A mouse forward genetics screen identifies INAUGURAL ARTICLE LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration Jessie Chua,1, Nancy A. Hongb,2, Claudio A. Masudac,3, Brian V. Jenkinsa, Keats A. Nelmsd, Christopher C. Goodnowd, Richard J. Glynnec, Hua Wub,4, Eliezer Masliahe, Claudio A. P. Joazeiroc,5, and Steve A. Kaya,6,7 aDepartment of Biochemistry, Institute for Childhood and Neglected Diseases, The Scripps Research Institute, ICND216, 10550 North Torrey Pines Road, La Jolla, CA 90237; bPhenomix Corporation, 5871 Oberlin Drive, Suite 200, San Diego, CA 92121; cGenomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121; dPhenomix Australia, Pty., Ltd., Level 3 Building 117, Australian Phenomics Facility, Garran Road, Acton, ACT 2601, Australia; and eDepartment of Neurosciences, University of California San Diego, School of Medicine, La Jolla, CA 92093 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2008. Contributed by Steve A. Kay, December 19, 2008 (sent for review November 13, 2008) A mouse neurological mutant, lister, was identified through a ENU generated a rat model for human Usher syndrome type 1B genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen. (9). Further, ENU-induced mutation in dynein led to progressive Homozygous lister mice exhibit profound early-onset and progres- motor neuron degeneration in mice (10). Although no human sive neurological and motor dysfunction. lister encodes a RING disease has yet been mapped to dynein itself, mutation in the finger protein, LISTERIN, which functions as an E3 ubiquitin ligase dynein activator, dynactin, causes degeneration of lower motor in vitro. Although lister is widely expressed in all tissues, motor and neurons in humans (11). Thus, the ENU dynein model correctly sensory neurons and neuronal processes in the brainstem and predicted that errors in a particular machinery would give rise to spinal cord are primarily affected in the mutant. Pathological signs human disease. include gliosis, dystrophic neurites, vacuolated mitochondria, and Here, we report the identification and characterization of a accumulation of soluble hyperphosphorylated tau. Analysis with a mutant mouse model of neurodegeneration, and cloning of the different lister allele generated through targeted gene trap inser- affected gene, lister. Histological analysis of lister mutants shows tion reveals LISTERIN is required for embryonic development and several pathological biomarkers seen in human neurodegenera- confirms that direct perturbation of a LISTERIN-regulated process tive diseases. The lister mouse uncovers a pathway involved in causes neurodegeneration. The lister mouse uncovers a pathway neurodegeneration and may serves as a model for understanding involved in neurodegeneration and may serves as a model for the molecular mechanisms underlying human neurodegenera- understanding the molecular mechanisms underlying human neu- tive disorders. rodegenerative disorders. Results Through ENU mutagenesis screening, we have identified a eurodegenerative disorders are a group of diseases with recessive mutation that manifests as a progressive movement distinct etiology and molecular bases. These diseases pre- N disorder. Newborn homozygous mutants are found with the dominately occur as sporadic cases, and the causes are complex expected Mendelian ratio, and although initially show no sig- and multifactorial. Nevertheless, causative mutations for some nificant differences in general appearance or body weight, these cases of neurodegenerative disorders, such as Alzheimer’s dis- animals exhibit age-dependent and often asymmetrical progres- ease (AD), Amyotrophic Lateral Sclerosis (ALS) and Parkin- sive weakness of hind limbs, bradykinesia and eventually loss of son’s disease (PD) have been identified. Importantly, some of locomotor ability (Movie S1). Because of the ‘‘tilting’’ or ‘‘list- the linked genes are also involved prominently in the pathology ing’’ phenotype, we have named the mutant ‘‘lister.’’ One of the of sporadic forms of these diseases. For example, mutation in ␣ ␣ earliest neurological symptoms apparent in homozygous lister -synuclein results in early onset familial PD (1), and -synuclein mice is the loss of hind limb extension reflex, which is charac- is a major component of Lewy bodies and of Lewy neuritic pathology in both familial PD and sporadic PD (1–4). Identifi- cation of causative genes for neurodegenerative disorders also Author contributions: J.C., N.A.H., C.A.M., C.A.P.J., and S.A.K. designed research; J.C., allows the development of genetic mouse models that are C.A.M., B.V.J., and H.W. performed research; K.A.N., C.C.G., R.J.G., and E.M. contributed valuable tools for providing insights into the pathogenesis and new reagents/analytic tools; J.C., N.A.H., C.A.M., B.V.J., and S.A.K. analyzed data; and J.C., NEUROSCIENCE N.A.H., C.A.P.J., and S.A.K. wrote the paper. molecular pathways leading to degeneration. To date, the genetic or molecular basis of the majority of The authors declare no conflict of interest. neurological disorders remains largely undetermined (5–7). For 1Present address: La Jolla Laboratories, Pfizer Inc, 10646 Science Center Drive, San Diego, CA 92121. example, Cu/Zn Superoxide dismutase (SOD1) mutations ac- Ϸ 2Present address: Forward Ventures, 9393 Towne Centre Drive, Suite 200, San Diego, count for only 2% of all ALS patients (6). To identify other CA 92121. causal genes or risk factors involved in neurodegeneration, we 3Present address: Instituto de Bioquímica Me´dica, Programa de Biologia Molecular e performed a forward genetic screen, using ENU-mediated ran- Biotecnologia, Universidade Federal do Rio de Janeiro, 21941–590, Rio de Janeiro, Brazil. dom mutagenesis. ENU is a powerful DNA alkylating agent in 4Present address: Novartis Institute for Biomedical Research, Inc., Cambridge, MA 02139. mouse spermatogonial germ cells. Because ENU primarily in- 5Present address: Department of Cell Biology, CB168, The Scripps Research Institute, 10550 duces DNA point mutations that can result in partial loss- or North Torrey Pines Road, La Jolla, CA 90237. gain-of-function phenotypes that predominate in human dis- 6Present address: Division of Biological Science, UCSD, 9500 Gilman Drive, La Jolla, eases (8), this strategy for identifying mouse disease models is CA 92093. potentially more productive for modeling human neurodegen- 7To whom correspondence should be addressed. E-mail: [email protected]. erative disorders than traditional gene-targeting approaches in This article contains supporting information online at www.pnas.org/cgi/content/full/ mice. Indeed, this approach has generated many models of 0812819106/DCSupplemental. human disease. For example, mutation in Myo7a induced by © 2009 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812819106 PNAS ͉ February 17, 2009 ͉ vol. 106 ͉ no. 7 ͉ 2097–2103 Downloaded by guest on September 24, 2021 Fig. 1. Progressive impairment of neuronal and motor functions associated with weight loss and reduced life span in lister mutant mice. (A) Loss of hind limb extension reflex in lister/lister mutant manifested by hind limb clenching when lifted by the tail. (B) Motor function of lister/lister mutants (white bars, n ϭ 8) compared with heterozygous (gray bars, n ϭ 12) and wild-type littermates (black bars, n ϭ 12) measured by accelerated rotarod test. ϩ/ϩ versus lister/lister mice: P Ͻ 0.0005 at 3 weeks of age, P Ͻ 0.02 at 6 weeks of age, P Ͻ 0.0005 at 9 weeks of age. lister/ϩ (n ϭ 12) versus ϩ/ϩ mice (n ϭ 10): P ϭ 0.127 at 63 weeks of age. (C) Survival curve of homozygous lister mutants (white circles, n ϭ 25) compared with heterozygous and wild-type littermates (black circles, n ϭ 25). (D) Body weight curve for ϩ/ϩ (black circles, n ϭ 14), lister/ϩ (gray circles, n ϭ 32) and lister/lister mice (white circles, n ϭ 14). terized by spasmodic grasping movements and flexing of the hind starting from intermediate stage (Ϸ6 weeks of age) (Fig. S2 limbs (Fig. 1A). The onset and progression of the symptoms A–C). Mild astrogliosis is also seen in thalamus and hypothala- varies among individual animals but consistently worsens over mus (data not shown). Along the spinal cord, GFAP-reactive time when compared with age-matched wild-type or heterozy- astrocytes are first detected in the ventral horn and intermediate gous animals (Fig. S1A). Balance and motor coordination were zone of lumbar sections, and then found in the thoracic and tested by an accelerating rotarod paradigm. lister/lister mice have cervical sections as disease progresses, indicating neuronal in- markedly reduced performance by 3 weeks of age, the earliest jury in these areas (Fig. 2 A and B). Gallyas silver staining time at which a reliable behavioral assessment could be per- revealed dystrophic neurites in lister/lister brainstems and spinal formed. Heterozygotes remain indistinguishable from wild-type cords (Fig. 2 C and D). These silver-positive structures occur mice for up to a year (Fig. 1B). The progression of motor deficits after phenotype onset and increase in density as disease was also monitored by a righting reflex test (Fig. S1B) and grip progresses. Another neuropathology observed in both brain and strength test (data not shown). In combination, these assays spinal cord of symptomatic homozygous lister mice are hyper- demonstrate an age-dependent decrease in motor function of phosphorylated forms of the microtubule-associated protein, lister homozygotes that progresses until a terminal stage gener- tau, as recognized by phosphorylation-specific antibodies AT-8 ally between 2 to 3 months of age (Fig. 1C). There is no clear and AT-180 (Fig. 2E Left). Both phospho-antibody reactivity gender effect with respect to onset of symptoms or lifespan (data and protein migration were altered upon treatment with phos- not shown). Starting at Ϸ3 weeks of age, lister/lister mice appear phatase (Fig.