Hypothesis

In aged basal forebrain cholinergic neurons, the decline of calbindin‑D28K does not merely weaken calcium buffering; it actively re‑wires ER‑mitochondrial calcium microdomains to sustain a calcineurin‑NFAT transcriptional program that represses autophagy genes. This active suppression preserves a damaged intracellular state by preventing autophagic clearance, creating a feed‑forward loop of calcium dysregulation and neuronal vulnerability.

Mechanistic reasoning

1. Loss of calbindin shifts calcium flux – Without calbindin’s high‑affinity cytosolic buffering, calcium entering the cytosol via voltage‑gated channels or IP3R channels is less sequestered, raising the probability that it reaches nearby mitochondria through ER‑mitochondrial contact sites (MAMs). Elevated mitochondrial Ca2+ uptake via MCU inhibits autophagy, as shown by increased Beclin‑1 and decreased p62 when MCU is blocked [2].
2. Sustained calcineurin activation – Moderate, prolonged mitochondrial Ca2+ overload activates the phosphatase calcineurin, which dephosphorylates NFAT transcription factors, permitting their nuclear translocation. NFAT can bind promoters of autophagy regulators (e.g., TFEB, LC3) and recruit repressive complexes, lowering autophagy initiation. This links calcium handling directly to transcriptional repression, a step not addressed in the cited works.
3. Feedback via lysosomal stress – Autophagy suppression leads to accumulation of damaged proteins and organelles, which perturb lysosomal Ca2+ release (via TRPML1) and further impair lysosomal acidification, exacerbating the block [2]. Lysosomal dysfunction also amplifies ER calcium leak through IP3R sensitization, closing the loop.

Testable predictions

• Prediction 1: In aged cholinergic neurons, nuclear NFAT levels will be elevated compared with young counterparts, and NFAT inhibition (e.g., with VIVIT peptide) will restore autophagy markers (LC3‑II, p62 decline) without altering calbindin expression.
• Prediction 2: Overexpressing calbindin in aged neurons will reduce mitochondrial Ca2+ uptake (measured with mito‑GCaMP), decrease calcineurin activity, and increase autophagic flux, even when MCU remains active.
• Prediction 3: Disrupting ER‑mitochondrial tethers (e.g., knocking down mitofusin‑2) will blunt the calcium transfer that sustains calcineurin activation, rescuing autophagy despite calbindin loss.
• Prediction 4: Chronic low‑dose calcineurin inhibition in aged mice will ameliorate calcium‑induced hyperexcitability (reduced ER Ca2+ release via RyR) and slow cholinergic neuron degeneration in Alzheimer’s‑model backgrounds.

Experimental approach

• Use transgenic mice with cholinergic‑specific calbindin knockdown or overexpression, combined with AAV‑delivered NFAT reporter (NFAT‑GFP) to quantify nuclear translocation.
• Measure autophagy flux via tandem mCherry‑GFP‑LC3 and lysosomal pH (LysoSensor) in isolated basal forebrain neurons.
• Employ mito‑GCaMP6f and ER‑targeted D1ER sensors to map calcium microdomains at MAMs during spontaneous activity.
• Apply pharmacological tools: VIVIT (calcineurin inhibitor), Ru360 (MCU blocker), Xestospongin B (IP3R antagonist) and assess rescue of autophagy and neuronal survival.
• Correlate findings with histological markers of Alzheimer’s pathology (phospho‑tau, amyloid‑β) in the same animals.

Falsifiability

If nuclear NFAT does not increase in calbindin‑deficient aged cholinergic neurons, or if NFAT inhibition fails to restore autophagy without altering calcium buffering, the hypothesis would be refuted. Likewise, if manipulating ER‑mitochondrial contacts does not affect calcineurin activity or autophagy despite altered calcium fluxes, the proposed mechanistic link would be unsupported.