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Human amyotrophic lateral sclerosis excitability phenotype screen: Target discovery and validation.

ABSTRACT: Drug development is hampered by poor target selection. Phenotypic screens using neurons differentiated from patient stem cells offer the possibility to validate known and discover novel disease targets in an unbiased fashion. To identify targets for managing hyperexcitability, a pathological feature of amyotrophic lateral sclerosis (ALS), we design a multi-step screening funnel using patient-derived motor neurons. High-content live cell imaging is used to evaluate neuronal excitability, and from a screen against a chemogenomic library of 2,899 target-annotated compounds, 67 reduce the hyperexcitability of ALS motor neurons carrying the SOD1(A4V) mutation, without cytotoxicity. Bioinformatic deconvolution identifies 13 targets that modulate motor neuron excitability, including two known ALS excitability modulators, AMPA receptors and Kv7.2/3 ion channels, constituting target validation. We also identify D2 dopamine receptors as modulators of ALS motor neuron excitability. This screen demonstrates the power of human disease cell-based phenotypic screens for identifying clinically relevant targets for neurological disorders.

SUBMITTER: Huang X

PROVIDER: S-EPMC8209673 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Amyotrophic lateral sclerosis (ALS) is underpinned by an oligogenic rare variant architecture. Identified genetic variants of ALS include RNA-binding proteins containing prion-like domains (PrLDs). We hypothesized that screening genes encoding additional similar proteins will yield novel genetic causes of ALS. The most common genetic variant of ALS patients is a G4C2-repeat expansion within C9ORF72. We have shown that G4C2-repeat RNA sequesters RNA-binding proteins. A logical consequence of this is that loss-of-function mutations in G4C2-binding partners might contribute to ALS pathogenesis independently of and/or synergistically with C9ORF72 expansions. Targeted sequencing of genomic DNA encoding either RNA-binding proteins or known ALS genes (n = 274 genes) was performed in ALS patients to identify rare deleterious genetic variants and explore genotype-phenotype relationships. Genomic DNA was extracted from 103 ALS patients including 42 familial ALS patients and 61 young-onset (average age of onset 41 years) sporadic ALS patients; patients were chosen to maximize the probability of identifying genetic causes of ALS. Thirteen patients carried a G4C2-repeat expansion of C9ORF72. We identified 42 patients with rare deleterious variants; 6 patients carried more than one variant. Twelve mutations were discovered in known ALS genes which served as a validation of our strategy. Rare deleterious variants in RNA-binding proteins were significantly enriched in ALS patients compared to control frequencies (p = 5.31E-18). Nineteen patients featured at least one variant in a RNA-binding protein containing a PrLD. The number of variants per patient correlated with rate of disease progression (t-test, p = 0.033). We identified eighteen patients with a single variant in a G4C2-repeat binding protein. Patients with a G4C2-binding protein variant in combination with a C9ORF72 expansion had a significantly faster disease course (t-test, p = 0.025). Our data are consistent with an oligogenic model of ALS. We provide evidence for a number of entirely novel genetic variants of ALS caused by mutations in RNA-binding proteins. Moreover we show that these mutations act synergistically with each other and with C9ORF72 expansions to modify the clinical phenotype of ALS. A key finding is that this synergy is present only between functionally interacting variants. This work has significant implications for ALS therapy development.

Project description:ImportanceAmyotrophic lateral sclerosis (ALS) is a common adult-onset neurodegenerative disease characterized by selective loss of upper and lower motor neurons. Patients with ALS have persistent peripheral and central inflammatory responses including abnormally functioning T cells and activated microglia. However, much less is known about the inflammatory gene profile of circulating innate immune monocytes in these patients.ObjectiveTo characterize the transcriptomics of peripheral monocytes in patients with ALS.Design, setting, and participantsMonocytes were isolated from peripheral blood of 43 patients with ALS and 22 healthy control individuals. Total RNA was extracted from the monocytes and subjected to deep RNA sequencing, and these results were validated by quantitative reverse transcription polymerase chain reaction.Main outcomes and measuresThe differential expressed gene signatures of these monocytes were identified using unbiased RNA sequencing strategy for gene expression profiling.ResultsThe demographics between the patients with ALS (mean [SD] age, 58.8 [1.57] years; 55.8% were men and 44.2% were women; 90.7% were white, 4.65% were Hispanic, 2.33% were black, and 2.33% were Asian) and control individuals were similar (mean [SD] age, 57.6 [2.15] years; 50.0% were men and 50.0% were women; 90.9% were white, none were Hispanic, none were black, and 9.09% were Asian). RNA sequencing data from negative selected monocytes revealed 233 differential expressed genes in ALS monocytes compared with healthy control monocytes. Notably, ALS monocytes demonstrated a unique inflammation-related gene expression profile, the most prominent of which, including IL1B, IL8, FOSB, CXCL1, and CXCL2, were confirmed by quantitative reverse transcription polymerase chain reaction (IL8, mean [SE], 1.00 [0.18]; P = .002; FOSB, 1.00 [0.21]; P = .009; CXCL1, 1.00 [0.14]; P = .002; and CXCL2, 1.00 [0.11]; P = .01). Amyotrophic lateral sclerosis monocytes from rapidly progressing patients had more proinflammatory DEGs than monocytes from slowly progressing patients.Conclusions and relevanceOur data indicate that ALS monocytes are skewed toward a proinflammatory state in the peripheral circulation and may play a role in ALS disease progression, especially in rapidly progressing patients. This increased inflammatory response of peripheral immune cells may provide a potential target for disease-modifying therapy in patients with ALS.

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Human amyotrophic lateral sclerosis excitability phenotype screen: Target discovery and validation.

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