Understanding multisystem proteinopathy gene (dys)function in immune cells through multi-omic analyses of human stem cell models

Multisystem proteinopathies (MSP) represent a collection of dominantly inherited disorders that can manifest as amyotrophic lateral sclerosis (ALS) (affecting motor neurons), frontotemporal dementia (FTD) (affecting cells of the brain), inclusion body myopathy (IBM) (affecting muscle), Paget's disease of bone (PDB) (affecting the bone), or in combinations, where multiple cell types/tissues are affected. Genome-wide association studies have identified MSP-associated variants in genes including; VCP, PFN1, HNRNPA1, HNRNPA2B1, MATR3, OPTN, ANXA11, TIA1 and SQSTM1. Defining the biology of why patients with identical mutations in the same gene develop distinct clinical phenotypes, affecting different tissues, even sometimes within the same families remains a priority. The existence of modifier genes is an obvious possibility, but the high prevalence of pleiotropy even among closely related family members argues that other stochastic factors, perhaps at the cellular level, may be at work. In this regard, it is notable that many of these 'MSP genes' are highly expressed in macrophages - tissue-specific immune sentinels that act as effectors of the innate immune response. This is particularly pertinent given there is an emerging inflammatory component common to all MSP disorders. Despite this, the study of how MSP genes influence macrophage function has been overlooked, in part due to the difficulties of genetically manipulating primary human macrophages.
In work leading up to this project, the supervisory team has established methods for CRISPR gene editing of iPSCs, and the conversion to of iPSCs to a host of MSP-relevant cells including macrophages of the blood, brain and bone, and motor neurons. In this studentship, we propose to further utilise these cutting-edge tools (CRISPR editing, stem cell culture and conversion) to understand how MSP gene deregulation modifies macrophage function. We hypothesise that this
will deliver new insights into the etiological relationship between seemingly distinct age-related degenerative diseases of muscle, bone, and brain
Experimental plan
1. CRISPR gene editing to knock-out all nine MSP genes that are shared across the disease spectrum in control iPSC lines.
2. Conversion of iPS cells to myeloid factories, that can subsequently undergo differentiation tohighly relevant cell types, including a pan-macrophage (iMac) and tissue-specific residents of the bone (osteoclast) and brain (microglia).
3. Molecular and phenotypic screening of iPSC-derived models. Primary screening of MSP gene-mediated dysfunction will take place in iMacs. We will first assess whether MSP gene knockout lines induce the protein pathology that typifies MSP (the cytoplasmic accumulation of TDP-43).
Concurrently we will complete quantitative proteomics to define protein deregulation in MSP iMacs.
We will stage protein network deregulation against macrophage (dys)function, screening phagocytic capacity of MSP mutant lines (uptake of yeast and bacterial particles) using standard and imaging flow cytometry and by quantifying cytokine production (ELISA) in response to activating stimuli (i.e bacterial cell wall mimics).
4. Establishing tissue-specific iPS macrophage culture. We will then explore the conversion of MSP gene knock-out lines to osteoclasts and microglia, to validate common and distinct molecular dysregulation, established in 3.
5. Development of advanced stem cell models. Given the motor neuron component to anumber of the MSP disorders, we will investigate the co-culture of iPSC-derived macrophage models with iPSC-motor neurons, to understand how macrophage deregulation may influence motor neuron homeostasis/function