Screening for Protein Turnover Modulators in Human Neurons

100%

Screening for Protein Turnover Modulators in Human Neurons

Thèse n. deux mille vingt-six présentée le onze mai deux mille vingt-six à la Faculté des sciences de la vie laboratoire de Davis Suter programme doctoral en Molecular Sciences École polytechnique fédérale de Lausanne pour l'obtention du grade de Docteur ès Sciences par acceptée sur proposition du jury :

Abstract

Disturbed protein homeostasis is a hallmark of aging and many neurodegenerative disorders, reflecting imbalances in protein synthesis and clearance. The interplay of these processes defines protein turnover and maintains a functional proteome. Protein turnover regulation remains challenging to quantify, particularly in post-mitotic human neurons and at a scale compatible with small molecule or genetic screening. In this PhD thesis, we established a live-cell imaging platform based on a fluorescent timer to perturb and quantify protein turnover in stem cell-derived human neurons and to identify pharmacological modulators in a disease-relevant context.

We generated human induced neurons expressing the fluorescent timer which allows quantitative determination of protein turnover. We then implemented the timer in the context of a high-content imaging workflow suitable for phenotypic screening. A primary screen of six thousand small molecules identified compounds that enhanced protein turnover. The dose-response profiling of the prioritized candidates yielded a set of robust modulators spanning diverse target classes. From this group, we selected three hits: CI-994, CGP-52411 and AS-252424. We performed RNA-seq and LFQ proteomics of drug-treated neurons, which converged on translation- and ribosome-associated signatures, whereas the abundance of proteasome and canonical autophagy components remained largely unchanged, suggesting that turnover enhancement can arise from functional regulation of existing protein quality control machinery rather than increased abundance.

To relate turnover modulation to proteotoxic stress, selected compounds were tested in an a-synuclein (aSyn) preformed-fibril seeding model in mouse primary neurons and human dopaminergic neurons. Pathology was quantified by high-content imaging of phosphorylated aSyn (pS129) and confirmed by immunoblotting. All three compounds suppressed the pS129 pathology in a concentration-dependent manner without altering fibril uptake, consistent with modulation of early aggregation and/or neuronal stress responses.

In summary, this work describes a scalable single-cell imaging framework for protein turnover-focused screening in human neurons, provides candidate small-molecule modulators, and establishes an experimental basis to dissect how neuronal protein turnover can be tuned under basal and proteotoxic conditions. While the causal links between protein turnover modulation, long-term neuronal health, and neuroprotection require further study, the platform generalizes beyond Parkinson's disease and enables systematic exploration of proteostasis-targeting strategies across diverse conditions.

Abstract

Résumé

1 Introduction

Introduction

One point one. Proteome and Protein Turnover

One point one point one. Protein Production

From genes to proteins

Introduction

Synthesis of proteins

One point one. Proteome and Protein Turnover

Protein synthesis pathologies

Introduction

One point one. Proteome and Protein Turnover

One point one point two. Protein Clearance

Introduction

Degradation via Ubiquitin-Proteasome System

One point one. Proteome and Protein Turnover

Proteasome

Introduction

One point one. Proteome and Protein Turnover

Autophagy-lysosome pathway

Introduction

Protein degradation pathologies

One point one. Proteome and Protein Turnover

Introduction

One point one point three. Pathways Governing Protein Turnover

One point one. Proteome and Protein Turnover

Coordination of Protein Synthesis and Degradation

Introduction

Mechanistic/Mammalian Target of Rapamycin pathway

One point one. Proteome and Protein Turnover

Introduction

Integrated Stress Response

Unfolded Protein Response

One point two. Monitoring Protein Turnover

One point two point one. Mathematical Representation of Protein Turnover

Introduction

One point two point two. Current Methods of Monitoring

Measuring protein synthesis by nascent-chain labeling

One point two. Monitoring Protein Turnover

Fluorescent reporters and tag-based pulse-chase approaches

Tandem Fluorescent Timer as a tool of protein turnover quantification

Introduction

One point two Monitoring Protein Turnover

Introduction

One point three Neuronal Proteostasis: experimental models and approaches

Introduction

One point three Neuronal Proteostasis: experimental models and approaches

One point three point two Experimental Models of Proteinopathies

Introduction

One point three Neuronal Proteostasis: experimental models and approaches

Introduction

One point four. Modulating Protein Turnover

Introduction

One point four point one. Protein Quality Control as a Therapeutic Target

One point four. Modulating Protein Turnover

Introduction

One point five. Aims of the thesis

Two Fluorescent timer-based screening of protein turnover modulators in human neurons

Two point two. Results

Abstract

Introduction

Results

High-throughput, phenotypic screening identifies protein turnover modulators in human neurons

The secondary screening reveals potent enhancers of protein turnover

Protein turnover modulators enhance expression of ribosomal protein genes

Protein turnover modulators suppress seeding initiation and pS129 pathology in a neuronal aSyn aggregation assay

CI-nine-hundred-ninety-four suppresses seeded aSyn aggregation in human iPSC-derived dopaminergic neurons

Discussion

Limitations of this study

Materials and Methods

Cell lines used in this study

Cell culture and maintenance

Neuronal induction

Neuronal two D long-term differentiation

Differentiation to neurons

Live cell imaging

Immunofluorescence for validation of differentiation

Image processing

Quantification of MCFT half-life

Screening window coefficient

Primary screening

Primary screening data curation

Secondary screening

Selected compounds

Label Free Quantitative Proteomics

Cell culture

Sample collection

Sample preparation

Mass Spectrometry

Data analysis

Statistical Analysis

RNA sequencing Cell culture

Sample collection

Sample preparation

Sequencing

Data analysis

Expression, purification, and fibrillization of mouse aSyn

Primary hippocampal neuron cultures

Differentiation of NGN two-iPSCs into dopaminergic neuronal cultures

Treatment of primary hippocampal neurons or induced dopaminergic with mouse aSyn PFF and small-molecule modulators

Immunocytochemistry and high-content imaging analysis of aSyn PFF-treated primary neuronal cultures

Biochemical analysis of PFF internalization and aSyn pS one hundred twenty-nine pathology

Statistical analysis of aSyn-related experiments

Figures

Supplementary Tables

Three Core passive and facultative mTOR-mediated mechanisms coordinate mammalian protein synthesis and decay

Three point one. Contributions

Three point two. Results Core passive and facultative mTOR-mediated mechanisms coordinate mammalian protein synthesis and decay

Highlights

Cell Systems Article

Naive mESCs display a distinct, biphasic adaptation mode

Physiological variability in protein turnover recapitulates cell-type-specific adaptation

Sustained changes in mTOR signaling drive robust adaptation in mESCs

Proteome composition and dynamics upon passive and mTOR-driven adaptation

Cell Systems

Limitations of the study

Cell Systems Article

STARMETHODS

Generation of hESC-derived neurons (iNGNs)

Differentiation of hESCs into astrocytes

Immunofluorescence for characterization of astrocyte-enriched cell culture

Immunofluorescence for characterization of pluripotency of mESC

Snapshot imaging of the MCFT upon CHX/MYCi/INK one hundred twenty-eight steady-state treatments

Snapshot imaging of the MCFT upon steady-state amino acids depletion

Live imaging of the MCFT upon CHX treatment/release

Live imaging of the MCFT upon MYCi treatment/release

Dox pulse-chase experiment

Flow cytometry

Imbalance computation

N-Hydroxysuccinimide (NHS ester) labeling and imaging

ELISA for p seventy S six K

Sample preparation and sequencing

Label-free Mass Spectrometry experiments Sample collection

Mass spectrometry

Sample collection

Sample preparation for Mass Spectrometry

Computation of protein half-life

Image pre-processing

Quantification of k and kdeg from SNAP pulse-chase labeling

Quantification of S and k from the MCFT

Quantification of kdil from time-lapse movies

ETHICS STATEMENT

Four Discussion

Four point one. Perturbing protein turnover in neurons

Discussion

Four point two. Primary screen uncovers multiple compound groups that shift neuronal protein turnover

Four point two Primary screen uncovers multiple compound groups that shift neuronal protein turnover

Discussion

Four point two point one. AS-two hundred fifty-two four twenty-four as a protein turnover enhancer

Four point two. Primary screen uncovers multiple compound groups that shift neuronal protein turnover

Four point two point two. CGP-five-two-four-eleven as a protein turnover enhancer

Discussion

Four point two Primary screen uncovers multiple compound groups that shift neuronal protein turnover

Four point two point three. CI-nine hundred ninety-four as a protein turnover enhancer

Discussion

Four point two Primary screen uncovers multiple compound groups that shift neuronal protein turnover

Four point two point four. Common features of three distinct modulators of neuronal protein turnover

Discussion

Four point three. Limitations of this study

Four point four Passive adaptation in human cells

Four point four. Passive adaptation in human cells

Discussion

Four point five Conclusions & Outlook

Four point five. Conclusions & Outlook

Discussion

Supplemental Tables

Supplemental Text for: Coordination of protein synthesis and degradation in mammalian cells

One point one Modeling of the MCFT

One point two Steady-state behavior of the MCFT

One point three Scaling factor &

One point four Inference of mOrange two maturation rate

Two Hierarchical Bayesian algorithm for inferring the dynamics of time variable rates

Two point two Likelihood

Two point three Data retrodiction

Two point four. Test on synthetic data

Three. Passive adaptation model

Three point two. Model formalization

Three point three. In-silico passive adaptation model simulation

Three point four. Modelling the adaptation of protein decay to changes in protein synthesis

Three point four point two. Perfect adaptation model

Three point six Including the ubiquitination pathway into the passive adaptation model

Four Mass spectrometry data analysis

Four point two Predicting changes in protein relative amount for pskdeg

Four point two point two The short-lived protein limit

Four point two point three The long-lived protein limit

Five Dynamic SILAC analysis

Five point two Determination of protein decay rate

Overview

This PhD thesis focuses on developing a scalable imaging method to assess protein turnover in human neurons, exploring compounds that can enhance this process and their potential impacts on neurodegenerative disease contexts. It identifies several small molecules as candidates for further investigation.

Key Points

1A live-cell imaging platform was created to study protein turnover in human neurons
2A primary screen of 6,000 small molecules identified compounds that increase protein turnover
3The study highlights three specific compounds: CI-994, CGP-52411, and AS-252424
4Results suggest enhanced protein turnover relates to functional regulation of existing quality control machinery
5The platform enables exploration of proteostasis-targeting strategies beyond Parkinson's disease.

Details

Authors: Michael Shoujie Sun, Benjamin Martin, Joanna Dembska, Ekaterina Lyublinskaya, Cedric Deluz, David M. Suter
Category: Health and Medicine

PDF
Why art? The role of arts in arts and health
This article addresses the complex relationship between the arts and health, questioning the role of the arts in improving mental and physical well-being. It explores various theoretical constructs and empirical evidence while emphasizing the need for a coherent definition of art to understand its effects on health.
PDF
Oncogenesis
This article examines the role of p52-ZER6 in the metabolic reprogramming of tumor cells, highlighting its impact on the pentose phosphate pathway and tumor progression. The study provides insights into the biochemical processes involved and points to potential therapeutic targets for cancer treatment.
PDF
From library to landscape: integrative annotation workflows for compound libraries in drug repurposing
This article details the creation of two automated annotation workflows designed for the integration of chemical and bioactivity data from various public repositories to support drug repurposing projects.
PDF
Comprehensive Hemodialysis and Nephrology Lecture
This document is a detailed lecture series on hemodialysis and nephrology, discussing the fundamentals of dialysis, historical milestones, advanced modalities, and complications associated with the treatment, aiming to enhance understanding for medical trainees and professionals.
PDF
ShiftScan: A tool for rapid analysis of high-throughput differential scanning fluorimetry data and compound prioritization
This document presents ShiftScan, a standalone tool designed for the rapid analysis of differential scanning fluorimetry (DSF) data in drug discovery, enabling efficient filtering and prioritization of compounds based on thermal transition patterns. It aims to assist researchers in managing large datasets and improving the identification of potential drug candidates.