Viral expression of ALS-linked ubiquilin-2 mutants causes inclusion pathology and behavioral deficits in mice

Molecular Neurodegeneration

Nikolaus R. McFarland¹  Todd E. Golde²  Natalia Ravelo¹  Corey Moran²  Nicolas Lara²  Zoe Siemienski²  Pedro E. Cruz²  Amanda Sacino²  Paramita Chakrabarty²  Hyo-Jin Park¹  Awilda M. Rosario²  Carolina Ceballos-Diaz² 

[1] Department of Neurology, College of Medicine, University of Florida, 1149 S Newell Dr, L3-100, Gainesville 32610, FL, USA;Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, 1275 Center Dr, Gainesville 32610, FL, USA

关键词: Mouse model;    Somatic brain transgenesis;    Proteinopathy;    Amyotrophic lateral sclerosis (ALS);    Ubiquilin-2;   

Others  :  1218655
DOI  :  10.1186/s13024-015-0026-7

 received in 2015-02-17, accepted in 2015-06-30,  发布年份 2015

【 摘 要 】

Background

UBQLN2 mutations have recently been associated with familial forms of amyotrophic lateral sclerosis (ALS) and ALS-dementia. UBQLN2 encodes for ubiquilin-2, a member of the ubiquitin-like protein family which facilitates delivery of ubiquitinated proteins to the proteasome for degradation. To study the potential role of ubiquilin-2 in ALS, we used recombinant adeno-associated viral (rAAV) vectors to express UBQLN2 and three of the identified ALS-linked mutants (P497H, P497S, and P506T) in primary neuroglial cultures and in developing neonatal mouse brains.

Results

In primary cultures rAAV2/8-mediated expression of UBQLN2 mutants resulted in inclusion bodies and insoluble aggregates. Intracerebroventricular injection of FVB mice at post-natal day 0 with rAAV2/8 expressing wild type or mutant UBQLN2 resulted in widespread, sustained expression of ubiquilin-2 in brain. In contrast to wild type, mutant UBQLN2 expression induced significant pathology with large neuronal, cytoplasmic inclusions and ubiquilin-2-positive aggregates in surrounding neuropil. Ubiquilin-2 inclusions co-localized with ubiquitin, p62/SQSTM, optineurin, and occasionally TDP-43, but were negative for α-synuclein, neurofilament, tau, and FUS. Mutant UBLQN2 expression also resulted in Thioflavin-S-positive inclusions/aggregates. Mice expressing mutant forms of UBQLN2 variably developed a motor phenotype at 3–4 months, including nonspecific clasping and rotarod deficits.

Conclusions

These findings demonstrate that UBQLN2 mutants (P497H, P497S, and P506T) induce proteinopathy and cause behavioral deficits, supporting a “toxic” gain-of-function, which may contribute to ALS pathology. These data establish also that our rAAV model can be used to rapidly assess the pathological consequences of various UBQLN2 mutations and provides an agile system to further interrogate the molecular mechanisms of ubiquilins in neurodegeneration.

【 授权许可】

   
2015 Ceballos-Diaz et al.

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【 参考文献 】

• [1]Deng HX, Chen W, Hong ST, Boycott KM, Gorrie GH, Siddique N et al.. Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia. Nature. 2011; 477:211-5.
• [2]Synofzik M, Maetzler W, Grehl T, Prudlo J, Vom Hagen JM, Haack T et al.. Screening in ALS and FTD patients reveals 3 novel UBQLN2 mutations outside the PXX domain and a pure FTD phenotype. Neurobiol Aging. 2012; 33:2949 e2913-2947.
• [3]Fahed AC, McDonough B, Gouvion CM, Newell KL, Dure LS, Bebin M, Bick AG, Seidman JG, Harter DH, Seidman CE: UBQLN2 mutation causing heterogeneous X-linked dominant neurodegeneration. Annals of neurology 2014, 75(5):793-798.
• [4]Marin I. The ubiquilin gene family: evolutionary patterns and functional insights. BMC Evol Biol. 2014; 14:63. BioMed Central Full Text
• [5]Rothenberg C, Srinivasan D, Mah L, Kaushik S, Peterhoff CM, Ugolino J et al.. Ubiquilin functions in autophagy and is degraded by chaperone-mediated autophagy. Hum Mol Genet. 2010; 19:3219-32.
• [6]Lim PJ, Danner R, Liang J, Doong H, Harman C, Srinivasan D et al.. Ubiquilin and p97/VCP bind erasin, forming a complex involved in ERAD. J Cell Biol. 2009; 187:201-17.
• [7]Kim TY, Kim E, Yoon SK, Yoon JB. Herp enhances ER-associated protein degradation by recruiting ubiquilins. Biochem Biophys Res Commun. 2008; 369:741-6.
• [8]Mizusawa H, Nakamura H, Wakayama I, Yen SH, Hirano A. Skein-like inclusions in the anterior horn cells in motor neuron disease. J Neurol Sci. 1991; 105:14-21.
• [9]Kiernan MC, Vucic S, Cheah BC, Turner MR, Eisen A, Hardiman O et al.. Amyotrophic lateral sclerosis. Lancet. 2011; 377:942-55.
• [10]Gorrie GH, Fecto F, Radzicki D, Weiss C, Shi Y, Dong H, Zhai H, Fu R, Liu E, Li S, et al: Dendritic spinopathy in transgenic mice expressing ALS/dementia-linked mutant UBQLN2. Proceedings of the National Academy of Sciences of the United States of America 2014, 111(40):14524-14529.
• [11]Wu Q, Liu M, Huang C, Liu X, Huang B, Li N, Zhou H, Xia XG: Pathogenic Ubqln2 gains toxic properties to induce neuron death. Acta neuropathologica 2014, 129(3):417-428.
• [12]Kim J, Miller VM, Levites Y, West KJ, Zwizinski CW, Moore BD et al.. BRI2 (ITM2b) inhibits Abeta deposition in vivo. J Neurosci. 2008; 28:6030-6.
• [13]Chakrabarty P, Rosario A, Cruz P, Siemienski Z, Ceballos-Diaz C, Crosby K et al.. Capsid Serotype and Timing of Injection Determines AAV Transduction in the Neonatal Mice Brain. PLoS One. 2013; 8:e67680.
• [14]Fecto F, Yan J, Vemula SP, Liu E, Yang Y, Chen W et al.. SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis. Arch Neurol. 2011; 68:1440-6.
• [15]Sacino AN, Brooks M, Thomas MA, McKinney AB, McGarvey NH, Rutherford NJ et al.. Amyloidogenic alpha-synuclein seeds do not invariably induce rapid, widespread pathology in mice. Acta Neuropathol. 2014; 127:645-65.
• [16]Nolle A, van Haastert ES, Zwart R, Hoozemans JJ, Scheper W. Ubiquilin 2 is not associated with tau pathology. PLoS One. 2013; 8:e76598.
• [17]Fecto F, Siddique T. Making connections: pathology and genetics link amyotrophic lateral sclerosis with frontotemporal lobe dementia. J Mol Neurosci. 2011; 45:663-75.
• [18]Wu S, Mikhailov A, Kallo-Hosein H, Hara K, Yonezawa K, Avruch J. Characterization of ubiquilin 1, an mTOR-interacting protein. Biochim Biophys Acta. 2002; 1542:41-56.
• [19]Xia Y, Yan LH, Huang B, Liu M, Liu X, Huang C. Pathogenic mutation of UBQLN2 impairs its interaction with UBXD8 and disrupts endoplasmic reticulum-associated protein degradation. J Neurochem. 2014; 129:99-106.
• [20]Cassel JA, Reitz AB. Ubiquilin-2 (UBQLN2) binds with high affinity to the C-terminal region of TDP-43 and modulates TDP-43 levels in H4 cells: characterization of inhibition by nucleic acids and 4-aminoquinolines. Biochim Biophys Acta. 1834; 2013:964-71.
• [21]a multicenter controlled clinical trial in early Parkinson’s disease. Parkinson Study Group. Arch Neurol. 1989; 46:1052-60.
• [22]Ilieva H, Polymenidou M, Cleveland DW. Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. J Cell Biol. 2009; 187:761-72.
• [23]Zolotukhin S, Potter M, Zolotukhin I, Sakai Y, Loiler S, Fraites TJ et al.. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods. 2002; 28:158-67.
• [24]Sacino AN, Thomas MA, Ceballos-Diaz C, Cruz PE, Rosario AM, Lewis J et al.. Conformational templating of alpha-synuclein aggregates in neuronal-glial cultures. Mol Neurodegener. 2013; 8:17. BioMed Central Full Text
• [25]Lalonde R, Eyer J, Wunderle V, Strazielle C. Characterization of NFH-LacZ transgenic mice with the SHIRPA primary screening battery and tests of motor coordination, exploratory activity, and spatial learning. Behav Processes. 2003; 63:9-19.
• [26]Waxman EA, Duda JE, Giasson BI. Characterization of antibodies that selectively detect alpha-synuclein in pathological inclusions. Acta Neuropathol. 2008; 116:37-46.