1. Generation of dynactin p150glued conditional KO mice Since p150glued homozygous KO mice are early embryonic lethal, we decided to generate p150glued conditional KO mice in order to study the function of p150glued in postmitotic neurons. Mouse p150glued protein is encoded by the Dctn1 gene at chromosome 6. A 9.3 kb genomic DNA fragment carrying exons 28 of Dctn1 gene was isolated for genetic modifications. Briefly, a 1.4 kb genomic DNA fragment containing Dctn1 exons 2 to 4 was inserted between two tandem repeat loxP sites in order to build the conditional KO targeting vector. The targeted ES cell selection marker, neomycin (neo) resistance gene flanked with two FRT sites, was then inserted between the first loxP site and exon 2. The targeting vector was linearized and transfected into 129/SvJ ES cells, which were later subjected to G418 selection. The G418 resistant ES clones were picked and screened by Southern blot analysis for the correctly targeted clones. Two positive ES clones were expanded and injected into blastocysts, and the resulting male chimera mice were bred with WT C57BL/6J female mice to obtain Dctn1+/loxP-FRT-neo-FRT-ex2&4-loxP mice. These Dctn1+/loxP-FRT-neo-FRT-ex2&4-loxP mice were then crossed with FLP Tg mice 129S4/SvJaeSor-Gt(ROSA)26Sortm1(FLP1)Dym/J to remove the FRT-neo-FRT cassette. The resulting Dctn1+/loxP-ex2&4-loxP (Dctn1+/loxP) animals were intercrossed to generate homozygous Dctn1loxP/loxP animals. When Dctn1loxP/loxP mice were crossed with a line of inducible Cre Tg mice B6.Cg-Tg (CAG-cre/Esr1)5Amc/J, we turned off the expression of p150glued in the one month old creEsr1/Dctn1loxP/loxP mice by administrating the animals tamoxifen, which triggered the activation of Cre recombinase and deleted the exons 2-4 of Dctn1 gene. Western blot analyses demonstrated a nearly complete elimination of p150glued protein expression in brain tissues. Concomitantly, the expression level of alternative spliced dynactin p135 protein was up-regulated in p150glued conditional KO mouse brains. Interestingly, the p150glued conditional KO mice appeared normal and did not displayed any obvious behavioral phenotypes up to 18 months of age. These results indicate that p150glued may not perform any critical physiological functions in adult animals. 2. The loss of dynactin p150glued did not affect the complex formation of other dynactin components The vertebrate dynactin complex has been reported to migrate as a 19S protein heteromultimer resolved by 520% sucrose gradient sedimentation. To test the integrity of the dynactin complex in p150glued conditional KO mice, we performed sedimentation analysis on a 520% sucrose gradient using brain extracts from WT and KO mice. We found that the migration patterns of dynactin p135, p50, and Arp1 as well as DIC from p150glued conditional KO brains were the same as those from WT controls, indicating that the deletion of p150glued does not impair the formation of remaining dynactin complex with DIC. 3. The loss of dynactin p150glued altered the subcellular distribution of dynactin complex in primary cultured fibroblasts Since dynactin p135 mainly exists in neurons, we wondered whether the loss of p150glued may cause severe disruption of remaining dynactin protein complex in non-neuronal cells such as skin fibroblasts (FB). We derived primary cultured FBs from CreEsr1-positive or negative p150glued conditional mice and treated these cells with tamoxifen for four days. The presence of p150glued was almost completely abolished in tamoxifen-treated CreEsr1-positive p150glued conditional KO FBs. However, sucrose gradient sedimentation experiments revealed the same migration patterns of dynactin p50 and Arp1 as well as DIC from p150glued conditional KO FBs as cells from CreEsr1-negative controls. These observations are consistent with our earlier findings in brain tissues. We then examined the subcellular distribution of dynactin proteins in p150glued conditional KO FBs. In control CreEsr1-negative FBs, p50 and Arp1 staining typically presented as large patches adjacent to the nucleus By contrast, p50 and Arp1 staining in CreEsr1-positive FBs was evenly distributed in the entire intracellular space as small clusters. These results indicate that p150glued may serve as anchor points for perinuclear localization of the dynactin protein complex. 4. Generation of tetO-dynactin p150glued Tg mice We previously generated G59S p150glued knock-in mice to model an autosomal familial form of lower motor neuron disease. Interestingly, three more missense mutations of G71A, G71R, and Q74P have been recently identified in the same CAP-Gly domain of p150glued that cause Perry syndrome. To study the pathogenic mechanism of p150glued Perry mutations in the degeneration of nigrostriatal DA neurons, we decided to generate G71R p150glued inducible Tg mice that selectively express human G71R p150glued in the midbrain DA neurons. We have generated multiple founders of tetO-G71R p150glued Tg mice. As controls, tetO-WT p150glued and tetO-G59S p150glued Tg founder mice were also generated. At present, we are crossbreeding these tetO-p150glued mice with PITX3-IRES-tTA mice to drive the expression of WT, G71R or G59S p150glued in midbrain DA neurons. The redistribution of dynactin family proteins in p150glued-deficent FBs may reflect a disengagement of dynactin complex with dynein-mediated centripetal transport or a loss of p150glued as the anchor points for the attachment of dynactin proteins to the minus end of microtubules. Future studies will be planned to test these two possibilities. We will examine the dynein/dynactin-dependent retrograde transport of lysosomes and endosomes in p150glued conditional KO FBs by live imaging techniques, and also check the targeting of p150glued-lacking dynactin complex to the minus end of microtubule assemblies by co-immunostaining with gamma-tubulin and other microtubule minus end binding proteins. More importantly, we are going to evaluate how the loss of p150glued impacts the function of neurons. We speculate that p150glued may affect the ER-to-Golgi transport in neurons, and will test this hypothesis in primary cultured neurons prepared from p150glued conditional KO mice.
|Aliaga, Leonardo; Lai, Chen; Yu, Jia et al. (2013) Amyotrophic lateral sclerosis-related VAPB P56S mutation differentially affects the function and survival of corticospinal and spinal motor neurons. Hum Mol Genet 22:4293-305|