Motor Neuron Disease Occurring in a Mutant Dynactin Mouse Model Is Characterized by Defects in Vesicular Trafficking

The Journal of Neuroscience, February 27, 2008 • 28(9):1997–2005 • 1997 Neurobiology of Disease Motor Neuron Disease Occurring in a Mutant Dynactin Mouse Model Is Characterized by Defects in Vesicular Trafficking Fiona M. Laird,1,4* Mohamed H. Farah,1,4* Steven Ackerley,1,4*† Ahmet Hoke,2,3 Nicholas Maragakis,3 Jeffrey D. Rothstein,2,3 John Griffin,3 Donald L. Price,1,2,3,4 Lee J. Martin,1,2,4 and Philip C. Wong1,2,4 Departments of 1Pathology, 2Neuroscience, and 3Neurology, and 4Division of Neuropathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Amyotrophic lateral sclerosis (ALS), a fatal and progressive neurodegenerative disorder characterized by weakness, muscle atrophy, and spasticity, is the most common adult-onset motor neuron disease. Although the majority of ALS cases are sporadic, ϳ5–10% are familial, including those linked to mutations in SOD1 (Cu/Zn superoxide dismutase). Missense mutations in a dynactin gene (DCTN1) encoding the p150 Glued subunit of dynactin have been linked to both familial and sporadic ALS. To determine the molecular mechanism whereby mutant dynactin p150 Glued causes selective degeneration of motor neurons, we generated and characterized mice expressing either wild-type or mutant human dynactin p150 Glued. Neuronal expression of mutant, but not wild type, dynactin p150 Glued causes motor neuron disease in these animals that are characterized by defects in vesicular transport in cell bodies of motor neurons, axonal swelling and axo-terminal degeneration. Importantly, we provide evidence that autophagic cell death is implicated in the pathogenesis of mutant p150 Glued mice. This novel mouse model will be instrumental for not only clarifying disease mechanisms in ALS, but also for testing therapeutic strategies to ameliorate this devastating disease. Key words: dynactin; p150 Glued; ALS; motor neuron disease model; vesicular trafficking; autophagy Introduction ties of axons in the skin. Since this first report, additional muta- A family with a slowly progressive autosomal dominant lower tions in the dynactin gene (DCTN1) have been associated with motor neuron disease has been linked to a mutation in the motor neuron disease (Munch et al., 2004). p150 Glued subunit (G59S) of the dynactin complex (Puls et al., Dynactin p150 Glued is a critical component of a multiprotein 2003). This disease begins in early adult life with vocal cord pa- complex that differentially copurifies with dynein (Gill et al., ralysis (associated with breathing difficulties), facial weakness, 1991; Schroer and Sheetz, 1991; Schroer, 2004), the molecular and atrophy of muscles in the hands (Puls et al., 2003, 2005). motor required for fast retrograde transport of vesicles, or- Subsequently, weakness and atrophy appear in the distal lower ganelles, RNAs and proteins along microtubules (Schroer, 2004; extremities. There is evidence of degeneration of motor axons, Hirokawa and Takemura, 2005; Price et al., 2005). Although dy- and reduced numbers of motor neurons in the spinal cord and nactin has been postulated to enhance the processivity and effi- hypoglossal nucleus (Puls et al., 2005). Significantly, inclusions ciency of the dynein motor (LaMonte et al., 2002; Schroer, 2004), containing components of dynactin and dynein are evident in previous studies demonstrate in vitro that the dynein-dynactin motor neurons (Puls et al., 2005). Although no sensory deficits complexes exhibit bidirectional processive motility (Ross et al., are reported, examinations of skin biopsies disclose abnormali- 2006) and in vivo that dynactin is required for coordinated an- terograde and retrograde organelle movement (Haghnia et al., 2007). The disruption of the dynactin complex by overexpression Received Sept. 14, 2007; revised Dec. 13, 2007; accepted Jan. 6, 2008. of dynamitin (LaMonte et al., 2002) results in motor neuron This work was supported in part by The Muscular Dystrophy Association, The Robert Packard Center for Amyo- trophic Lateral Sclerosis Research, and National Institute of Neurological Disorder and Stroke Grant RO1 NS40014 disease in mice, but the exact pathogenic mechanism remains (P.C.W.). The Thy1.2 promoter was a gift from P. Caroni. We thank V. Nehus, Y. Pan, D. Prasad, Ashley Moon, and M. unknown. Bidirectional transport of vesicles between the endo- Polley for their technical support, and Drs. H. Cai, A. Ludolph, A. Kolodkin, and T. Lloyd for sharing of unpublished plasmic reticulum (ER) and Golgi apparatus have also been results. Our coauthor and friend, Dr. Steven Ackerley, passed away suddenly on January 14, 2008. With deep sorrow shown to involve dynein/dynactin mediated transport (Lee et al., and mourning, we dedicate this paper to his memory. *F.M.L., M.H.F., and S.A. contributed equally to this work. 2004). Disruption of dynein function also impairs clearance of †Deceased, Jan. 14, 2008. aggregate prone proteins by autophagy (Ravikumar et al., 2005), Correspondence should be addressed to Philip C. Wong, Department of Pathology, The Johns Hopkins University a lysosomal pathway for degrading damaged organelles and ag- School of Medicine, 558 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205-2196. E-mail: gregated proteins within the cell. Autophagy has been implicated [email protected]. DOI:10.1523/JNEUROSCI.4231-07.2008 in a number of neurodegenerative disorders, but considerable Copyright © 2008 Society for Neuroscience 0270-6474/08/281997-09$15.00/0 debate exists regarding the role of autophagy in pathogenesis of 1998 • J. Neurosci., February 27, 2008 • 28(9):1997–2005 Laird et al. • A Mutant Dynactin Mouse Model of Motor Neuron Disease specific diseases (Nixon, 2006). The dem- onstration that genetic deletions of autophagy genes in neurons are sufficient to cause neurodegeneration supports the view that disruption of basal autophagy participates in pathogenesis of some of these illnesses (Hara et al., 2006; Komatsu et al., 2006). Therefore, given the numerous roles that dynactin plays in intracellular and axonal transport, it is conceivable that alter- ation in dynactin function could impact on several critical cellular processes within various compartments of the motor neuron. Here, we document that a novel mouse model expressing human mutant p150 Glued dynactin exhibits clinical and pathological hallmarks of motor neuron disease. Importantly, we show that motor neuron degeneration in these mutant dynactin mice is associated with abnormalities in intracellular vesicular trafficking, axonal swelling and axo-terminal degeneration. Moreover, autophagic cell death is implicated in the pathogenesis of disease in mutant p150 Glued mice. Materials and Methods Generation of transgenic mice. We first engi- neered the G59S amyotrophic lateral sclerosis (ALS)-linked mutation (Puls et al., 2003) into the human dynactin p150Glued cDNA by PCR/ oligonucleotide primer-directed mutagenesis strategy. In addition, the full-length human cDNA encoding wild-type and mutant dynactin p150 Glued were modified to include a C-terminal epitope tag (FLAG) to facilitate identification of the transgenic product. Be- cause previous studies show that the Thy1.2 cassette works well for generation of mice with neuronal expression and was the strategy used for Figure 1. Motor neuron disease in mice expressing mutant but not wild-type p150 Glued. A, Schematic drawing of the con- the overexpression of dynactin p50 (LaMonte et structusedtogeneratetransgenicmice.Humanwild-typeormutantp150 Glued cDNAwithaFLAGtag(indicatedbyastripedbox) al., 2002), the resulting constructs were sub- were cloned into the Thy1.2 expression cassette at the XhoI site. B, Accumulation of mutant G59S dynactin in mice. Total protein cloned into the XhoI site of the mouse Thy1.2 (20 ␮g) extracted from nontransgenic, wild-type, and mutant dynactin mice were electrophoresed on 6% SDS-PAGE gels and expression cassette to generate expression con- immunoblotted using a monoclonal antibody recognizing the p150 Glued (top) or the FLAG (middle) epitope. Identical blot was structs, one encoding wild-type dynactin reprobed with antisera against actin (bottom). C, Kaplan-Meier plot of the onset of tremor for two mutant lines, M1 (gray line) p150 glued (Thy1.2p150WT), and the other G59S Ϯ Ϯ glued and M2 (black line). The average onset of tremor for M1 is 4.8 1.2 months and for M2 is 6.2 1.3months. D, Onset of the dynactin p150 (Thy1.2p150G59S). These reduction in grip strength for line M1 is plotted on a Kaplan-Meier graph. The black and gray dotted lines denote forelimb and vectors were initially tested in cultured cells by hindlimb grip strength, respectively. E, Kaplan-Meier plot for survival of two mutant lines; line M1 (gray) has a shorter lifespan transient transfection to verify their expression than line M2 (black). (data not shown). Subsequently, the purified in- serts from these vectors were submitted to the Indianapolis, IN). Protein extracts were sonicated before protein assay Transgenic Facility at Johns Hopkins University School of Medicine for and protein blot analysis. 20 ␮g of total protein extracts were loaded onto injection into C57BL/6;SJL hybrid mouse embryos. Positive founders a 6 or 16% Tris-glycine gel (Invitrogen, San Diego, CA) and transferred were identified by Southern blot analysis and two lines of mutant and glued to polyvinylidene difluoride membrane (Invitrogen). Immunoblotting three lines of wild-type dynactin p150 were established. F1 offspring, was performed using primary antibodies to p150

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