Aberrant axon branching via Fos-B dysregulation in FUS-ALS motor neurons

Abstract

Background The characteristic structure of motor neurons (MNs), particularly of the long axons, becomes damaged in the early stages of amyotrophic lateral sclerosis (ALS). However, the molecular pathophysiology of axonal degeneration remains to be fully elucidated. Method Two sets of isogenic human-induced pluripotent stem cell (hiPSCs)-derived MNs possessing the single amino acid difference (p.H517D) in the fused in sarcoma ( FUS ) were constructed. By combining MN reporter lentivirus, MN specific phenotype was analyzed. Moreover, RNA profiling of isolated axons were conducted by applying the microfluidic devices that enable axon bundles to be produced for omics analysis. The relationship between the target gene, which was identified as a pathological candidate in ALS with RNA-sequencing, and the MN phenotype was confirmed by intervention with si-RNA or overexpression to hiPSCs-derived MNs and even in vivo . The commonality was further confirmed with other ALS-causative mutant hiPSCs-derived MNs and human pathology. Findings We identified aberrant increasing of axon branchings in FUS -mutant hiPSCs-derived MN axons compared with isogenic controls as a novel phenotype. We identified increased level of Fos-B mRNA, the binding target of FUS, in FUS -mutant MNs. While Fos-B reduction using si-RNA or an inhibitor ameliorated the observed aberrant axon branching, Fos-B overexpression resulted in aberrant axon branching even in vivo . The commonality of those phenotypes was further confirmed with other ALS causative mutation than FUS . Interpretation Analyzing the axonal fraction of hiPSC-derived MNs using microfluidic devices revealed that Fos-B is a key regulator of FUS -mutant axon branching. Fund Japan Agency for Medical Research and development; Japanese Ministry of Education, Culture, Sports, Science and Technology Clinical Research, Innovation and Education Center, Tohoku University Hospital; Japan Intractable Diseases (Nanbyo) Research Foundation; the Kanae Foundation for the Promotion of Medical Science; and “Inochi-no-Iro” ALS research grant.

Omics Approach to Axonal Dysfunction of Motor Neurons in Amyotrophic Lateral Sclerosis (ALS)

“…FUS mutation causes axonal retention of the FUS protein prior to its aggregation, which is caused by poly(ADP-ribose) polymerase-dependent DNA response signaling (Naumann et al, 2018). The authors of this review also observed the accumulation of mutant FUS protein in the neurites of FUS-mutant induced pluripotent stem cell (iPSC)-derived MNs (Akiyama et al, 2019).…”

“…Increased synaptic activity or branching is considered desirable in the field of psychiatric disease (Shao et al, 2019). The authors of the present review identified aberrant axonal branching in FUS-mutant iPSC-derived MNs (Akiyama et al, 2019). The sensory axons branching in the presence of nerve growth factor (NGF) can be observed at sites marked by stalled mitochondria.…”

“…In the process of improving the dimensions of the well and materials, a novel microfluidic device was developed by the authors of this study ( Table 3). The device enabled comparison of two sets of isogenic FUSmutant iPSC-derived MNs generated using genome editing technology (Joung and Sander, 2013;Okano and Yamanaka, 2014), and provided observations of increased branching in FUSmutant MN axons compared with those in isogenic controls (Akiyama et al, 2019). This phenotype was confirmed using other ALS-causative mutations, including SOD1 and TARDBP.…”

miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro

“…In fact, iPSC-based approaches for studying the pathogenesis of neurological diseases, which are difficult to model with animals, are being explored, and research on therapeutic and preventive agents for such diseases has been accelerated. In particular, dementia including AD [39][40][41], Parkinson's disease [42,43], and ALS [43][44][45][46][47] are among the diseases that are actively studied so that the disease states can be further elucidated and therapeutic agents can be developed for intractable diseases. In terms of neural differentiation methods, the emergence of a neuronal induction method using the transient expression of Ngn2 greatly enhanced neuronal induction efficiencies [8].…”

Dysfunction in nonsense-mediated decay, protein homeostasis, mitochondrial function, and brain connectivity in ALS-FUS mice with cognitive deficits

“…FUS knockout mice do not develop ALS-like symptoms or pathology [ 36 – 38 ], suggesting that loss of FUS function is not sufficient to cause motor neuron diseases. Recent studies using human induced pluripotent stem cells (iPSCs)-derived motor neurons carrying FUS mutations [ 39 – 41 ], and various mouse models [ 28 , 29 , 34 , 37 , 38 , 42 , 43 ], indicate disease-linked FUS mutations use a “gain of toxicity” mechanism to drive ALS pathogenesis. It is worth mentioning that partial loss of FUS functions such as RNA misprocessing [ 23 , 34 , 44 ], dysfunctional paraspeckles [ 45 ], DNA damage repair [ 43 , 46 , 47 ], mitochondrial dysfunctions [ 48 ] and axonal translation [ 29 ], are also found in these cells and mouse models.…”