Digital color-coded molecular barcoding reveals dysregulation of common FUS and FMRP targets in soma and neurites of ALS mutant motoneurons

Abstract

Mutations in RNA binding proteins (RBPs) have been linked to the motor neuron disease amyotrophic lateral sclerosis (ALS). Extensive auto-regulation, cross-regulation, cooperation and competition mechanisms among RBPs are in place to ensure proper expression levels of common targets, often including other RBPs and their own transcripts. Moreover, several RBPs play a crucial role in the nervous system by localizing target RNAs in specific neuronal compartments. These include the RBPs FUS, FMRP, and HuD. ALS mutations in a given RBP are predicted to produce a broad impact on such delicate equilibrium. Here we studied the effects of the severe FUS-P525L mutation on common FUS and FMRP targets. Expression profiling by digital color-coded molecular barcoding in cell bodies and neurites of human iPSC-derived motor neurons revealed altered levels of transcripts involved in the cytoskeleton, neural projection and synapses. One of the common targets is HuD, which is upregulated because of the loss of FMRP binding to its 3′UTR due to mutant FUS competition. Notably, many genes are commonly altered upon FUS mutation or HuD overexpression, suggesting that a substantial part of the effects of mutant FUS on the motor neuron transcriptome could be due to HuD gain-of-function. Among altered transcripts, we also identified other common FUS and FMRP targets, namely MAP1B, PTEN, and AP2B1, that are upregulated upon loss of FMRP binding on their 3’UTR in FUS-P525L motor neurons. This work demonstrates that the impairment of FMRP function by mutant FUS might alter the expression of several genes, including new possible biomarkers and therapeutic targets for ALS.

HuD (ELAVL4) gain-of-function impairs neuromuscular junctions and induces apoptosis in familial and sporadic amyotrophic lateral sclerosis models

“…Moreover, knockdown of HuD decreased mutant FUS-induced apoptosis in vitro and, strikingly, knockdown of the related elav Drosophila gene completely rescued motor phenotypes induced by FUS in vivo. We have recently shown that in MNs FUS mutation or HuD overexpression exert similar effects in the expression of disease-linked genes (Garone et al, 2023). Altogether, these findings point to a crucial role for HuD in the cell autonomous mechanisms that underlie the death of MNs via a "dying back" pattern in FUS and, possibly, a subset of sporadic ALS cases with hallmarks of oxidative and proteotoxic stress.…”

“…This could be due to the combined effects of downregulation of miR-375, a HuD targeting miRNA (De Santis et al, 2017), and direct binding of mutant FUS to HuD 3'UTR, resulting in competition between mutant FUS and FMRP for HuD 3'UTR binding (De Santis et al, 2019;Garone et al, 2021). Increased HuD levels in FUS WT MNs produce changes in the transcriptome that are substantially similar to those exerted by the severe FUS P525L variant (Garone et al, 2023). A direct consequence of HuD upregulation in FUS mutant iPSCderived MNs and mouse spinal cord is an increase of the levels of two previously known HuD targets, Neuritin1 (NRN1; also known as cpg15) and GAP43 (Garone et al, 2021).…”

HuD (ELAVL4) gain-of-function impairs neuromuscular junctions and induces apoptosis inin vitroandin vivomodels of amyotrophic lateral sclerosis

“…Moreover, knockdown of HuD decreased mutant FUS-induced apoptosis in vitro and, strikingly, knockdown of the related elav Drosophila gene completely rescued motor phenotypes induced by FUS in vivo . We have recently shown that in MNs FUS mutation or HuD overexpression exert similar effects on the expression of disease-linked genes (Garone et al, 2023). Altogether, these findings point to a crucial role for HuD in the cell-autonomous mechanisms that underlie the death of MNs via a “dying back” pattern in FUS and, possibly, a subset of sporadic ALS cases with hallmarks of oxidative and proteotoxic stress.…”

“…In this context, we have previously shown that mRNA and protein levels of HuD, encoded by the ELAVL4 gene, are upregulated in MNs derived from FUS mutant human induced pluripotent stem cells (iPSCs) and in a mouse model (Fus-Δ14 knock-in) (Garone et al, 2021). Increased HuD levels in FUS WT MNs produce changes in the transcriptome that are substantially similar to those exerted by the severe FUS P525L variant (Garone et al, 2023). A direct consequence of HuD upregulation in FUS mutant iPSC-derived MNs and mouse spinal cord is an increase in the levels of its targets, including Neuritin1 (NRN1; also known as cpg15) and GAP43 (Garone et al, 2021).…”

“…In this work we aimed to assess if HuD dysregulation could underlie neuromuscular junctions (NMJs) defects in the context of ALS. Several genes that are commonly altered in FUS P525L MNs and HuD overexpressing MNs are involved in axon projection, cell junction and synapse formation (Garone et al, 2020; Garone et al, 2023). ALS is considered a distal axonopathy as early impairment of NMJs has been reported in both animal models and patients.…”