A Regulatory Circuitry Between Gria2, miR-409, and miR-495 Is Affected by ALS FUS Mutation in ESC-Derived Motor Neurons

Abstract

Mutations in fused in sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS). FUS is a multifunctional protein involved in the biogenesis and activity of several types of RNAs, and its role in the pathogenesis of ALS may involve both direct effects of disease-associated mutations through gain- and loss-of-function mechanisms and indirect effects due to the cross talk between different classes of FUS-dependent RNAs. To explore how FUS mutations impinge on motor neuron-specific RNA-based circuitries, we performed transcriptome profiling of small and long RNAs of motor neurons (MNs) derived from mouse embryonic stem cells carrying a FUS-P517L knock-in mutation, which is equivalent to human FUS-P525L, associated with a severe and juvenile-onset form of ALS. Combining ontological, predictive and molecular analyses, we found an inverse correlation between several classes of deregulated miRNAs and their corresponding mRNA targets in both homozygous and heterozygous P517L MNs. We validated a circuitry in which the upregulation of miR-409-3p and miR-495-3p, belonging to a brain-specific miRNA subcluster implicated in several neurodevelopmental disorders, produced the downregulation of Gria2, a subunit of the glutamate α‐amino‐3‐hydroxy‐5‐methyl-4-isoxazole propionic acid (AMPA) receptor with a significant role in excitatory neurotransmission. Moreover, we found that FUS was involved in mediating such miRNA repression. Gria2 alteration has been proposed to be implicated in MN degeneration, through disturbance of Ca2+ homeostasis, which triggers a cascade of damaging “excitotoxic” events. The molecular cross talk identified highlights a role for FUS in excitotoxicity and in miRNA-dependent regulation of Gria2. This circuitry also proved to be deregulated in heterozygosity, which matches the human condition perfectly.Electronic supplementary materialThe online version of this article (10.1007/s12035-018-0884-4) contains supplementary material, which is available to authorized users.

FUS ALS-causative mutations impair FUS autoregulation and splicing factor networks through intron retention

“…To enhance the confidence of our analysis and identify changes relevant across FUS NLS mutations, we took advantage of two publicly available mouse CNS datasets, where ALS-causative mutations in the endogenous Fus gene were expressed homozygously, and where FUS KO was used in parallel. The RNA-seq datasets (described in Supplementary Table S1A ) were (i) E18.5 brains from FUS-ΔNLS, a model of the R495X mutation that removes the entire NLS ( 47 ), along with a GeneTrap FUS-KO that results in a strong knockdown ( 32 ), together referred to as the ‘Dupuis samples’; and (ii) ES-derived motor neurons from mice homozygous for the FUS-P517L mutation, corresponding to human P525L ( 48 ) that mutates the critical proline residue of the NLS, paired with a FUS-KO ( 33 ), together referred to as the ‘Bozzoni samples’. The FUS KO construct used by the Bozzoni lab is a GeneTrap inserted into intron 12 ( 17 ), which leads to a partial FUS knockout ( Supplementary Figure S1B ).…”

“…FUS-ΔNLS and FUS KO mouse brain samples with their respective controls ( 32 ) were downloaded from SRA accession SRP070906. FUS P517L mutants, FUS KO and shared control samples from mouse motor neurons ( 33 ) were downloaded from SRA accession SRP111475. Data from a series of motor neuron differentiation experiments, where induced pluripotent stem cells with and without VCP mutations were differentiated to mature motor neurons with RNA-seq libraries created from cells taken at 0, 7, 14, 21 and 35 days following differentiation ( 34 ), were downloaded from the Gene Expression Omnibus with accession GSE98290.…”

An evolutionarily acquired microRNA shapes development of mammalian cortical projections

“…CSMN, along with LMN, degenerate in amyotrophic lateral sclerosis (ALS) (91)(92)(93). Interestingly, miR-409-3p and a second 12qF1 cluster miRNA (miR-495-3p) have recently been implicated in the degeneration of embryonic stem cell-derived LMN bearing a mutation specific to a juvenile-onset form of ALS (94). Given the role of the 12qF1 lncRNAs in LMN development discovered recently (89), and the key role of miR-409-3p in CSMN development discovered here, it would be interesting to investigate whether miR-409-3p and other 12qF1 noncoding RNAs also play roles in the pathogenesis of ALS in CSMN.…”

HOTAIRM1 regulates neuronal differentiation by modulating NEUROGENIN 2 and the downstream neurogenic cascade

“…S8A and ref. 59 ), which plays a relevant role in brain development and neurodegeneration [60][61][62] . FUS knockdown experiments in differentiating SH-SY5Y cells produced an 80% reduction of FUS mRNA and protein (Figs.…”