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Dendritic spinopathy in transgenic mice expressing ALS/dementia-linked mutant UBQLN2 George H. Gorriea,1, Faisal Fectoa,b,1, Daniel Radzickic, Craig Weissc,d, Yong Shia, Hongxin Dongb,e, Hong Zhaia, Ronggen Fua, Erdong Liua, Sisi Lia, Hasan Arrata, Eileen H. Bigiof,g, John F. Disterhoftc, Marco Martinac, Enrico Mugnainih, Teepu Siddiquea,b,h,2, and Han-Xiang Denga,2 aDivision of Neuromuscular Medicine, Davee Department of Neurology and Clinical Neurosciences, bInterdepartmental Neuroscience Program, cDepartment of Physiology, dBehavioral Phenotyping Core Facility, eDepartment of Psychiatry and Behavioral Sciences, fDivision of Neuropathology, Department of Pathology, gCognitive Neurology and Alzheimer Disease Center, and hDepartment of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611 Edited* by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved August 27, 2014 (received for review April 1, 2014) Mutations in the gene encoding ubiquilin2 (UBQLN2) cause amyo- using chloramphenicol acetyltransferase (CAT) reporter system trophic lateral sclerosis (ALS), frontotemporal type of dementia, or and constructed a 10.8-kb EcoRI/BamHI human UBQLN2 ge- both. However, the molecular mechanisms are unknown. Here, nomic DNA transgene with a P497H mutation, which was P497H we show that ALS/dementia-linked UBQLN2 transgenic mice identified in a large family with ALS and dementia (2) (Fig. S1 develop neuronal pathology with ubiquilin2/ubiquitin/p62- A and B). Transgenic mice were developed by microinjection positive inclusions in the brain, especially in the hippocampus, re- of the transgene into fertilized eggs. capitulating several key pathological features of dementia ob- We identified 13 transgenic founders and performed initial ge- served in human patients with UBQLN2 mutations. A major netic analysis to select a founder line with a relatively higher copy P497H feature of the ubiquilin2-related pathology in these mice, and rem- number of the transgene (Fig. S1C). The human UBQLN2 iniscent of human disease, is a dendritic spinopathy with protein transgene appeared to be integrated into the mouse Y chro- aggregation in the dendritic spines and an associated decrease in mosome in this line, because the transgene cotransmitted with dendritic spine density and synaptic dysfunction. Finally, we show the Y chromosome and all of the transgenic mice were males P497H that the protein inclusions in the dendritic spines are composed of (n > 200). The expression profiles of the human UBQLN2 several components of the proteasome machinery, including transgene and mouse endogenous ubqln2 showed similar ex- G76V Ub –GFP, a representative ubiquitinated protein substrate that pression levels with relatively higher expression in the brain and is accumulated in the transgenic mice. Our data, therefore, directly spinal cord, but much lower in the liver (Fig. S1D). link impaired protein degradation to inclusion formation that is as- P497H sociated with synaptic dysfunction and cognitive deficits. These Cognitive Deficits in UBQLN2 Transgenic Mice. Two major cat- data imply a convergent molecular pathway involving synaptic pro- egories of symptoms have been observed in patients with tein recycling that may also be involved in other neurodegenerative UBQLN2 mutations, which involve motor and cognitive func- disorders, with implications for development of widely applicable tions. We did not observe gross motor abnormalities through the P497H rational therapeutics. lifetime (>600 d, n > 40) of the UBQLN2 transgenic mice. Y-maze test for spontaneous alternation revealed good spatial rotein aggregates or inclusions with immunoreactivity to memory in the nontransgenic controls (78%), but near random Pubiquitin represent a common pathological hallmark in a arm selection (52%) for transgenic mice, although this did not broad range of late-onset neurodegenerative disorders, including reach statistical significance (Fig. 1A). The number of arm Alzheimer’s disease (AD), Parkinson disease (PD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) (1). Significance However, the molecular mechanisms underlying the formation of these inclusions and their relationship to neuronal dysfunction Mutations in the UBQLN2 gene, which encodes the ubiquitin- and degeneration are poorly understood. Mutations in UBQLN2, like protein ubiquilin2 (UBQLN2) have been shown to cause which encodes the ubiquitin-like protein ubiquilin2 (UBQLN2), ALS and ALS/dementia. Ubiquilin2 links familial and sporadic have been recently shown to cause a subset of ALS, FTD-type of forms of the disease through pathology observed in the spinal dementia, or both (2, 3). Notably, mutations within and in close cords of all ALS cases and in the brains of ALS/dementia cases proximity to the PXX domain of ubiquilin2 appear to have high with or without UBQLN2 mutations. In this communication, we penetrance as shown in familial cases (2). The distribution of develop and characterize a mouse model of mutant UBQLN2- ubiquilin2-positive inclusions in the brain and spinal cord is well linked dementia. We demonstrate that mutant mice develop correlated with cognitive and motor symptoms of patients with impairment in the protein degradation pathway, abnormal diverse etiology, including chromosome 9 open reading frame 72 protein aggregation, synaptic dysfunction, and cognitive defi- (C9ORF72)-linked cases (2, 4). The ubiquilin2-positive inclusions cits. This model provides a useful tool to further study de- appear to cover a wide range of protein inclusions, including mentia and develop rational therapies. those without TAR DNA binding protein (TDP43) immunore- activity (2, 4), suggesting a primary role for ubiquilin2 in inclusion Author contributions: T.S. and H.-X.D. designed research; G.H.G., F.F., D.R., C.W., Y.S., H.D., H.Z., R.F., E.L., S.L., H.A., E.H.B., J.F.D., M.M., E.M., T.S., and H.-X.D. performed re- formation and neurodegeneration. Therefore, understanding the search; G.H.G., F.F., C.W., H.D., E.H.B., J.F.D., M.M., E.M., T.S., and H.-X.D. analyzed data; pathophysiological basis of UBQLN2-linked dementia may provide and G.H.G., F.F., C.W., H.D., E.H.B., J.F.D., M.M., E.M., T.S., and H.-X.D. wrote the paper. mechanistic insight into the pathogenesis of neurodegenerative The authors declare no conflict of interest. disorders and allow for the design of rational therapies. To this end, *This Direct Submission article had a prearranged editor. UBQLN2 we developed and characterized mutant transgenic mice. 1G.H.G. and F.F. contributed equally to this work. 2To whom correspondence may be addressed. Email: [email protected] or Results [email protected]. UBQLN2P497H UBQLN2 Development of Transgenic Mice. Human is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. an intronless gene. We analyzed the UBQLN2 promoter activity 1073/pnas.1405741111/-/DCSupplemental. 14524–14529 | PNAS | October 7, 2014 | vol. 111 | no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1405741111 Downloaded by guest on October 4, 2021 entries was significantly higher for transgenic mice compared cognitive symptoms in patients with UBQLN2 mutations. In with controls (Fig. 1B). Similar performance in the Y maze has the spinal cord, we observed some ubiquilin2-immunoreactive been observed in the transgenic mice of AD (5). aggregates predominantly located around the small interneurons Temporal memory function was tested by a delay fear condi- in the Rexed lamina II of the dorsal horns (Fig. S2 A–D). The tioning task that assesses freezing behavior to the same context distribution pattern of these aggregates was similar to that in the and to a conditioned stimulus (CS) in a novel context 24 h after patients with UBQLN2 mutations (Fig. S2 E and F), and they G–I training. The transgenic mice showed significantly decreased were also immunoreactive for ubiquitin and p62 (Fig. S2 ). freezing in response to the context and CS compared with con- We did not observe skeinlike inclusions, the characteristic pa- trol mice (Fig. 1 C and D). thology of ALS, in anterior horn motor neurons, which might explain the lack of motor symptoms. Spatial memory was tested using the Morris water maze. The UBQLN2 time and distance to reach a visible platform did not show signifi- The peculiar pathology in ALS/dementia patients with mutations is the presence of ubiquilin2 aggregates, which is most cant differences between genotypes, suggesting equal visual acuity, prominent in the hippocampus, especially in the molecular layer of swimming ability, and motivation to reach the platform. In the the dentate gyrus (2). This pathology is also present in some ALS/ hidden platform version, mice showed an improvement in the time dementia patients without UBQLN2 mutations (2). Recently, to find the platform, but significant differences were found between this pathology has been shown to be a common feature in the the transgenic mice and controls in acquisition rates across all C9ORF72-linked ALS/dementia and frontotemporal lobar E F UBQLN2P497H training sessions (Fig. 1 and ), with mice taking degeneration with TDP43 positive inclusions (FTLD-TDP) more time and longer path lengths to reach the platform. cases (4), which account for over 10% of familial FTD and over 60% of familial ALS/dementia (3). We performed initial Ubiquilin2-Immunoreactive Inclusions in the Hippocampus and Other pathological analysis using 4-mo-old transgenic mice.

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