A genetic model of amyotrophic lateral sclerosis in …RESOURCE ARTICLE 652 dmm.biologists.org...

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INTRODUCTIONAmyotrophic lateral sclerosis (ALS) is a devastatingneurodegenerative disease that is characterized by progressivemotor neuron loss in the brain and spinal cord, leading to paralysisand death. The incidence of ALS is 1.6 per 100,000 andapproximately 5000 Americans are diagnosed with ALS each year(Hirtz et al., 2007), with only 20% of patients living for five yearsor more after diagnosis. Thus far, riluzole is the only FDA-approvedtreatment for the disease and extends life by only a few months(Bensimon et al., 1994). Approximately 10% of ALS cases are causedby mutations in specific genes, whereas the remaining 90% of ALScases are sporadic, with no known cause. Sporadic and familial ALS(FALS) share several clinically and pathologically indistinguishabledisease features, including progressive upper and lower motorneuron loss, muscle atrophy, paralysis and early death. However,it has been reported recently that TDP43-containing aggregates arepresent in sporadic ALS but not FALS (Mackenzie et al., 2007).Modeling ALS using identified genes, however, has the potentialto lend insight into common disease mechanisms. Mutations inthe superoxide dismutase (SOD1) gene account for 20% of FALScases (Deng et al., 1993; Rosen et al., 1993; Valdmanis and Rouleau,2008). To date, over 150 mutations in SOD1 have been discovered,including the point mutation G93R (Elshafey et al., 1994; Turnerand Talbot, 2008).

To gain an understanding of how mutations in SOD1 cause ALS,mouse models were generated and have been used extensively tostudy ALS onset, progression and therapeutics (Turner and Talbot,2008). These animals exhibit hindlimb tremor, progressiveweakness, locomotor deficits, paralysis and early death (Bruijn etal., 1997; Gurney et al., 1994; Wong et al., 1995). As useful as thesehave been, mouse models do have some drawbacks: the mice arehighly inbred, which may affect the disease progression andphenotypes; some lines express very high levels of mutant protein,which may not accurately reflect the human situation; and it isexpensive to perform drug screens in mice. Drosophila and C.elegans that overexpress human SOD1 mutations have also beengenerated (Mockett et al., 2003; Oeda et al., 2001; Watson et al.,2008). These animals do recapitulate some ALS phenotypes in thatthey show motoneuron damage (Watson et al., 2008), however, theydo not display motoneuron loss, paralysis or premature death. Tocomplement these models and to generate a vertebrate model ofALS that may be more amenable to cellular manipulation and todrug or genetic screening, we have generated zebrafish that carrymutant forms of Sod1.

The zebrafish is an excellent organism for modeling neurologicaldiseases owing to its conserved, yet simplified, vertebrate nervoussystem, the ability to make transgenic and targeted knockoutanimals, and the ease of making genetic mosaics (Doyon et al., 2008;Meng et al., 2008; Lieschke and Currie, 2007; Carmany-Rampeyand Moens, 2006). Furthermore, for the study of motoneurondiseases, the zebrafish offers accessible motoneurons that can bemanipulated in vivo (McWhorter et al., 2003; Lemmens et al., 2007;Boon et al., 2009; Kabashi et al., 2010), making them highlyrelevant for performing cell-autonomy studies, electrophysiologyand imaging. We generated transgenic zebrafish expressing mutantzebrafish Sod1 at moderate levels. We show that these fishrecapitulate the major phenotypes of ALS including neuromuscular

Disease Models & Mechanisms 3, 652-662 (2010) doi:10.1242/dmm.005538© 2010. Published by The Company of Biologists Ltd

1Center for Molecular Neurobiology and Department of Neuroscience,2Department of Physiology and Cell Biology and 3Department of Cellular andMolecular Biochemistry, The Ohio State University, Columbus OH 43210, USA4Present address: Department of Academic Neurology, University of Sheffield,Sheffield, S10 2RX, UK*These authors contributed equally to this work‡Authors for correspondence ([email protected]; [email protected])

SUMMARY

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that, for ~80% of patients, is fatal within five years of diagnosis. Tobetter understand ALS, animal models have been essential; however, only rodent models of ALS exhibit the major hallmarks of the disease. Here,we report the generation of transgenic zebrafish overexpressing mutant Sod1. The construct used to generate these lines contained the zebrafishsod1 gene and ~16 kb of flanking sequences. We generated lines expressing the G93R mutation, as well as lines expressing wild-type Sod1. Focusingon two G93R lines, we found that they displayed the major phenotypes of ALS. Changes at the neuromuscular junction were observed at larval andadult stages. In adulthood the G93R mutants exhibited decreased endurance in a swim tunnel test. An analysis of muscle revealed normal muscleforce, however, at the end stage the fish exhibited motoneuron loss, muscle atrophy, paralysis and premature death. These phenotypes were moresevere in lines expressing higher levels of mutant Sod1 and were absent in lines overexpressing wild-type Sod1. Thus, we have generated a vertebratemodel of ALS to complement existing mammal models.

A genetic model of amyotrophic lateral sclerosis inzebrafish displays phenotypic hallmarks of motoneurondiseaseTennore Ramesh1,4,*,‡, Alison N. Lyon1,*, Ricardo H. Pineda1, Chunping Wang1, Paul M. L. Janssen2, Benjamin D. Canan2,Arthur H. M. Burghes3 and Christine E. Beattie1,‡

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Disease Models & Mechanisms 653

A zebrafish model of ALS RESOURCE ARTICLE

junction defects, decreased endurance, motoneuron loss andmuscle pathology. Moreover, we find that the onset and progressionof the disease phenotypes is variable, which may reflect a morenatural state of the disease, as seen in humans.

RESULTSGeneration of transgenic sod1 zebrafishTo generate zebrafish expressing transgenic mutant Sod1, we choseto use a zebrafish genomic region containing the endogenous sod1promoter and sod1 gene. Because zebrafish live at a lowertemperature (28°C) than mammals (37°C), we thought it would bemore prudent to use the fish sod1 gene and regulatory regions toguard against any temperature sensitivity. SOD1 is highly conservedbetween human, mouse and zebrafish (supplementary material Fig.S1), with 77% identity between the zebrafish and human aminoacid sequence. Furthermore, of the 72 amino acids within SOD1that are mutated in FALS, 88% (63 out of 72) are conserved inzebrafish, compared with 90% (65 out of 72) in the mouse and 72%(52 out of 72) in the fly. We mutated Sod1 by changing glycine 93to arginine (G93R); this mutation affects a conserved amino acidthat is often mutated in FALS (http://alsod.iop.kcl.ac.uk) (Elshafeyet al., 1994).

To generate transgenic fish expressing mutant Sod1, we identifieda bacterial artificial chromosome (BAC) containing the zebrafishsod1 gene (accession number AL837524). Since this BAC containedother genes, we isolated a 21-kb region containing the 5-kb sod1gene and flanking sequences (11.7 kb upstream and 4.5 kbdownstream). This amount of upstream sequence is consistent withmouse and rat transgenic Sod1 models (11.5-14.5 kb) (Gurney etal., 1994; Nagai et al., 2001). The point mutation G93R wasgenerated and the wild-type sod1 or mutant sod1 21-kb genomicregion were recombineered into a BAC vector backbone (pBAC5).To track transgene expression, we utilized the zebrafish heat shockprotein 70 (hsp70) promoter (Halloran et al., 2000), which we usedto drive the fluorescent protein DsRed. We added the hsp70 thatwas driving DsRed to the engineered pBAC5, generating the finalconstruct: Tg(sod1:sod1G93R or WT; hsp70:DsRed) (Fig. 1A). TheDNA construct was injected into early one-cell stage embryos. At48 hours post-fertilization (hpf), the injected embryos (F0) wereheat shocked and those with high mosaic DsRed expression at 72hpf were kept and grown to adulthood. Since integration into thegermline is random, these F0 fish were raised to adulthood andoutcrossed to identify those that incorporated the construct intotheir germline. F1 larvae from these outcrosses were heat shockedand analyzed for DsRed expression. F1 animals carrying thetransgene, as identified by DsRed expression (supplementarymaterial Fig. S2), were grown to adulthood and used to establishlines (F2 generation). Tg(sod1:sod1;hsp70:DsRed) fish will hereafterbe referred to as either WT (transgenic wild-type Sod1) or G93R(transgenic mutant Sod1 with a point mutation at G93R), followedby the line designation [e.g. Ohio State (os) #].

We next characterized copy number and Sod1 protein levels intwo WT lines and two G93R lines (Fig. 1B-D). Quantitative PCR(qPCR) was performed to determine sod1 gene dosage incomparison to non-transgenic fish, and western blots wereperformed to examine Sod1 steady-state protein levels. Onefounder line, G93R os10, with a high transgene copy number (~165copies) showed a threefold increase in steady-state Sod1 protein

levels in the brain and a fourfold increase in the spinal cord at oneyear of age compared with non-transgenic, wild-type siblings (nTg)(Fig. 1B-D). Another G93R line, os6, had approximately ninecopies of the transgene and Sod1 protein levels were 2.5-fold higherthan in non-transgenics (Fig. 1B,D). In WT lines, the number ofcopies of the transgene expressing wild-type Sod1 varied from twoto 42, and the increase in protein levels varied from 1.9- to 3.3-fold. Thus, WT os4 protein levels were similar to those in G93Ros10 animals, and WT os3 protein levels were similar to those inG93R os6 animals. The finding that the G93R os10 line showsincreased Sod1 protein in the spinal cord, brain, muscle and liver(Fig. 1C) is consistent with the ubiquitous expression of Sod1 andindicates that the transgene is expressed in a manner consistentwith endogenous sod1. The disease characterization that isdiscussed here was performed most extensively on G93R os10 fish.

Fig. 1. Generation of zebrafish transgenics that overexpress wild-typeand mutant Sod1. (A)BAC recombineering was utilized to generate thetransgenic construct from the zebrafish sod1 gene and surrounding regulatorysequences. The hsp70 promoter was utilized to drive the fluorescent proteinDsRed to track transgenesis. (B)Transgene copy number was approximated byquantitative PCR (n6 per line). (C)Western blots for Sod1 and -actin with10g of protein from various tissues from G93R os10 and non-transgenicclutch-mate controls (nTg). B, brain; SC, spinal cord; M, muscle; L, liver. (D)Foldchange of Sod1 steady-state protein levels in brain homogenates fromtransgenic lines over nTg control fish. A total of three to six separate sampleswere quantitated for each line from fish that were 12±2 months of age. Sod1levels were determined by densitometry and normalized to -actin. Data aremean ± s.e.m.

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A zebrafish model of ALSRESOURCE ARTICLE

G93R larvae and adult fish have abnormal neuromuscularjunctionsOne of the earliest hallmarks of disease in mouse models of ALSis neuromuscular junction (NMJ) defects that are seen as early asone to two months of age, before overt behavioral phenotypes (Freyet al., 2000; Schaefer et al., 2005; Fischer et al., 2004). We firstexamined motor axon outgrowth in G93R os10 embryos and foundthis to be indistinguishable from motor axon outgrowth in nTgs(supplementary material Fig. S3). An examination of NMJs in larvaland adult fish, however, did reveal abnormalities (Fig. 2). Using SV2as a pre-synaptic marker and -bungarotoxin (BTX) as a post-synaptic marker, whole-mount immunostaining and confocalmicroscopy was performed on 11 days post-fertilization (dpf)larvae. A small but significant reduction in SV2 and BTXcolocalization was observed using Pearson’s correlation coefficients.Furthermore, a significant reduction in the area occupied by BTX(post-synaptic volume) in G93R os10 larvae compared with nTgcontrols was observed.

Despite larval NMJ alterations, these fish survived and grew toadulthood. In 12-month-old fish, numerous NMJ abnormalities wereobserved. Not only was colocalization of SV2 and BTX reduced

significantly, but there was also a reduction in the area of the NMJ(NMJ volume). These quantitative measures are consistent withobservations that NMJ synaptic boutons were shorter and morepunctate in G93R os10 adults compared with nTg controls, whichshowed longer stretches of boutons (Fig. 2C and supplementarymaterial Fig. S4). We did not observe gross denervation across allmuscles, even in end-stage fish. This may reflect differences inzebrafish muscle innervation, which is polyneuronally innervatedthroughout life and may allow more compensatory innervation tooccur. However, these data do indicate that there are changes at theNMJ in zebrafish overexpressing mutant Sod1.

G93R adult fish develop progressive motor abnormalitiesTo examine motor output, fish were subjected to whole-bodyexercise using a swimming tunnel. To measure endurance in fish,critical swimming speed (Ucrit) was determined by monitoring theability to swim against an increasing current over time (Pagala et al.,1998; Plaut, 2000). Fish were placed in a swimming tunnel and awater current was applied. Zebrafish naturally swim against thiscurrent. Every five minutes the current was increased until the fishcould no longer swim against the current. Adult (10 months old)

Fig. 2. Abnormal NMJs are observed in larval and adult G93R os10 fish. (A)Whole-mount antibody labeling with synaptic vesicle 2 (SV2, red) for pre-synapticvesicles and -bungarotoxin (BTX, green) for post-synaptic terminals from nTg (top) and G93R os10 (bottom) 11-dpf larvae. (B)Quantitation of NMJ properties inlarval (11 dpf) G93R os10 zebrafish in comparison to nTg controls. (C)Adult NMJs were examined in trunk sections by immunolabeling with antibodies to SV2and -BTX. (D)Quantitation of NMJ properties in adult (12 month) G93R os10 zebrafish and nTg controls (*P<0.02, n10 sections from three larvae or adult fishper genotype). Bars, 20m.

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WT os4 and nTg fish swam for similar lengths of time (Fig. 3A).When 12-month G93R os10 adults were tested, we found that theywere compromised in their ability to swim against increasing current(Fig. 3B). Furthermore, a subset of G93R os6 fish (eight of 22, 36%of those tested) also showed significant endurance defects at 16months of age (Fig. 3C). Fish that showed endurance defects in theswim tunnel test did not recover and continued to perform poorlyover time. WT os3 fish were unaffected compared with nTg controlsat 24 months of age (Fig. 3D). Thus, both G93R lines were unableto maintain swimming when subjected to increasing current. Thisis in contrast to WT and nTg fish that could swim against strongercurrents. Therefore, the presence of mutant Sod1 in zebrafish resultsin a reduced ability to perform in this swimming test.

Muscle physiology in G93R adultsTo determine whether the motor abnormalities observed in G93Ros10 fish at 12 months of age were caused by changes in skeletalmuscle contractile properties, we examined the optimal force andfatigability of trunk skeletal muscle. Adult G93R os10 fish andsibling controls were anesthetized and optimal force measurementswere obtained by directly stimulating across the body trunk musclefrom behind the gills to the tail. No difference in maximum twitchforce was detected between nTg and G93R os10 fish indicating thatthe muscle contractile properties were intact (Fig. 4A). Todetermine whether the muscle fatigued in a similar manner,repeated stimulations of 4 milliseconds at 0.2 Hz was applied tothe muscle. The response of skeletal muscle to repeated stimulationdid produce significantly different responses between nTg and G93Ros10 fish, as indicated by the difference in slope upon repeatedstimulation (Fig. 4B,C). We found that G93R os10 fish exhibitedbetter fatigue resistance than nTg body muscle. This is similar towhat was found in mice overexpressing mutant SOD (Derave etal., 2003) and suggests a common muscle phenotype in these two

species. These data also support the hypothesis that the majorityof the movement impairments (i.e. decreased swim tunnelperformance and partial paralysis) in G93R os10 fish are the resultof defects in the neural input to the muscle, as opposed to defectsin intrinsic muscle properties.

Examination of pathology in G93R fish reveals motoneuron lossand muscle atrophyG93R os10 fish exhibited a progressive deficiency in locomotion withsome fish showing intermittent paralysis over time. Some fishexhibited signs of more progressive paralysis, including severelydecreased movement for several consecutive days (supplementarymaterial Movie 1). These fish were considered end-stage and weresacrificed for pathological analysis. Cellular pathology in ALSincludes spinal cord motoneuron loss and degeneration. To analyzethis in zebrafish overexpressing mutant Sod1, spinal cords from end-stage G93R os10 fish were processed for choline acetyl transferase(ChAT) labeling to examine motoneurons (Fig. 5A). For this analysis,only ChAT-positive cells that were ≥10 m in diameter were countedto ensure that only motoneurons were included in these counts. Weobserved a significant reduction in motor neuron number with end-stage fish having ~38% loss compared with nTg siblings (Fig. 5B).

SOD1 mouse models of ALS have revealed mitochondrialvacuolization in spinal cord motor neurons (Dal Canto and Gurney,1995; Wong et al., 1995). Consistent with these findings, electronmicroscopy (EM) on spinal cords from end-stage G93R os10 fishrevealed that spinal cord motoneurons were compromised.Shrunken motoneurons containing vacuolated mitochondria withdisintegrating cristae were consistently observed (Fig. 6A).Furthermore, EM examination of fast trunk muscle also revealeddefects. G93R os10 fish had myofibrillar and mitochondrialdegeneration, and extensive collagen deposition, suggesting muscleatrophy and degeneration at end-stage (Fig. 6B). Therefore, fish

Fig. 3. G93R mutant fish exhibit reducedendurance. (A)Ten-month-old adult WTos4 and nTg siblings were subjected toincreasing current (4.1 cm/second steps)every 5 minutes until fatigue. Criticalswimming speed (Ucrit, cm/s), a measure ofthe velocity at which a fish can maintainswimming for a set period, was notsignificantly different between WT os4 andnTg siblings (n5 nTg and n9 WT os4).(B)Ucrit values between G93R os10 adultsand nTg controls were significantlydifferent at 12 months of age (*P<0.0001,n15 for each genotype). (C)AffectedG93R os6 fish at 16 months of age cannotmaintain swimming for as long as nTgcontrols (*P<0.001, n10 nTg and n8G93R os6). (D)WT os3 fish are unaffectedat 24 months of age in comparison to nTgfish (n9 nTg and n11 WT os3). Data aremean ± s.d.

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overexpressing mutant Sod1 exhibited motoneuron loss, and bothmotoneuron and muscle degeneration, consistent with ALSphenotypes.

Reduced survival in G93R linesTo address whether fish overexpressing mutant Sod1 had a shorterlife span, we analyzed survival over time (Fig. 7). Both G93R os10and G93R os6 fish exhibited significantly shorter survival incomparison to WT os3 and nTg controls. G93R os10 and G93Ros6 fish did not exhibit reduced survival at 7±2 or 14±2 months ofage in comparison to nTg or WT os3 fish. However, by 18±1 monthsof age, significant differences in survival between the G93R linesand WT os3 or nTg fish were observed. A significant reduction insurvival for G93R os6 and G93R os10 fish was observed at 22±2and 27±2 months of age in comparison to WT os3 or nTg fish. WTos3 and nTg survival did not differ significantly at any age. Priorto death, many of the G93R os6 and G93R os10 fish exhibited

severely decreased movement or partial paralysis. However, no nTgor WT os3 fish exhibited behavioral abnormalities or paralysis.These data indicate that fish overexpressing mutant Sod1 exhibitedreduced rates of survival.

DISCUSSIONHere we describe the development and characterization of transgeniczebrafish overexpressing mutant Sod1 as an approach to model FALS.Fish overexpressing mutant Sod1 have NMJ defects, spinal cordmotoneuron loss, muscle degeneration, decreased endurance, partialparalysis and premature death. Many of the cellular pathologies areconsistent with those seen in mice and rats overexpressing SOD1,supporting the idea that overexpression of SOD1 in vertebrateanimals results in the development of common pathologies. However,we would not expect all aspects of disease phenotypes to be identicalacross species owing to differences in the genetics of laboratorystrains and animal physiology. For example, zebrafish laboratorystrains are not highly inbred owing to the negative impact ofinbreeding on reproduction (Guryev et al., 2006; Nechiporuk et al.,1999). Thus, disease onset and the time between onset and death,which is very stereotyped in some inbred mouse lines, is much morevariable in the G93R zebrafish lines, and may be because of geneticheterogeneity within strains. This is not unlike FALS patients, whoshow phenotypic variability even when carrying the same SOD1mutation (Aggarwal and Nicholson, 2005; Hand et al., 2002). NMJdenervation in SOD1 mutant mice is variable and muscle dependent,with fast muscle regions affected first and more dramatically andslow regions spared (Frey et al., 2000). Only around 8% of synapsesat the sternomastoid muscle are denervated in symptomatic G93Amice and around 70% are denervated at end-stage (Schaefer et al.,2005), suggesting a progressive loss of NMJ synapses. Fish thatoverexpress mutant Sod1 show pre-symptomatic changes at NMJs;this was more severe in adults, supporting progressive NMJ defects.However, zebrafish expressing mutant Sod1 do not exhibit the samedegree of NMJ abnormality or motoneuron loss as rodent models.Adult zebrafish, however, have the capacity to regeneratemotoneurons (Reimer et al., 2008), and zebrafish muscle ispolyneuronally innervated throughout life unlike in mice and humans(Westerfield et al., 1986). Thus, it is possible that compensatorymechanisms such as collateral innervation and motoneuronregeneration occur in these animals and may affect the phenotype.

Transient overexpression of human mutant SOD1 RNA inzebrafish was reported to cause motor axon outgrowth defects suchas shorter axons or branched axons during embryonic development(Lemmens et al., 2007). The transgenic lines expressing mutantSod1 reported here did not show defects in motor axon outgrowth.Lemmens et al. (Lemmens et al., 2007) reported a 6.1% decreasein axonal length [approximately 101 m in SOD1(wt) versusapproximately 95 m in SOD1(G93A) embryos] in theSOD1(G93A)-injected embryos compared with SOD1(wt)-injectedembryos. Because we did not measure axons directly, we may nothave detected such a subtle result. It is more likely, however, thatthis difference may simply reflect the high levels of RNA used inthe transient study compared with the steady, lower levels of mutantSod1 present in the transgenics.

Loss of muscle strength is a hallmark phenotype of ALS. BothG93R os6 and os10 fish displayed a significant decrease inendurance, as shown by their performance in the swim tunnel test.

Fig. 4. Contractile muscle properties of G93R mutant fish are intact, butthe response to repeated stimulation is significantly altered. (A)Musclesof G93R mutant fish are capable of producing similar maximum twitch force asnTg siblings (n9 per genotype, P0.816). (B)When subjected to repeated 4 msstimuli at 0.2 Hz, the muscles of G93R mutant fish fatigue more slowly thanthose of nTg siblings. The average of the percentage of maximum forceachieved (CSA dimension) is plotted against time and linear regressionapplied to the data (n8 per genotype). (C)The slope values of linearregression of the percentage of maximal force achieved versus time inresponse to repeated stimulation were significantly different. G93R mutantfish are able to maintain significantly larger forces for a longer period of timein comparison to nTg sibling controls (n8 per genotype, P0.018).

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Endurance tests measure the ability of an animal to sustain a forceover time and, although it is difficult to relate aquatic to non-aquatictests, the swim tunnel is a close approximation of a treadmill. Bothmuscle integrity and cardiovascular health contribute to endurance.In SOD mutant mice, both neural and vascular defects are earlyphenotypes (Zhong et al., 2008). Thus, we contend that theneuromuscular defects observed in G93R fish certainly contributeto the decreased endurance, however we cannot rule out a role forthe cardiovascular system.

Analysis of muscle force properties supports the hypothesis thatalterations in the neural component of the neuromuscular systemcontribute to movement defects because we found that the intrinsicforce generated by body muscle in nTg and G93R os10 fish wasindistinguishable. We did find areas of muscle fiber degeneration byEM suggesting that there are regions of compromised muscle fibers.However, when the whole fish is analyzed, the muscle functionsnormally. This supports the idea that the defects in swimmingendurance are not the result of changes in the ability of muscle togenerate force and indicate that the problem is neural as opposed tomuscular. One area where there was a difference was in how themuscle responded to repeated stimulation as a way to test fatigue. Inthese tests, the G93R os10 line was actually more resistant to fatigue.This result is consistent with mouse ALS SOD models (Derave et al.,2003; Sharma and Miller, 1996). One possible explanation for whythese fish are more resistant to fatigue may be to do with the fact thatrepetitive stimulation exhausts muscle, which can cause oxygen radicaldamage. Higher levels of Sod may help curb this damage.

Here, we have generated a vertebrate model of FALS and showthat zebrafish overexpressing mutant Sod1 exhibit the hallmarkphenotypes of ALS. We suggest that this model will be useful forfurther investigation into early changes in the neuromuscularsystem and motoneuron physiology during disease progression.Moreover, zebrafish are highly amenable to chimeric analysis andthus will offer a tool to address the continued debate concerningthe cell autonomy of FALS.

MATERIALS AND METHODSAnimalsAdult and larval zebrafish (Danio rerio) were maintained at TheOhio State University fish facility at 28.5°C and bred according toestablished procedures (Westerfield, 2000). Animal protocols wereapproved by the Ohio State University Committee on Use and Careof Animals.

The transgenic lines reported here are available by [email protected]. If there is sufficient interest, they will bedeposited in the zebrafish international resource center (ZIRC:http://zebrafish.org/zirc/home).

Transgene constructsThe zebrafish sod1 gene was isolated by BLASTing the zebrafishgenome with the mouse cDNA, and a BAC containing the zebrafishsod1 gene was identified (DanioKey BAC library, BAC accessionnumber AL837524). Since this BAC contained other genes, weisolated a fragment of the BAC containing an 11.7-kb upstreamregion, the sod1 gene with its five exons spanning 5.06 kb, and 4.5kb of downstream sequences. We recombineered this into pBAC5,into which we had added ISceI meganuclease sites to increase therate of transgenesis (Grabher et al., 2004; Liu et al., 2003). Thezebrafish heat shock protein 70 (hsp70) promoter and the DsRed(Discosoma sp. red fluorescent protein) marker gene were alsointroduced into the pBAC5 by recombineering. A 3-kb fragmentcontaining the last two exons of sod1 was then excised from theengineered pBAC5 and mutagenesis was performed using theStratagene site-directed mutagenesis kit. The primers for the G93Rmutation were: forward, 5�-GACCGCTGATGCCAGT CGTG -TTGCAAAAATTG-3� and reverse, 5�-CAATTTTTG CAACAC -GACTGGCATCAGCGGTC-3�. The mutagenized fragment wasthen recombineered back into the engineered pBAC5. The finalconstruct [Tg(sod1:sod1G93R or WT; hsp70:DsRed)] was then cutwith ISceI meganuclease (New England Biolabs) and injected intozebrafish embryos at the single cell stage.

Generation of transgenics and quantitative PCRAdult wild-type hybrids of AB* and LF strains, designated AB*LF,were used to generate transgenics. Each ISceI-digested BAC wasdiluted in buffer containing 1� enzyme buffer, 1 U/ml of ISceI, PhenolRed and 1� injection buffer (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA,100 mM NaCl, 30 M spermine and 70 M spermidine) to a finalconcentration of DNA ranging from 80-100 ng/l, and 1 nl of DNAsolution was injected into early one-cell stage embryos. At 48 hpf,the embryos were heat shocked for 1 hour at 37°C in a standardthermocycler (Bio-rad Peltier Thermal Cycler PTC-100) andexamined at 72 hpf under a fluorescence microscope. Only thoseembryos with the strongest DsRed expression levels, and with a highnumber of cells expressing DsRed, were grown to adulthood andsubsequently screened as potential F0s. The potential F0s were bred

Fig. 5. Motor neuron loss was observed in G93R os10 spinal cord at the disease end-stage. (A)ChAT immunohistochemistry of anterior spinal cord sectionsfrom adult G93R os10 fish and nTg siblings demonstrates reduced numbers of positively labeled motor neurons (denoted by arrows) in end-stage fish comparedwith controls. (B)ChAT-positive motoneurons (≥10m diameter) from anterior spinal cord sections (20m thickness) of adult end-stage fish were blinded togenotype and counted (*P<0.0001, n40 sections per fish, three fish per genotype). Data are mean ± s.e.m. Bar, 200m.

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to AB*LF fish, and offspring were subjected to heat shock and screenedfor DsRed expression. DsRed-positive embryos were isolated, raisedto adulthood, and bred to generate founder transgenic fish (F1s). Nowthat the DNA had integrated into the germ line, the DsRed was in allcells. Transgenic F1s were bred to generate the individual transgenic

lines. In total, approximately 500 embryos per line were injected and50 highly chimeric fish were grown to adulthood. Among these, 10-30% gave rise to transgenic F1s.

Transgene copy number was determined by quantitative PCR(qPCR). Genomic DNA was isolated from the tail fin using theDNeasy blood and tissue kit (Qiagen). Primers were designed tounique genomic sequences for sod1: forward, 5�-TAAAG -GGCCACAAATCACAC-3� and reverse, 5�-TTTCAGTGACA -ATCCGTACAG-3�; and -actin: forward, 5�-ATGAGACCAC -CTTCAACTCC-3� and reverse, 5�-TGAAATCACTGCAAGCA -AACTG-3�. qPCRs using sod1 and -actin primers were run atvarious primer concentrations with a standard dilution of genomicDNA from AB*LF fish in order to identify a concentration thatyielded the optimal cT spread. Each reaction contained 0.3 M offorward and reverse primer, 5 ng genomic DNA, and iQ SYBRGreen Supermix (Bio-rad). qPCR was performed under thefollowing conditions: one cycle at 94°C for 5 minutes; 45 cycles of94°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds;and then a step down from 94°C to 4°C, by 0.5°C every 10 seconds,using an iCycler (Bio-rad). To determine copy number, thedifference in cT of sod1 and cT of -actin was given the value X(DcT). 2X was then calculated for each sample. The fold changebetween the non-transgenic control and transgenic line wasdetermined [DDcT2X(AB*LF)–X(Tg)]. Following the methods ofAlexander et al. (Alexander et al., 2004) and assuming two copiesin the non-transgenic control fish, sod1 gene copy number of thetransgenic was calculated [2(DDcT)] (Alexander et al., 2004).

Swimming endurance testCritical swimming speed, Ucrit, which is the maximum velocity afish can sustain for a set period, was measured using a simplifiedwater tunnel (AccuScan Instruments Inc.) (Plaut, 2000). Adultzebrafish were individually introduced into the water flow chamberof a swim tunnel. The water current flow rate was graduallyincreased to 4.1 cm/sec, and the fish were subjected to this currentfor 5 minutes. Subsequently, every 5 minutes, the flow rate wasincreased by 4.1 cm/sec increments until the fish could no longermaintain swimming and fell into a mesh net at the end of the water

Fig. 6. Pathological changes were observed in G93R os10 spinal cord andmuscle at the disease end-stage. (A)Electron microscopy of a spinal cordmotoneuron from nTg sibling (left) and G93R os10 (right) adult end-stage fish.Numerous vacuolated mitochondria were observed in G93R os10motoneurons (top) and were readily apparent at higher magnification(bottom). (B)Electron microscopy of trunk muscle from nTg sibling (left) andG93R os10 (right) fish also revealed severe abnormalities (top). Themitochondria in G93R os10 muscle also appeared to be affected, both innumber and integrity, and cristae appeared to be reduced in number andlacked the normal organization (bottom). Bars, 2m (A,B; top panels); 150 nm(A,B; bottom panels).

Fig. 7. G93R mutant fish have reduced survival. The percentage of survivingfish ± 95% confidence intervals is shown. To track the survival of more than1000 fish, the numbers of fish were routinely counted. Each time fish werecounted, they were placed into an age bracket: 7±2, 14±2, 18±1, 22±2 and27±2 months of age. At any given age, several independent clutches of fishwere counted for each line. See Materials and methods for details on thenumber of fish counted for each line at each age bracket.

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flow chamber. The fish was allowed two opportunities to re-enterthe highest achieved current by pausing the time, reducing thecurrently flow, and slowly increasing the current back to the fatiguevelocity. If the fish could not sustain swimming in this current, thetime was recorded when the fish stopped swimming. The criticalswimming speed was calculated based on the formula: UcritUi +(UiiTi/Tii), where Uithe highest velocity maintained for a wholeinterval (cm/sec), Uiithe velocity increment (4.1 cm/sec), Tithetime elapsed at fatigue velocity (minutes) and Tiithe time interval(5 minutes) (Brett, 1964; Plaut, 2000).

Force measurements in zebrafishThe contractile strength of intact zebrafish was assessed followinganesthesia by tricaine. The whole fish was mounted at slack lengthin a circulating bath containing oxygenated water (26°C) from thezebrafish housing tank. One end of the fish was held in place byinserting a hook (connected to a force transducer) through the body,just caudal to the gills. A second hook, connected to a linear micro-manipulator, was placed through the body at the tail. Electricalstimulation was delivered using two parallel platinum-iridiumelectrodes positioned along the length of the bath on either sideof the fish. The fish was stretched from slack length and subjectedto a 4-ms stimulus at 1-minute intervals until optimum length wasreached, defined as the length at which maximum twitch force wasachieved, and forces were recorded and stored digitally. After a five-minute rest period, the fish was subjected to a series of 4-ms stimuliat 0.2 Hz until the force had declined by at least 50% of the initialvalue. All force measurements are expressed per unit of cross-sectional area (mN/mm2), as done previously for similar skeletalmuscle experiments (Martin et al., 2009). Cross-sectional area(CSA) was calculated using the following equation: CSA(musclemass in g)/[(optimal fiber length in cm) � (muscle density ing/cm3)], where muscle density is 1.06 g/cm3.

Western blotsAdult zebrafish were deeply anesthetized in fish water, with 2%tricaine, and decapitated. Tissues (brain, spinal cord, muscle and liver)were removed and homogenized with a TissueMiser homogenizer(Fisher Scientific) in homogenizing buffer (50 mM Tris, 10 mMEDTA, pH 7.2, 10% SDS) with protease inhibitors (Roche). Tissuewas boiled and centrifuged at 16,000 � g for 5 minutes at 4°C. Thetotal soluble protein concentrations were determined by the Dcprotein assay according to manufacturer’s directions (Bio-Rad).Western blots were performed as described (Schagger, 2006). Briefly,protein lysates were separated on 7.5% tricine SDS-polyacrylamidegels and transferred to poly-vinylidene difluoride (PVDF) membranes(Immobilon P, Millipore). Blots were blocked for 1 hour with 5% milkin tris-buffered saline with 0.1% Tween20 (TBST) and, subsequently,probed with rabbit anti-SOD1 antibody (1:5000 in blocking buffer;Santa Cruz) or mouse anti-actin (1:1000; Abcam) overnight (ON) at4°C. Following four washes with TBST for 5 minutes each, blots wereincubated with the appropriate secondary antibody (goat anti-mouseIgG-HRP or goat anti-rabbit IgG-HRP, 1:5000 in 5% milk/TBST,Jackson ImmunoResearch Laboratories, Inc.). After five washes withTBST for 5 minutes each, signal was detected by chemiluminescence(Amersham Western Blotting Detection Reagents, GE Healthcare)by exposures with an Omega 12iC Molecular Imagine System(UltraLum, Inc.) and by film. Densitometry was performed on the

digital images using Ultraquant Image acquisition and analysissoftware V6.0 (UltraLum, Inc.). For subsequent blotting, membraneswere stripped with Restore Plus western blot stripping buffer(Thermo Scientific) for 15 minutes at room temperature (RT) andwashed five times with TBST for 5 minutes per wash prior toblocking, as described above. Quantitation was performed bynormalizing the signal from each lane on the Sod1 blot to the -actin signal in each lane.

ImmunohistochemistryWhole-mount staining for synaptic vesicle 2 (SV2) and -bungarotoxin (BTX) was performed on 11-dpf zebrafish larvae asfollows. Larvae were fixed in 4% paraformaldehyde (PFA) ON at4°C and permeabilized by successive incubations in dH2O for 5minutes, ice-cold acetone for 7 minutes, and dH2O for 5 minutes.Larvae were incubated in PBDT buffer (1% DMSO, 1% BSA, 0.5%TritonX-100, 1� PBS) with 5% normal goat serum (NGS) for 60minutes, followed by incubation with Alexa 488-conjugated -BTX(1:100 in 5% NGS/PBDT, Molecular Probes) for 30 minutes at RT.Larvae were washed three times in PBDT for 15 minutes per washand incubated in primary mouse monoclonal anti-SV2 antibody(1:50 in 5% NGS/PBDT, Developmental Studies Hybridoma Bank)at 4°C ON. Larvae were then washed six times with PBST (0.5%TritonX-100, 1� PBS) for 15 minutes per wash and incubated inAlexa 633-conjugated goat anti-mouse antibody (1:400 in 2%NGS/PBDT, Chemicon International) for 4 hours at RT followedby six 15-minute washes in PBST and clearing in PBS:glycerol. Thelarvae were mounted onto slides with Vectashield (Vector Labs) andimaged using confocal microscopy (TCS SL, Leica Microsystems).

To examine synapses in adult fish, adult zebrafish (12 months)were anesthetized in tricaine, decapitated, and fixed in 4% PFA ONat 4°C. Fish were embedded in OCT (Tissue-Tek) and snap frozenin isopentane. Cryostat serial cross sections (16 m) of the caudalregion of the fish were collected on superfrost plus slides(ColePalmer) and stored at –80°C until processed further. Sectionswere rehydrated for 5 minutes in PBDT buffer with 5% NGS,incubated with Alexa 488-conjugated -BTX (1:100 in 5%NGS/PBDT) for 30 minutes at RT, and washed three times for 15minutes each in PBDT. This was followed by incubation in primarymouse monoclonal anti-SV2 antibody (1:50 in 5% NGS/PBDT) at4°C ON. Sections were washed four times with PBST for 15 minuteseach and incubated in Alexa 633-conjugated goat anti-mouseantibody (1:400, Chemicon International) for 2 hours at RT.Sections were washed six times with PBST, mounted usingVectashield (Vector Labs) and imaged using a Leica TCS SLconfocal microscope (Leica Microsystems). Image stacks of 16 mthickness (0.6 m/section) were obtained, and then processed usingImageJ Software (National Institutes of Health) to quantitatefluorescence intensity and identify co-localization of BTX and SV2signals. Additionally, the overall NMJ density (Ncoloc) and pre- andpost-synaptic volume were calculated using Costes’ colocalizationquantification and quantitative immunocolocalization program(Intensity colocalization analysis) (Costes et al., 2004; Li et al., 2004).

ChAT immunohistochemistryChAT immunohistochemistry was performed as described(Clemente et al., 2004). Briefly, fresh frozen sections (20 m) of spinalcord were collected from end-stage fish that had been fixed in 4%

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PFA in 0.1 M phosphate buffer (PB) (pH 7.4) for 10 minutes. Thesections were washed twice in 0.1 M PB (pH 7.4) for 5 minutes perwash. To decrease background staining, the sections were incubatedin 2% H2O2/PBS for 20 minutes. Sections were washed three timesin PBDT with 0.2% Tween20 for 5 minutes, incubated in PBDT with0.2% Tween and 10% normal donkey serum (NDS) for 2 hours atRT, followed by incubation in goat polyclonal anti-ChAT antibody(1:125 in PBDT with 0.2% Tween and 10% NDS, ChemiconInternational) for 2 days at 4°C. Sections were washed four timeswith PBST with 0.2% Tween20 for 15 minutes each and incubatedin biotinylated donkey anti-goat secondary antibody (1:250 in PBDTwith 5% NDS, Santa Cruz Biotechnology) for 2 hours at RT. Sectionswere washed four times with PBST with 0.2% Tween20 for 15 minuteseach and washed twice with 0.01 M PB (pH 7.4) for 15 minutes.Sections were incubated in Vectastain RTU reagent (Vector Labs)for 90 minutes and were washed four times with 0.01M PB (pH 7.4)for 15 minutes each. The reaction product was visualized with 0.025%3,3-diaminobenzidine (DAB) and 0.0033% H2O2 in 0.01 M PB (pH7.4) or 0.2 M Tris (pH 7.6). The course of the reaction was monitoredand stopped by washing in PBST. Sections were serially dehydratedfor 10 minutes each in 70% and 100% EtOH, cleared in xylene for15 minutes, and mounted with Permount (Fisher Scientific). Sectionsfrom the dorsal fin region were blinded to genotype and each sectionwas counted for ChAT-positive motor neurons. A minimum of 40serial sections were counted from three fish for each genotype.

Survival analysisNumerous tanks of WT os3, G93R os6, G93R os10 and nTg fishwere followed from the age of 3 months, which is when the zebrafishreached adulthood, until 27.5 months of age. To track the survivalof more than 1000 fish, the numbers of fish were countedperiodically. Each time fish were counted, they were placed intoan age bracket: 7±2, 14±2, 18±1, 22±2 and 27±2 months of age. Atany given age, several independent clutches of fish were countedfor each line. At the respective ages listed above, for nTg fish n55,147, 516, 628 and 520; for WT os3 fish n34, 34, 95, 155 and 89;for G93R os6 n44, 101, 169, 152 and 154; and for G93R os10 n49,147, 333, 370 and 230. Fish were also monitored for abnormalbehavior, such as severely reduced movement or paralysis. Thepercentage of surviving fish was calculated for each age bracket. AZ-test for two proportions was used to determine significance with99% confidence. All data are reported as the percentage of survivingfish at the 95% confidence intervals.

Statistical analysisStatistics were performed using SPSS 10. Motor axons were analyzedusing a non-parametric analysis using the Mann-Whitney test.Image analysis for NMJ analysis was performed using NIH ImageJsoftware and quantitative analysis of images was performed usingcolocalization analysis and an intensity colocalization analysis plugin(Costes et al., 2004; Li et al., 2004). The values from the individualimages were compared between control and transgenics using anunpaired t-test. Critical swimming speed was analyzed using anunpaired t-test. Survival data was reported as the percentage of fishthat died within a given age bracket with 95% confidence intervals.A Z-test for two proportions was performed to determine thesignificance of survival data at 99% confidence. All P values reportedare two-tailed and the significance was set at P<0.05.

ACKNOWLEDGEMENTSWe thank Tera Lindquist for the motor axon analysis and the fish facility staff forexcellent fish care. This work was supported by the ALS Association (grant number822 to C.E.B. and A.H.M.B.) with additional support from The National Institutes ofHealth (grant number RO1NS050414 to C.E.B. and P30NS045758), Dave EltschlagerALS fund, ACM business solutions and Accuscan Instruments, Inc. Deposited inPMC for release after 12 months.

COMPETING INTERESTSThe authors declare no competing financial interests.

AUTHOR CONTRIBUTIONST.R. initiated the project, designed and made the transgenic DNA constructs, andgenerated the lines. He also developed the protocols for analysis, performed theinitial characterization, and designed and developed the behavioral analysissystem for measuring the swim strength. A.N.L. performed all of the western blotsand qPCR. She also helped analyze the antibody labeling, performed the swimtunnel tests, and was involved in the muscle physiology. R.H.P. performedantibody labeling for synapses and the following analysis. C.W. helped to identify

RESOURCE IMPACT

BackgroundAmyotrophic lateral sclerosis (ALS), one of the most common neuromusculardiseases, is a devastating incurable illness that leads to progressive paralysis andpremature death, usually within 5 years of diagnosis. Drug treatment to slow ALSprogression is limited to one drug, riluzole, which only prolongs survival by a fewmonths. Very little is known about the molecular causes of ALS. The symptomsand progression of ALS are very similar in both sporadic and familial forms of thedisease, leading to the notion that although there may be multiple initial causes,there could be convergence upon a common disease pathway.

Around 2% of ALS cases are due to mutations in the superoxide dismutase(SOD1) gene. SOD1 protects the body from free radical damage. Mice that aredefective in SOD1 activity develop very similar symptoms to humans witheither sporadic or familial ALS. Animal models in which SOD1 has beenmutated have therefore been developed as vehicles to study both the onsetand progression of ALS but, to date, only rodent models show the majorphenotypes of ALS, limiting both the questions that can be addressed and thetypes of experiments that can be performed. Rodents are also impractical as invivo models for high throughput whole-organism drug screens, which areessential for a disease where multiple tissues and cell types are affected.

ResultsThe authors present a zebrafish model of familial ALS based on a commonmutation, G93R, in familial SOD1-derived human ALS. They introduced the G93Rmutation into zebrafish sod1 and generated transgenic lines expressing eitherG93R sod1 or wild-type sod1 protein. The highest expressing line had three- tofourfold more Sod1 protein in the spinal cord than normal fish. Fishoverexpressing mutant Sod1 recapitulated the major phenotypes of ALS.Neuromuscular junction defects were detected during larval stages and weremuch more severe in adult animals. Adults also underwent motoneuron cell loss,muscle degeneration and early death, and showed decreased muscle endurancewhen subjected to a swim tunnel test. Maintenance of overall muscle integritysuggested that there was a neural, as opposed to muscular, defect.

Implications and future directionsThis study provides a new vertebrate model of familial ALS that exhibits themajor hallmarks of motoneuron disease. Zebrafish are an excellent organismfor modeling neurological diseases as they have a conserved, yet simplified,vertebrate nervous system. They are particularly good for the study ofmotoneuron diseases, with accessible motoneurons that can be manipulatedin vivo, making them highly relevant for performing cell-autonomy studies,electrophysiology and imaging. Importantly, this demonstration that zebrafishare a suitable model for ALS means that they can now be used both for large-scale genetic screening to identify ALS-causing genes, and as a platform forthe development and screening of novel drugs.

doi:10.1242/dmm.005660

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and establish the transgenic lines. P.M.L.J. and B.D.C. performed the musclephysiology. A.H.M.B. contributed to the design of the transgenic constructs. C.E.B.oversaw the project, analyzed all data and wrote the manuscript.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.005538/-/DC1

Received 8 February 2010; Accepted 20 March 2010.

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