Mutant Presenilin 1 Dysregulates Exosomal Proteome Cargo Produced by Human-Induced Pluripotent Stem Cell Neurons

Abstract

The accumulation and propagation of hyperphosphorylated tau (p-Tau) is a neuropathological hallmark occurring with neurodegeneration of Alzheimer’s disease (AD). Extracellular vesicles, exosomes, have been shown to initiate tau propagation in the brain. Notably, exosomes from human-induced pluripotent stem cell (iPSC) neurons expressing the AD familial A246E mutant form of presenilin 1 (mPS1) are capable of inducing tau deposits in the mouse brain after in vivo injection. To gain insights into the exosome proteome cargo that participates in propagating tau pathology, this study conducted proteomic analysis of exosomes produced by human iPSC neurons expressing A246E mPS1. Significantly, mPS1 altered the profile of exosome cargo proteins to result in (1) proteins present only in mPS1 exosomes and not in controls, (2) the absence of proteins in the mPS1 exosomes which were present only in controls, and (3) shared proteins which were upregulated or downregulated in the mPS1 exosomes compared to controls. These results show that mPS1 dysregulates the proteome cargo of exosomes to result in the acquisition of proteins involved in the extracellular matrix and protease functions, deletion of proteins involved in RNA and protein translation systems along with proteasome and related functions, combined with the upregulation and downregulation of shared proteins, including the upregulation of amyloid precursor protein. Notably, mPS1 neuron-derived exosomes displayed altered profiles of protein phosphatases and kinases involved in regulating the status of p-tau. The dysregulation of exosome cargo proteins by mPS1 may be associated with the ability of mPS1 neuron-derived exosomes to propagate tau pathology.

Small but Mighty—Exosomes, Novel Intercellular Messengers in Neurodegeneration

“…A total of 1117 proteins were identified in both exosome groups and 733 proteins were common to both populations of exosomes. Among differentially associated proteins with mPS1 were phosphatases and protein kinases and their protein levels associated with mPS1 exosomes was significantly lower as compared to control neurons [179]. Furthermore, these exosomes contained distinct proteins absent in control exosomes.…”

“…Furthermore, these exosomes contained distinct proteins absent in control exosomes. Specifically, the distinct proteins were those involved in extracellular matrix structure and function suggesting another mechanism for propagation of tau pathology in AD [179].…”

Host neuronal PRSS3 interacts with enterovirus A71 3A protein and its role in viral replication

“…The EV-A71 viral stock (Thai clinical isolate, genotype B5 designated as TUCU001/B5 isolate) was propagated in 90–95% confluence of RD cell monolayer. The viral titers were determined by CCID50 method as described previously 59 .…”

“…Both HEK293T and RD cells were maintained in complete DMEM and plated at seeding density of 1 × 10 4 cells/well into wells of 96-well plate. EV-A71 viral stock with titer 10 6.125 CCID50/100 μL was ten-fold serially diluted from 10 –1 to 10 –8 CCID50 determined in HEK293T and RD cells as described previously 59 . The permissiveness of HEK293T cells to EV-A71 infection was determined by comparing CCID50 to those derived from the RD cells.…”

Distinct Cleavage Properties of Cathepsin B Compared to Cysteine Cathepsins Enable the Design and Validation of a Specific Substrate for Cathepsin B over a Broad pH Range

“…Analysis of neuronal and microglial cells by proteomics MS utilized protocols that we have previously described elsewhere. , To summarize, approximately 10 8 cells were collected from 90% confluent flasks with PBS washing and centrifugation at 500 g . Cell pellets were lysed by dounce homogenization in ice-cold 100 mM Tris pH 7.4, 50 mM NaCl, 1 mM EDTA, and protease inhibitors [10 μM pepstatin A, 10 μM leupeptin, 10 μM chymostatin, 100 μM AEBSF (Millipore, Burlington, MA), and 10 μM E64c (Bachem, Torrance, KA)].…”