Neurons, particularly large primary projection neurons such as motor neurons, rely heavily on microtubule-based long distance transport of organelles, vesicles, and molecules for their function and survival (63). The intracellular transport of materials across microtubules is mediated by the kinesin or dynein motor protein complex. Defects in intracellular transport are often associated with neurological disorders such as AD, Huntingtons disease, ALS, and likely PD. Protein deposition inside axons may compromise axonal transport and eventually lead to neuronal death. Alternatively, mutations in genes encoding components of motor protein complexes often cause deficits in axonal transport and neurological disorders (64). For example, two missense mutations in the cytoplasmic dynein heavy chain have been shown to cause decreased axonal retrograde transport and neuron degeneration in chemically induced mutant mice (65). Over-expression of dynamitin, the p50 subunit of the dynein/dynactin motor complex, disrupts the dynein/dynactin complex and produces a late-onset, progressive motor neuron degeneration in transgenic mice (20). A missense mutation in dynactin p150glued (DCTN1) has been linked to a slowly progressive, autosomal dominant form of lower motor neuron disease without sensory symptoms (10;66). The mutation, a single-basepair change in the p150glued gene (957C-T), results in an amino acid substitution of serine (S) for glycine (G) at position 59 (G59S) at the conserved CAP-Gly (cytoskeleton-associated protein, glycine-rich) domain of p150glued protein (Schroer, 2004). Recently, three more missense mutations (G71A, G71R, and Q74P) in dynactin p150glued have been identified as the cause of Perry syndrome, a type of PD with severe mental depression and central hypoventilation (67). P150glued, the p150 subunit of dynactin complex, exists as a dimer that binds directly to both microtubules and cytoplasmic dynein intermediate chain (DIC) (Fig. 8B). Functional expression studies showed that the CAP-Gly domain had a critical role in the initiation and persistence of dynein-dependent movement of the mitotic spindle and nucleus but was otherwise dispensable for dynein-based movement. The function also appeared to be context-dependent, e.g., during mitosis, indicating that CAP-Gly activity may only be necessary when dynein needs to overcome high force thresholds to produce movement. Further evidence for a critical role for p150glued in vivo comes from our study of p150glued KO mice, which died at early embryonic stages (11). Previous studies suggest that the G59S substitution occurring in p150glued partially compromises the association of p150glued with microtubules in an in vitro assay, which may contribute to the degeneration of motor neurons in G59S p150glued knock-in and Tg mice (68). The Perry mutations may cause similar structural alterations of the CAP-Gly domain and the dysfunction of p150glued protein. However, it is very intriguing to imagine how the different missense mutations within the same CAP-Gly domain of p150glued cause a rather selective loss of nigrastriatal DA neurons than spinal motor neurons. Therefore, we are interested in delineating the pathogenic properties of the motor neuron disease and Perry syndrome-causing missense mutations in the CAP-Gly domain of p150glued protein. We plan to generate G59S and G71R p150glued Tg mice that selectively over-express these two mutations in either midbrain DA neurons or spinal motor neurons and cross-examine the progression of midbrain DA and spinal motor neuron degeneration induced by G59S and G71R p150glued. This study will present us a unique opportunity to explore the molecular pathogenic mechanisms of the selective vulnerability typically associated with different types of neurodegenerative diseases. Previous studies indicate that the CAP-Gly domain is critical in the initiation and persistence of dynein-dependent movement of the mitotic spindle and nucleus during cell proliferation. Accordingly, genetic deletion of the CAP-Gly domain of p150glued results in early embryonic lethality of p150glued homozygous KO mice (11). However, the CAP-Gly domain seems dispensable for dynactins activity in postmitotic cells. A replacement of p150glued with p135, which lacks the N-terminal CAP-Gly domain of p150glued, seems to have no effect on the vesicle transport in cells (69). These observations raise the questions about how the G59S and G71R mutations within the CAP-Gly domain affect the function of dynactin in postmitotic neurons. One possible scenario is that the CAP-Gly domain of p150glued may have some specialized activities in neurons. To test this hypothesis, in Aim 1, we will generate and characterize p150glued conditional KO mice that selectively delete the CAP-Gly domain-containing full-length p150glued protein in postmitotic neurons to evaluate its impact on neuron development and degeneration. By crossbreeding p150glued conditional KO mice with various Cre Tg mice, we will be able to delete p150glued in any given type of neurons, such as DA and spinal motor neurons. Perry syndrome is a form of autosomal dominant PD associated with depression, weight loss and central hypoventilation (67). Previous neuropathological studies on autopsy brains of Perry syndrome revealed substantial loss of nigrostriatal DA neurons with no or few Lewy bodies and no tau inclusions. Recently, three missense mutations (G71A, G71R, and Q74P) in the conserved CAP-Gly domain of dynactin p150glued have been identified to cause Perry syndrome. Therefore, in Aim 2, we will generate G71R p150glued Tg mice that selectively express human G71R p150glued in the midbrain DA neurons to investigate how this mutation causes the death of nigrostriatal DA neurons. We will first generate tetO-G71R p150glued Tg mice and then crossbreed them with PITX3-IRES-tTA mice to achieve the selective expression of G71R p150glued in midbrain DA neurons. As controls, we will also develop tetO-WT p150glued mice. As discussed earlier, the G59S mutation in CAP-Gly domain of dynactin p150glued causes a type of slow progressive spinal motor neuron disease (10). Given that both G59S and G71R mutations are located in the CAP-Gly domain of p150glued it is very intriguing to speculate how the G59S mutation only significantly affects the function and survival of spinal motor neurons but not the nigrostriatal DA neurons. Therefore, in Aim 3, we will generate PITX3-IRES-tTA/tetO-G59S p150glued double Tg mice to selectively over-express G59S p150glued in the midbrain DA neurons to cross-examine the progression of DA neuron degeneration with PITX3-IRES-tTA/tetO-G71R p150glued mice. In parallel, we will also generate tetO-dynactin p150glued/Rosa-LNL-tTA/HB9-cre triple Tg mice to selectively express exogenous WT and mutant dynactin p150glued in spinal motor neurons to evaluate the pathogenic properties of G59S and G71R in motor neurons.
