Frontotemporal Dementia Caused by CHMP2B Mutation Is Characterised by Neuronal Lysosomal Storage Pathology

Acta Neuropathol (2015) 130:511–523 DOI 10.1007/s00401-015-1475-3 ORIGINAL PAPER Frontotemporal dementia caused by CHMP2B mutation is characterised by neuronal lysosomal storage pathology Emma L. Clayton1 · Sarah Mizielinska1 · James R. Edgar3 · Troels Tolstrup Nielsen4 · Sarah Marshall1 · Frances E. Norona1 · Miranda Robbins1 · Hana Damirji1 · Ida E. Holm5,6 · Peter Johannsen7 · Jørgen E. Nielsen4,7,8 · Emmanuel A. Asante2 · John Collinge1,2 · The FReJA consortium · Adrian M. Isaacs1 Received: 7 July 2015 / Revised: 1 September 2015 / Accepted: 2 September 2015 / Published online: 10 September 2015 © The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Mutations in the charged multivesicular body controls, indicating that they are a specific pathology protein 2B (CHMP2B) cause frontotemporal dementia caused by mutant CHMP2B. Ultrastructural analysis and (FTD). We report that mice which express FTD-causative immuno-gold labelling confirmed that they are derived mutant CHMP2B at physiological levels develop a novel from the endolysosomal system. Consistent with these lysosomal storage pathology characterised by large neu- findings, CHMP2B mutation patient brains contain mor- ronal autofluorescent aggregates. The aggregates are an phologically similar autofluorescent aggregates. These early and progressive pathology that occur at 3 months aggregates occur significantly more frequently in human of age and increase in both size and number over time. CHMP2B mutation brain than in neurodegenerative disease These autofluorescent aggregates are not observed in or age-matched control brains. These data suggest that lys- mice expressing wild-type CHMP2B, or in non-transgenic osomal storage pathology is the major neuronal pathology in FTD caused by CHMP2B mutation. Recent evidence E. L. Clayton and S. Mizielinska contributed equally to this work. suggests that two other genes associated with FTD, GRN and TMEM106B are important for lysosomal function. Frontotemporal Dementia Research in Jutland Association Our identification of lysosomal storage pathology in FTD (FreJA) members are listed in the acknowledgements. caused by CHMP2B mutation now provides evidence that endolysosomal dysfunction is a major degenerative path- Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1475-3) contains supplementary way in FTD. material, which is available to authorized users. * Adrian M. Isaacs 7 Memory Clinic, Department of Neurology, Danish Dementia [email protected] Research Centre, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark 1 Department of Neurodegenerative Disease, UCL Institute 8 Department of Cellular and Molecular Medicine, Section of Neurology, Queen Square, London WC1N 3BG, UK of Neurogenetics, The Panum Institute, University 2 MRC Prion Unit, UCL Institute of Neurology, Queen Square, of Copenhagen, Copenhagen, Denmark London WC1N 3BG, UK 3 Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK 4 Neurogenetics Research Laboratory, Department of Neurology, Danish Dementia Research Centre, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark 5 Laboratory for Experimental Neuropathology, Department of Pathology, Randers Hospital, 8930 Randers NØ, Denmark 6 Institute of Clinical Medicine, Aarhus University, 8000 Aarhus C, Denmark 1 3 512 Acta Neuropathol (2015) 130:511–523 Keywords CHMP2B · FTD · ESCRT · Lysosome · MVBs fuse with autophagosomes to form hybrid com- Lysosomal storage disorder partments termed amphisomes, which then fuse with lysosomes, enabling degradation [5, 27]. Mutant CHMP2BIn- tron5 has been shown to affect the maturation of both Introduction endosomes and autophagosomes [10, 22, 25, 38], impli- cating impaired lysosomal degradation as a key pathway Frontotemporal dementia (FTD) is the second most com- in FTD caused by CHMP2B mutation. Importantly, accu- mon form of young-onset dementia [17, 32]. FTD is char- mulating evidence suggests that progranulin, a common acterised by atrophy of the frontal and temporal lobes, genetic cause of FTD, and TMEM106B a major risk factor which results in alterations in personality, behaviour or for FTD also have roles in endolysosomal function [7, 9, language [26, 28]. FTD also shows clinical overlap with 16, 21, 34, 36, 37], with progranulin mutations leading to amyotrophic lateral sclerosis (ALS) and FTD and ALS pathology reminiscent of lysosomal storage disorders [16]. share common pathologies and genetic causes [3]. We have previously shown that transgenic mice express- Mutations in a number of genes are known to cause ing endogenous levels of human C-terminally truncated familial FTD. Mutations in the genes that encode tau mutant CHMP2BIntron5 show progressive gliosis and p62 (MAPT), progranulin (GRN) and C9orf72 are the most inclusion pathology, which are also observed in CHMP2B common causes of FTD, whilst rare mutations have been mutation patient brain [14, 19]. Here we report, using identified in valosin-containing protein (VCP), TDP-43 immunoblotting and light and electron microscopy, that (TARDBP), fused in sarcoma (FUS) and charged multi- these CHMP2BIntron5 mice additionally develop a separate vesicular body protein 2B (CHMP2B) [33]. It is currently and distinct progressive lysosomal storage pathology that unclear how defects in such diverse genes result in specific is characterised by large autofluorescent aggregates. These degeneration of neurons of the frontal and temporal lobes. autofluorescent aggregates are not seen in CHMP2Bwild-type A mutation in CHMP2B is responsible for an autoso- transgenics, or non-transgenic control mice. Importantly, mal dominant form of inherited FTD (termed FTD-3) in we also report increased autofluorescent aggregates in the a Danish cohort [24, 35]. The mutation occurs in a splice frontal cortex of FTD-3 patients with the CHMP2B muta- acceptor site, resulting in the production of two variants of tion, when compared to neurodegenerative disease controls. C-terminally truncated CHMP2B proteins: CHMP2BIntron5 These data indicate that impaired lysosomal storage is a in which the final 36 amino acids are replaced with a single novel pathological pathway in the aetiology of FTD caused valine residue, or CHMP2B∆10 in which the final 36 amino by CHMP2B mutation, and provide evidence that lysoso- acids are replaced with 29 nonsense residues [35]. A sub- mal degradation is a key pathway in FTD pathogenesis. sequent study in a Belgian FTD cohort identified a familial FTD patient with a distinct truncation mutation CHMP- 2BQ165X that leads to the loss of the final 49 amino acids, Materials and methods providing further evidence that C-terminal truncations of CHMP2B lead to FTD [20, 41]. Mice CHMP2B functions as a subunit of the endosomal sorting complex required for transport III (ESCRT-III). The mutant CHMP2BIntron5 expressing mouse line Tg153 ESCRTs are multi-subunit complexes, highly conserved was used and maintained as a homozygous line as previously from yeast to mammals, which mediate the bending and described [14]. The full-length human CHMP2BWild-type fission of membranes [18]. Membrane manipulation by mouse line Tg168 was maintained as a hemizygous line ESCRT complexes occurs through the sequential recruit- and non-transgenic littermates used for controls as previ- ment of the multimeric complexes ESCRTS 0 through III, ously described [14]. which function to scaffold membrane deformation, and finally recruit VPS4 to fission the deformed membranes. Mouse brain immunofluorescence and pathology This process of membrane bending is used for several bio- quantification logically diverse but topologically similar processes: during the final stages of cell division, during virus egress from Mouse brains were immersion fixed in 10 % buffered for- cells, and importantly for this study, during the maturation mal saline, embedded in paraffin wax and sections cut at of endosomes [18]. Maturing endosomes acquire numerous 3–4 μm thickness. Antigen retrieval was performed by intraluminal vesicles (ILVs) during their progress to late microwaving for 20 min in 0.1 M citrate buffer. Primary endosomes, which are also known as multivesicular bodies antibodies were as follows: p62 (GP62-C, PROGEN), (MVBs). MVBs ultimately fuse with lysosomes to allow β-III tubulin (ab78078, Abcam), Iba1 (019-19741, Wako), degradation of the endosomal content [18, 27]. In addition, Iba1 (ab5076, Abcam), GFAP (Z0334, DAKO), SMI-94 1 3 Acta Neuropathol (2015) 130:511–523 513 (Ab24567, Abcam) and CAII (ab6621, Abcam). Alexa autofluorescent object identification were performed as Fluor-conjugated secondary antibodies (Life Technolo- above for mouse sections. Quantification of pathology was gies) were used to visualise the staining. Images were col- performed on all cases by a researcher blinded to the sam- lected using a 40 lens with 1.4 NA on a Zeiss LSM 710, ple origin. Autofluorescent objects were manually catego- × or a 63 lens with a 1.4 NA on a Zeiss LSM 510 confo- rised based on their characteristic morphology (see Figs. 7, × cal microscope. Spectral imaging with subsequent linear S8), enabling differentiation of granular lipofuscin from the unmixing was used to separate the autofluorescence from large, dense autofluorescent aggregates. Autofluorescent the emission of each of the Alexa Fluor-labelled second- objects were localised to either microglia (identified by ary antibodies. Quantification was performed blinded on Iba1 positive

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