, 2007), suggesting that most cells recognize the decreased stability of the GSK126 protein and can effectively target the polypeptide for degradation. However, motor neurons may not effectively induce a stress response to protein misfolding (Batulan et al., 2003), leaving them vulnerable to the dominant-negative effect of the G59S mutation. In contrast, the distinct morphology of dopaminergic neurons may make these cells uniquely vulnerable to defects in the initiation of retrograde transport. The immense axonal arborizations of dopaminergic neurons (Matsuda et al., 2009) suggest that the loss-of-function effects of the Perry mutations may critically

affect this cell type. Thus, our data inform normal dynein-dynactin function as well as the selective vulnerabilities of discrete populations of neurons to specific perturbations in the cellular function of these proteins. The two distinct mechanisms we propose for the pathogenesis of HMN7B and Perry syndrome highlight the specialized function of a single domain of dynactin and provide a model for the function of the CAP-Gly domain of p150Glued in neurons. Dorsal root ganglia (DRGs) were dissected from adult mice less than 1 year old and treated with 20 U/ml papain, followed by

2 mg/ml collagenase II and 2.4 mg/ml dispase II. Neurons were then dissociated in HBSS, supplemented with 5 mM HEPES and 10 mM D-glucose (pH 7.35), and purified through a 20% Percoll gradient for 8 min at 1,000 × g. DRG neurons were transfected with either HDAC inhibitor DNA, siRNAs B3GAT3 or both using the basic neuron SCN nucleofector kit (Lonza) and then plated onto 0.01% poly-L-lysine and 20 μg/ml laminin coated coverslips or glass-bottom dishes (FluoroDish, World Precision Instruments) and grown for 2–4 days in F-12 medium supplemented with 10% heat-inactivated FBS and 100 U/ml penicillin-streptomycin.

Live-cell imaging was done in Hibernate-A (Brain Bits, Springfield IL) supplemented with 2% B27 supplement (Invitrogen) and 2 mM GlutaMax (GIBCO). All experiments involving animals were approved by the IACUC at the University of Pennsylvania. Images of LAMP1-RFP motility (Figures 1 and 7) were acquired 366 ms per frame for 360 frames. Images were acquired in epifluorescence on an inverted Leica DMI-6000/CTR-7000HS microscope with an Apochromat 63× 1.4 NA oil-immersion objective in a temperature-controlled chamber (37°C) with an ORCA R2 (Hamamatsu) camera using LAS-AF (Leica) software. Fixed cells were imaged in epifluorescence, as described above. Neurons were imaged with a 1.6× magnifier. Photobleaching of LAMP1-RFP (Figures 5A, 8C, and S4) and FRAP of EGFP or EGFP-p150Glued (Figure 4A) was performed in a temperature-controlled chamber (37°C) using the 561 nm or 488 nm laser, respectively, at 100% power for 25 cycles on the Ultraview Vox (PerkinElmer) spinning disk confocal system with an Ultraview Photokinesis (PerkinElmer) unit on an inverted Nikon Ti microscope with apochromat 100× 1.