Genetic ablation of dynactin p150Glued in postnatal neurons causes preferential degeneration of spinal motor neurons in aged mice

Background: Dynactin p150(Glue)d, the largest subunit of the dynactin macromolecular complex, binds to both microtubules and tubulin dimers through the N-terminal cytoskeleton-associated protein and glycine-rich (CAP-Gly) and basic domains, and serves as an anti-catastrophe factor in stabilizing microtubules in neurons. P150(Glued) also initiates dynein-mediated axonal retrograde transport. Multiple missense mutations at the CAP-Gly domain of p150(Glued) are associated with motor neuron diseases and other neurodegenerative disorders, further supporting the importance of microtubule domains (MTBDs) in p150Glued functions. However, most functional studies were performed in vitro. Whether p150Glued is required for neuronal function and survival in vivo is unknown. Methods: Using Cre-loxP genetic manipulation, we first generated a line of p150(Glued) knock-in mice by inserting two LoxP sites flanking the MTBD-coding exons 2 to 4 of p150(Glued)-encoding Dctn1 gene (Dctn1(LoxP/)), and then crossbred the resulting Dctn1LoxP/ mice with Thy1-Cre mice to generate the bigenic p150(Glued) (Dctn(1LoxP/) LoxP; Thy1-Cre) conditional knockout (cKO) mice for the downstream motor behavioral and neuropathological studies. Results: P150(Glued) expression was completely abolished in Cre-expressing postnatal neurons, including corticospinal motor neurons (CSMNs) and spinal motor neurons (SMNs), while the MTBD-truncated forms remained. P150(Glued) ablation did not affect the formation of dynein/dynactin complex in neurons. The p150(Glue)d cKO mice did not show any obvious developmental phenotypes, but exhibited impairments in motor coordination and rearing after 12 months of age. Around 20% loss of SMNs was found in the lumbar spinal cord of 18-month-old cKO mice, in company with increased gliosis, neuromuscular junction (NMJ) disintegration and muscle atrophy. By contrast, no obvious degeneration of CSMNs, striatal neurons, midbrain dopaminergic neurons, cerebellar granule cells or Purkinje cells was observed. Abnormal accumulation of acetylated a-tubulin, and autophagosome/lysosome proteins was found in the SMNs of aged cKO mice. Additionally, the total and cell surface levels of glutamate receptors were also substantially elevated in the p150(Glued)-depleted spinal neurons, in correlation with increased vulnerability to excitotoxicity. Conclusion: Overall, our findings demonstrate that p150(Glued) is particularly required to maintain the function and survival of SMNs during aging. P150(Glued) may exert its protective function through regulating the transportation of autophagosomes, lysosomes, and postsynaptic glutamate receptors in neurons.