A previous study suggests that the G59S substitution occurring in p150glued partially compromises the association of p150glued with microtubules in an in vitro assay. However, how this mutation affects the function of the dynein/dynactin complex and contributes to motor neuron degeneration is unclear. To further study the pathobiological mechanism of this type of motor neuron disease, our specific aims are:? Aim 1: To generate and characterize p150glued G59S knock-in mice.? Aim 2: To examine any alterations in axonal transport of spinal motor neurons derived from p150glued G59S knock-in mice.? Aim 3: To further investigate the role of p150glued in dynein-mediated retrograde transport. ? ? To study how the G59S mutation in dynactin p150glued affects the function of the dynein/dynactin complex and contributes to motor neuron degeneration, we generated p150glued G59S knock-in mice. We found that the G59S mutation destabilizes p150glued and disrupts the function of dynein/dynactin complex, resulting in early embryonic lethality of homozygous knock-in mice. Heterozygous knock-in mice, which developed normally, displayed MND-like phenotypes after 10 months of age, including excessive accumulation of cytoskeletal and synaptic vesicle proteins at neuromuscular junctions, loss of spinal motor neurons, increase of reactive astrogliosis, and shortening of gait compared with wild-type littermates and age-matched p150glued heterozygous knockout mice. Our findings indicate that the G59S mutation in p150glued abrogates the normal function of p150glued and accelerates motor neuron degeneration. This work has been published by the Journal of Neuroscience (Lai et al., 2007), and highlighted in the This Week in the Journal (TWIJ) by the Journal of Neuroscience.? ? Because the loss of dynactin p150glued leads to early embryonic lethality, we decided to generate dynactin p150glued conditional knockout (CKO) mice to investigator whether dynactin p150glued is required in dynein-mediated retrograde transport. To generated dynactin p150glued CKO mice, we modified the DCTN1 gene locus by flanking the exons 2-4 of DCTN1 gene with a pair of Loxp sites. To genetically inhibit the expression of dynactin p150glued at different developmental stages of neurons, we then crossed the dynactin p150glued CKO mice with three different lines of Cre transgenic mice in which the Cre recombinase is under the control of nestin, CaMKII, and chick β-actin (CAG) promoters, respectively. For the CAG-Cre transgenic mice, the transgene insert contains a fusion product involving Cre recombinase and a mutant form of the mouse estrogen receptor (Esr1) ligand binding domain. The mutant mouse estrogen receptor does not bind natural ligand at physiological concentrations but will bind the synthetic ligand, 4-hydroxytamoxifen. Restricted to the cytoplasm, the Cre/Esr1 protein can only gain access to the nuclear compartment after exposure to tamoxifen. A systematic analysis of neuropathological and behavioral phenotypes of these mutant mice will allow us to reveal the role of dynactin p150glued in neuron differentiation, migration, synaptogenesis, and survival. We have successfully deleted dynactin p150glued from adult mice using the Cre/Esr1 system. To our surprise, inhibition of dynactin p150glued in adult mice does not cause any overt phenotypes. It appears that p135, an alternative translational variant of dynactin p150glued may compensate for the loss of dynactin p150glued. Whether the loss of dynactin p150glued affects the neuron development remains to be investigated. ? ? ? Our present study demonstrates for the first time that a mouse model carrying a motor neuron disease (MND)-linked G59S substitution in the dynactin p150glued subunit develops many symptoms related to ALS and MND, such as motor neuron degeneration, reactive astrogliosis, and abnormal gait. Because Dctn1+/m mice contain one copy of wild-type allele and one copy of G59S mutant allele, it faithfully replicates the genetic mutation in humans and may serve as a useful animal model for studying the pathogenic mechanism of MND and testing potential therapeutics. We provided evidence to further demonstrate that p150glued is required for the cellular functions of cytoplasmic dynein. Dctn1m/m and Dctn1-/- mice died before 8.5 dpc, similar to cytoplasmic dynein heavy chain knockout mice. The G59S mutation in p150glued does not affect the integrity of the dynein/dynactin complex, except for inducing self-aggregation when over-expressed in cell lines. In fact, we observed around a 50% reduction of p150glued in Dctn1+/m mouse brains and failed to detect any p150glued from Dctn1m/m mouse embryos, suggesting that the G59S mutation in p150glued leads to a rapid degradation of mutant p150glued. Because age-matched Dctn1+/- mice did not develop any of these neuropathological abnormalities as observed in Dctn1+/m mice, our data indicate that a 50% reduction of p150glued alone is not sufficient to cause motor neuron degeneration at the age having been examined. Neither does the increase of p135 account for the motor behavioral and neuropathological deficits observed in Dctn1+/m mice, because a comparable increase of p135 was also found in Dctn1+/- mice. The G59S mutation may exert additional stress to motor neurons through further disruption of the function of dynein/dynactin complex. A conditional p150glued knockout mouse model that selectively removes p150glued from adult motor neurons will be useful to address whether dynein/dynactin-mediated retrograde transport is essential for the survival of motor neurons. In addition, real time imaging of dynein/dynactin-mediated axonal retrograde transport will be employed to investigate potential axonal trafficking defects in both p150glued knock-in and conditional KO mice.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Intramural Research (Z01)
Project #
1Z01AG000946-01
Application #
7732367
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2008
Total Cost
$155,329
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Yu, Jia; Lai, Chen; Shim, Hoon et al. (2018) Genetic ablation of dynactin p150Glued in postnatal neurons causes preferential degeneration of spinal motor neurons in aged mice. Mol Neurodegener 13:10
Lai, Chen; Lin, Xian; Chandran, Jayanth et al. (2007) The G59S mutation in p150(glued) causes dysfunction of dynactin in mice. J Neurosci 27:13982-90
Chandran, Jayanth; Ding, Jinhui; Cai, Huaibin (2007) Alsin and the molecular pathways of amyotrophic lateral sclerosis. Mol Neurobiol 36:224-31