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doi:10.1093/brain/awt043 Brain 2013: 136; 1488–1507 | 1488 BRAIN A JOURNAL OF NEUROLOGY A zebrafish model of CLN2 disease is deficient in tripeptidyl peptidase 1 and displays progressive neurodegeneration accompanied by a reduction in proliferation Fahad Mahmood,1 Sonia Fu,2 Jennifer Cooke,1 Stephen W. Wilson,2 Jonathan D. Cooper3 and Claire Russell1 1 Department of Comparative Biomedical Sciences, Royal Veterinary College, London, NW1 0TU, UK 2 University College London, Department of Cell and Developmental Biology, London WC1E 6BT, UK 3 Paediatric Storage Disorders Laboratory, Department of Neuroscience, Centre for the Cellular Basis of Behaviour, King’s Health Partners Centre for Neurodegeneration, James Black Centre, Institute of Psychiatry, King’s College London, London SE5 9NU, UK Correspondence to: Dr Claire Russell, Royal Veterinary College, Royal College Street, London, NW1 0TU, UK E-mail: [email protected] Tripeptidyl peptidase 1 (TPP1) deficiency causes CLN2 disease, late infantile (or classic late infantile neuronal ceroid lipofuscinosis), a paediatric neurodegenerative disease of autosomal recessive inheritance. Patients suffer from blindness, ataxia, epilepsy and cognitive defects, with MRI indicating widespread brain atrophy, and profound neuron loss is evident within the retina and brain. Currently there are no effective therapies for this disease, which causes premature death in adolescence. Zebrafish have been successfully used to model a range of neurological and behavioural abnormalities. The aim of this study was to characterize the pathological and functional consequences of Tpp1 deficiency in zebrafish and to correlate these with human CLN2 disease, thereby providing a platform for drug discovery. Our data show that homozygous tpp1sa0011 mutant (tpp1sa0011 / ) zebrafish display a severe, progressive, early onset neurodegenerative phenotype, characterized by a significantly small retina, a small head and curved body. The mutant zebrafish have significantly reduced median survival with death occurring 5 days post-fertilization. As in human patients with CLN2 disease, mutant zebrafish display storage of subunit c of mitochondrial ATP-synthase, hypertrophic lysosomes as well as localized apoptotic cell death in the retina, optic tectum and cerebellum. Further neuropathological phenotypes of these mutants provide novel insights into mechanisms of pathogenesis in CLN2 disease. Secondary neurogenesis in the retina, optic tectum and cerebellum is impaired and axon tracts within the spinal cord, optic nerve and the posterior commissure are disorganized, with the optic nerve failing to reach its target. This severe neurodegenerative phenotype eventually results in functional motor impairment, but this is preceded by a phase of hyperactivity that is consistent with seizures. Importantly, both of these locomotion phenotypes can be assayed in an automated manner suitable for high-throughput studies. Our study provides proof-of-principle that tpp1sa0011 / mutants can utilize the advantages of zebrafish for understanding pathogenesis and drug discovery in CLN2 disease and other epilepsies. Keywords: tripeptidyl peptidase 1; TPP1; CLN2 disease; late infantile neuronal ceroid lipofuscinosis; lysosomal storage disorder; zebrafish; model Abbreviation: CLN = ceroid lipofuscinosis neuronal; SCMAS = subunit c of mitochondrial ATP synthase; TUNEL = terminal deoxynucleotidyl transferase mediated dUTP nick end labelling Received June 29, 2012. Revised December 13, 2012. Accepted January 7, 2013 ß The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected] Zebrafish model of CLN2 disease Introduction The neuronal ceroid lipofuscinoses are a group of mostly autosomal recessive inherited lysosomal storage disorders with an estimated prevalence of 1 in 100 000 in the UK (www.orpha. net/orphacom/cahiers/dosc/GB/Prevalence_of_rare_diseases_by_ alphabetical_list.pdf). These progressive neurodegenerative diseases are the most common cause of childhood dementia in the UK (Verity et al., 2010) and are traditionally classified according to age of onset: congenital, infantile, late infantile, juvenile and adult onset. Genes for nearly all forms of neuronal ceroid lipofuscinosis have been identified and they are used in the new nomenclature system. For example, when the ceroid-lipofuscinosis, neuronal 2 (CLN2) gene is mutated the most common disease onset is late infantile, hence the disease is called CLN2 disease, late infantile (previously called late infantile neuronal ceroid lipofuscinosis). The CLN genes predominantly encode lysosomal hydrolases, transmembrane proteins or lysosomal transmembrane proteins, with at least one gene remaining to be identified (Siintola et al., 2007; Mole et al., 2011). The clinical signs of CLN2 disease, late infantile manifest between years 2 and 4 postnatally (Goebel and Wisniewski, 2004). Presenting symptoms are usually seizures accompanied by developmental regression leading eventually to psychomotor retardation, ataxia and spasticity (Santavuori, 1988; Williams et al., 2006). Visual failure is also apparent leading to blindness towards latter stages of the disease (Santavuori, 1988). MRI scans invariably show widespread cortical and cerebellar atrophy with enlarged sulci, as well as gliosis and demyelination of white matter affecting the internal capsule and optic radiation (Boustany, 1992; Autti et al., 1997a, b; Seitz et al., 1998). Furthermore, macular degeneration and bilateral optic nerve atrophy are prominent on ophthalmological examination (Traboulsi et al., 1987). The majority of patients become refractory to anti-epileptic drugs and there are no effective therapies for this condition, which causes death in the early teens. Mutations in the CLN2 gene at location 11p15 affects the function of the lysosomal serine protease tripeptidyl peptidase 1 (encoded by the TPP1 gene) (EC 3.4.14.9) and these mutations are responsible for CLN2 disease (Sleat et al., 1997; Rawlings and Barrett, 1999; Vines and Warburton, 1999). TPP1 is an N-terminal exopeptidase, with limited endopeptidase activity, of the sedolisin family that functions by utilizing a Glu272, Asp276, Ser475 catalytic triad (Junaid and Pullarkat, 2001; Walus et al., 2005). It is synthesized as a 46–48 kDa mature peptide from a 66–68 kDa precursor (Ezaki et al., 1999; Lin et al., 2001). Furthermore, TPP1 is part of the mannose 6-phosphate glycoproteome and is thus targeted to the lysosome (Sleat et al., 2008a). So far 98 mutations and 24 polymorphisms have been identified worldwide in the CLN2 disease-causing gene (neuronal ceroid lipofuscinoses mutation resource: www.ucl.ac.uk/ncl). As a result of TPP1 deficiency, cells from patients with CLN2 disease accumulate storage material including subunit c of mitochondrial ATP synthase (SCMAS), which may be a direct substrate of TPP1, although other protein and lipid components also accumulate (Palmer et al., 1992; Ezaki et al., 2000). Brain 2013: 136; 1488–1507 | 1489 Amongst the vertebrates, one mouse model (Sleat et al., 2004) and one canine model (Awano et al., 2006) exist for CLN2 disease. Both models reflect the clinical phenotype of CLN2 disease and provide valuable insights into CLN2 disease pathogenesis. However, both model systems are relatively slow to develop, are inaccessible in the early stages of development and it is costly and impractical to generate large numbers of animals for drug discovery. Therefore assessment of early disease pathogenesis has not been performed and high-throughput drug screening for therapeutics development is expensive. An alternative model organism, that develops externally, is relatively cheap to house and breed and is amenable to high-throughput drug discovery, is the zebrafish, Danio rerio. Zebrafish have been successfully used to model a range of metabolic, neurological and behavioural abnormalities including lysosomal storage diseases, neurodegeneration, epilepsy and retinal degeneration (Lockwood et al., 2004; Milan et al., 2006; Anichtchik et al., 2008; Flinn et al., 2009; Sallinen et al., 2009; Moro et al., 2010). The amenability of zebrafish to large-scale mutagenesis has facilitated the generation of mutants in genes orthologous to those causing human disease. The transparent, externally developing zebrafish embryos display rapid development with highly characteristic phenotypes that can be studied from the earliest stages of development. Furthermore, the ability of adult pairs to lay 100–200 embryos per week coupled with high stocking densities enables high-throughput assessment of drug efficacy and toxicity (Goldsmith, 2004; Barros et al., 2008; Rihel et al., 2010). In addition, the zebrafish tpp1 gene (Ensembl I.D. ENSDARG00000042793) is 62% identical to the human orthologue whereas the encoded Tpp1 protein is 67% homologous including the conserved Glu360, Asp272, Ser475 catalytic triad (Wlodawer et al., 2003). According to Ensembl, zebrafish Tpp1 is synthesized from 13 exons as a 557 amino acid pro-peptide, and therefore has a similar exon structure and size as seen in humans. The sequence also indicates that the zebrafish propeptide, like its human homologue, is likely to be targeted to the lysosome after removal of a 19 amino acid signal peptide. Given all the aforementioned features, zebrafish provide an excellent novel, and complementary, platform for modelling human TPP1 deficiency. In the present study we demonstrate that embryonic and larval zebrafish homozygous for a premature stop codon mutation in exon 3 of the tpp1 gene (tpp1sa0011 / ) recapitulate the pathological and behavioural features of the human condition. tpp1sa0011 / mutants show phenotypic resemblance to human CLN2 disease including early onset retinal and cerebellar degeneration, as well as motor defects and reduced survival. Furthermore, the degenerative changes correlate with localized apoptosis in the CNS and are accompanied by ubiquitous accumulation of SCMAS in cells and enlarged lysosomes, as seen in human CLN2 disease. In addition, novel defects in neurodevelopment are described, including reduced proliferation in the CNS and disorganized axon pathways, including the optic nerve, spinal motor nerves and the posterior commissure. Finally, we demonstrate a period of increased locomotion that indicates seizures before impaired swimming ability that progresses to complete loss of locomotion, which can be monitored using a digital video-tracking system. This 1490 | Brain 2013: 136; 1488–1507 zebrafish mutant thus provides not only a faithful model of human CLN2 disease, but also a platform for screening of compounds using potentially high-throughput assays such as locomotion, to identify any potentially therapeutic non-toxic compounds. Such compounds could be used in further preclinical trials in mouse or canine models of CLN2 disease, eventually leading to application in human patients. Materials and methods Generation and maintenance of mutant and morphant zebrafish Zebrafish were housed in a multi-rack aquarium system at the Royal Veterinary College and kept on a constant 14/10 h light/dark cycle at 27-29 C (Westerfield, 2007). tpp1sa0011 carrier and tpp1hu3587 carrier zebrafish were created by N-ethyl-N-nitrosourea mutagenesis in the Tübingen strain and were obtained from the Sanger Institute (Cambridge, UK: www.sanger.ac.uk/Projects/D_rerio/zmp/) as an outcross to wild-type Tupfel long fin. To generate tpp1 morphant embryos, 2 ng morpholino anti-sense oligonucleotides (Bill et al., 2009) against tpp1 messenger RNA were pressure-injected into 1-2 cell stage wild-type TupLF strain zebrafish embryos using a glass capillary injection needle. Morpholino sequences used were: tpp1 ATG morpholino (gcaacccgCATtgctttcgtgttcg; ATG is shown in capitals; Gene Tools) or tpp1 SPL morpholino (gcaaatgtctagtttggacttacAC; exon2 is in capitals; Gene Tools). Morpholino buffer [58 mM NaCl; 0.7 mM KCl; 0.4 mM MgSO4; 0.6 mM Ca(NO3)2; 5 mM HEPES; pH 7.6] (Nasevicius and Ekker, 2000; Bill et al., 2009) injection was used as a control. To prevent pigmentation, zebrafish were raised in water containing 0.003% 1-phenyl-2-thiourea from 24 h post fertilization. Ethical approval for zebrafish experiments was obtained from the Royal Veterinary College and the Home Office (UK) under the Animal (Scientific Procedures) Act 1986. Complementary DNA preparation, cloning and sequencing Total RNA extraction of zebrafish embryos was performed as described Westerfield (2007). For complementary DNA synthesis, 1 ng to 5 mg of total RNA was incubated with 250 ng of random primers and 0.8 mM (each) dNTPs at 65 C for 5 min. This was before further incubating the reaction mix in first strand buffer, 10 mM dTT and RNAsinÕ (400 U) at 25 C for 2 min. Finally 200 U of SuperscriptÕ II Reverse Transcriptase (Invitrogen) was added and the reaction mix was incubated at 25 C for 10 min before 42 C for 50 min. The resulting complementary DNA was used directly in a standard PCR reaction in 50 ml using GoTaqÕ (Promega). Primers used were: tpp1 AF1 gaggtgcacgaacacgaaa (forward primer in the 5’UTR); tpp1 AR2 caacccgtccaagaaatgtc (reverse primer in exon 3); tpp1 AR1 tatggcttgacgtgctcct (reverse primer in exon 6). Ten microlitres of the PCR product was separated by electrophoresis using VisiGelTM (Stratagene) for good band separation and visualized on a Bio-Rad UV Transilluminator. PCR products were cloned directly into pCRII-TOPO vector (Invitrogen) and transformed into chemically competent TOP10 Escherichia coli (Invitrogen) as per the manual. White colonies were cultured and their plasmids mini-prepped (Qiagen QIA Spin Miniprep Kit). Plasmid DNA was quantified using a NanoDropÕ ND1000 and sequenced using standard primers at the Wolfson Institute of Biomedical Research (UCL). F. Mahmood et al. Sequences were aligned to tpp1 genomic and transcript sequences using CLC Free Workbench software. Genotyping the tpp1sa0011 and tpp1hu3587 alleles Genomic DNA was extracted as described (Rehbein and Bogerd, 2007). The caudal fin clips of adult fish were obtained after anaesthesia in MS222 (Tricaine) and dissolved in 400 ml of lysis buffer (100 mM Tris-HCl; 200 mM NaCl; 0.2% SDS; 5 mM EDTA; 100 mg/ml proteinase K; double distilled H2O) overnight at 55 C. This was followed by proteinase K inactivation at 80 C for 30 min before quenching at 4 C. DNA was precipitated with 500 ml of isopropanol before centrifugation at 6500 rpm for 40 min. The resulting pellet of DNA was washed twice with 70% ethanol and suspended in 250 ml double distilled H2O. The same procedure was followed for 72 to 120 h post fertilization embryonic genomic DNA extractions with the exception that each embryo was suspended in only 50 ml of lysis buffer and DNA was precipitated using 60 ml of isopropanol. Genomic DNA was then used for PCR. The primers used to PCR amplify the region about exon 3 of the tpp1sa0011 allele were: Forward (tpp1-1-2a), tgtaaaacgacggccagt CAATAACCTGCCTAGTCCAAAC (lower case is M13f sequence, upper case is in introns 2–3); and reverse (tpp1-1-3a), aggaaacagctatgaccatTTGATGACTCGGCTGAATAG (lower case is a non-standard M13r sequence, upper case is in intron 3-4). The primers used to PCR amplify the region about exon 11 of the tpp1hu3587 allele were: forward (tpp1-9-2), TTGGCTTGTTGTGAAGACTC (in introns 10–11); and reverse (tpp1-9-3), GCAGCTTACATTGCTTATTGG (in introns 11–12). The PCR was performed using the IMMOLASETM DNA Polymerase kit (Bioline): 1 Hi-Spec additive, 1 Imo-buffer, 2.5 mM MgCl2, 0.25 mM dNTP mix, 1 unit of IMMOLASETM DNA Polymerase (5 U/ml), 0.4 mM of each primer and 50 ng of genomic DNA in a final volume of 25 ml (annealing temperature = 56.5 C). PCR products were subsequently purified using the QIAGEN PCR Purification Kit and sequenced at the Wolfson Institute of Biomedical Research (UCL). Western blotting Western blotting was performed as previously described (Link et al., 2006; Westerfield, 2007). Briefly, 48 h post fertilization embryos were dechorionated before yolk sac removal by vigorous pipetting in cold Ringers + solution (116 mM NaCl, mM KCl, 1.8 mM CaCl2, 5 mM HEPES; 0.1 mM EDTA, 0.3 mM phenylmethylsulphonyl fluoride, pH 7.2). The samples were homogenized in 1 SDS sample buffer (0.125 M Tris-HCl pH 6.8, 20% glycerol, 10% b-mercaptoethanol, 4% SDS) and proteins were denatured at 95 C for 5 min. Protein quantification was performed by NanoDropÕ spectrophotometry and 50 mg of protein sample as well as 5 ml of ColorPlusTM prestained protein ladder (NEB P7711) markers were loaded onto a 4% polyacrylamide stacking gel (4% ProtoGelTM; 0.125 M Tris-HCl pH6.8, 0.1% SDS, 0.06% ammonium persulphate, 0.12% TEMED) and run on a 10% polyacrylamide gel (10% ProtoGelTM), 0.375 M TRIS-HCl pH8.4, 0.1% SDS, 0.1% ammonium persulphate; 0.04% TEMED). The electrophoresed proteins were transferred onto a methanol saturated polyvinylidene difluoride membrane using wet-transfer methodology (transfer buffer: 25 mM Tris-base pH 8.5, 0.2 M glycine, 20% methanol). The polyvinylidene difluoride membrane was probed with rabbit anti-TPP1 (human) (gift from Dr J. Tynnelä) (Ezaki et al., 1999) primary antibody (1:900) overnight at 4 C in 5% bovine serum albumin/Tris-buffered saline 0.1% Tween Goat anti-rabbit horseradish peroxidase-conjugated Zebrafish model of CLN2 disease secondary antibody (1:3000, Dako, P0048) was subsequently applied for 1.5 h at room temperature. The blot was developed using the ECL immunoblotting protocol (GE Healthcare). Horseradish peroxidase conjugated anti-GAPDH (1:4000) (gift from Dr M. Campanella) (Abcam, ab9482) was used as a loading control. Tpp1 activity assay Tpp1 activity was measured as previously described (Sleat et al., 2008b). Zebrafish at 96 h post fertilization were suspended in substrate buffer (150 mM NaCl, 100 mM sodium citrate, 1 g/l TritonTM X-100, pH 4.0) at a ratio of one embryo per 10 ml. The embryos were homogenized using a pestle and the crude protein suspension was quantified using NanoDropÕ spectrophotometry. Total protein (6 mg) was added to 0.25 mM Arg-Ala-Phe-ACC (from 25 mM stock in dimethyl sulphoxide; gift from Professor P. Lobel) (Sleat et al., 2008b) in substrate buffer (as above) to a final reaction volume of 50 ml in a single well of a black 96-well plate. The reaction mixture was incubated in the dark for 90 mins before termination by addition of 25 ml 10% SDS. The fluorescence values were determined with a Mithras fluorescence plate reader using a 360 nm excitation and 460 nm emission filter. The final fluorescence values were subtracted from the initial fluorescence (prior to addition of protein) to correct for background histofluorescence. Whole mount immunofluorescence Immunofluorescence staining was performed as previously described by (Westerfield, 2007). 1-phenyl-2-thiourea-treated embryos were digested in 0.25% trypsin for 8 min on ice or 10 mg/ml proteinase K for 1 h at room temperature following fixation in 4% paraformaldehyde/PBS. The embryos were subsequently washed in PBS + 0.8% TritonTM X-100 and blocked in 10% normal goat serum: 1% dimethyl sulphoxide in PBS + 0.8% TritonTM X-100 blocking solution for 1 h at room temperature. This was followed by incubation with primary antibody at 4 C overnight [anti-acetylated mouse alpha-tubulin (1:1000, Sigma, T6793), rabbit anti-pH3 (1:1000, Upstate, 06-570), mouse anti-HuC/D (1:1000) (Molecular Probes, A21271), rabbit anti-SCMAS (1:1000) (gift from Dr J. Tyynelä) (Suopanki et al., 2000), or rabbit anti-GFAP (gift from Sam Nona and John Scholes) (Heath and Xavier, 2009)] in blocking solution. Following several washes, the embryos were incubated at 4 C overnight in an appropriate fluorescent secondary antibody [goat anti-rabbit Alexa FluorÕ 488, (1:200, Invitrogen, A10034) or goat anti-mouse Alexa FluorÕ 488 (1:200, Invitrogen, A11001)]. Fluorescently-labelled embryos were imaged by scanning laser confocal microscopy (LeicaTM SP5) and images subsequently manipulated using Adobe Photoshop. All images for the same antibody were acquired and manipulated in exactly the same way. Immunofluorescence staining of zebrafish embryo sections Tpp1 immunofluorescence staining was performed as described (Westerfield, 2007). Briefly, 1-phenyl-2-thiourea-treated zebrafish embryos were fixed in 4% paraformaldehyde/PBS overnight at 4 C before 30% sucrose impregnation. The embryos were subsequently mounted and frozen in TissueTek O.C.T. (VWR, 25608-930) in liquid nitrogen before being transverse-sectioned into 10 mm slices on a cryostat and collected onto SuperfrostÕ PLUS slides. Following a 10 min wash in PBS, the sections were permeabilized in 100% Brain 2013: 136; 1488–1507 | 1491 methanol for 10 min before three 5 min washes in PBS + 0.1% Tween-20. The sections were blocked for 1 h with 1.5% normal goat serum in PBS + 0.1% Tween-20, before overnight incubation with rabbit anti-TPP1 (1:600) (see above) in blocking solution at 4 C. After a further three washes for 5 min in PBS + 0.1% Tween-20, the sections are incubated in secondary antibody [goat anti-rabbit Alexa488 FluorÕ (as above)] in PBS + 0.1% Tween-20 for 1 h in the dark at room temperature. The sections were mounted in 70% glycerol before imaging using the Leica SP5 Confocal. TUNEL assay Apoptosis was detected by a terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) assay (Gavrieli et al., 1992; Williams and Holder, 2000). Dechorionated 1-phenyl-2thiourea-treated embryos between 48 and 72 h post fertilization were fixed in 4% paraformaldehyde/PBS overnight at 4 C followed by washing in PBS + 0.1% TritonTM X-100 and subjected to proteinase K digestion according to the age of the embryo (48 h post fertilization, 10 ng/ml for 1 h; 72 h post fertilization, 15 ng/ml for 1 h in PBS + 0.1% TritonTM X-100). Subsequently, the embryos were fixed for 20 min in 4% paraformaldehyde and blocked in Equilibration Buffer (MilliporeTM) for 1 h before overnight incubation at 37 C in TdT reaction mix (Reaction Buffer 2, ChemiconTM and TdT enzyme, ChemiconTM, in a ratio of 2:1) and 0.3% TritonTM X-100. Subsequent to several washes in PBS + 0.1% TritonTM X-100 the embryos were blocked in MA Block [150 mM NaCl 100 mM malic acid, 2% blocking reagent (Boehringer Mannheim), pH7.5] for 2 h at room temperature and then incubated overnight in anti-DIG-alkaline phosphatase Fab fragments (1:3000 in MA Block). The DNA fragments were resolved using the chromogenic substrate NBT (18.75 mg/ml)/BCIP (9.4 mg/ml) (Roche) [20 ml in 1 ml Buffer B3 (0.1 M Tris-HCl, 50 mM MgCl2, 0.1 M NaCl, 0.1% Tween20)]. TUNEL-labelled embryos were imaged using a LeicaTM DM IRB inverted microscope. The number of apoptotic cell bodies in TUNEL stained embryos were quantified within the whole eye, the forebrain, mid-hindbrain and spinal cord (somites 14–20) using ImageJ software. Locomotion analysis Individual 96 h post fertilization tpp1sa0011 / or wild-type fish were placed in 50 ml of aquarium water within a single well of a 96-well plate, mounted on the stage of a DMI-6000 brightfield microscope. One hundred and seventy second-long video recordings of individual fish were obtained with the Leica DFC-350 FX camera (frame rate: 20 frames/s) with an exposure time of 82 ms. A diffuser fitted within the microscope ensured even light spread. The well chamber temperature was kept constant at 28 C and humidified during the recording period using 5% CO2 (200 bar pharmaceutical grade, BOC) delivered at 1 l/min. For locomotion tracking of 72 h post fertilization larvae, individual tpp1sa0011 / or wild-type fish were placed in 50 ml of aquarium water within a single well of a 96-well plate, mounted on a Nikon SMZ1500 stereomicroscope and recorded for 20 min using a Digital Sight DS-2 Mv camera (30 frames/s) in a room kept at 25 C. Various movement parameters of individual zebrafish were quantified using Ethovision XT (Noldus) software. Movement bouts were defined as the period between which the average velocity of zebrafish exceeded the user-defined Start Velocity of 0.4 mm/s and remained moving until the user defined Stop Velocity of 0.2 mm/s over a predefined averaging interval of five samples. This was taken to represent darting behaviour. 1492 | Brain 2013: 136; 1488–1507 All statistical analysis was performed using GraphPad Prism 5.00 except survival assessment, which was performed using SPSS v7. Results Zebrafish tpp1 mutant homozygotes show outward signs of neurodegeneration Zebrafish carriers of the tpp1sa0011 and tpp1hu3587 mutant alleles (tpp1sa0011 + / and tpp1hu3587 + / , respectively) generated by Nethyl-N-nitrosourea mutagenesis were obtained and verified by PCR and direct sequencing of genomic DNA from adult fish fin-clips. The tpp1sa0011 allele has a single T4A base pair change in exon 3 (Fig. 1C and D), resulting in an in-frame, premature stop codon within the tpp1 gene and transcript (Fig. 1B). The tpp1hu3587 allele has a single T4A base pair change (Fig. 1F and G) that encodes an in-frame stop codon in exon 11 (Fig. 1E). A comparison of the tpp1 transcripts generated from the wild-type, tpp1sa0011 and tpp1hu3587 alleles is shown in Fig. 1A. matings (n = 10) gave approximately All tpp1sa0011 + / one-quarter of embryos (28%) with an abnormal phenotype at 72 h post fertilization, indicating autosomal recessive inheritance (Fig. 1I). We named these embryos ‘putative tpp1sa0011 / ’, and their normal looking siblings ‘wild-type’. More detailed inspection revealed that the abnormal phenotype could be first observed at 48 h post fertilization, following a period of apparently normal development, after which severe developmental abnormalities appeared, characterized by an increasingly curved body, small eyes and a shrunken head (Fig. 2A and C). Furthermore, by 72 h post fertilization there was no detectable jaw as well as signs of pericardial oedema (Fig. 2B and D). At later stages the swim bladder was not detectable in tpp1sa0011 / embryos (data not shown). The progressive reduction in eye size is an early and prominent feature that we show to be statistically significantly different in putative tpp1sa0011 / embryos compared with wild-type. The mean retinal area of putative tpp1sa0011 / embryos was significantly different from wild-type siblings (P 5 0.001, unpaired t-test) (n = at least four each) (Fig. 2L). By 72 h post fertilization this difference was even greater (P 5 0.001, unpaired t-test) as the putative tpp1sa0011 / showed little change since 48 h post fertilization but the wild-type retinal area increased (Fig. 2L) (n = at least four each). Importantly, genotyping confirmed that putative tpp1sa0011 / larvae (n = 13) were homozygous for tpp1sa0011 (Fig. 1D). We next examined the phenotype of a second zebrafish tpp1 mutant, by examining offspring from incrossing fish heterozygous for the tpp1hu3587 allele (tpp1hu3587 + / ). Only 9.4% (n = 6 crosses) (data not shown) of tpp1hu3587 + / offspring displayed a similar morphological phenotype to putative tpp1sa0011 / (Fig. 2E and F), which is less than expected for autosomal recessive inheritance and suggests a weaker allele. We raised the offspring to 3 months old, genotyped them tpp1hu3587 + / (Fig. 1G) and found that most tpp1hu3587 / zebrafish had surand tpp1sa0011 + / were vived. Finally, when tpp1hu3587 + / F. Mahmood et al. inter-crossed, approximately one-quarter of the offspring (Supplementary Fig. 1G) displayed an abnormal phenotype similar to that displayed by tpp1sa0011 / larvae, but their disease progression was more protracted. Morphological abnormalities were first evident in these compound mutant fish at 72 h post fertilization and by 120 h post fertilization larvae had small eyes and brain as well as loss of jaw and swim bladder compared with normal siblings (Fig. 2G and Supplementary Fig, 1A–F). Furthermore, genotyping confirmed that morphologically abnormal larvae were heterozygous for tpp1sa0011 and tpp1hu3587 alleles (n = 3) (Supplementary Fig. 1H and I). To further confirm that the abnormal phenotype seen in tpp1sa0011 homozygotes is due to mutation in tpp1 and no other genes, we examined if the phenotype was recapitulated in Tpp1 morpholino knock-down embryos. Injecting 2 ng of tpp1 SPL morpholino (aberrantly splicing the tpp1 messenger RNA transcript) or tpp1 ATG morpholino (inhibiting tpp1 messenger RNA translation) into wild-type fish resulted in an abnormal phenotype beginning around 48 h post fertilization including increasingly smaller eyes and head, curvature within the body axis, a reduced jaw (Fig. 2H–K) and no swim bladder (data not shown), similar to that seen in tpp1sa0011 / zebrafish. Western blotting confirmed the specificity of the TPP1 antibody for the zebrafish Tpp1 protein and a loss of Tpp1 in tpp1 mutants and morphants. A band of 46 kDa, the expected size for mature Tpp1 (Ezaki et al., 1999), was observed in wild-type 48 h post fertilization embryos from a tpp1sa0011 + / incross. Near complete absence of TPP1 protein was observed in abnormal 48 h post fertilization tpp1sa0011 / embryos from the same tpp1sa0011 + / incross, and also in wild-type embryos injected with 2 ng of the tpp1 ATG morpholino (Fig. 1J). To further demonstrate that tpp1 knockdown results in the morphologically abnormal phenotype, tpp1 SPL morpholino injected embryos raised to 24 h post fertilization were assayed for aberrant tpp1 splicing. Reverse transcriptase-PCR using a forward primer against the 5’-UTR region (TPP1 AF1) and a reverse primer either in exon 3 (TPP1 AF2) or exon 6 (TPP1 AF6) gave additional bands in tpp1 SPL morpholino injected fish (Fig. 1H) compared with uninjected controls. The extra bands were sequenced and two novel transcripts were found, one corresponding to the retention of intron 2, which contains a stop codon, and one corresponding to the loss of exon 2 (Fig. 1A). Therefore the tpp1 SPL morpholino causes two abnormal tpp1 transcripts to be produced. We next measured Tpp1 activity in whole protein derived from abnormal 96 h post fertilization tpp1sa0011 / mutants and wild-type siblings from a tpp1sa0011 + / incross using the fluorogenic substrate Arg-Ala-Phe-ACC (Sleat et al., 2008b). As any maternally derived Tpp1 protein would be found in the lysosomes, and because zygotically derived mutant Tpp1 would not reach the lysosome as it does not have a signal sequence, we decided to use a crude whole protein extract rather than a lysosomal preparation. Tpp1 activity was significantly reduced (P 5 0.0001) in putative tpp1sa0011 / embryos compared with wild-type embryos after 90 min incubation with 6 mg of crude protein extract (Fig. 1K). Lastly, immunofluorescence staining for Tpp1 was performed in 48 h post fertilization wild-type and tpp1sa0011 / embryos to confirm Tpp1 deficiency in situ. Tpp1 is present throughout the Zebrafish model of CLN2 disease Brain 2013: 136; 1488–1507 | 1493 Figure 1 Manipulations of tpp1 cause Tpp1 deficiency. (A) Diagram illustrating the tpp1 gene transcript in wild-type (WT), tpp1sa0011 / sa0011 mutant and tpp1 SPL morphants. The horizontal lines indicate untranslated regions whereas coloured blocks indicate exons. tpp1 and tpp1hu3587 predicted peptide sequences indicate the presence of a premature stop within peptides derived from these transcripts whereas tpp1 SPL morpholino (MO) causes the retention of intron 2 in some transcripts (MO T1) and excision of exon 2 in other transcripts (MO T2). Genomic DNA extracted from adult fin clips (B and C) or embryos (D) was PCR amplified and sequenced in the region of exon 3 of the tpp1 gene to identify wild-type (B), tpp1sa0011 + / heterozygous carriers (C) and tpp1sa0011 / homozygous mutants (D). The (continued) 1494 | Brain 2013: 136; 1488–1507 wild-type embryo but we focused on the retina as it has the most overtly recognizable structures in these sections and is clinically relevant. We detected Tpp1 in all layers of the wild-type retina, particularly in the outer nuclear layer and ganglion cell layer, whereas Tpp1 levels were markedly absent in 48 h post fertilization tpp1sa0011 / retina (Fig. 3) in correlation with western blot analysis. Taken together, these data confirm that the homozygous tpp1sa0011 / phenotype is caused by mutation in tpp1, and not by any other mutations that may be present in the tpp1sa0011 + / carriers. Importantly, the tpp1sa0011 / phenotype is fully penetrant and consistent with a phenotype expected for a neurodegenerative disease. Tpp1 deficient zebrafish die prematurely Humans with the late infantile form of CLN2 disease die in or before the second decade of life (Chang et al., 2011). Therefore, we sought to determine when Tpp1 deficient zebrafish died by performing Kaplan-Meier survival analysis. The tpp1sa0011 / zebrafish started to die from 4 days post fertilization and none were surviving at 7 days post fertilization. All wild-type and tpp1hu3587 / embryos were alive at 7 days post fertilization at which point the experiment was halted (Fig. 1L). The median survival for tpp1sa0011 / larvae was calculated as 5 days post fertilization and log-rank (Mantel–Cox) analysis to test the null hypothesis of no difference in survival functions between wild-type and tpp1sa0011 / larvae gave a P-value 50.0001. Thus the mutant phenotype can be correlated directly with significantly reduced survival prospects. Ubiquitous storage of subunit c of mitochondrial ATP synthase in tpp1sa0011 / mutants Storage material accumulation, in particular stored subunit c of mitochondrial ATP synthase (SCMAS) within lysosomes, is characteristic of CLN2 disease pathology (Palmer et al., 1992, 1997; Westlake et al., 1995). Forty-eight hours post fertilization tpp1sa0011 / mutants had higher levels of SCMAS throughout F. Mahmood et al. the embryo, including within the eye, head, spinal cord, and prominently within the muscle fibres compared with wild-type siblings (Fig. 4A–D). We were concerned whether such ubiquitous signal could be background histofluorescence or due to accumulation of autofluorescent storage material (attributable to ceroid and lipofuscin accumulation) in tpp1sa0011 / zebrafish so we performed the same experiment without the anti-SCMAS primary antibody. Images demonstrate that the SCMAS staining in the CNS and muscle is neither background nor autofluorescence (Fig. 4I–L). To ascertain if storage is intralysosomal, LysoTrackerÕ Red was used to label lysosomes, demonstrating the presence of enlarged lysosomes throughout the CNS as well as in yolk cells (not shown), but not in the muscle, in 48 h post fertilization zebrafish compared with wild-type siblings tpp1sa0011 / (Fig. 4E–H). These data demonstrate that storage of SCMAS is present throughout tpp1sa0011 / mutants and that the storage material accumulation at 48 h post fertilization correlates with Tpp1 deficiency and the visible onset of neurodegeneration, but is not associated with detectable autofluorescence at this stage. However, lysosomal enlargement is predominantly seen in the CNS. Neuropathology in Tpp1 deficient zebrafish Selective neuronal loss in the cerebral cortex, cerebellum and retina is another hallmark of CLN2 disease pathology (Nardocci et al., 1995; Petersen et al., 1996; Autti et al., 1997a; Lavrov et al., 2002; Sleat et al., 2004; Chang et al., 2008, 2011; Cooper et al., 2011). As Tpp1 deficient embryos have a smaller head and eye, we examined their CNS for a neuronal deficit in comparison with wild-type siblings. A profound absence of HuC/ D-positive differentiated neurons was observed in 48 h post fertilization tpp1sa0011 / (Fig. 5A and E; n = 5 each) and tpp1 SPL morpholino injected wild-type (Supplementary Fig. 2A and D) retina, optic tectum and cerebellum when compared with wild-type (n = 5) that was also evident at 72 h post fertilization (data not shown). We did not detect such overt loss of neurons in other structures including the thalamus, telencephalon and olfactory bulbs using this method. Figure 1 Continued tpp1hu3587 + / carrier offspring was raised to adulthood and sequencing of exon 11 revealed that wild-type siblings (E), tpp1hu3587 + / heterozygous carriers (F) and tpp1hu3587 / homozygous mutants (G) can survive to adulthood. Reverse transcriptase-PCR of tpp1 SPL morpholino injected wild-type zebrafish (H, Lane 1) confirmed mis-splicing of tpp1 messenger RNA transcript compared with the normal tpp1 transcript (H, Lane 2). (I) Proportion of wild-type to tpp1sa0011 / mutants obtained over 10 crosses of tpp1sa0011 + / carriers shows autosomal recessive inheritance of the tpp1sa0011 / phenotype. Mean percentage of tpp1sa0011 / obtained was 28%. Western blotting (J) confirmed the near complete absence of 46 kDa mature Tpp1 in tpp1sa0011 / mutants as well as complete Tpp1 absence in tpp1 ATG morpholino injected 48 h post fertilization wild-type embryos. TPP1 enzyme activity was measured for 90 min in 96 h post fertilization tpp1sa0011 / mutants and wild-type sibling crude protein extracts using the Arg-Ala-Phe-ACC substrate (K, n = 7). TPP1 activity was significantly diminished in tpp1sa0011 / mutants compared to wild-type (P 5 0.0001). Kaplan-Meier survival analysis (G), comparing wild-type, tpp1sa0011 / and tpp1hu3587 / embryos (15 of each), shows the probability of tpp1sa0011 / mutant survival compared with wild-type siblings. tpp1sa0011 / mutants started to die at 4 days post fertilization and all had died by 7 days post fertilization. The median survival was determined as 5 days post fertilization. All 15 wild-type siblings and tpp1hu3587 / embryos survived beyond 7 days post fertilization at which point the experiment was concluded. Log rank (Mantel-Cox) test applied to the null hypothesis that there is no difference between survival functions (P 5 0.0001). Black arrows = site of base pair change. hpf = hours post fertilization. Zebrafish model of CLN2 disease Brain 2013: 136; 1488–1507 | 1495 Figure 2 Similarity of phenotypes obtained after manipulation of tpp1. Bright-field images show wild-type siblings (A and B), tpp1sa0011 / larvae (C and D), tpp1hu3587 / (E and F), tpp1sa0011 + / and tpp1hu3587 + / cross (G), tpp1 ATG morpholino (H and I) and tpp1 SPL morpholino (J and K). All tpp1 manipulations cause small eyes, a small head and loss of jaw compared with wild-type siblings. The offspring from a tpp1hu3587 + / incross display a variably penetrant phenotype. At 48 h post fertilization offspring displayed a range of phenotypes from seeming wild-type to a typical tpp1 mutant phenotype. Quantification of mean retinal area at several time points (L) shows significantly impaired retinal growth over time in tpp1sa0011 / mutants compared with wild-type (unpaired t-test P 5 0.001 at 48 h and 72 h post fertilization; P = 0.01 at 54 h post fertilization) (n = 10 at 48 h post fertilization, n = 4 at both 54 h post fertilization and 72 h post fertilization). Dashed line indicates outline of the eye. Error bars = SEM; scale bar 300 mm. FB = forebrain; hpf = hours post fertilization; MO = morpholino; WT = wild-type. 1496 | Brain 2013: 136; 1488–1507 Figure 3 Tpp1 absence in tpp1sa0011 F. Mahmood et al. / mutant retinal cells. Ten micrometre-thick retinal sections of 48 h post fertilization wild-type mutants (C and D) were immunolabelled with rabbit anti-TPP1. Tpp1 is expressed throughout the siblings (A and B) and tpp1 wild-type retina (A) with prominent expression in the ganglion cell layer, inner plexiform layer and the outer nuclear layer (B). In the tpp1sa0011 / mutant retina (C), no obvious Tpp1 protein is observed (D) and the retinal architecture is abnormal owing to widespread pathology. BF = brightfield; GCL = ganglion cell layer; INL = inner nuclear layer; IPL = inner plexiform layer; ONL = outer nuclear layer; OPL = outer plexiform layer; n = 6 sections each for wild-type and tpp1sa0011 / mutant. Scale bars: brightfield = 100 mm; immunofluorescence = 10 mm. sa0011 / Astrocytosis is also a significant and early pathological feature of CLN2 disease in the mouse model (Chang et al., 2008) and when we looked at glial fibrillary acidic protein (GFAP) expression (a marker of astrocytosis) we found localized GFAP immunoreactiity in the thalamus and cerebellum of 48 h post fertilization tpp1sa0011 / zebrafish that was absent in wild-type siblings (Fig. 5B and F). Apoptosis is evident in Tpp1 deficient zebrafish The absence of neurons in specific parts of the CNS of tpp1sa0011 / zebrafish could reflect cell death or a defect in the development of neurons. As TUNEL staining of CNS tissue obtained at autopsy from patients with CLN2 disease has Zebrafish model of CLN2 disease Brain 2013: 136; 1488–1507 | 1497 / embryos. Forty-eight hours post fertilization wild-type siblings (A, B, E, F, I, I’, J and J’) and tpp1sa0011 / (C, D, G, H, K, K’, L and L’) were immunostained with rabbit SCMAS (A–D), LysoTrackerÕ Red (E–H), or using the same procotol as for the SCMAS immunofluorescence, but without the primary antibody (I–K and I’–K’). No significant SCMAS staining observed in the head (A) or tail (B) of wild-type siblings. Extensive SCMAS storage is observed throughout tpp1sa0011 / mutants including within the retina and forebrain in the head (C), and muscle fibres in the tail (D). LysoTrackerÕ Red staining revealed small punctate lysosomes in the retina, forebrain and hindbrain of wild-type siblings (E), with no significant staining in the tail (F). In tpp1sa0011 / embryos, lysosomes are greatly enlarged in the retina, forebrain and hindbrain (G) but not in the tail (H). Immunofluorescence performed without primary antibody shows no significant background immunofluorescence or autofluorescence in wild-type siblings (I and J) or tpp1sa0011 / (K and L). Brightfield images of tissues shown in I–L are shown in I’–L’. FB = forebrain; m = muscle fibres; R = retina; OV = otic vesicle; WT = wild-type. n = 6 each for wild-type and tpp1sa0011 / mutants. Scale bar = 100 mm. Figure 4 Storage material accumulates in tpp1sa0011 1498 | Brain 2013: 136; 1488–1507 F. Mahmood et al. Figure 5 Tpp1 knockdown induces early onset neurodegeneration. Anti-HuC/D (A and E), anti-GFAP (B and F), anti-phosphohistone 3 (PH3) (C and G) and anti-acetylated alpha tubulin (D and H) immunofluorescence was performed on 48 h post fertilization wild-type siblings (A–D) and tpp1sa0011 / mutants (E–H). HuC/D reveals differentiated neurons and shows a missing cerebellum, retina and optic tectum in tpp1sa0011 / mutants (E) compared with wild-type siblings (A). GFAP labels few glia in the brain of wild-type siblings (B), but is evident in the thalamus and cerebellum of tpp1sa0011 / mutants (F). Phosphohistone 3 labels cell proliferation and shows impaired proliferation in the CNS of tpp1sa0011 / mutants (G) compared with wild-type siblings (C). Acetylated alpha-tubulin labels axons and reveals defects within the optic nerve, posterior commissure and trochlear nerve in tpp1sa0011 / (H) compared with wild-type siblings (D). AC = anterior commissure; C = cerebellum; FB = forebrain; ON = optic nerve; OT = optic tectum; PC = posterior commissure; R = retina; SOT = supraoptic tract; TPOC = tract of the post optic commissure; T = thalamus; IV = trochlear nerve. (n = 5 for wild-type and tpp1sa0011 / mutants for each stain). Scale bar = 100 mm. To quantify cell proliferation in specific CNS regions of wild-type (continued) Zebrafish model of CLN2 disease previously shown selective neuronal cell death in the cerebral cortex, cerebellum and retina (Lane et al., 1996; Kurata et al., 1999; Mitchison et al., 2004), we examined cell death in tpp1sa0011 / zebrafish using the TUNEL assay. Examination of embryos derived from tpp1sa0011 + / heterozygote in-crosses at 24 h, 30 h and 42 h post fertilization did not reveal TUNEL-positive bodies greater than expected for the particular stage of development in any of the embryos (data not shown) (Cole and Ross, 2001). However, when compared with wild-type siblings from 48 h to 54 h post fertilization, mutants possessed a large number of TUNEL-positive apoptotic bodies selectively localized in the retina, optic tectum, cerebellum and throughout the spinal cord (Fig. 6A–H). However by 72 h post fertilization, although there were numerous apoptotic bodies in the skin throughout the length of the tpp1sa0011 / mutant, only a few were seen within neural tissue. We quantified the TUNEL positive bodies in the retina, forebrain, mid/hindbrain and the spinal cord (somites 14–20) from 48 h to 72 h post fertilization (n = 4 each, wild-type and tpp1sa0011 / mutant). The Mann–Whitney U-test, comparing wild-type and tpp1sa0011 / embryos at 48 h and 54 h post fertilization gave P 5 0.0286 and at 72 h post fertilization P = 1 (Fig. 6I). Therefore Tpp1 deficient zebrafish show profound early onset, localized cell death in a subset of CNS regions homologous to some of those affected in human CLN2 disease. Moreover, increased cell death in tpp1sa0011 / mutants is first visible at 48 h post fertilization consistent with the onset of morphological abnormalities and also suggests that apoptosis is an early, not late, feature of CLN2 disease. The combined pathological features of tpp1sa0011 / zebrafish indicate that it is a good model for human CLN2 disease. We next sought to examine tpp1sa0011 / zebrafish for novel defects that have not yet been described in other models of CLN2 disease. Reduction of cell proliferation in the zebrafish tpp1sa0011 / central nervous system Altered CNS cell proliferation has not yet been examined in animal models of CLN2 disease despite being implicated in other forms of neuronal ceroid lipofuscinoses including CLN3 disease, juvenile (Kay et al., 2006; Vantaggiato et al., 2009; Weimer et al., 2009). We investigated if the marked absence of differentiated neurons correlated with impaired cell proliferation or if proliferation was enhanced as an attempt to repair the CNS. Phosphohistone H3 labelling of Tpp1 deficient zebrafish was Brain 2013: 136; 1488–1507 | 1499 performed between 48 h post fertilization and 72 h post fertilization (n = 5 each) and the number of proliferative cells were quantified in four different regions [retina, forebrain, mid-hindbrain and spinal cord (somite 14-20)] of wild-type and tpp1sa0011 / zebrafish at each stage. There was no difference in the level of proliferative neuronal precursors before 48 h post fertilization (data not shown). At 48 h post fertilization cell proliferation within the tpp1sa0011 / (Fig. 5C and G) and tpp1 SPL morpholino injected wild-type (Supplementary Fig. 2C and F) retina, forebrain, midbrain-hindbrain boundary and spinal cord was significantly reduced compared with wild-type zebrafish. The difference in proliferation of cells between wild-type and tpp1sa0011 / retina, forebrain and midbrain-hindbrain boundary, but not spinal cord, was significant at 54 h post fertilization and 72 h post fertilization (Fig. 5I–L). These data show that reduced cell proliferation is extensive from phenotypic onset to beyond 72 h post fertilization, lasting longer than apoptosis in tpp1sa0011 / zebrafish. Axon tracts are disrupted in Tpp1 deficient zebrafish Axonal disorganization and white matter tract degeneration is widespread both in the CNS of patients with CLN2 disease (Goebel et al., 1999), as well as in the murine model of CLN2 disease (Sleat et al., 2004; Cabrera-Salazar et al., 2007). Despite white matter being reduced in patients with CLN2 disease and animals models, it is not known whether these defects are a consequence of neuronal loss of whether there are primary defects in axon growth, guidance and fasciculation. In addition, by 24 h post fertilization zebrafish possess the primary axonal (scaffold) tracts including the anterior commissure, supraoptic tract, the tract of the post optic commissure, posterior commissure and medial longitudinal fasciculus, which further mature by 48 h post fertilization (Wilson et al., 1990). The tpp1sa0011 / zebrafish model is therefore well suited to investigating axon tract formation in CLN2 disease. Neurodevelopmental defects of axon guidance in Tpp1 deficient embryos were seen in some of these tracts when stained with an antibody to acetylated alpha-tubulin. Within both 48 h post fertilization tpp1sa0011 / zebrafish (Fig. 5D and H) and tpp1 SPL morpholino injected wild-type embryos (Supplementary Fig. 2B and E) the primary axon tracts begin to look disorganized compared with wild-type siblings. The posterior commissure is highly de-fasciculated from the optic tectum to the anterior tegmentum, and the optic nerve is almost completely absent with no fibres apparently reaching the optic tectum after crossing at the optic chiasm. In addition, the trochlear nerve, which lies Figure 5 Continued and tpp1sa0011 / mutants, phosphohistone 3-positive cells from 5 -mm thick z-slices of immunostained wild-type and tpp1sa0011 / mutants were quantified in specific CNS regions between 48 h and 72 h post fertilization. Overall cell proliferation in the CNS is greater in wild-type versus mutant between 48–72 h post fertilization (G). Regionally, cell proliferation within the retina, forebrain, and mid-hindbrain is greater in wild-type compared with tpp1sa0011 / mutants at 48 h (H), 54 h (I) and 72 h post fertilization (J), whereas in spinal cord (somite 14–20) wild-type proliferation is only greater than tpp1sa0011 / mutants at 48 h post fertilization. P-values were determined by the Mann–Whitney U test that compared the median number of proliferative cells between wild-type and mutant in each region, at each time point. n = 4 for both wild-type and tpp1sa0011 / mutants at each time period. Error bar = SEM. hpf = hours post fertilization; WT = wild-type. 1500 | Brain 2013: 136; 1488–1507 Figure 6 Neurodegeneration is evident in tpp1sa0011 F. Mahmood et al. / mutants in comparison with wild-type siblings. The TUNEL assay was performed in 1-phenyl-2-thiourea (PTU)-treated embryos to detect apoptosis at various time periods and apoptotic bodies were quantified over several equivalent planes of foci in each embryo. At 48 h post fertilization no apoptotic bodies are detected in the eye or CNS tissue of wild-type siblings (A and G) compared with tpp1sa0011 / , which has a large number of apoptotic bodies localized in the retina, optic tectum, hindbrain and rostral portion of the spinal cord (D and H). At 54 h post fertilization few apoptotic bodies are present in the wild-type sibling retina and apoptotic bodies accumulate in large cells on the surface of the forebrain and midbrain regions (B), compared with tpp1sa0011 / mutants, which has a slightly increased number of apoptotic bodies (E). At 72 h post fertilization the wild-type sibling has an increased number of positively stained cells outside the CNS (C), as do tpp1sa0011 / mutants (F). Quantification of TUNEL-positive bodies in wild-type siblings versus tpp1sa0011 / mutants (I) demonstrates a significantly increased median number of total apoptotic bodies present in the tpp1sa0011 / CNS versus wild-type siblings at 48 h and 54 h post fertilization but not at 72 h post fertilization. Mann–Whitney U test, P = 0.0286 at 48 h and 54 h post fertilization, whereas P = 1 at 72 h post fertilization (n = 4 at all time stages for both wild-type and mutant) (error bar = SEM). FB = forebrain; HB = hindbrain; hpf = hours post fertilization; OT = optic tectum; R = retina; SC = spinal cord; WT = wild-type. Scale bar = 100 mm. Zebrafish model of CLN2 disease immediately rostral to the cerebellum, is completely absent in Tpp1 deficient zebrafish. Observations of later stages in 54 h and 72 h post fertilization tpp1sa0011 / larvae further confirm the complete absence of tectal fibres and trochlear nerve, with no sign of late onset growth (data not shown). Furthermore, alpha-tubulin staining at 42 h post fertilization, before the onset of an abnormal phenotype in tpp1sa0011 / embryos, did not show any defects in primary axon tract formation (data not shown). These results imply that primary axon tract formation in Tpp1 deficient zebrafish is not significantly impaired and axon loss is secondary to degenerative changes. The pathological phenotype of our tpp1sa0011 / zebrafish confirms severe neurodegenerative changes consistent with cases of human CLN2 disease. We subsequently decided to investigate functional of traits the tpp1sa0011 / larvae, particularly locomotion, in order to investigate the potential of this model for high-throughput automated drug screening. Tpp1 deficient zebrafish have abnormal locomotion that can form the basis of high-throughput drug screening We sought to characterize the locomotion defects in tpp1sa0011 / zebrafish in more detail with a view to finding parameters that could form the basis of an automated assay suitable for high-throughput drug discovery. First we analysed the touch-evoked escape response. Zebrafish develop mechanosensory neuromasts and are touch responsive as early as 24 h post fertilization (Ghysen and Dambly-Chaudiere, 2004). Furthermore, by 48 h post fertilization a single stimulus to the head or tail is sufficient to evoke a highly characteristic motile response consisting of alternating C-shaped body movements, propelling the fish in a straight line (Brustein et al., 2003). tpp1sa0011 / embryos 48 h post fertilization and older were less responsive to touch than wild-type siblings and after repeated prodding with a pipette tip, responded with circular motion due to their curved body (data not shown). Secondly, we observed free-swimming locomotion of tpp1sa0011 / mutants and found movement that may be related to seizure activity. Wild-type siblings at 72 h post fertilization rarely move an appreciable distance and mostly flick their tail while remaining in the same location. We found, however, that tpp1sa0011 / mutants at this stage have an aberrant swimming pattern with many turns and appear to move further and cover a larger area than their wild-type siblings (Fig. 7A and Table 1). Although these turns could be attributed to abnormal body shape, the greater distance moved is likely to reflect seizures. We statistically analysed several movement parameters (summarized in Table 1; n = 8 per genotype), being careful to establish new criteria for a movement bout that corresponded to a period of prolonged locomotion (averaged over 10 s with a start velocity of 4.5 mm/s and a stop velocity of 2 mm/s; Fig. 7B) and found that tpp1sa0011 / mutants had a significantly greater mean velocity and moved further, with more frequent and longer movement bouts than their wild-type siblings. Supplementary Video 1 shows a clip of the tpp1sa0011 / mutant shown in Fig. 7A. This Brain 2013: 136; 1488–1507 | 1501 mutant clearly shows a sustained period (6 s) of abnormal swimming activity that is similar to a Stage III seizure (Baraban et al., 2005) and was frequently seen in tpp1sa0011 / mutants, yet never seen in wild-type zebrafish at this stage. Finally, we noted that by 96 h post fertilization all tpp1sa0011 / larvae show repetitive twitching most likely caused by sustained muscle contractions (data not shown). Ethovision XT (Noldus) software was then used to quantitatively assess several behavioural parameters of 96 h post fertilization tpp1sa0011 / larvae compared with wild-type siblings at 96 h post fertilization which usually move an appreciable amount. The mean distance moved, velocity and proportion of time spent in motion by wild-type in a 170 s recording period was significantly greater (Mann-Whitney U, P = 0.0023, 0.0023 and 0.0012, respectively) in wild-type siblings than tpp1sa0011 / larvae (Fig. 8B–D). Furthermore the number of movement bouts (in this case averaged over 5 s with a start velocity of 0.4 mm/s and a stop velocity of 0.2 mm/s) was used to assess darting behaviour in wild-type and tpp1sa0011 / larvae. The frequency of movement bouts and mean distance moved during each movement bout were significantly lower in tpp1sa0011 / larvae compared with wild-type (Mann–Whitney U, P = 0.0042 and 0.0047, respectively) (Fig. 8E and F) whereas no significant differences in movement bout velocity were detected (data not shown). Discussion This study demonstrates that zebrafish can be used to model CLN2 disease. This is an early onset, autosomal recessive condition in which afflicted patients are homozygous for a mutation in the CLN2 disease-causing gene (TPP1) causing reduced or absent TPP1 enzyme activity (Sleat et al., 1997). Consistent with this, approximately one-quarter of all offspring obtained from zebrafish tpp1sa0011 + / display an abnormal early onset neurodegenerative phenotype and genotyping confirms the presence of an in-frame premature stop codon in exon 3 of the tpp1 gene. The most common mutations in CLN2 disease are null (exon 6: R208X in 58 families) or frameshift (intron 5: IVS5-1G4C in 67 families) whereas early TPP1 mutations, including the null mutation Q66X in exon 3, affect only a few families (Kousi et al., 2012) (www.ucl. ac.uk/ncl). Nonetheless the effect of these common mutations would result in an absence of TPP1 due to early degradation of the mis-formed protein, similar to the lack of Tpp1 in tpp1sa0011 / zebrafish. Therefore tpp1sa0011 / zebrafish are a representative stable mutant model of most cases of CLN2 disease. Importantly, Tpp1 activity does not appear to be completely absent in tpp1sa0011 / as predicted by the genotype. This could be due to a small amount of non-specific Arg-Ala-PheACC breakdown owing to the use of total protein from whole zebrafish for the enzyme assay. However, it is just as likely that the small amount of full-length Tpp1 we detected by western blot with protein from tpp1sa0011 / zebrafish is able to break down the substrate. This small amount of full length Tpp1 found in tpp1sa0011 / could be due to either perdurance of maternaldeposited protein, translation of maternally-deposited transcript or stop codon read-through. In support of this, others have 1502 | Brain 2013: 136; 1488–1507 F. Mahmood et al. / mutants. Motion tracking plots reveal that at 72 h post fertilization tpp1sa0011 / mutants (A, left) appear to have increased movement compared to wild-type siblings (A, right). This was quantitatively assessed using Ethovision XT software analysis of 20 min motion movies (Table 1; n = 8 wild-type siblings and 8 tpp1sa0011 / at 72 h post fertilization). Panel B demonstrates that the criteria used to reveal movement bouts picks up periods of prolonged locomotion. Figure 7 Locomotion is abnormal in 72 h post fertilization tpp1sa0011 Table 1 Quantitative assessment of 20 min motion videos using Ethovision XT software Total distance moved (mm) Mean movement velocity (mm/s) Maximum movement velocity (mm/s) Toatal time spent moving (s) Frequency of movement bouts Duration of movement bouts (s) n = 8 wild-type siblings and 8 tpp1sa0011 * = P 5 0.05. / at 72 h post fertilization. Wild-type sibling tppsa0011 182.553 0.165 46.646 189.917 4.5 5.804 969.143 0.812 42.511 596.795 24.5 54.375 / P-value 0.0003* 0.0019* 0.6454 0.065 0.02* 0.0281* Zebrafish model of CLN2 disease Figure 8 Locomotion is impaired in 96 h post fertilization tpp1sa0011 Brain 2013: 136; 1488–1507 | 1503 / mutants. Motion tracking plots reveal that at 96 h post fertilization tpp1 mutants (A; lower) appear to have impaired movement compared with wild-type (WT) siblings (A; upper). This was quantitatively assessed using Ethovision XT software analysis of 170 s motion movies (n = 6 wild-type siblings and 7 tpp1sa0011 / at 96 h post fertilization). Mean duration of time spent in motion (D) is significantly lower in tpp1sa0011 / mutants compared with wild-type (Mann–Whitney U, P = 0.0012), with tpp1sa0011 / mutants spending 5 10% of recording time in motion. The mean total distance moved (B) was significantly lower in tpp1sa0011 / mutants (8.3 mm) compared with wild-type (108.1 mm) (Mann–Whitney U, P = 0.0023). Mean velocity (C) of tpp1sa0011 / was also significantly lower compared with wild-type (Mann–Whitney U, P = 0.0023). The number of movement bouts (F) and distance moved as a result of each movement bout (E) in 96 h post fertilization (hpf) tpp1sa0011 / mutants was significantly lower compared with 96 h post fertilization wild-type (Mann–Whitney U, P = 0.0042 and 0.0047, respectively). Error bars = SEM. sa0011 / reported that the mature protein has a half-life of 20 h (Golabek et al., 2003), suggesting that maternal contribution could still be present at 48 h post fertilization, the age of the embryos used for western blot analysis. These results further imply that even a small amount of Tpp1 activity may be insufficient to prevent neurodegeneration in early developing zebrafish. The genotype-phenotype correlation of tpp1sa0011 / zebrafish is further strengthened by observing tpp1sa0011 + / and tpp1hu3587 + / inter-cross offspring. tpp1hu3587 / mutants are only mildly affected compared with tpp1sa0011 / mutants, with few developing early onset neurodegeneration and most reaching reproductive maturity. However, tpp1sa0011 /tpp1hu3587 trans-heterozygotes have a similar phenotype to tpp1sa0011 / mutants albeit with mild retinal degeneration and delayed disease onset of 24 h. Finally, genotype–phenotype correlation of tpp1sa0011 / mutants was further established by morpholino knockdown experiments targeting the tpp1 gene. Injection of two different morpholinos in wild-type embryos resulted in a similar phenotype to tpp1sa0011 / mutants thereby implying a clear link between tpp1 gene defect, and a concomitant lack of Tpp1 activity, with early onset neurodegeneration. The consequence of absent TPP1 is the build up of storage material inside lysosomes (Ezaki et al., 1999, 2000; Ezaki and Kominami, 2004). In human brain biopsy samples and leucocyte culture the accumulation of subunit c of mitochondrial ATP synthase (SCMAS) has been shown by immunohistochemistry (Hall et al., 1991; Palmer et al., 1992). We have demonstrated the ubiquitous storage of SCMAS in tpp1sa0011 / mutants throughout the body, most prominently in the CNS and muscles. Furthermore, MRI scans of patients with CLN2 disease show selective degeneration in the cerebral cortex, cerebellum and retina with the basal ganglia relatively preserved (Autti et al., 1997b; 1504 | Brain 2013: 136; 1488–1507 D’Incerti, 2000). Consistent with these observations, the optic tectum, cerebellum and retina lack mature neurons in 48 h post fertilization tpp1sa0011 / and we have shown this to be mediated by a combination of apoptotic cell death and aberrant cell proliferation well before these fish die. In wild-type zebrafish, the number of apoptotic bodies in the CNS remains low at 48, 54 and 72 h post fertilization, whereas the number of apoptotic bodies in tpp1sa0011 / zebrafish is much greater than in wild-type at 48 and 54 h post fertilization, but similar to wild-type at 72 h post fertilization. This demonstrates an early increase in apoptosis in tpp1sa0011 / . In contrast, proliferation in the CNS of wild-type zebrafish is greatest at 48 and 54 h post fertilization and there is relatively little at 72 h post fertilization. In tpp1sa0011 / zebrafish, proliferation is reduced at all stages assayed, demonstrating a sustained reduction in proliferation, predominantly affecting the retina and around the midbrain-hindbrain boundary where the cerebellum is developing. The role of apoptosis, however, remains controversial in CLN2 disease despite several studies showing TUNEL-positive bodies in the CNS of patients, molecular evidence for upregulation of apoptotic factors has not been forthcoming (Kurata et al., 1999; Kim et al., 2009). Additionally, it is likely that necrosis and autophagy may also play a role in mediating cell death (Mitchison et al., 2004). The zebrafish CLN2 disease model presented here thus provides a useful system for further investigating the various mechanisms mediating cell death in CLN2 disease. In addition, the majority of the specific regions that are affected in CLN2 disease, late infantile are prominently targeted in tpp1sa0011 / leading to deficits representative of the human condition. However, we did not detect neuronal loss in the thalamus and telencephalon but it is possible that more sensitive methods, such as stereology, would demonstrate that these regions are also affected. Interestingly, Tpp1 (previously known as Cln2) mutant mice do not suffer from visual problems or retinal degeneration even up until late stages of the disease (Sleat et al., 2004), so in this case the zebrafish model appears relatively more representative of the human condition. The phenotype manifested in the zebrafish is extremely severe and rapidly progressive which may reflect their rapid external development and relatively simple brain structure as well as overall shorter lifespan (Kimmel, 1993; Kimmel et al., 1995). The early timing of the pathology manifested in tpp1sa0011 / shows the critical requirement of Tpp1 for zebrafish neurodevelopment. The highly defasciculated posterior commissure in 48 h post fertilization tpp1sa0011 / and disorganized or absent trochlear nerve and tectal projections indicate a localized defect in mid-hindbrain axon guidance that is probably secondary to loss of neurons either resulting directly in a loss of axons or indirectly in a lack of guidance cues. Furthermore phosphohistone H3 staining demonstrates a loss of proliferative cells at the onset of secondary neurogenesis (Mueller and Wullimann, 2003) in 48 h post fertilization tpp1sa0011 / embryos. Thus it could be that there are few mature neurons present in the regions of the affected CNS and non-proliferating neural progenitors are subjected to apoptosis, perhaps owing to an inability to divide or that mature and proliferative cells are equally affected. In addition, the earliest TPP1 expression in human foetal samples was observed at 12 weeks F. Mahmood et al. gestation and some neurons in the cerebral cortex expressed TPP1 only post-natally with TPP1 expression reaching adult levels at 2 years of age (Kida et al., 2001). Thus the onset of TPP1 expression in humans is more gradual throughout development. Although this may explain the delay in manifestation of pathological consequences of TPP1 deficiency in humans, it may also be explained by continued maternal contribution of TPP1 protein across the placenta until birth. The zebrafish develops externally and hence a placental source of maternal contribution does not exist. Lastly, humans express TPP2, a highly related enzyme, which is not found in zebrafish. Therefore the presence of TPP2 may also contribute to the later onset of CLN2 disease in humans compared with zebrafish. Nonetheless, the zebrafish CLN2 disease model can be used to further investigate the early pathological consequences of TPP1 deficiency. Zebrafish larvae are highly motile displaying a variety of stereotypical movement behaviours from early development (Brustein et al., 2003). From as early as 17 h post fertilization the embryonic zebrafish undergo spontaneous tail coiling, followed by the development of the touch response by 21 h post fertilization and finally the first swimming behaviour is elicited at 27 h post fertilization (Brustein et al., 2003). Furthermore, three typical swimming behaviours are elicited between 48 h and 96 h post fertilization including cyclic swimming, slow starts and fast-startle responses, which all depend upon an upright posture being adopted by the zebrafish (Müller and van Leeuwen, 2004). By 96 h post fertilization the zebrafish larva is freely swimming, able to change direction spontaneously and swim towards targets (Granato et al., 1996). In contrast with wild-type zebrafish, the tpp1sa0011 / larvae have impaired escape response by 48 h post fertilization, abnormal, seizure-like motility by 72 h post fertilization and impaired locomotion by 96 h post fertilization. Most tpp1sa0011 / larvae have a permanently curved body. Some larvae are able to flick their tail to the left and the right and swim in fairly straight lines but most larvae are incapable of making the alternate C-shaped contractions that are required for motion in a straight line. As a result, tpp1sa0011 / zebrafish swim in circles and have a restricted range of motion. At 72 h post fertilization tpp1sa0011 / zebrafish exhibit activity that may be seizure related, although it will require electrophysiological recordings to determine if these are epileptiform events. Similarly, the repetitive twitching may represent myoclonus and this needs conformation using electromyography. By 96 h post fertilization, nearly all ability to move is lost in tpp1sa0011 / mutants, reflecting the loss of mobility seen in patients over time. The motor consequences of Tpp1 deficiency in tpp1sa0011 / larvae therefore closely reflect the human CLN2 disease condition. In summary, tpp1sa0011 / zebrafish do recapitulate many features of CLN2 disease. A comparison of the currently available CLN2 disease models and the extent to which they demonstrate the specific features of CLN2 disease, presented in Table 2, which shows that no model has yet been shown to have all features of the disease. Although the study of disease mechanisms and development of experimental therapies will rely on the use of all three models, the zebrafish has a unique role in providing a platform for drug discovery. Zebrafish model of CLN2 disease Brain 2013: 136; 1488–1507 | 1505 Table 2 A comparison of the zebrafish tpp1sa0011 mutant with the hallmarks of CLN2 disease, late infantile and the mouse and long-haired dachshund dog models of CLN2 disease Clinical signs Movement changes over time Vision Lifespan Pathology Brain/retina White matter Ventricles Microglia Astrocytes Ceroid and lipofuscin (autofluorescence) SCMAS Periodic acid-Schiff Ultrastructure Human CLN2 disease, late infantile Mouse Cln2 (Tpp1) mutants Long-haired Dachshund Zebrafish tpp1sa0011 Ataxia, seizures, myoclonus, loss of motor function Motor deficits, tremors, seizures (infrequent), hyperactivity Not described Reduced Ataxia, myoclonus, seizures, hyperactivity Blindness Reduced Bouts of seizure-like excessive movement, loss of motor function Degeneration Reduced Reduced Enlarged ventricles Microglial activation Astrocytosis Accumulation Atrophy in thalamocortical and cerebellar systems, less in retina Reduced Not described Microglial activation Astrocytosis Accumulation Atrophy in cerebellum and retina, not described in cortex Reduced Enlarged ventricles Not described Astrocytosis Accumulation SCMAS accumulation Periodic acid-Schiff-positive storage bodies Not described Periodic acid-Schiff-positive storage bodies Curvilinear profiles Curvilinear profiles Not described Periodic acid-Schiff-positive storage bodies Curvilinear profiles Blindness Reduced Atrophy in cortex/cerebellum, retina Atrophy in optic tectum, cerebellum and retina, Reduced Not described Not described Astrocytosis No accumulation SCMAS accumulation Not described Not described Reference: Mole et al., 2011. Conclusions and future work The rapidly progressive pathological-behavioural CLN2 disease phenotype in tpp1sa0011 / zebrafish combined with the ability to automate assessment of movement provides the ideal platform for high-throughput drug screening with the aim of developing therapeutic or palliative compounds for CLN2 disease. Firstly, individual zebrafish embryos can be placed in a single well of a 96-well plate and compounds applied at various time-points and concentrations (Goldsmith, 2004; Barros et al., 2008; Rihel et al., 2010). Furthermore, movement behaviour at 48 h post fertilization and 96 h post fertilization as well as enhanced survival prospects can be used as a marker for phenotypic improvement in tpp1sa0011 / mutants. Since the tpp1sa0011 / model possesses an early null mutation, premature termination codon read-through compounds could be tested such as those that have entered phase 3 trials for Duchenne muscular dystrophy and cystic fibrosis. Previous research has suggested that 10–15% of the normal level of TPP1 would be sufficient to prevent neuronal ceroid lipofuscinoses [reviewed in Sleat et al. (2008b)], so this could be a promising avenue to pursue. In addition, screening of FDA-approved and other compound libraries, could help identify anti-apoptotic, anti-spasticity and survival enhancing drugs that improve function and reduce morbidity in patients with CLN2 disease. Any efficacious and safe therapy identified can be further validated in higher vertebrates such as existing TPP1 deficient mouse and canine models before trial in humans. There are several aspects of this model that remain to be characterized, or have only been partially characterized, that could provide further mechanistic insights or markers for drug testing. The potential of the zebrafish as a model organism for human disease is just beginning to be realized. Our zebrafish CLN2 disease model provides the first stable genetic zebrafish model of a childhood neurodegeneration and epilepsy and provides proof of principle that zebrafish can be used to model such disorders. Acknowledgements For providing the zebrafish knockout allele tpp1hu3587 we thank the Hubrecht laboratory and the Sanger Institute Zebrafish Mutation Resource, (ZF-MODELS Integrated Project; contract number LSHG- CT-2003–503496; funded by the European Commission) also sponsored by the Wellcome Trust [grant number wild-type 077047/Z/05/Z]. For providing the zebrafish knockout allele tpp1sa0011 we thank the Sanger Institute Zebrafish Mutation Resource, sponsored by the Wellcome Trust [grant number wild-type 077047/Z/05/Z]. Thanks also to Andrew Hibbert (RVC Imaging Unit) for advice on making digital movies and Elizabeth Bate (Tracksys) for locomotion anaylsis. 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