UBQLN2/Ubiquilin 2 Mutation and Pathology in Familial Amyotrophic Lateral Sclerosis Kelly L

Neurobiology of Aging 33 (2012) 2527.e3–2527.e10 www.elsevier.com/locate/neuaging

UBQLN2/ubiquilin 2 mutation and pathology in familial amyotrophic lateral sclerosis Kelly L. Williamsa,b, Sadaf T. Warraicha,b, Shu Yanga, Jennifer A. Solskia, Ruvini Fernandoa, Guy A. Rouleauc, Garth A. Nicholsona,b,d, Ian P. Blaira,b,* a Northcott Neuroscience Laboratory, ANZAC Research Institute, Sydney, New South Wales, Australia b Sydney Medical School, University of Sydney, New South Wales, Australia c Department of Medicine, University of Montreal, Montreal, Canada d Molecular Medicine Laboratory, Concord Hospital, New South Wales, Australia Received 9 March 2012; received in revised form 18 May 2012; accepted 20 May 2012

Abstract

Amyotrophic lateral sclerosis (ALS) shows clinical and pathological overlap with frontotemporal dementia that includes the presence of hallmark ubiquitinated inclusions in affected neurons. Mutations in UBQLN2, which encodes ubiquilin 2, were recently identified in X-linked juvenile and adult-onset ALS and ALS/dementia. As part of an established exome sequencing program to identify disease genes in familial ALS, we identified a novel missense UBQLN2 mutation (c.1460CϾT, p.T487I) in 2 apparently unrelated multigenerational ALS families with no evidence of frontotemporal dementia. This mutation segregated with the disease and was absent in 820 healthy controls and all public single nucleotide polymorphism databases. The UBQLN2 p.T487I mutation substitutes a highly conserved residue and is located immediately upstream of a PXX region where all previous mutations have been identified. Immunostaining of spinal cord from a patient with UBQLN2 p.T487I mutation showed colocalization of ubiquilin 2 with ubiquitin in all neuronal inclusions examined and frequent colocalization with TAR DNA-binding protein 43 (TDP-43) and fused in sarcoma protein (FUS). To examine ubiquilin 2 pathology in broader ALS, we showed that ubiquilin 2 pathology also extends to ALS with a FUS mutation. These data further support the importance of ubiquilin 2 in the pathogenesis of ALS. © 2012 Elsevier Inc. All rights reserved.

Keywords: Amyotrophic lateral sclerosis; Exome sequencing; Mutation; UBQLN2; Ubiquilin 2

1. Introduction brains and spinal cords of familial and sporadic patients (Mackenzie et al., 2010). TAR DNA-binding protein 43 Amyotrophic lateral sclerosis (ALS) is a late-onset fatal (TDP-43) has been shown to be a principal component of disorder characterized by the rapidly progressive degenera- these inclusions (Neumann et al., 2006). tion of the upper and lower motor neurons. Frontotemporal Most ALS cases occur sporadically but approximately dementia (FTD) is observed in about 20% of ALS cases, 10% of cases show familial inheritance (FALS). To date, and as many as 50% of ALS cases show some frontotem- mutations in SOD1, TARDBP, and FUS account for approx- poral impairment (reviewed by Neumann et al., 2007). A pathological hallmark of ALS and ALS-FTD is the presence imately 25% of FALS (Kwiatkowski et al., 2009; Rosen et of ubiquitin positive neuronal cytoplasmic inclusions in the al., 1993; Sreedharan et al., 2008; Vance et al., 2009). Expansions of a hexanucleotide repeat in C9ORF72 cause ALS and FTD linked to chromosome 9p21 and may account for approximately a third of FALS with outbred European * Corresponding author at: Northcott Neuroscience Laboratory, ANZAC ancestry (DeJesus-Hernandez et al., 2011; Renton et al., Research Institute, Concord Hospital, NSW 2139, Australia. Tel.: ϩ61 2 9767 9118; fax: ϩ61 2 9767 9101. 2011). Rare mutations in OPTN, VCP, SQSTM1 (encoding E-mail address: [email protected] (I.P. Blair). p62), and FIG4 are also believed to account for a small

0197-4580/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2012.05.008 2527.e4 K.L. Williams et al. / Neurobiology of Aging 33 (2012) 2527.e3–2527.e10 percentage of FALS cases (Chow et al., 2009; Fecto et al., expansion was performed using the method described by 2011; Johnson et al., 2010; Maruyama et al., 2010). Rare Renton et al. (2011). mutations in TARDBP, FUS, and VCP have also been iden- Validation and screening of the UBQLN2 p.T487I mu- tified in FALS patients with FTD. tation was performed by polymerase chain reaction (PCR) Deng et al. (2011) recently identified a missense muta- amplification of a 487 base pair amplicon encompassing the tion in the ubiquilin 2 gene (UBQLN2) in a large X-linked mutation. PCR products were sequenced using Big-Dye dominant ALS-dementia family. Subsequent mutation anal- terminator sequencing and an ABI 3730XL DNA analyzer ysis in a cohort of 188 families without male-to-male trans- (Applied Biosystems, Foster City CA, USA). Primer se- mission identified 4 additional UBQLN2 missense muta- quences and amplification conditions are available on re- tions. Two of these segregated with the disease in quest. multigenerational ALS-FTD families and 2 were found in Single nucleotide polymorphism (SNP) genotyping of single patients each from small families with classic ALS. controls was performed using a custom TaqMan SNP geno- Ubiquilin 2 pathology was demonstrated in affected tissues typing assay according to the manufacturer’s instructions of cases with UBQLN2 mutations. Ubiquilin 2 was also (Life Technologies, Carlsbad, CA, USA) and analyzed us- shown to be a component of spinal cord neuronal inclusions ing a Viia 7 real time PCR system (Life Technologies). SNP in a subset of ALS patients that included sporadic ALS, genotyping for haplotype analysis was performed by PCR FALS, and ALS-dementia cases (Deng et al., 2011; Fecto and Big-Dye terminator sequencing as described above. and Siddique, 2011). Microsatellite marker genotypes were determined by PCR As part of an established exome sequencing program to and analysis using an ABI 3730XL DNA analyzer (Applied identify disease genes in an Australian familial ALS cohort Biosystems). without dementia, we identified a novel UBQLN2 mutation Conservation of ubiquilin 2 orthologs was examined by that segregated in 2 apparently unrelated ALS pedigrees and aligning sequences from a variety of species (Entrez protein demonstrated pathological features of ubiquilin 2 in patient database, ncbi.nlm.nih.gov) using ClustalW2, Version neuronal tissues and nonneuronal cells. 2.0.12 (www.ebi.ac.uk/Tools/msa/clustalw2/). 2.3. Cell culture 2. Methods Fibroblasts were cultured from skin biopsies taken from 2.1. Participants the inner forearm of individual IV: 18 (Fig. 1A) and 2 neurologically normal age-matched controls. Fibroblasts were One hundred FALS patients, 6 related “married-in” con- seeded at a density of 20,000 cells per well on 12-mm diameter trols, and 190 unrelated controls were recruited through thickness number 1.5 glass coverslips in Dulbecco’s Modified neurogenetic clinics at Concord Hospital, Sydney, as well as Eagle’s Medium (Sigma-Aldrich, St Louis, MO, USA) con- at the Molecular Medicine Laboratory, Concord Hospital. taining 2 mM L-glutamine, 100 U/mL penicillin, 100 ␮g/mL An additional 770 healthy controls were obtained from the streptomycin, and 10% (vol/vol) heat-inactivated fetal bovine Australian MND DNA Bank. Most families were of Euro- serum (Sigma-Aldrich, St Louis, MO, USA). Cells were main- pean descent. All patients were diagnosed with definite or tained in a humidified 37 °C incubator with 5% CO2. probable ALS according to El Escorial criteria (Brooks et al., 2000). Patients and family members provided informed 2.4. Immunohistochemistry and immunofluorescence written consent regulated by the human research ethics Immunohistochemistry and immunofluorescence were committee of the Sydney South West Area Health Service. performed on spinal cord tissue from 2 ALS patients using All families had previously been screened for mutations in a mouse monoclonal anti-ubiquilin 2 antibody (Novus Bi- known ALS genes including TARDBP, SOD1, FUS, OPTN, ologicals, St Louis, MO, USA), rabbit polyclonal anti- VCP, ANG, FIG4, DCTN1, and CHMP2B. TDP-43 antibody (Proteintech, Chicago, IL, USA), rabbit 2.2. Genetic analysis polyclonal anti-FUS antibody (Abcam, Cambridge, UK) and rabbit polyclonal antiubiquitin antibody (Dako, Genomic DNA was extracted from peripheral blood using Glostrup, Germany). Spinal cord sections (5-␮m) were standard protocols. The genomic DNA of family FALS5 in- deparaffinized with xylene, and rehydrated with a descend- dividual IV: 10 was derived from formalin-fixed, paraffin- ing series of diluted ethanol and water. Antigens were re- embedded kidney tissue using an FFPE Repli-G kit (Qiagen trieved by heating sections in 10 mM citrate buffer (pH 6.0). Valencia, CA, USA). The exomes of 106 individuals (ALS Endogenous peroxidase activity was blocked for immuno- patients, obligate carriers, and related “married-in” controls) histochemical studies using 3% hydrogen peroxide in meth- from 65 ALS families were captured using TruSeq exome anol. Nonspecific background was blocked with either 1.5% Enrichment kit and sequenced with HiSeq2000 sequencing goat serum (Vector Laboratories, Burlingame, CA, USA) system (Illumina, San Diego, CA, USA). for immunohistochemistry, or 1% bovine serum albumin Analysis of the C9ORF72 hexanucleotide repeat and (Sigma-Aldrich, St Louis, MO, USA) for immunofluores- K.L. Williams et al. / Neurobiology of Aging 33 (2012) 2527.e3–2527.e10 2527.e5

Fig. 1. A novel UBQLN2 mutation in familial amyotrophic lateral sclerosis (ALS). Pedigree of family FALS5 (A) and FALS14 (B) showing segregation of the UBQLN2 c.1460CϾT missense mutation resulting in p.T487I. Affected individuals are indicated by filled symbols; unaffected individuals by open symbols. Individuals for whom DNA was available are indicated with an asterisk. The sex of individuals in recent generations has been omitted for confidentiality. (C) DNA sequence traces from a healthy control and a male carrying a hemizygous mutation. The arrowhead indicates the mutated nucleotide and the codon is underlined. (D) Alignment of ubiquilin 2 protein sequences from different species demonstrates conservation of the substituted residue. An arrow indicates the p.T487I mutation. Sequences include NP_038472.2 (human), XP_003317539.1 (chimpanzee), NP_001192611.1 (cattle), XP_549029.2 (dog), XP_001914714.1 (horse), NP_061268.2 (mouse), and NP_001101721.1 (rat). (E) Diagram of the predicted structural and functional domains of ubiquilin 2. The 624aa protein is predicted to contain a ubiquitin-like domain (UBL; aa.33–103), 4 heat-shock-chaperone-binding motifs (STI1; aa.178–206, 208–247, 379–426, 430–462), 12 PXX repeats (aa.491–526) and an ubiquitin-associated domain (UBA; aa.582–620). The p.T487I mutation, indicated by a red arrow, is located immediately upstream of the PXX domain where previously reported mutations (indicted by black arrows) are located. cence. Sections were incubated at room temperature for 1 blocked for 1 hour in 1% bovine serum albumin (Sigma Al- hour with the primary antibodies (1:1000 for anti-TDP-43, drich). Fibroblasts were incubated at 4 °C overnight with a 1:100 anti-FUS,1:100 antiubiquitin or 1:100 for anti-ubiqui- primary mouse monoclonal anti-ubiquilin 2 antibody (1:500, lin 2). Novus Biologicals, Littleton, CO, USA), followed by Alexa Biotinylated goat anti-rabbit or anti-mouse IgG were used Fluor 488 goat anti-mouse secondary antibody (1:250; Life as secondary antibodies for immunohistochemical staining. Technologies). Cells were then stained at 4 °C overnight with The avidin-biotin complex detection system (Vector Labora- a primary rabbit polyclonal anti–TDP-43 antibody (1:500, Pro- tories) with 3,3’-diaminobenzidine as chromogen was used to teintech, Chicago, IL, USA), followed by Alexa Fluor 555 detect the immunoreactive signal. The slides were counter- donkey anti-rabbit secondary antibody (1:250; Life Technolo- stained with hematoxylin and coverslipped using Di-N-Butyle gies). Phthalate in xylene (DPX). For colocalization studies in fibroblasts, cells were seeded 2.5. Confocal microscopy in 24-well plates at a density of 20,000 cells per well. After 48 hours, 5 ␮M of proteasome inhibitor MG-132 (Merck, Bil- Confocal fluorescence imaging was performed using an lerica, MA, USA) was added and cells were cultured for an Olympus (Center Valley, PA, USA) Fluoview FV300 laser additional 24 hours. For immunofluorescence staining, cells scanning confocal microscope. Images were acquired with were washed twice with Dulbecco’s phosphate-buffered saline an inverted 60ϫ (1.4 numerical aperture) oil immersion (DPBS) (Life Technologies) and fixed with 4% (wt/vol) para- objective. Emission spectra were defined by band pass fil- formaldehyde (Sigma-Aldrich) in DPBS supplemented with ters (510–530 nm and 565–610 nm). Images were taken 0.2 M sucrose for 15 minutes at 37 °C. Fixed cells were using sequential mode to avoid crosstalk between 2 dyes. permeabilized with 0.5% (vol/vol) Triton X-100 (Sigma Al- Immunohistochemistry imaging was performed with drich) in DPBS for 15 minutes at room temperature and CellSens v1.4.1 program (Olympus), using a 40ϫ objective. 2527.e6 K.L. Williams et al. / Neurobiology of Aging 33 (2012) 2527.e3–2527.e10

Table 1 Summary of clinical features seen in amyotrophic lateral sclerosis families FALS5 and FALS14 with UBQLN2 mutation p.T487I Patient Sex Age at onset, y Duration of disease Age at death, y Site of onset Cognitive impairment/ frontotemporal dementia FALS5 I:1 F 43 12 y 55 Unknown No II:2 M Unknown Unknown 55 Unknown No II:3 F Unknown Unknown 39 Unknown No II:4 M 55 3 y 58 Unknown No III:2 F 73 3 y 76 Unknown No III:6 F 55 6 mo 56 Limb No III:8a F 76 1 y 77 Unknown Intellectual falloff III:12 F 41 7 y 48 Bulbar No IV:2 F 51 2 y 53 Unknown No IV:9a F 49 4 y 53 Bulbar No IV:10a M 34 1 y 35 Bulbar No IV:17 M 36 8 y 44 Bulbar No IV:18a F 58 Alive at 3 y — Limb (arms) No IV:22 F 35 3 y 38 Limb (arms) No FALS14 I:1 F 52 4 y 55 Bulbar No II:2a F 30 11 y 41 Limb (hands) No II:4a M 33 3 y 36 Limb (hands) No Key: F, female; M, male. a Individuals for whom DNA was available.

To count the number of cells that showed ubiquilin (www.ncbi.nlm.nih.gov/projects/SNP/), 1000 Genomes Project 2/TDP-43 colocalization, 6 images were randomly acquired. data (November 2010 release), the exome Variant Server (evs.gs- The number of cells with ubiquilin 2/TDP-43 colocalization .washington.edu/EVS/), or an additional 820 Australian healthy was counted and presented as a ratio to the total number of controls. The UBQLN2 T487 residue is highly conserved (Fig. cells in a visual field. 1D), and is located immediately upstream of 5 previously reported UBQLN2 mutations (Deng et al., 2011)(Fig. 1E). 2.6. Statistical analysis 3.2. Clinical features of family FALS5 Statistical analysis was performed using 2-tailed Student t tests (p Ͻ 0.05) (GraphPad Software 5.01, La Jolla, CA, A summary of clinical data from 17 affected individuals USA). All values were mean Ϯ standard error of the mean. (5 male and 12 female) from families FALS5 and FALS14 (Fig. 1A and B) is shown in Table 1. All were diagnosed 3. Results with ALS. No individuals were diagnosed with FTD al- though 1 individual showed possible frontotemporal impair- 3.1. Genetic analysis ment. The age of disease onset ranged from 30 to 76 years. Male individuals appeared to show an earlier age of onset As part of an established exome sequencing program to Ϯ Ϯ identify new disease genes in an Australian familial ALS (39.5 10.4 years) than female (51.2 14.3 years), how- cohort, we identified a novel nonsynonymous variant in ever this was not statistically significant. Duration of disease UBQLN2 (c.1460CϾT, p.T487I), in the exomes of 4 af- ranged from 6 months to 12 years, and the mean duration was not statistically different for male and female individ- fected individuals from 2 apparently unrelated families with Ϯ Ϯ no male-to-male transmission (families FALS5 and uals (3.8 3.0 years and 4.8 4.0 years respectively). The FALS14; Fig. 1 and Supplementary Fig. 1). Genotyping of mutation shows high penetrance in these families. Addi- this mutation in the 2 extended families confirmed segrega- tional clinical information and details of postmortem exam- tion of the mutation in 15 individuals with ALS (Fig. 1A– ination are given in the Supplementary material. C). The mutation was absent in all unaffected individuals 3.3. Ubiquilin 2 pathology in ALS patient spinal cord older than 42 years (where DNA was available). Haplotype analysis using polymorphic microsatellite markers and da- Immunohistochemical staining was performed in post- tabase SNPs that flank the mutation demonstrated that the 2 mortem spinal cord tissue from an ALS patient with the families share a disease haplotype at this locus, indicative of p.T487I mutation in UBQLN2 (individual IV: 10, Fig. 1A) an ancestral founder mutation (Supplementary Table 1). and a patient diagnosed with ALS-FTD with a FUS Genotyping also confirmed that these families were nega- p.R521C mutation (patient previously described in Blair et tive for the C9ORF72 hexanucleotide repeat expansion. The al., 2010: ALS53, individual IV: 5). Staining in UBQLN2 UBQLN2 c.1460CϾT variant was not present in dbSNP p.T487I patient showed compact and skein-like inclusions K.L. Williams et al. / Neurobiology of Aging 33 (2012) 2527.e3–2527.e10 2527.e7

Fig. 2. Immunostaining of spinal motor neurons from a patient with the UBQLN2 p.T487I mutation. The compact and skein-like inclusions are immunoreactive for antibodies against TDP-43 (A, B, C), ubiquilin 2 (D, E), ubiquitin (F, G) and FUS (H, I). Scale bar ϭ 10 ␮m. in spinal motor neurons that were immunoreactive for and I). The inclusions present in the FUS p.R521C patient ubiquilin 2 (Figs. 2D and E, and 3C). Pathological inclu- were immunoreactive for ubiquilin 2 (Fig. 3C) and FUS sions present in the UBQLN2 p.T487I patient were positive (Fig. 3B) but not TDP-43 (Fig. 3A). for ubiquitin (Fig. 2F and G), and some of these inclusions Multiple-labeling immunoﬂuorescence was performed in were immunoreactive for TDP-43 and FUS (Fig. 2A–C, H, patient spinal cord to establish whether ubiquilin 2 colocalized

Fig. 3. Immunostaining of spinal motor neurons from a familial amyotrophic lateral sclerosis (ALS) patient with FUS R521C mutation. Immunohistochem- istry showed (A) normal nuclear TDP-43 staining, (B) FUS positive inclusions, and (C) ubiquilin 2 positive skein-like inclusions. Scale bar ϭ 10 ␮m. 2527.e8 K.L. Williams et al. / Neurobiology of Aging 33 (2012) 2527.e3–2527.e10 with other ALS proteins. Ubiquilin 2 colocalized with ubiqui- al., 2011). Despite this difference, the mean duration of tin in all spinal motor neuron inclusions examined from the disease is similar. UBQLN2 p.T487I patient. TDP-43 and FUS also showed fre- All previously reported UBQLN2 missense mutations in quent colocalization with ubiquilin 2 (Supplementary Fig. 2). ALS and ALS/dementia replace a proline residue and are The spinal motor neurons of the FUS p.R521C patient showed located in a unique PXX repeat region of the gene (Deng et ubiquilin 2 immunoreactive inclusions that colocalized with al., 2011). Recent mutation analysis of UBQLN2 in a large FUS and ubiquitin (Supplementary Fig. 3). French ALS cohort identified a novel deletion within the PXX repeat region in a FALS patient, which did not seg- 3.4. Ubiquilin 2 colocalized with TDP-43 in fibroblasts regate with the disease and was subsequently found in a To further study the association between ubiquilin 2 and control individual (Millecamps et al., 2012). The UBQLN2 TDP-43 in ALS, we studied the effect of the proteasome p.T487I mutation described in the current study substitutes inhibitor MG-132 on the colocalization of ubiquilin 2 with a highly conserved residue immediately upstream of the TDP-43 using ALS patient (UBQLN2 p.T487I) and control PXX repeat region. This mutation changes a polar un- fibroblasts. Under normal cell culture conditions (in the charged threonine to a larger hydrophobic amino acid iso- absence of MG-132), ubiquilin 2 showed diffuse cytoplas- leucine, potentially disrupting the ubiquilin 2 protein struc- mic staining in both patient and control cells, while TDP-43 ture. remained almost exclusively nuclear (Supplementary Fig. A pathological hallmark of ALS and ALS-FTD is the 4A and D). After treatment with MG-132 for 24 hours, presence of ubiquitin-positive neuronal cytoplasmic inclu- immunofluorescent staining showed cytoplasmic inclusions sions in the brains and spinal cords of familial and sporadic in both patient and control fibroblasts that were positive for patients (Mackenzie et al., 2010). Previous immunohisto- either ubiquilin 2 alone (Supplementary Fig. 4B and E) or chemical studies of tissues from ALS/dementia patients both ubiquilin 2 and TDP-43 (Supplementary Fig. 4C and with UBQLN2 p.P506T mutation showed skein-like ubiqui- F). Patient fibroblasts showed significantly more cytoplas- lin 2 positive inclusions in the spinal cord, and more prom- mic inclusions that were positive for both ubiquilin 2 and inently in the hippocampus (Deng et al., 2011; Fecto and TDP-43 than control fibroblasts (Supplementary Fig. 5). Siddique, 2011). Our analysis of the spinal cord from a patient with classic ALS and UBQLN2 p.T487I mutation showed both compact and skein-like ubiquilin 2 positive 4. Discussion inclusions, often coexisting, in spinal motor neurons. We We identified a novel UBQLN2 missense mutation that also demonstrated that ubiquilin 2 consistently colocalized segregates with disease in 2 multigenerational families with with ubiquitin in spinal motor neuron inclusions indicating classic ALS, substitutes a highly conserved residue, and is this is likely to be the primary pathology in ALS cases with absent in a large number of control individuals. The 2 UBQLN2 mutations. Ubiquilin 2 has been reported as a families were apparently unrelated, however they share a common component of neuronal inclusions in a variety of disease haplotype at this locus, providing further evidence ALS cases, suggesting that like ubiquitin, it may be a that this is a pathogenic ancestral founder mutation. ALS general feature of ALS and ALS-FTD (Fecto and Siddique, patient spinal cord with this mutation showed compact and 2011). TDP-43 and other ALS linked proteins, including, skein-like pathological inclusions that were immunoreactive FUS, p62, and optineurin, have also variably been shown to for ubiquilin 2. Together with previous reports of juvenile be components of these inclusions (Arai et al., 2006; Kwi- and adult-onset ALS and ALS-FTD, this further supports atkowski et al., 2009; Maruyama et al., 2010; Neumann et the role of UBQLN2 in ALS. al., 2006; Vance et al., 2009). We showed that a subset of Unlike previously reported families associated with spinal motor neuron inclusions from the case with UBQLN2 UBQLN2 mutations, the Australian family had no diagnoses p.T487I mutation also showed colocalization of ubiquilin 2 of FTD. Indeed, only 1 of 17 cases was documented with with TDP-43 and/or FUS indicating that these latter mole- any frontotemporal impairment. In contrast, FTD was a cules are downstream in the pathological cascade and are common feature in previously reported large families with more general markers for inclusions in ALS with UBQLN2 UBQLN2 mutations (Deng et al., 2011). UBQLN2 muta- mutation. tions may cause classic ALS with or without FTD. Similar In addition to patients with UBQLN2 mutations, ubiqui- phenotypic variability is also seen among patients with lin 2 pathology was previously shown to extend to patients mutations in TARDBP and FUS, and those with expansions with other forms of ALS including sporadic ALS, FALS in C9ORF72 (Mackenzie et al., 2010; Stewart et al., 2012). with mutations in SOD1 or TARDBP, and FALS with un- The families with UBQLN2 mutation described here known cause (Deng et al., 2011). We further explored the showed only adult-onset ALS (30–76 years). Previously role of ubiquilin 2 in ALS and show that ALS caused by a described families with UBQLN2 mutation showed a FUS mutation can now be added to the subtypes of ALS broader range of disease onset and included cases with associated with ubiquilin 2-positive inclusions. juvenile onset ALS and ALS/FTD (16–71 years) (Deng et The ubiquilins are a family of ubiquitin-like proteins that K.L. Williams et al. / Neurobiology of Aging 33 (2012) 2527.e3–2527.e10 2527.e9 that deliver ubiquitinated proteins to the proteasome for 570957) and a Bill Gole Fellowship (to SY) and Peter degradation. Previously reported mutations in ubiquilin 2 Stearne grant from the Motor Neurone Disease Research were shown to impair this proteasome-mediated degrada- Institute of Australia. tion process (Deng et al., 2011). We disrupted the proteasome pathway in ALS patient and control fibroblasts and Appendix A. Supplementary data showed that cells with the UBQLN2 p.T487I mutation had Supplementary data associated with this article can be significantly more cytoplasmic inclusions that were positive found, in the online version, at http://dx.doi.org/10.1016/j. for both ubiquilin 2 and TDP-43. This further supports the neurobiolaging.2012.05.008. notion that impaired proteasome mediated degradation is a pathological mechanism. In addition, ubiquilin 2 was a constant marker of inclusions in these cells, whereas References TDP-43 staining of inclusions was less frequent. This suggests that recruitment of TDP-43 to these aggregates is Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H., secondary to that of ubiquilin 2. Our data suggest that Mann, D., Tsuchiya, K., Yoshida, M., Hashizume, Y., Oda, T., 2006. experimentally observable pathological features extend be- TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. yond neuronal cells and this may prove useful in the devel- Biochem. Biophys. Res. Commun. 351, 602–611. opment of experimental tools and clinical biomarkers. 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Neuron Disord. 1, 293– ubiquitinated inclusions that cannot be degraded (Kwiat- 239. kowski et al., 2009; Neumann et al., 2006; Sreedharan et al., Chow, C.Y., Landers, J.E., Bergren, S.K., Sapp, P.C., Grant, A.E., Jones, J.M., Everett, L., Lenk, G.M., McKenna-Yasek, D.M., Weisman, L.S., 2008; Vance et al., 2009). Mutant ubiquilin 2 has been Figlewicz, D., Brown, R.H., Meisler, M.H., 2009. Deleterious variants shown to impair protein degradation (Deng et al., 2011). of FIG4, a phosphoinositide phosphatase, in patients with ALS. Am. J. This suggests a convergence of 2 mechanistic pathways in Hum. Genet. 84, 85–88. ALS, which may lead researchers to novel therapeutic strat- DeJesus-Hernandez, M., Mackenzie, I.R., Boeve, B.F., Boxer, A.L., Baker, egies. M., Rutherford, N.J., Nicholson, A.M., Finch, N.A., Flynn, H., Adam- son, J., Kouri, N., Wojtas, A., Sengdy, P., Hsiung, G.Y., Karydas, A., The identification of a novel mutation in UBQLN2 in 2 Seeley, W.W., Josephs, K.A., Coppola, G., Geschwind, D.H., Wszolek, ALS families, coupled with observed ubiquilin 2 pathology, Z.K., Feldman, H., Knopman, D.S., Petersen, R.C., Miller, B.L., Dick- further supports the importance of ubiquilin 2 in the path- son, D.W., Boylan, K.B., Graff-Radford, N.R., Rademakers, R., 2011. ological cascade leading to ALS. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256. Disclosure statement Deng, H.X., Chen, W., Hong, S.T., Boycott, K.M., Gorrie, G.H., Siddique, N., Yang, Y., Fecto, F., Shi, Y., Zhai, H., Jiang, H., Hirano, M., All authors have no actual or potential conflicts of inter- Rampersaud, E., Jansen, G.H., Donkervoort, S., Bigio, E.H., Brooks, est. B.R., Ajroud, K., Sufit, R.L., Haines, J.L., Mugnaini, E., Pericak- Vance, M.A., Siddique, T., 2011. Mutations in UBQLN2 cause dom- Patients and family members provided informed written inant X-linked juvenile and adult-onset ALS and ALS/dementia. Na- consent in accordance with protocols approved by the hu- ture 477, 211–215. man research ethics committee of the Sydney South West Fecto, F., Siddique, T., 2011. Making connections: pathology and genetics Area Health Service. link amyotrophic lateral sclerosis with frontotemporal lobe dementia. J. Mol. Neurosci. 45, 663–675. Fecto, F., Yan, J., Vemula, S.P., Liu, E., Yang, Y., Chen, W., Zheng, J.G., Acknowledgements Shi, Y., Siddique, N., Arrat, H., Donkervoort, S., Ajroud-Driss, S., Sufit, R.L., Heller, S.L., Deng, H.X., Siddique, T., 2011. SQSTM1 The authors thank patients and family members for their mutations in familial and sporadic amyotrophic lateral sclerosis. Arch. Neurol. 68, 1440–1446. ongoing participation and support, C. Cecere for assistance Johnson, J.O., Mandrioli, J., Benatar, M., Abramzon, Y., Van Deerlin, with family recruitment, the Australian MND DNA Bank V.M., Trojanowski, J.Q., Gibbs, J.R., Brunetti, M., Gronka, S., Wuu, J., for assistance in sample collection, and J. Manavis and P.C. Ding, J., McCluskey, L., Martinez-Lage, M., Falcone, D., Hernandez, Blumbergs from Hanson Institute Centre for Neurological D.G., Arepalli, S., Chong, S., Schymick, J.C., Rothstein, J., Landi, F., Diseases, Institute of Medical and Veterinary Science, Ad- Wang, Y.D., Calvo, A., Mora, G., Sabatelli, M., Monsurrò, M.R., Battistini, S., Salvi, F., Spataro, R., Sola, P., Borghero, G., Galassi, G., elaide, Australia for providing FUS patient tissues. This Scholz, S.W., Taylor, J.P., Restagno, G., Chiò, A., ITALSGEN Con- work was supported by grants from the National Health and sortium, 2010. Exome Sequencing Reveals VCP Mutations as a Cause Medical Research Council of Australia (1004670, 511941, of Familial ALS. 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