pd-vatpase-lysosomal-compensation-r1

Confirmatory replication of a self-generated OmiCairn finding. In a prior OmiCairn run on LRRK2 G2019S isogenic iPSC-derived dopaminergic (iPSC-DA) neurons (NanoString Human Neuropathology panel), nine CLEAR-network V-ATPase subunit genes — ATP6V0C, ATP6V0D1, ATP6V0E1, ATP6V0E2, ATP6V1A, ATP6V1D, ATP6V1E1, ATP6V1G2, ATP6V1H — were PREDOMINANTLY UPregulated in LRRK2 G2019S vs isogenic corrected controls (8/9 up at the donor level, mean log2FC = +0.485), and the direction was not comparison-specific (also +0.452, 7/9 up vs an independent healthy control). We interpret this as a COMPENSATORY transcriptional response: failing lysosomal acidification in PD-genotype DA neurons drives TFEB/CLEAR-network upregulation of the V-ATPase proton pump. The original cohort was small (NanoString, donor-level permutation power-limited), so the direction is a SUPPORTED but under-powered lab-KB hypothesis, not an established fact.

This run PRE-REGISTERS THE OBSERVED DIRECTION (V-ATPase UP) and tests it in INDEPENDENT cohorts and modalities. Cohorts: (1) GSE167335 — the LRRK2 G2019S isogenic iPSC-DA NanoString panel (independent replication of the exact original modality, where the panel covers the V-ATPase subunits it measures); (2) ArrayExpress E-MTAB-7303 (Lang

2020) — iPSC-DA neurons from GBA-N370S PD patients vs controls vs idiopathic PD, bulk + single-cell RNA-seq, the GBA1-mutant arm; and

(3) Smajić GSE157783 — postmortem human substantia-nigra/midbrain snRNA-seq (idiopathic PD vs control), the postmortem in-vivo arm and the blind held-out validation cohort. This pre-registers a clear honesty guard: we are testing — NOT assuming — that the compensatory UP-direction replicates; a clean reversal or null in independent data is the headline.

Hypotheses (pre-registered — directions are TESTED, not assumed):

H1 (primary — independent LRRK2 replication of V-ATPase UP): In GSE167335 (LRRK2 G2019S isogenic iPSC-DA), the 9 measured CLEAR-network V-ATPase subunits are UPregulated in LRRK2 G2019S relative to isogenic corrected controls, replicating the prior observed +0.485 mean log2FC direction. Decision rule: donor-level (NOT replicate-level — collapse differentiation replicates to donor means to avoid pseudo-replication, exactly as the prior finding required) mean log2FC across the V-ATPase set, with a donor-label permutation test (report the minimum achievable permutation p given n) + per-gene 95% bootstrap CI. H1 REPLICATES if the mean direction is positive AND ≥6/9 subunits are individually up with the donor-level permutation p at its achievable floor. A reversal (net down) or a null is reported verbatim as failure-to-replicate.

H2 (secondary — GBA1-mutant direction in an independent modality): The compensatory V-ATPase UP-direction generalises from LRRK2 to the OTHER major lysosomal-membrane PD gene, GBA1. In E-MTAB-7303 (GBA-N370S iPSC-DA vs control), the V-ATPase / CLEAR-network module is UPregulated in GBA-N370S DA neurons. Decision rule: per-sample pseudobulk (single-cell arm) and bulk DE for the V-ATPase module; report module-level effect size + FDR + 95% CI. H2 supports the compensation model if GBA-N370S shows positive module shift (FDR<0.05, CI excluding 0). The competing model (V-ATPase DOWN with failing lysosome) is equally reportable; do NOT tune toward UP.

H3 (secondary — postmortem-SN direction + held-out blind validation): A V-ATPase / CLEAR-network module score, LOCKED from the iPSC-DA arms (the 9-gene set + any weights), is scored WITHOUT refitting on the held-out Smajić GSE157783 postmortem SN/midbrain atlas (idiopathic PD vs control), restricted to the dopaminergic compartment. Report the PD-vs-control module effect + 95% CI and, as a classification readout, AUC + 1000× bootstrap CI for separating PD from control donors on the module score. The lock file is frozen before touching GSE157783. A null/opposite postmortem direction is informative (iPSC compensation may not survive to end-stage postmortem tissue) and is reported as such.

H4 (secondary — DA-neuron selectivity): The V-ATPase compensatory shift is SELECTIVE for the dopaminergic compartment rather than a global neuronal/glial effect. In the cohorts with multiple cell types (E-MTAB-7303 single-cell; GSE157783 SN), compare the V-ATPase module shift in DA neurons vs non-DA neurons vs glia. Decision rule: per-donor module score × cell-class interaction; report the DA-vs-other contrast effect + p + 95% CI. H4 supports selectivity if the DA shift significantly exceeds the non-DA shift. A non-selective (global) shift is an informative alternative and reported plainly.

H5 (secondary — honest-failure / sensitivity + power decomposition): Decompose the replication into: (i) the achievable donor-label-permutation power floor at each cohort's n (the original limitation — state the minimum p reachable and whether the cohort can even support a significant call); (ii) sensitivity to the V-ATPase gene-set definition (the 9-gene CLEAR subset vs a broader TFEB/CLEAR target set vs V0-only vs V1-only); (iii) normalisation / batch sensitivity (NanoString housekeeping panel vs bulk vs single-cell pseudobulk). State explicitly that an under-powered null (cannot reject) is NOT the same as a replicated reversal, and report which cohorts are merely under-powered vs genuinely contradictory.

A negative or partial result is fully informative. If independent cohorts show V-ATPase DOWNregulation (true lysosomal failure rather than compensation), or only an under-powered null, THAT is the headline — the self-generated compensatory-UP hypothesis must be reported as not-replicated rather than retuned (gene-set cherry-picking, replicate-level inflation) until it reads UP.

The research question comes from the topic's current research brief. All attempts at a topic share it; earlier attempts may have started from an earlier wording.