Modeling sporadic ALS in iPSC-derived motor neurons identifies a potential therapeutic agent

Abstract

Amyotrophic lateral sclerosis (ALS) is a heterogeneous motor neuron disease for which no effective treatment is available, despite decades of research into SOD1-mutant familial ALS (FALS). The majority of ALS patients have no familial history, making the modeling of sporadic ALS (SALS) essential to the development of ALS therapeutics. However, as mutations underlying ALS pathogenesis have not yet been identified, it remains difficult to establish useful models of SALS. Using induced pluripotent stem cell (iPSC) technology to generate stem and differentiated cells retaining the patients' full genetic information, we have established a large number of in vitro cellular models of SALS. These models showed phenotypic differences in their pattern of neuronal degeneration, types of abnormal protein aggregates, cell death mechanisms, and onset and progression of these phenotypes in vitro among cases. We therefore developed a system for case clustering capable of subdividing these heterogeneous SALS models by their in vitro characteristics. We further evaluated multiple-phenotype rescue of these subclassified SALS models using agents selected from non-SOD1 FALS models, and identified ropinirole as a potential therapeutic candidate. Integration of the datasets acquired in this study permitted the visualization of molecular pathologies shared across a wide range of SALS models.

Single-cell RNA-seq analysis of human iPSC-derived motor neurons resolves early and predictive ALS signatures

“…We also aimed to establish the robustness of any signal across two different scRs platforms: the Illumina Bio-Rad Single-Cell Sequencing Solution (DDSEQ) and the 10X (TENEX) Genomics Chromium (Table S1 and Figure 1G). Immunostaining and quantification of day 18 cultures indicated no significant differences in ISL1 and SMI-32 positive MNs between ALS and control suggesting that an overt disease phenotype such as cell death has not manifested at this relatively early differentiation time point ( Figure S3B) as shown in previous studies (Fujimori et al, 2018;Sareen et al, 2013). In total, we analyzed 21,702 cells that passed quality control filters.…”

“…Similarly, these genes also distinguished C9orf72 ALS patient derived, HB9-RFP positive MNs from control samples, and further distinguished isogenic control samples in which one or two copies of the C9orf72 HRE were targeted into the genome with CRISPR-Cas9 (Shi et al, 2018) ( Figure 7F). Finally, this panel of genes distinguished control subject iPSC-MN cultures from sporadic and familial ALS subjects, including those with variants in FUS, SOD1, and TARDBP (Fujimori et al, 2018) ( Figure 7F). Among the six genes quantifiable by RNA in all expression data sets tested, ELAVL3 was the only gene quantifiable as a protein when analyzing the NeuroLINCS proteomics data sets at 18 and 90 days of differentiation.…”

“…As variable molecular readouts caused by genetic background is becoming increasingly acknowledged by experimentalist in human iPSC disease modeling, experimental design must account for the genetic backgrounds of several individual subjects as well as isogenic controls in order to isolate reproducible diseaserelated effects (Fujimori et al, 2018;Kiskinis et al, 2014;Shi et al, 2018). In line with this outlook, we incorporated iPSC lines from several ALS and control subjects into this study, and repeatedly assayed MN differentiations with the goal of detecting reproducible transcriptional signatures in distinct cellular subpopulations.…”

A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation

“…Indeed, it is known that ALS patient-derived motor neurons with mutations in TDP-43 11 have shorter neurites compared to those from healthy donors (Fujimori et al, 2018). To evaluate neuronal protective activity of Hero proteins, we measured the total neurite length of the transfected neurons.…”

Lipid-derived electrophiles induce covalent modification and aggregation of Cu,Zn-superoxide dismutase in a hydrophobicity-dependent manner

“…[42][43][44] Yet, the molecular mechanism leading to the formation of SOD1 aggregates is still not clearly understood. Some studies suggest that lipids, in particular, lipid peroxidation and its electrophilic products may play an important role in motor neuron degeneration 45 and SOD1 aggregation. 46,47 Previously we reported that secosterol aldehydes are present in tissues isolated from ALS rat model and are effective in inducing SOD1 aggregation 41 .…”