DIV10 WT neuron. As lifelong interphase cells, neurons face an array of unique challenges. A key challenge is regulating nuclear pore complex (NPC) biogenesis and localization, the mechanisms of which are largely unknown. Here, we identify neuronal maturation as a period of strongly upregulated NPC biogenesis. We demonstrate that the AAA+ protein torsinA, whose dysfunction causes the neurodevelopmental movement disorder DYT-TOR1A (DYT1) dystonia, coordinates NPC spatial organization without impacting total NPC density. We generated an endogenous Nup107-HaloTag mouse line to directly visualize NPC organization in developing neurons and find that torsinA is essential for proper NPC localization. In the absence of torsinA, the inner nuclear membrane buds excessively at sites of mislocalized nascent NPCs, and formation of complete NPCs is delayed. Our work demonstrates that NPC spatial organization and number are independently determined and identifies NPC biogenesis as a process vulnerable to neurodevelopmental disease insults.
Primary Cell Culture. Brains were isolated from P0 pups into ice cold, filtered dissection buffer (6.85 mM sodium chloride, 0.27mM potassium chloride, 0.0085mM sodium phosphate dibasic anhydrous, 0.011mM potassium phosphate monobasic anhydrous, 33.3mM D-glucose, 43.8mM sucrose, 0.277mM HEPES, pH 7.4). After removing the cerebellum and the meninges, cortices were dissected out, placed into a microcentrifuge tube, cut into small pieces with dissection forceps, and incubated in 50µL papain (2mg/mL; BrainBits) and 10µL DNase I (1mg/mL; Worthington Biochemical) for 30min at 37 °C. 500µL BrainPhys Neuronal Medium (Stemcell Technologies) and 10µL additional DNase I were added, and cortices were triturated using P1000 and P200 pipet tips, then centrifuged at 125xg for 5min. Supernatant was discarded and pellets were triturated and centrifuged three more times. Pelleted neurons were resuspended in BrainPhys Neuronal medium with SM1 supplement, then plated onto 35mm #1.5 glass-bottom dishes (MatTek Life Sciences) or 12mm circular #1.5 coverslips (Neuvitro) coated with polyethylenimine (100 µg/ml; Polysciences). Neurons were incubated in 5% CO2 at 37 °C, with half media change every four days.
