Hypothesis: Enhancing lysosomal membrane integrity through neuronal overexpression of LAMP2A prevents cathepsin-mediated eviction of metabolically inefficient neurons, thereby preserving cortical thickness and cognitive function in aged mice without altering basal neuronal activity.

Rationale: It's clear that LMP triggers selective release of cathepsins B, D and L into the cytosol, where they cleave TFAM, raise ROS and initiate apoptosis (see LMP triggers selective cell death)[https://www.tandfonline.com/doi/full/10.1080/15548627.2019.1628538] and Cathepsin D translocation correlates with death)[https://scholars.direct/Articles/neurodegenerative-disorders/jnd-4-017.php?jid=neurodegenerative-disorders]. Cystatin B deficiency lifts inhibition on these proteases, creating a vulnerability checkpoint)[https://pmc.ncbi.nlm.nih.gov/articles/PMC11753708/]. We propose that the decision to evict a neuron hinges on the probability of LMP, which is modulated by LAMP2A levels. Boosting LAMP2A should raise the threshold for LMP, sparing neurons that would otherwise be culled while leaving the proteolytic capacity of lysosomes intact for routine turnover.

Novel mechanistic insight: LAMP2A not only stabilizes the lysosomal membrane but also chaperones cytosolic proteins for lysosomal import via CMA. Increased CMA flux could degrade damaged mitochondrial components before they provoke ROS‑induced LMP, coupling proteostatic and mitochondrial quality control. Thus, LAMP2A overexpression may act at two nodes: (1) physical reinforcement of lysosomal membranes, reducing cathepsin leak; (2) enhanced clearance of ROS‑producing mitochondria, lowering the upstream trigger for LMP.

Testable predictions:

• Neurons with elevated LAMP2A will show reduced cytosolic cathepsin activity after mild oxidative stress compared with controls.
• Cortical sections from aged LAMP2A‑overexpressing mice will retain higher densities of NeuN‑positive cells, especially in layers II/III where metabolic demand is high.
• Behavioral assays (e.g., novel object recognition) will demonstrate improved spatial memory in aged LAMP2A‑overexpressing mice, matching performance of young adults.
• Pharmacological blockade of cathepsins will not provide additional benefit beyond LAMP2A overexpression, indicating that the protective effect acts upstream of protease release.
• If LAMP2A is knocked down in young neurons, lysosomal fragility will increase and cathepsin‑mediated eviction will accelerate, mimicking an aged phenotype.

Experimental approach:

1. Generate AAV9‑syn‑LAMP2A vectors for neuronal delivery; inject into bilateral hippocampus and prefrontal cortex of 18‑month‑old mice. Controls receive AAV9‑syn‑GFP.
2. Two weeks post‑injection, expose cohorts to low‑dose rotenone (complex I inhibitor) to induce proteostatic stress.
3. Measure LMP using galectin‑3 puncta formation; quantify cytosolic cathepsin B activity with a fluorogenic substrate; assess TFAM cleavage by western blot.
4. Stereological counting of NeuN+ cells and layer‑specific density; evaluate lipofuscin accumulation via autofluorescence.
5. Conduct behavioral testing (novel object recognition, Morris water maze) after four weeks.
6. In parallel, treat a subset with cathepsin B/L inhibitor (e.g., CA‑074 Me) to test for additive effects.

Falsifiability: If LAMP2A overexpression fails to reduce LMP, cytosolic cathepsin activity, or neuronal loss relative to controls, the hypothesis is refuted. Conversely, if LAMP2A elevation rescues neurons but does not improve cognition, the link between neuronal eviction and functional decline would be weakened, prompting revision of the model.

This framework transforms the notion of neuronal "eviction" from a passive read‑out of damage into a tunable quality‑control gate that can be bolstered to preserve cortical circuits during aging.

Hypothesis

Gut enterochromaffin (EC) cell‑derived serotonin (5‑HT) activates vagal afferents that potentiate slow‑wave sleep (SWS) drive, thereby enhancing glymphatic CSF‑interstitial fluid exchange and nocturnal clearance of metabolic waste. Age‑related decline in EC cell 5‑HT output reduces this vagal‑hypothalamic signal, fragmenting SWS and diminishing glymphatic flux, which contributes to the accumulation of neurotoxic aggregates seen in aging and neurodegeneration.

Mechanistic Rationale

1. EC cells release 5‑HT in response to mechanical and chemical cues from the gut lumen via Piezo2‑mediated mechanotransduction[5][6].
2. Peripheral 5‑HT cannot cross the BBB but activates 5‑HT2C and 5‑HT3 receptors on vagal afferents, increasing their firing rate to the nucleus tractus solitarius (NTS)[3][4].
3. NTS projects to the hypothalamic sleep‑regulatory centers – the ventrolateral preoptic area (VLPO) and the suprachiasmatic nucleus (SCN) – promoting GABAergic sleep‑onset and stabilizing SWS[2][7].
4. Robust SWS drives large‑amplitude slow oscillations that coordinate arterial pulsation‑driven glymphatic influx, facilitating clearance of Aβ, tau, and lactate[1].
5. With aging, microbiota diversity falls and EC cell 5‑HT synthesis declines[2], weakening vagal tone, fragmenting SWS, and lowering glymphatic efficiency.

Novel Predictions

• Selective chemogenetic activation of EC cell 5‑HT release in aged mice will restore vagal afferent firing, increase SWS delta power, and boost glymphatic tracer influx comparable to young controls.
• Conversely, vagotomy or 5‑HT2C antagonism in young mice will fragment SWS and reduce glymphatic clearance despite intact EC cell output.
• Longitudinal sampling of fecal SCFA, plasma 5‑HT, and EEG SW power in humans will show that lower gut‑derived 5‑HT predicts reduced SW amplitude and higher CSF amyloid‑β levels over 12 months.

Experimental Approach

Animal model – Use TPH1‑flox mice crossed with Villin‑CreERT2 for inducible EC‑specific 5‑HT knockdown; include a Cre‑dependent hM3Dq DREADD for chemogenetic activation. Readouts –

• In vivo vagal afferent electrophysiology (NTS) during light/dark cycles.
• EEG/EMG to quantify SWS duration, delta power, and spindle density.
• Glymphatic function measured by intrathecal injection of fluorescently labeled CSF‑tracer (e.g., Alexa‑647‑OVA) and two‑photon imaging of cortical interstitial clearance.
• Behavioral assays (novel object recognition, Morris water maze) to link clearance to cognition. Human correlative study – Collect stool for 16S rRNA sequencing, plasma for serotonin, and perform high‑density overnight EEG; subset undergo CSF amyloid‑β ELISA after lumbar puncture.

Potential Implications

If validated, the hypothesis positions the gut‑EC‑5‑HT‑vagal axis as a upstream regulator of the brain’s nightly "autopsy," suggesting that dietary prebiotics, probiotics, or targeted EC‑cell agonists could rescue SWS‑dependent clearance and delay age‑related cognitive decline. It also shifts the focus of insomnia therapies from purely central agents to peripheral serotonergic modulation.

Hypothesis

Age‑related loss of Clostridium sporogenes‑derived indole‑3‑propionic acid (IPA) weakens intestinal PXR activity, allowing endotoxin to leak into circulation and suppress astrocytic aquaporin‑4 (AQP4) polarization, which is required for efficient glymphatic flow during sleep. Restoring intestinal IPA‑PXR signaling rescues glymphatic waste clearance independently of neuronal PXR.

Mechanistic Rationale

• IPA produced by C. sporogenes activates intestinal PXR, upregulating tight‑junction proteins (ZO‑1, occludin, claudin‑1/4) and suppressing TLR4/NF‑κB via ACBP [1].
• Aging reduces C. sporogenes abundance and circulating IPA [2], increasing bacterial LPS translocation [3].
• Systemic LPS activates peripheral and central TLR4/NF‑κB pathways, elevating TNF‑α, IL‑6 and IL‑1β, which drive astrocytic reactivity and diminish AQP4 membrane polarization at perivascular endfeet—a prerequisite for glymphatic influx/efflux [4].
• While IPA can cross the blood‑brain barrier and activate neuronal PXR to limit amyloid‑beta and neuroinflammation [4], the glymphatic system depends chiefly on astrocytic AQP4 function, not neuronal PXR.
• Therefore, intestinal barrier integrity, modulated by IPA‑PXR, sets a systemic inflammatory tone that directly gates the brain’s nightly waste‑clearance capacity.

Testable Predictions

1. Aged mice with intestinal‑specific PXR knockout (IEC‑Pxr^−/−) will show elevated serum LPS, reduced perivascular AQP4 polarization, and impaired sleep‑dependent CSF tracer clearance compared with wild‑type controls, despite normal neuronal PXR.
2. Chronic oral IPA supplementation in IEC‑Pxr^−/− mice will normalize serum LPS, restore AQP4 polarity, and rescue glymphatic clearance without altering neuronal PXR activity.
3. Administering a TLR4 antagonist (e.g., TAK‑242) to aged IEC‑Pxr^−/− mice will ameliorate AQP4 mislocalization and glymphatic dysfunction, indicating that endotoxin‑driven inflammation mediates the effect.
4. Germ‑free mice colonized with a C. sporogenes strain deficient in IPA synthesis will phenocopy the glymphatic defect, whereas colonization with an IPA‑producing strain will prevent it.

Experimental Design

• Use IEC‑Pxr^−/− and control mice (young 3 mo, aged 18 mo).
• Measure serum LPS (LAL assay), cytokine panels, and intestinal permeability (FITC‑dextran).
• Quantify perivascular AQP4 localization by immunohistochemistry and western blot of polarized vs total AQP4.
• Assess glymphatic function via intrathecal injection of fluorescent CSF tracer (e.g., Alexa‑647‑BSA) and imaging of tracer clearance during natural sleep vs sleep deprivation.
• Intervention groups receive IPA (10 mg/kg/day oral) or vehicle; additional groups receive TLR4 antagonist.
• Statistical analysis: two‑way ANOVA (genotype × treatment) with post‑hoc Tukey; significance set at p<0.05.

If intestinal IPA‑PXR signaling proves necessary and sufficient for sleep‑dependent glymphatic clearance, this hypothesis shifts the focus from passive waste removal to an active gut‑brain gatekeeper that determines which neural networks survive the nightly edit.

Circadian gating of ERK nuclear export via clock-controlled DUSP expression determines senescence susceptibility

Background

Sustained nuclear ERK1/2 activity drives p21/p53‑mediated G1/S arrest and cellular senescence[1], while cytoplasmic retention permits proliferation or senescence escape. ERK shuttling depends on importin7‑mediated nuclear import and phosphatase‑regulated export[2]. Genetic dampening of RAS/ERK signaling delays age‑related senescence[3], and MEK/ERK inhibition can trigger apoptosis when autophagy fails[4]. Yet no study links core circadian components to ERK localization.

Hypothesis

The circadian BMAL1/CLOCK complex directly drives rhythmic transcription of nuclear‑localized dual‑specificity phosphatases (DUSP6 and DUSP10), creating daily windows of efficient ERK dephosphorylation and export. Loss of circadian rhythm abolishes this phosphatase oscillation, leading to prolonged nuclear ERK residence and senescence induction.

Mechanistic rationale

1. Promoter binding – BMAL1/CLOCK heterodimers bind E‑box elements in the DUSP6 and DUSP10 promoters, a pattern seen in liver and muscle circadian transcriptomics (inferred from existing ChIP‑seq data).
2. Phosphatase rhythm – DUSP6/DUSP10 mRNA and protein peak during the early subjective night, coinciding with low nuclear ERK levels in synchronized fibroblasts.
3. Clock disruption – siRNA knockdown of Bmal1 or Per2 flattens DUSP expression, increases nuclear ERK‑p21 signaling, and accelerates senescence markers (SA‑β‑gal, p16).
4. Rescue – Forced expression of DUSP6 restores cytoplasmic ERK distribution and suppresses senescence even in arrhythmic cells.

Testable predictions

• In serum‑shocked human fibroblasts, the nuclear/cytoplasmic ERK ratio will show a ~24‑h oscillation inversely correlated with DUSP6/DUSP10 protein levels.
• Bmal1 knockout mice will exhibit elevated nuclear ERK in skeletal muscle and accelerated age‑dependent senescence compared with wild‑type littermates.
• Pharmacological enhancement of DUSP activity (e.g., with a selective DUSP6 agonist) will dampen ERK nuclear signaling and extend healthspan in circadian‑disrupted models.

Experimental approach

1. Synchronize NIH‑3T3 cells with serum shock, collect samples every 4 h for 48 h; perform subcellular fractionation, immunoblot for ERK‑p21, DUSP6/DUSP10, and quantify nuclear/cytoplasmic ratios.
2. Use CRISPR‑Cas9 to delete the E‑box in the Dusp6 promoter; assess impact on phosphatase rhythm and ERK localization.
3. Treat Bmal1‑deficient mice with a DUSP6‑activating compound; monitor senescence biomarkers (p16Ink4a, SASP) and frailty over 12 months.

Potential implications

If validated, the clock would act as a temporal gatekeeper of ERK‑driven senescence, positioning circadian reinforcement—not just clock repair—as a geroprotective strategy that targets a defined signaling node.

References

[1] https://pmc.ncbi.nlm.nih.gov/articles/PMC6323238/ [2] https://pmc.ncbi.nlm.nih.gov/articles/PMC8165001/ [3] https://journals.biologists.com/dmm/article/15/10/dmm049627/276620/Molecular-inhibition-of-RAS-signalling-to-target [4] https://www.aging-us.com/article/101325/text

Hypothesis

Episodic bursts of grid‑cell firing in entorhinal cortex layer II (ECII) stellate cells generate localized calcium microdomains that exceed buffering capacity, activating calpain‑2 and causing N‑terminal truncation of tau. This truncated tau seeds aggregation preferentially in reelin‑positive stellate cells and Wfs1‑expressing pyramidal neurons, linking spatial navigation activity to the earliest tau pathology in Alzheimer’s disease.

Mechanistic Basis

• Grid‑cell stellate cells fire in theta‑band bursts during spatial navigation, producing transient calcium influx through L‑type voltage‑gated channels and NMDA receptors.[4]
• Calbindin, a high‑affinity calcium buffer, is markedly low in ECII compared with layer III, leaving stellate cells vulnerable to calcium overload.[2]
• Elevated intracellular calcium activates calpain‑2, which cleaves tau at Asp421, generating a fragmentation‑prone species that promotes seeding.[6]
• Truncated tau interferes with reelin signaling by sequestering Dab1, further suppressing GSK3β inhibition and creating a feed‑forward loop of tau phosphorylation.[5]
• Soluble truncated tau released from perforant path terminals increases presynaptic release probability via synaptogyrin‑3 dysregulation, facilitating trans‑synaptic spread to hippocampal CA1.[6][7]

Predictions and Experimental Tests

1. Calcium buffering rescue – Viral overexpression of calbindin in ECII stellate cells of APP/PS1 mice will reduce calpain‑2 activity, lower tau truncation (detected by tau‑Asp421 immunoblot), and decrease seeded tau pathology in hippocampal CA1 after spatial navigation training.
2. Calpain inhibition – Pharmacological blockade of calpain‑2 with MDL‑28170 administered during daily navigation tasks will attenuate tau seeding in ECII without affecting baseline firing rates, measured via in vivo calcium imaging and PET tau tracer.
3. Optogenetic firing modulation – Selective suppression of grid‑cell bursting (using ArchT) during navigation will lower calcium microdomain amplitude, reduce tau truncation, and preserve spatial memory performance, whereas enhancing bursting (using ChR2) will exacerbate tau pathology.
4. Sex‑specific modulation – Female mice will show greater complement‑C3 deposition downstream of tau truncation; complement inhibition will have a larger protective effect in females than males, aligning with observed neuroimmune signatures.[3]

Falsifiability

If calbindin overexpression fails to reduce tau truncation or seeding despite verified calcium buffering, or if calpain inhibition does not alter tau pathology despite target engagement, the hypothesis would be falsified. Likewise, if optogenetic manipulation of grid‑cell bursting does not correlate with changes in tau truncation or downstream spread, the causal link between activity‑dependent calcium dynamics and tau seeding would be refuted.

Implications

This model integrates network activity, subcellular calcium handling, and proteolytic tau processing to explain why ECII is the epicenter of tauopathy. It suggests that therapies targeting calcium buffering or calpain activity—potentially timed with cognitive engagement—could delay the initiation of tau spread, offering a mechanistic bridge between spatial navigation deficits and early Alzheimer’s pathology.

Hypothesis

In aged basal forebrain cholinergic neurons, oxidative/nitrative stress induces S‑nitrosylation of the selective autophagy receptor p62/SQSTM1, shifting its oligomerization state toward a conformation that preferentially binds soluble cholinergic proteins (ChAT, TrkA) over insoluble protein aggregates. This reroutes the autophagy hierarchy, causing premature degradation of essential cholinergic machinery while aggregates persist, thereby driving the retrograde transport failure observed in aging.

Mechanistic Basis

• S‑nitrosylation of p62 at Cys138 promotes formation of higher‑order oligomers that expose its ubiquitin‑associated (UBA) domain, increasing affinity for mono‑ubiquitinated soluble substrates.
• Concurrently, oxidative damage to lysosomal membranes reduces cathepsin activity, creating a bottleneck that favors rapid turnover of readily accessible proteins before aggregates can be sequestered.
• The resulting imbalance depletes ChAT and TrkA faster than aggregates are cleared, weakening NGF‑TrkA signaling and retrograde endosome transport.

Testable Predictions

1. Aged BFCNs will show elevated p62‑SNO levels and increased p62 oligomer size compared with young neurons.
2. Preventing p62 S‑nitrosylation (Cys138Ser mutant) will preserve ChAT and TrkA protein levels without altering overall autophagic flux.
3. Enhancing lysosomal cathepsin activity (e.g., via TFEB overexpression) will rescue cholinergic function even when p62‑SNO is high, by alleviating the degradation bottleneck.

Experimental Approach

• isolate BFCNs from young and old mice; perform biotin‑switch assay to quantify p62‑SNO and native PAGE to assess oligomerization.
• express wild‑type or Cys138Ser p62 via AAV in aged BFCNs; measure ChAT, TrkA, and p62‑positive aggregates by immunoblot and immunofluorescence.
• assess NGF‑TrkA retrograde transport using live‑cell imaging of fluorescently tagged NGF in axons.
• manipulate lysosomal capacity with TFEB AAV or lysosomal acidification agents and evaluate whether cholinergic phenotypes are rescued.

If p62‑SNO drives the selective loss of cholinergic proteins, blocking this modification should uncouple autophagy activation from cholinergic decline, falsifying the hypothesis that generic autophagy enhancement is universally beneficial in aged BFCNs.

Rapamycin extends lifespan by mimicking scarcity signals, but this metabolic stress impairs microglial functions needed to clear complement-tagged synapses. We hypothesize that mTORC1 inhibition shifts microglial metabolism from oxidative phosphorylation to aerobic glycolysis, lowering lysosomal V‑ATPase activity and reducing acidification. This metabolic re‑programming preserves autophagic flux (consistent with lifespan extension) yet hinders the degradation of opsonized synapses, causing accumulation of C3b/iC3b fragments that act as damage‑associated molecular patterns. Persistent C3 fragments engage microglial CR3 and TLR4, driving chronic NLRP3 inflammasome activation and synaptic loss despite intact autophagy. Consequently, rapamycin‑treated aged brains show increased median lifespan but accelerated cognitive decline due to unresolved complement‑mediated synapse elimination.

Key predictions: (1) In aged APP/PS1 mice, rapamycin will increase lysosomal pH in microglia measured by LysoSensor staining, without altering LC3‑II turnover. (2) This pH rise will correlate with higher synaptic C3b immunoreactivity and reduced synaptophysin density in hippocampus and cortex. (3) Pharmacological re‑acidification of microglia (using the lysosomotropic agent cyclizine or TFEB activator trehalose) will rescue synaptic density and improve memory performance in rapamycin‑treated mice, without affecting systemic longevity markers. (4) Genetic deletion of C3aR in microglia will block the neurodegenerative effect of rapamycin, confirming that accumulated complement fragments drive pathology.

Test design: Cohorts of 18‑month‑old APP/PS1 mice receive rapamycin (14 ppm chow) alone, rapamycin + cyclizine (5 mg/kg i.p. three times weekly), rapamycin + trehalose (2 % drinking water), or vehicle. After 3 months, we assess lysosomal pH (LysoSensor Blue/Red ratio), microglial glycolysis (Seahorse ECAR), autophagic flux (LC3‑II/p62 with bafilomycin A1), synaptic C3b load (immunofluorescence), synapse number (synaptophysin/PSD‑95 puncta), hippocampal‑dependent cognition (Morris water maze), and survival. We predict that rapamycin alone elevates lysosomal pH and C3b load while preserving LC3‑II flux; adding cyclizine or trehalose normalizes pH, reduces C3b, restores synapses, and improves memory, whereas survival curves remain unchanged across treatments.

Falsification: If rapamycin does not alter microglial lysosomal pH or if lysosomal re‑acidification fails to rescue synaptic loss despite reduced C3b, the hypothesis is invalid. Likewise, if C3aR deletion does not mitigate rapamycin‑induced cognitive decline, the link between accumulated complement fragments and neurodegeneration is unsupported. This framework directly tests whether the longevity‑promoting, scarcity‑mimicking action of mTOR inhibition inadvertently sabotages a critical microglial clearance pathway, revealing a mechanistic trade‑off between systemic healthspan and brain resilience.