Multi-modal proteomic characterization of lysosomal function and proteostasis in progranulin-deficient neurons

Abstract

Background Progranulin (PGRN) is a lysosomal glycoprotein implicated in various neurodegenerative diseases, including frontotemporal dementia and neuronal ceroid lipofuscinosis. Over 70 mutations discovered in the GRN gene all result in reduced expression of the PGRN protein. Genetic and functional studies point toward a regulatory role for PGRN in lysosome functions. However, the detailed molecular function of PGRN within lysosomes and the impact of PGRN deficiency on lysosomes remain unclear. Methods We developed multifaceted proteomic techniques to characterize the dynamic lysosomal biology in living human neurons and fixed mouse brain tissues. Using lysosome proximity labeling and immuno-purification of intact lysosomes, we characterized lysosome compositions and interactome in both human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (i3Neurons) and mouse brains. Using dynamic stable isotope labeling by amino acids in cell culture (dSILAC) proteomics, we measured global protein half-lives in human i3Neurons for the first time. Results Leveraging the multi-modal proteomics and live-cell imaging techniques, we comprehensively characterized how PGRN deficiency changes the molecular and functional landscape of neuronal lysosomes. We found that PGRN loss impairs the lysosome’s degradative capacity with increased levels of v-ATPase subunits on the lysosome membrane, increased hydrolases within the lysosome, altered protein regulations related to lysosomal transport, and elevated lysosomal pH. Consistent with impairments in lysosomal function, GRN-null i3Neurons and frontotemporal dementia patient-derived i3Neurons carrying GRN mutation showed pronounced alterations in protein turnover, such as cathepsins and proteins related to supramolecular polymerization and inherited neurodegenerative diseases. Conclusion This study suggested PGRN as a critical regulator of lysosomal pH and degradative capacity, which influences global proteostasis in neurons. Beyond the study of progranulin deficiency, these newly developed proteomic methods in neurons and brain tissues provided useful tools and data resources for the field to study the highly dynamic neuronal lysosome biology. Graphical Abstract

A specific and portable gene expression program underlies antigen archiving by lymphatic endothelial cells

“…From this analysis, we find that the Ag-high programs overlap with ontology terms suggestive of increased endosomal-lysosomal function (Figure S3A), and this is most significant for cLECs and fLECs. This includes lysosome factors with broad roles in protein homeostasis (Ctsd 25 , Grn 26 , Lgmn 27 , Prcp 28 ), lipid metabolism (Psap 29 ), and lysosome acidification (Atp6v1a 30 ). In addition, expression of the clathrin adaptor protein Dab2 31 may indicate increased endocytic activity.…”

Multiomics Evaluation of Human iPSCs and iPSC-Derived Neurons

“…Human iPSCs and iPSC-derived glutamatergic neurons were routinely cultured in our lab using the i 3 Neuron technology as described previously . Briefly, WT and GRN –/– iPSCs with the stably expressed Dox-inducible NGN2 transcription factor were maintained in Essential 8 medium (Gibco) in 10 cm dishes coated with Matrigel (Corning) . Neuron differentiation was initiated by replating iPSCs in neuronal induction medium (DMEM/F12 medium, N2 supplement, nonessential amino acid (NEAA), Glutamax, doxycycline) for 3 days.…”

“…24 Briefly, WT and GRN −/− iPSCs with the stably expressed Dox-inducible NGN2 transcription factor were maintained in Essential 8 medium (Gibco) in 10 cm dishes coated with Matrigel (Corning). 22 Neuron differentiation was initiated by replating iPSCs in neuronal induction medium (DMEM/F12 medium, N2 supplement, nonessential amino acid (NEAA), Glutamax, doxycycline) for 3 days. Day 3 neurons were dissociated by Accutase (Gibco) and seeded on 6-well dishes (2 million neurons per well) coated by poly ornithine (Sigma).…”

“…18−21 Our recent study also found that PGRN deficiency neutralizes lysosome pH and influences global protein homeostasis in human neurons. 22 PGRN is involved in multiple organelles and various types of biomolecules, but an integrated proteomics, lipidomics, and metabolomics evaluation has not been reported.…”

“…Here we conducted an integrated multiomics study that can identify and quantify proteins, lipids, and metabolites from the same sample with optimal confidence, accuracy, and reproducibility. To understand the molecular profiles of iPSCs and neurons in both healthy and disease states, we used the advanced i 3 Neuron technology (integrated, inducible, isogenic) which stably integrates the doxycycline (Dox)-inducible Neurogenin 2 (NGN2) transcription factor into the iPSC line and differentiates all iPSCs into a pure population of cortical glutamatergic neurons in 2 weeks. , This i 3 Neuron platform provides significantly improved neuron viability, purity, and speed compared to traditional neuron differentiation methods, enabling a scalable and reproducible production of human neurons for omics studies. − We first compared the proteomics, lipidomics, and metabolomics profiles of healthy wild-type (WT) human iPSCs and i 3 Neurons. We then investigated how genetic mutations that cause neurodegeneration alter the multiomics molecular profiles of iPSCs and neurons in the context of the GRN gene and PGRN deficiency.…”