Fecal ATX(N) profiling reveals early genotype-associated molecular signatures in APP/PS1 mice

Reliable and minimally invasive approaches for studying Alzheimer’s disease (AD)-associated molecular changes are needed. Fecal material represents an accessible biological matrix that may contain molecular information associated with systemic or gastrointestinal processes linked to disease-related phenotypes. Here, we investigated whether ATX(N)-related immunoreactivities can be detected in fecal extracts from APP/PS1 mice and whether their levels differ from wild-type (WT) controls at age-defined stages of amyloid-driven pathology.

Fecal samples were collected from independent cohorts of WT and APP/PS1 mice at 1 and 6 months of age and analyzed using semi-quantitative Western blot and dot blot assays targeting amyloid-β, total Tau, phosphorylated Tau, neurodegeneration-associated, synaptic, and inflammatory markers. Exploratory ROC analyses were performed to assess discrimination between APP/PS1 and WT mice. Pre-analytical stability was evaluated under prolonged storage and repeated freeze–thaw conditions.

Cerebral amyloid and plaque-associated Tau pathology were characterized histologically at 3, 6, and 9 months. Aβ-, Tau-, phosphorylated Tau-, NfL-, synaptic-, and inflammation-associated immunoreactivities were detectable in fecal extracts. At 1 month, APP/PS1 mice showed increased fecal levels of several Aβ and phosphorylated Tau immunoreactivities, as well as NfL and synaptophysin, compared with WT mice.

Exploratory ROC analyses indicated discriminatory capacity for several markers at 1 month, including Aβ42 and pTauThr181, whereas Iba1 showed the highest AUC estimate at 6 months. Selected fecal immunoreactivities remained relatively stable under the tested storage and freeze–thaw conditions. These findings support the feasibility of detecting multiple ATX(N)-related immunoreactivities in fecal extracts from APP/PS1 mice and identify genotype-associated molecular differences at early and later stages of amyloid-driven pathology.

The authors thank the GIN animal facility and the GIN imaging core facility for their excellent technical support. Moreaud (Service de Neurologie – Centre Mémoire de Ressources et de Recherche (CMRR) at Grenoble Alpes University Hospital, CHUGA) for coordinating participant recruitment and fecal sample collection for the FECALZ clinical study. Le Gouellec for providing access to the Lumipulse platform and for technical support.

Finally, we sincerely thank all volunteers and participants who contributed to this study. This work was supported by INSERM, University Grenoble Alpes and by the French National Research Agency in the framework of the "Investissements d’avenir” program (ANR-15-IDEX-02). MS received a fourth-year doctoral fellowship from France Alzheimer.