N1-methylnicotinamide Mediates Multi-Target Cognitive Deficit Relief via AMPK-Lysosome Axis Regulation

Alzheimer's Disease (AD) progresses via interconnected Aβ aggregation, tau hyperphosphorylation, neuroinflammation, and lysosomal failure, while current single-target drugs only relieve cognitive symptoms without halting pathogenesis. Gut microbiota-derived N1-methylnicotinamide (MNAM) is associated with lower AD risk, yet its comprehensive multilevel regulatory mechanism remains unclear. This study systematically elucidated MNAM's neuroprotective network using in vitro neuronal/microglial injury models and two AD-relevant mouse models.

In silico prediction and LC-MS verified MNAM's BBB permeability. TMT proteomics, siRNA knockdown and pharmacological rescue validated the CAB39L-AMPK-lysosomal axis. Immunofluorescence, enzyme assays and molecular docking assessed MNAM's CTSB inhibitory capacity. 16S rRNA sequencing and metabolomics characterized gut microbiota and NAD+ metabolic remodeling.

In vitro, MNAM crossed the blood-brain barrier, improved neuronal viability, reduced ROS and pro-inflammatory cytokines (IL-1β, TNF-α). In AD-like mice, it alleviated cognitive deficits, tau hyperphosphorylation, Aβ deposition, microglial activation, and restored SOD activity. MNAM upregulated CAB39L to activate AMPK, stabilized lysosomes, and restrained CTSB leakage. It reshaped protective intestinal flora, suppressed hepatic NNMT, and restored NAD+ balance. In LPS-challenged mice, MNAM selectively alleviated microglial activation and IL-1β accumulation.

MNAM targets multiple AD pathological nodes (Aβ deposition, tau phosphorylation, neuroinflammation, lysosomal dysfunction, gut microbiota-metabolite interactions) via the CAB39L-AMPK-lysosomal axis and gut microbiota/NAD+ metabolism regulation, which is distinct from single-target drugs. Its ability to cross the BBB and regulate host-microbiota metabolic crosstalk provides a novel mechanistic basis for AD therapy, supporting a shift from single-target to multi-target intervention strategies.

MNAM targets the CAB39L-AMPK-lysosomal axis, inhibits CTSB, and remodels gut microbiota-NAD+ metabolism to concurrently suppress core AD pathologies. This metabolite serves as a promising multi-target disease-modifying candidate for AD, warranting further validation in transgenic AD models and human neurons.