive plaques (C and D) versus 3xTgAD mice. C: The pictures were taken in the subiculum of the hippocampus. E and F: Immunofluorescence for collagen IV and 6E10 indicates increased vascular Aβ deposition in 9-month-old Tg-SwDI–M₁R^−/− mice when compared with their littermates. G: Quantification of Aβ accumulation in the brain vasculature of Tg-SwDI and Tg-SwDI–M₁R^−/− mice. H and I: No changes were found in the expression of human APP transgene, αAPP fragment C83, ADAM10, ADAM17, or BACE1 in the brains of 18-month-old 3xTgAD-M₁R^−/− mice when compared with 3xTgAD mice. However, 3xTgAD-M₁R^−/− mice show increased βAPP fragment C99 levels. J: PKC activity is disrupted in the 3xTgAD-M₁R^−/− mouse brains. The values represent the mean ± SEM (N = 4 to 8). ^†P < 0.05 and ^††P < 0.01 compared with 3xTgAD mice. Scale bars: 100 μm (B and C); 50 μm (E and F).

Figure 4

M₁R genetic ablation exacerbates τ pathological features in 3xTgAD mice via GSK3β activation. A: Increased somatodendritic phosphorylated τ (S212/T214) deposits in 3xTgAD-M₁R^−/− mice when compared with 3xTgAD mice. IHC was performed with the AT100 antibody. Scale bar = 100 μm. B and C: Deletion of M₁R is also associated with higher levels of phosphorylated τ at residues S199/S202/T205 (AT8) and T181 (AT270) but not at residues S396/S404 (PHF-1). The total τ level was changed in 3xTgAD-M₁R^−/− mice when compared with 3xTgAD mice, as indicated by the HT7 immunoblot. D and E: No changes were found in the levels of CDK5, p35/p25, GSK3αβ, and PP2A. However, 3xTgAD-M₁R^−/− mice present significantly higher levels of activated GSK3β when compared with their littermates, as indicated by the reduction in the phosphorylated GSK3β (S9). The values represent the mean ± SEM (N = 5 to 8). ^†P < 0.05 compared with 3xTgAD mice. GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

M₁R genetic ablation enhances the inflammatory response in 3xTgAD mice. The number of GFAP⁺ (A and B) and CD45⁺ (A and C) cells was significantly higher in 3xTgAD-M₁R^−/− mice when compared with 3xTgAD mice. Colocalization of astrocytes (GFAP⁺ cells) (D) and microglia (Iba-1–positive cells) (E) with Aβ deposits (6E10) in the brains of 3xTgAD and 3xTgAD-M₁R^−/− mice. The values represent the mean ± SEM (N = 5). **P < 0.01 compared with nTg mice; ^††P < 0.01 compared with 3xTgAD mice. Scale bar = 100 μm (A, D, and E).