Hypothesis

The selective autophagy hierarchy in neurons is not static; it is driven by temporally ordered pulses of gut‑derived metabolites that act as checkpoint signals. When the pulse sequence is disrupted—by altered timing, amplitude, or ratio of SCFAs versus tryptophan derivatives—neurons mis‑prioritize organelle clearance, leading to premature accumulation of damaged mitochondria or protein aggregates and accelerating age‑related decline.

Mechanistic Basis

1. Metabolite pulse encoding – SCFAs (acetate, propionate, butyrate) peak during fasting phases and activate AMPK, biasing autophagy toward mitophagy and lipophagy. Tryptophan‑derived indoles rise after feeding and stimulate mTORC1 inhibition via TFEB nuclear translocation, favoring aggrephagy and synucleinphagy. The natural circadian feeding‑fasting cycle creates a repeating SCFA‑tryptophan pattern that neurons interpret as a "what to eat first" signal.
2. Checkpoint integration – Neuronal AMPK and mTORC1 converge on the ULK1 complex, which assigns cargo receptors (e.g., BNIP3 for mitochondria, p62/SQSTM1 for aggregates) based on the prevailing metabolite signal. This creates a hierarchy: SCFA‑high → mitophagy first; tryptophan‑high → aggrephagy second.
3. Failure mode – Chronic low‑SCFA/high‑tryptophan (as seen in Western diets) keeps mTORC1 partially active, suppressing the early mitophagy window. Mitochondria escape clearance, ROS rises, and secondary damage overwhelms later aggrephagy capacity. Conversely, intermittent SCFA spikes without tryptophan dampen aggrephagy, allowing α‑synuclein or amyloid‑β to accumulate.

Testable Predictions

• Disrupting the temporal order of metabolite administration (e.g., giving SCFAs during the fed state) will invert the autophagy substrate preference in cultured neurons, measurable by increased mitochondrial mass and decreased p62/SQSTM1 puncta.
• Mice fed a diet that flattens the SCFA‑tryptophan circadian amplitude will show earlier onset of mitochondrial ROS markers and later onset of aggregate pathology compared with controls on a matched‑calorie diet preserving the natural pulse.
• Restoring the pulse pattern via timed prebiotic/probiotic supplementation will rescue both mitophagy and aggrephagy fluxes and extend healthspan in aged mice, even if total metabolite exposure remains unchanged.

Experimental Design

1. In vitro – Differentiate human iPSC‑derived neurons; treat with SCFA or indole pulses in either physiological order (SCFA→indole) or reversed order. Measure mitophagy (mt‑Keima assay) and aggrephagy (GFP‑LC3‑α‑synuclein clearance) at 4‑h intervals.
2. In vivo – Use C57BL/6J mice assigned to three groups: (a) control chow with natural feeding rhythm, (b) chow supplemented with butyrate in the drinking water continuously (flattened SCFA pulse), (c) chow with timed butyrate bolus only during the dark phase (restored pulse). Monitor fecal metabolites, neuronal mitophagy (TOM20‑LC3 colocalization), hippocampal p62 accumulation, and behavioral aging markers over 12 months.
3. Readouts – Western blot for phospho‑AMPK, phospho‑mTOR, LC3‑II/I, p62; immunofluorescence for mitochondrial mass (MitoTracker) and aggregate load (ThioS); fecal SCFA and indole quantification via GC‑MS.

If the hypothesis holds, reversing the metabolite pulse will selectively impair the corresponding autophagy subtype, and reinstating the natural pulse will rescue both, confirming that timing—not mere presence—of gut signals encodes the autophagy hierarchy.