Optogenetic modulation of TDP-43 oligomerization fast-forwards ALS-related pathologies in the spinal motor neurons

Abstract

19Cytoplasmic aggregation of TDP-43 characterizes degenerating neurons in most cases 20 of amyotrophic lateral sclerosis (ALS), yet the mechanisms and cellular outcomes of 21 TDP-43 pathology remain largely elusive. Here, we develop an optogenetic TDP-43 22 variant (opTDP-43), whose multimerization status can be modulated in vivo through 23 external light illumination. Using the translucent zebrafish neuromuscular system, we 24 demonstrate that short-term light stimulation reversibly induces cytoplasmic opTDP-43 25 mislocalization, but not aggregation, in the spinal motor neuron, leading to an axon 26 outgrowth defect associated with myofiber denervation. In contrast, opTDP-43 forms 27 pathological aggregates in the cytoplasm after longer-term illumination and seeds 28 non-optogenetic TDP-43 aggregation. Furthermore, we find that an ALS-linked 29 mutation in the intrinsically disordered region (IDR) exacerbates the light-dependent 30 opTDP-43 toxicity on locomotor behavior. Together, our results propose that 31 IDR-mediated TDP-43 oligomerization triggers both acute and long-term pathologies of 32 motor neurons, which may be relevant to the pathogenesis and progression of ALS. 33Discussion 308

Non-neuromodulatory Optogenetic Tools in Zebrafish

“…In dark-state, opTDP-43z was detected mainly in cell nuclei, but started relocalizing to and aggregating in the cytosol after a blue-light stimulus. Light-induced clustering of opTDP-43z expressed in neurons perturbed axonal outgrowth, increased myofiber denervation frequency and seeded nonoptogenetically induced delocalization of TDP-43 (Asakawa et al, 2019).…”

“…CRY2 self-clusters when illuminated, which is an undesirable feature for its function in the CRY2/CIB1 heterodimer but was found useful to induce protein relocalization, leading to the development of the CRY2olig protein clustering system (Bugaj et al, 2013;Lee et al, 2014). Functionality of this system in zebrafish was demonstrated by expression of mRFP1tagged CRY2olig, which clustered in vivo upon blue-light stimulation (Asakawa et al, 2019). To study the effects of transactivation response element DNA-binding protein 43 (TDP-43) oligomerization in amyotrophic lateral sclerosis in zebrafish, the zebrafish tardp gene was inserted into the mRFP1-CRY2olig construct and named opTDP-43z.…”