Induction Of Chronic Stress Reveals An Interplay Of Stress Granules And TDP-43 Pathological Aggregates In Human ALS Fibroblasts And iPSC-Neurons

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases characterized by the presence of neuropathological aggregates of phosphorylated TDP-43 (P-TDP-43). The RNAbinding protein TDP-43 is also a component of stress granules (SG), cytoplasmic foci forming to arrest translation under sub-lethal stress conditions. Although commonly considered as distinct structures, a link between SG and pathological TDP-43 inclusions may occur despite evidence that TDP-43 pathology directly arises from SG is still under debate. Primary fibroblasts and iPSC-derived neurons (iPSC-N) from ALS patients carrying mutations in TARDBP (n=3) and C9ORF72 (n=3) genes and from healthy controls (n=3) were exposed to oxidative stress by sodium arsenite. SG formation and cell response to stress was evaluated and quantified by immunofluorescence and electron microscopy analyses. We found that, not only an acute, but also a chronic oxidative insult, better mimicking a persistent condition of stress as in neurodegeneration, is able to induce SG formation in primary fibroblasts and iPSC-N. Importantly, only upon chronic stress, we observed TDP-43 recruitment into SG and the formation of distinct P-TDP-43 aggregates, very similar to the abnormal inclusions observed in ALS/FTD autoptic brains. Moreover, in fibroblasts, cell response to stress was different in control compared with mutant ALS cells, probably due to their different vulnerability. A quantitative analysis revealed also differences in terms of number of SG-forming cells and SG size, suggesting a different composition of foci in acute and chronic stress. In condition of prolonged stress, SG and P-TDP-43 aggregate formation was concomitant with p62 increase and autophagy dysregulation in both ALS fibroblasts and iPSC-N, as confirmed by immunofluorescence and ultrastructural analyses. We found that exposure to a chronic oxidative insult promotes the formation of both SG and P-TDP-43 aggregates in patient-derived cells, reinforcing the idea that SG fail to properly disassemble, interfering with the protein quality control system. Moreover, we obtained a disease cell model recapitulating ALS/FTD P-TDP-43 aggregates, which represents an invaluable bioassay to study TDP-43 pathology and develop therapeutic strategies aimed at disaggregating or preventing the formation of pathological inclusions.

RNA-binding proteins as a common ground for neurodegeneration and inflammation in amyotrophic lateral sclerosis and multiple sclerosis

“…These mismanagements of oxidative stress and inefficient protective mechanisms will result in misfolding and mislocalization of nuclear RBPs such as FUS, TDP-43, or co-aggregates with poly (GR) dipeptides Table 1 (Kino et al, 2011;Patel, 2016;Cook et al, 2020;Ratti et al, 2020). TDP-43 actively participates in the generation of cytoplasmic inclusions such as P-Bodies and stress granules (SGs; Li et al, 2013;Guo et al, 2018) and causes damage to different organelles including mitochondria (Islam, 2017).…”

“…Due to elevated ROS, TDP-43 protein clumps in ALS patients (Ratti et al, 2020;Zuo et al, 2021). Furthermore, SGs formation is induced (Ratti et al, 2020) due to the increased accumulation of TDP-43, this increase in SGs impairs nucleocytoplasmic transport, permitting the buildup of nucleocytoplasmic transport factors and RNA/protein complexes in the cytoplasm (Zhang et al, 2018;Chen and Cohen, 2019). TDP-43 mutations are one of the additional causes of SGs in ALS for example Q331K, A315T, or Q343R (Liu-Yesucevitz et al, 2010).…”

Exploring the potential of mindfulness-based therapy in the prevention and treatment of neurodegenerative diseases based on molecular mechanism studies

“…Chronic stress may promote the loss of nigrostriatal cells in PD, hastening the disease's course (van der Heide et al, 2021), and stressful conditions may intensify the condition's motor symptoms, such as tremor. Additionally, in human ALS fibroblasts and pluripotent stem cell iPSC-motoneurons, prolonged stress stimulates the production of stress granules and pathogenic TDP-43 aggregates, accelerating the course of ALS (Ratti et al, 2020).…”

An optimized workflow to generate and characterize iPSC-derived motor neuron (MN) spheroids

“…Thus, iPSCs can be derived from healthy individuals or patients with MNDs. In a disease context, patient-specific iPSCs retain the genetic background of the patient and when differentiated into MNs, they can display characteristics of the diseases in vitro (6,7). Remarkably, in cases where a monogenic mutation is identified as the primary cause of the disease, CRISPR-Cas9 genome editing, can be used to correct the mutation, generating isogenic controls that facilitate precise genotype and phenotype correlations (8)(9)(10)(11).…”