Selective cholinergic denervation, independent from oxidative stress, in a mouse model of Alzheimer’s disease

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Abstract

Alzheimer’s disease (AD) is characterized by increases in amyloid-β (Aβ) peptides, neurofibrillary tangles, oxidative stress and cholinergic deficits. However, the selectivity of these deficits and their relation with the pathology or oxidative stress remain unclear. We therefore investigated amyloidosis-related changes in acetylcholine (ACh) and serotonin (5-HT) innervations of hippocampus and parietal cortex by quantitative choline acetyltransferase (ChAT) and 5-HT immunocytochemistry, in 6, 12/14 and 18 month-old transgenic mice carrying familial AD-linked mutations (hAPPSw,Ind). Further, using manganese superoxide dismutase (MnSOD) and nitrotyrosine immunoreactivity as markers, we evaluated the relationship between oxidative stress and the ACh deficit in 18 month-old mice. Thioflavin-positive Aβ plaques were seen in both regions at all ages; they were more numerous in hippocampus and increased in number (>15-fold) and size as a function of age. A majority of plaques exhibited or were surrounded by increased MnSOD immunoreactivity, and dystrophic ACh or 5-HT axons were seen in their immediate vicinity. Counts of immunoreactive axon varicosities revealed significant decreases in ACh innervation, with a sparing of the 5-HT, even in aged mice. First apparent in hippocampus, the loss of ACh terminals was in the order of 20% at 12/14 months, and not significantly greater (26%) at 18 months. In parietal cortex, the ACh denervation was significant at 18 months only, averaging 24% across the different layers. Despite increased perivascular MnSOD immunoreactivity, there was no evidence of dystrophic ACh varicosities or their accentuated loss in the perivascular area. Moreover, there was virtually no sign of tyrosine nitration in ChAT nerve terminals or neuronal cell bodies. These data suggest that aggregated Aβ exerts an early, non-selective and focal neurotoxic effect on both ACh and 5-HT axons, but that a selective, plaque- and oxidative stress-independent diffuse cholinotoxicity, most likely caused by soluble Aβ assemblies, is responsible for the hippocampal and cortical ACh denervation.

Section snippets

Animals

Experiments were approved by the Animal Ethics Committee of the respective institutions, and abided by the guidelines of the Canadian or American Council for Animal Care aimed at minimizing the number of animals used and their suffering. We used hAPP transgenic mice overexpressing the FAD-linked mutations hAPPSw,Ind carrying the 670/671KM→NL (Sw) double mutation and the 717V→F mutation (Ind; line J20; Mucke et al., 2000). Mice were screened for transgene expression by Touchdown PCR with

ACh and 5-HT innervations in wild-type mouse hippocampus and parietal cortex

In keeping with earlier reports in mouse (Wong et al 1999, Aznavour et al 2002), fine varicose ChAT-immunostained fibers pervaded all layers of the dorsal hippocampus and parietal cortex, as illustrated here in an 18 month-old wild-type mouse (Fig. 2A, C). ChAT-positive cell bodies were rare in hippocampus (not shown), and the overall regional density of the ACh innervation was comparable (15–20 m of axons per mm3 of tissue) between CA1, CA3 and DG (Fig. 2A; see Fig. 4, Fig. 5, Fig. 6). In

Early and non-selective neurotoxicity of Aβ plaques in the hAPPSw,Ind mouse

A first finding of this study was that neuritic plaques, as soon as they appeared, were locally neurotoxic to both ACh and 5-HT axons in hAPPSw,Ind mouse brain. This was evidenced by the presence of swollen and distorted ChAT- and 5-HT-immunostained axons in the vicinity or within the core of plaques in 6 month-old mice, an age at which the plaques were still rare and minuscule, and when there was no measurable denervation. This clearly indicates that aggregated Aβ species exert a local,

Conclusions

Our study shows that the plaque-associated, non-selective neurodegeneration and the diffuse, selective ACh denervation detected here in hAPPSw,Ind transgenic mice are independent events, most likely secondary to increased levels of aggregated and soluble forms of Aβ peptides, respectively. Our results also indicate that oxidative stress is not a primary factor in the diffuse ACh deficit. In view of the selectivity and regional progression of this ACh denervation in hippocampus, and later

Acknowledgments

We thank Dr. L. Mucke (Gladstone Inst of Neurological Disease and Dept of Neurology, UCSF, CA, USA) for the hAPPSw,Ind transgenic mouse breeders, and Drs. C. Cozzari (Rome, Italy) and B. K. Hartman (Minneapolis, MN, USA) for their generous gift of monoclonal anti-ChAT antibody. We are also grateful to Mrs. N. Serluca and A. Yanes for technical support, Drs. T. Yeo and F. Longo (VA San Francisco Medical Center and Dept of Neurology, UCSF) for help with the ChAT assay, and Ms L. Michel for

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