Pathogenic Mechanisms of Dynactin p150glued in ALS and Parkinson's Disease

National Institute On Aging

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Abstract

1. Generation of Dynactin p150glued Conditional KO Mice To study the function of p150glued in postmitotic neurons, we generated p150glued conditional KO mice since complete p150glued homozygous KO mice are embryonic lethal. The Dctn1 gene on chromosome 6 encodes the mouse p150glued protein, and a 9.3 kb genomic DNA fragment containing exons 28 of Dctn1 was used for genetic modifications. To create the conditional KO targeting vector, a 1.4 kb genomic DNA fragment containing Dctn1 exons 2 to 4 was inserted between two loxP sites, and a neomycin (neo) resistance gene flanked with FRT sites was inserted between the first loxP site and exon 2. After linearizing the targeting vector and transfecting it into 129/SvJ ES cells, G418 selection was performed, and correctly targeted ES clones were identified by Southern blot analysis. Positive ES clones were injected into blastocysts, and male chimeras were bred with WT C57BL/6J females to obtain Dctn1+/loxP-FRT-neo-FRT-ex2&4-loxP mice. These mice were then crossed with FLP Tg mice 129S4/SvJaeSor-Gt(ROSA)26Sortm1(FLP1)Dym/J to remove the FRT-neo-FRT cassette, resulting in Dctn1+/loxP-ex2&4-loxP (Dctn1+/loxP) animals. Homozygous Dctn1loxP/loxP mice were generated by intercrossing Dctn1+/loxP animals. To induce the p150glued knockout, tamoxifen was administered to one-month-old CreEsr1/Dctn1loxP/loxP mice, which activated Cre recombinase and deleted exons 2-4 of the Dctn1 gene. Western blot analyses confirmed a nearly complete elimination of p150glued protein expression in brain tissues, with a concurrent up-regulation of the alternative spliced dynactin p135 protein. Interestingly, up to 18 months of age, p150glued conditional KO mice displayed normal behavior and did not exhibit any obvious behavioral phenotypes, suggesting that p150glued may not have critical physiological functions in adult animals. Integrity of the Dynactin Complex in p150glued Conditional KO Mice To assess the integrity of the dynactin complex in p150glued conditional KO mice, sedimentation analysis was performed on a 520% sucrose gradient using brain extracts from both WT and KO mice. The migration patterns of dynactin p135, p50, Arp1, and DIC from p150glued conditional KO brains were identical to those from WT controls, indicating that the deletion of p150glued does not impair the formation of the remaining dynactin complex with DIC. Subcellular Distribution of Dynactin Complex in Primary Cultured Fibroblasts To examine whether the loss of p150glued affects the remaining dynactin protein complex in non-neuronal cells such as skin fibroblasts (FB), primary cultured FBs were derived from CreEsr1-positive or negative p150glued conditional mice and treated with tamoxifen for four days. Tamoxifen-treated CreEsr1-positive p150glued conditional KO FBs showed almost complete abolishment of p150glued presence. However, sedimentation experiments revealed that the migration patterns of dynactin p50, Arp1, and DIC in p150glued conditional KO FBs were similar to those in CreEsr1-negative controls, consistent with the findings in brain tissues. Interestingly, the subcellular distribution of dynactin proteins in CreEsr1-positive FBs differed from that in control FBs, suggesting that p150glued may serve as anchor points for perinuclear localization of the dynactin protein complex. Generation of TetO-Dynactin p150glued Tg Mice To study the pathogenic mechanism of p150glued Perry mutations in the degeneration of nigrostriatal DA neurons, we aimed to generate G71R p150glued inducible Tg mice selectively expressing human G71R p150glued in midbrain DA neurons. Multiple founders of tetO-G71R p150glued Tg mice were successfully generated, and tetO-WT p150glued and tetO-G59S p150glued Tg founder mice were also produced as controls. These tetO-p150glued mice are being crossbred with PITX3-IRES-tTA mice to drive the expression of WT, G71R, or G59S p150glued in midbrain DA neurons. In summary, future studies will further investigate the dynein/dynactin-dependent retrograde transport of lysosomes and endosomes in p150glued conditional KO FBs using live imaging techniques. Additionally, targeting of p150glued-lacking dynactin complex to the minus end of microtubule assemblies will be examined by co-immunostaining with gamma-tubulin and other microtubule minus end binding proteins. Moreover, the impact of p150glued loss on the function of neurons will be evaluated, focusing on the ER-to-Golgi transport in primary cultured neurons prepared from p150glued conditional KO mice. These investigations will provide valuable insights into the role of p150glued in various cellular processes and its relevance to neurodegenerative diseases.