Rapid iPSC inclusionopathy models shed light on formation, consequence and molecular subtype of α-synuclein inclusions

Abstract

In neurodegenerative proteinopathies, intracellular inclusions are histopathologically and ultrastructurally heterogeneous but the significance of this heterogeneity is unclear. Patient- derived iPSC models, while promising for disease modeling, do not form analogous inclusions in a reasonable timeframe and suffer from limited tractability and scalability. Here, we developed an iPSC toolbox that utilizes piggyBac-based or targeted transgenes to rapidly induce CNS cells with concomitant expression of misfolding-prone proteins. The system is scalable and amenable to screening and longitudinal tracking at single-cell and single-inclusion resolution. For proof-of- principle, cortical neuron alpha-synuclein inclusionopathy models were engineered to form inclusions spontaneously or through exogenous seeding by alpha-synuclein fibrils. These models recapitulated known fibril- and lipid-rich inclusion subtypes in human brain, shedding light on their formation and consequences. Genetic-modifier and protein-interaction screens identified sequestered proteins in these inclusions, including RhoA, that were deleterious to cells when lost. This new iPSC platform should facilitate biological and drug discovery for neurodegenerative proteinopathies.

A dual hit of α-synuclein internalization and immune challenge leads to the formation and maintenance of Lewy body-like inclusions in human dopaminergic neurons

“…This has allowed us to study the stages involved in their formation and their morphology. Our findings bring together two separate fields of inquiry into the pathophysiology of PD: (1) the internalization, propagation, seeding, overexpression, and incorporation of α-syn in LBs 81 ; (2) the involvement of the immune system in PD 23 .…”

“…Although IFN-γ secretion by microglia was detected and has been reported previously 83, 84 , it is likely a combination of microglia-secreted factors driving this effect in vitro , including IL-1β. In addition to microglia, we believe that neuronal exposure to IFN-γ may also occur through immune cell secretions, following their infiltration into the brain 45 , as immune cells are robust producers and secretors of IFN-γ 81 . In fact, aging microglia have been shown to facilitate immune system infiltration into the brain 85 .…”

“…In fact, aging microglia have been shown to facilitate immune system infiltration into the brain 85 . Exposure to IFN-γ can induce antigen presentation in neurons 81 , which may lead neurons to be further vulnerable to the immune system via autoimmune-mediated mechanisms 86 , and perhaps more likely to form inclusions as a response to α-syn upregulation and oxidative stress 87, 88 . Overall, our goal is not to implicate any one specific cell type for the secretion of proinflammatory cytokines, or that one specific cytokine is responsible for inclusion formation.…”

Deep sequencing of proteotoxicity modifier genes uncovers a Presenilin-2/beta-amyloid-actin genetic risk module shared among alpha-synucleinopathies

“…Notably, however, normalization here is to Gapdh. We previously showed that this underestimates true neuronal αS content 58 by several-fold in the human brain (where there are co-resident glia). SNCA-high neurons have approximately 2-fold higher protein levels than 4-copy SNCA neurons, thus likely a closer surrogate for true neuronal expression levels in human brain (Figure 3B).…”

“…A tractable synucleinopathy hiPSC model is established with transgenic SNCA expression iPSC modeling for neurodegenerative proteinopathies suffers from poor reproducibility and tractability, and low levels of endogenous αS. Recently, we 58 and others 59,60 have begun to establish a suite of tractable models to study different aspects of neurodegenerative disease biology. We extended this development by engineering the WTC11 iPSC line that was selected for engineering by the Allen Institute for Cell Science (https://www.allencell.org/cell-catalog.html).…”