Amyotrophic lateral sclerosis (ALS) is a fatal non‐cell‐autonomous neurodegenerative disease characterized by the loss of motor neurons (MNs). Mutations in CRMP4 are associated with ALS in patients, and elevated levels of CRMP4 are suggested to affect MN health in the SOD1G93A‐ALS mouse model. However, the mechanism by which CRMP4 mediates toxicity in ALS MNs is poorly understood. Here, by using tissue from human patients with sporadic ALS, MNs derived from C9orf72‐mutant patients, and the SOD1G93A‐ALS mouse model, we demonstrate that subcellular changes in CRMP4 levels promote MN loss in ALS. First, we show that while expression of CRMP4 protein is increased in cell bodies of ALS‐affected MN, CRMP4 levels are decreased in the distal axons. Cellular mislocalization of CRMP4 is caused by increased interaction with the retrograde motor protein, dynein, which mediates CRMP4 transport from distal axons to the soma and thereby promotes MN loss. Blocking the CRMP4‐dynein interaction reduces MN loss in human‐derived MNs (C9orf72) and in ALS model mice. Thus, we demonstrate a novel CRMP4‐dependent retrograde death signal that underlies MN loss in ALS.

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Mechanical events and alterations in neuronal morphology that accompany neuronal activity have been observed for decades. However, no clear neurophysiological role, nor an agreed molecular mechanism relating these events to the electrochemical process, has been found. Here we hypothesized that intense, yet physiological, electrical activity in neurons triggers cytoskeletal depolymerization. We excited the sciatic nerve of anesthetized mice with repetitive electric pulses (5, 10, and 100 Hz) for 1 and 2 min and immediately fixed the excised nerves. We then scanned the excised nerves with high-resolution transmission electron microscopy, and quantified cytoskeletal changes in the resulting micrographs. We demonstrate that excitation with a stimulation frequency that is within the physiological regime is accompanied by a significant reduction in the density of cytoskeletal proteins relative to the baseline values recorded in control nerves. After 10 Hz stimulation with durations of 1 and 2 min, neurofilaments density dropped to 55.8 and 51.1% of the baseline median values, respectively. In the same experiments, microtubules density dropped to 23.7 and 38.5% of the baseline median values, respectively. These changes were also accompanied by a reduction in the cytoskeleton-to-cytoplasm contrast that we attribute to the presence of depolymerized electron-dense molecules in the lumen. Thus, we demonstrate with an in vivo model a link between electrical activity and immediate cytoskeleton rearrangement at the nano-scale. We suggest that this cytoskeletal plasticity reduces cellular stiffness and allows cellular homeostasis, maintenance of neuronal morphology and that it facilitates in later stages growth of the neuronal projections.

29Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease 30 with selective dysfunction; it causes the death of motor neurons (MNs). In spite of 31 some progress, currently no effective treatment is available for ALS. Before such 32 treatment can be developed, a more thorough understanding of ALS pathogenesis is 33 required. Recently, we demonstrated that ALS-mutated muscles contribute to ALS 34 pathology via secretion of destabilizing factors such as Sema3A; these factors trigger 35 axon degeneration and Neuromuscular Junction (NMJ) disruption. Here, we focus on 36 the molecular mechanism by which muscle contribute to MNs loss in ALS. We 37 identified CRMP4 as part of a retrograde death signal generated in response to 38 muscle-secreted Sema3A, in ALS-diseased MNs. Exposing distal axons to Sema3A 39 induces CRMP4-dynein complex formation and MN loss in both mouse (SOD1 G93A ) 40 and human-derived (C9orf72) ALS models. Introducing peptides that interfere with 41 CRMP4-dynein interaction in MN axons profoundly reduces Sema3A-dependent MN 42 loss. Thus, we discovered a novel retrograde death signal mechanism underlying MN 43 loss in ALS. 44 45 46 Summary 47 Maimon et al. identify a novel retrograde death mechanism that contribute to MN loss 48 in ALS, in which CRMP4-Dynein complex is form and retrogradely move along the 49 axon.50 51 52 53 105 Strittmatter, 2007). The canonical signaling events of CRMPs during Semaphorin 106 signaling involve their phosphorylation via GTPase activity, which further leads to 107 microtubule destabilization and axon retraction (Sasaki et al., 2002; Yamashita and 108 5 Goshima , 2012; Balastik et al., 2015). In addition to their role in mediating Sema3A 109 intrinsic responses, CRMPs were also reported to bind dynein and kinesin, and 110 modulate their function (Arimura et al., 2009; Rahajeng et al., 2010). Several studies 111 further demonstrated the involvement of CRMPs in neurodegenerative diseases 112 (Charrier et al., 2003; Yamashita and Goshima, 2012; Nagai, Baba and Ohshima, 113 2017). Specifically, CRMP4 expression levels were previously found to be elevated in 114 the SOD1 G93A mouse spinal cord, and were suggested to hamper MN health (Duplan 115 et al., 2010; Valdez et al., 2012; Nagai et al., 2015). However, the mechanism underline 116 the involvement of CRMP4 mediating cell death and its role in other ALS model is still 117 elusive. Moreover, mutations in CRMP4 were associated with ALS patients (Blasco et 118 al., 2013). 119 120 Here, we identified a novel retrograde death signal, triggered by Sema3A, which 121 facilitates MN loss in both murine and human ALS models. This process is mediated 122 by the formation of CRMP4-Dynein complex along diseased axons. Interfering with the 123 binding of CRMP4 to Dynein by using short blocking peptides rescues Sema3A-124 dependent MN loss in these ALS models. 125 126 Results 127 128 SOD G93A MNs and muscle co-cultures enhance MN loss 129 MN cell death is a key process in ALS pathology. Taking into consideratio...

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