Biology subjects

Munoz-Estrada, J.

Publications and source records attributed to Munoz-Estrada, J..

5 recordsLinked to original sources

bioRxiv2026

Temporal Proteomics Links the APP London Mutation to Early Cholesterol Remodeling Cell Programs in Human Neurons

Familial Alzheimer's disease (AD) mutations in amyloid precursor protein (APP) alter amyloid-beta secretion profiles, but their effects on neuronal protein and lipid homeostasis remain incompletely understood. To identify early, cell-autonomous phenotypes associated with AD, we used isogenic KOLF2.1J iPSC-derived NGN2 neurons (iNeurons) carrying a heterozygous or homozygous APP-London mutation (V717I) and conducted a longitudinal proteomic study. Results revealed that maturation was the dominant source of proteomic variation, while genotype-dependent effects were pronounced in homozygous APP-London iNeurons. At four weeks, homozygous APP-London iNeurons showed increased APP abundance by proteomics and immunoblot, and increased alpha-CTF signal by immunoblotting. However, alpha-CTF/FL-APP and alpha-CTF/FL-APP ratios from immunoblot did not differ significantly from revertant controls, leaving preferential CTF accumulation unresolved and instead indicating increased steady-state abundance of APP-derived species. Proteomics remodeling in APP-London mutants converged on synaptic, endosomal, and calcium signaling pathways. Notably, homozygous APP-London iNeurons showed a distinct accumulation of cholesterol-biosynthetic enzymes and altered abundance of cholesterol transporters, an increased free/total cellular cholesterol without corresponding induction of SREBP2 target transcripts Pharmacological perturbation of cholesterol metabolism altered A beta 40 and Abeta 42 secretion in a compound-specific manner and produced minimal changes in bulk cellular cholesterol, indicating that A beta secretion was not simply determined by total cholesterol abundance. Finally, we identified SYT4 as a potential gamma-secretase target or associated factor that warrants further validation. These findings identify cholesterol dyshomeostasis as an early metabolic phenotype of iNeurons carrying the APP-London mutation, extending the molecular phenotype beyond altered A beta secretion.

neuroscience↗

bioRxiv2026

The p53-p21-Cyclin D2 regulatory axis drives metabolic reprogramming and a distinct senescent macrophage senotype during aging and MASLD.

Aging drives chronic disease in part through senescent cells, including macrophages, which fuel inflammation. Senescent macrophages are functionally heterogeneous: canonical p16-high macrophages promote tumorigenesis or, in other contexts, disease tolerance, whereas we previously identified a distinct p21-high, p16-low senotype that drives metabolic dysfunction-associated steatotic liver disease (MASLD). The molecular basis of this senotype has remained undefined. Using genetic and multi-omic approaches, we show that a p53-p21-dependent program actively represses p16 and is required for senescent macrophage viability. We identify Cyclin D2 as a non-canonical downstream effector that redistributes from the nucleus to mitochondria and lipid droplets, where it partners with MIC60 to drive metabolic reprogramming and modulates AKT1-mTORC1 signaling that sustains the SASP. Cyclin D2-p21 double-positive macrophages accumulate with aging and MASLD in mice and in human liver cirrhosis, and can be selectively depleted by senolytic treatment. Together, these findings define a druggable p53-p21-Cyclin D2 axis that specifies macrophage senotype.

immunology↗

bioRxiv2026

Robotic perturbation proteomics and AI agents enable scalable drug mechanism discovery

Large-scale mass spectrometry-based proteomic screening could reveal cellular mechanisms of drug action at systems resolution but remains limited by experimental complexity and the difficulty of extracting insight from high-dimensional datasets. Here, we describe an end-to-end platform that combines semi-automated sample preparation, rapid LC-MS/MS, and AI agent-based data analysis to enable scalable proteomic screening. In a screen of 172 compounds in HepG2 cells, we generated 1,232 proteomes with more than 8,700 quantified proteins in approximately three weeks. Agentic AI reduced data analysis and interpretation time to less than one day while translating proteomic measurements into structured mechanism-oriented summaries and experimentally testable hypotheses. Guided by this framework, we validated: (1) a cholesterol-lowering effect of methylene blue in vitro and (2) an association between loratadine exposure and increased circulating iron in matched electronic health record analyses. This work establishes a scalable platform for generating proteomic drug perturbation data and automatically converting that data into mechanistic insights and candidate translational hypotheses using AI.

systems biology↗

bioRxiv2025

Extended NGN2 Expression in iPSCs Dramatically Enhances Purity of Neuronal Cultures

iPSC-derived neuronal cultures can provide valuable insights into the pathogenesis of neurological disease. However, single-cell iPSC clones expressing NGN2 and mCherry exhibit spontaneous loss of mCherry fluorescence, raising questions about the homogeneity of neurons derived from what appear to be heterogeneous iPSCs. We find that mCherry silencing does not influence iNeurons with two lines of evidence. First, using single-cell proteomics, we found that spontaneous mCherry silencing does not drive heterogeneity in iPSCs. Second, bulk proteomics and immunofluorescence analysis indicated that iNeurons from iPSCs expressing or lacking mCherry both resemble cortical glutamatergic neurons. The primary confounding factor in iNeuron generation was that suboptimal neuronal conversion led to cell aggregates comprised of actively proliferating neuronal progenitor cells and astrocytes as the culture developed. Our results indicate that extended NGN2 dosage substantially improves neuron purity. HighlightsO_LIEF1-mCherry undergoes spontaneous silencing at the AAVS1 locus C_LIO_LISingle-cell proteomics reveals heterogeneity in edited iPSCs independent of mCherry silencing C_LIO_LImCherry silencing has minimal impact on NGN2-mediated neuronal differentiation. C_LIO_LIExtended NGN2 induction produces more homogenous neuronal cultures C_LI eTOC blurbExpression of a mCherry reporter under the EF1 promoter and integrated into the AAVS1 locus is silenced in undifferentiated iPSCs, whereas CAG-driven rtTA3G remains active for NGN2-induced neuronal differentiation. Optimizing NGN2 induction in iPSC cultures is crucial for generating homogeneous neuronal cultures, as suboptimal conditions result in heterogeneous populations enriched with neural progenitor cells (NPCs) and astrocytes, and a disrupted neuronal organization.

biochemistry↗

bioRxiv2023

TTBK2 mutations associated with spinocerebellar ataxia type 11 disrupt peroxisome dynamics and ciliary localization of SHH signaling proteins

Frameshift mutations in Tau Tubulin Kinase 2 (TTBK2) cause spinocerebellar ataxia type 11 (SCA11), which is characterized by the progressive loss of Purkinje cells and cerebellar atrophy. Previous work showed that these TTBK2 variants generate truncated proteins that interfere with primary ciliary trafficking and with Sonic Hedgehog (SHH) signaling in mice. Nevertheless, the molecular mechanisms underlying the dominant interference of mutations remain unknown. Herein, we discover that SCA11-associated variants contain a bona fide peroxisomal targeting signal type 1. We find that their expression in RPE1 cells reduces peroxisome numbers within the cell and at the base of the cilia, disrupts peroxisome fission pathways, and impairs trafficking of ciliary SMO upon SHH signaling activation. This work uncovers a neomorphic function of SCA11-causing mutations and identifies requirements for both peroxisomes and cholesterol in trafficking of cilia-localized SHH signaling proteins. In addition, we postulate that molecular mechanisms underlying cellular dysfunction in SCA11 converge on the SHH signaling pathway. SUMMARYMolecular mechanisms underlying spinocerebellar ataxia type 11 are not well understood. In this study, we identified a neomorphic function of the mutated gene (TTBK2) associated with this disease highlighting a functional inter-organelle interaction between peroxisomes and cilia.

cell biology↗