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PTL-1 regulates neuronal integrity and lifespan inC. elegans

Yee Lian Chew1, Xiaochen Fan1, Jurgen Gotz2 and Hannah R. Nicholas1,*1School of Molecular Bioscience, University of Sydney, New South Wales, 2006, Australia2Centre for Ageing Dementia Research (CADR), Queensland Brain Institute (QBI), University of Queensland, St Lucia Campus (Brisbane),Queensland, 4072, Australia

*Author for correspondence (hannah.nicholas@sydney.edu.au)

Accepted 18 February 2013Journal of Cell Science 126, 2079–2091� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.jcs124404

SummaryProtein with tau-like repeats (PTL-1) is the sole Caenorhabditis elegans homolog of tau and MAP2, which are members of the

mammalian family of microtubule-associated proteins (MAPs). In mammalian neurons, tau and MAP2 are segregated, with tau beingmainly localised to the axon and MAP2 mainly to the dendrite. In particular, tau plays a crucial role in pathology, as elevated levels leadto the formation of tau aggregates in many neurodegenerative conditions including Alzheimer’s disease. We used PTL-1 in C. elegans to

model the biological functions of a tau-like protein without the complication of functional redundancy that is observed among themammalian MAPs. Our findings indicate that PTL-1 is important for the maintenance of neuronal health as animals age, as well as in theregulation of whole organism lifespan. In addition, gene dosage of PTL-1 is crucial because variations from wild-type levels are

detrimental. We also observed that human tau is unable to robustly compensate for loss of PTL-1, although phenotypes observed in tautransgenic worms are dependent on the presence of endogenous PTL-1. Our data suggest that some of the effects of tau pathology resultfrom the loss of physiological tau function and not solely from a toxic gain-of-function due to accumulation of tau.

Key words: Tau, Protein with tau-like repeats, PTL-1, Caenorhabditis elegans, Neurodegenerative diseases, Alzheimer’s disease, Aging

IntroductionTau is a neuronal microtubule-associated protein (MAP) that is

predominantly localised to axons (Weingarten et al., 1975; Kosik

and Finch, 1987). In mammals, tau has a role in regulating

microtubule assembly, stability, and organisation (Cleveland

et al., 1977b; Chen et al., 1992; Harada et al., 1994). Mutations in

the MAPT locus, which encodes tau, are associated with an

increased risk of several neuronal disorders including Parkinson’s

disease and corticobasal degeneration (reviewed by Wade-

Martins, 2012). Furthermore, the pathological significance of

tau is demonstrated by the presence of neurofibrillary tangles

(NFTs), composed of fibrillar aggregates of this protein, in the

brains of individuals suffering from neurodegenerative disorders

such as Alzheimer’s disease (Lee et al., 2001; Iqbal et al., 2010).

It is only partly understood how tau exerts its toxic effects,

whether in fibrillar form (Ittner et al., 2008), or before the

formation of tangles due to the loss of some important

physiological functions (Gomez-Isla et al., 1997; Lee and

Leugers, 2012). Despite the significant advances made using

tau transgenic and knockout models (reviewed by Gotz and

Ittner, 2008; Gotz et al., 2010; Ittner et al., 2011; Ke et al., 2012),

these studies are complicated by the presence of other neuronal

MAPs such as MAP2, which may share several physiological

roles. In fact, tau knockout mice do not display overt defects in

neuronal development or function (Harada et al., 1994; Dawson

et al., 2001; Tucker et al., 2001).

Caenorhabditis elegans has one putative homolog in the tau/

MAP2/MAP4 family of MAPs, called protein with tau-like

repeats (PTL-1) (Goedert et al., 1996; McDermott et al., 1996).

PTL-1 contains a high level of sequence homology to tau/MAP2/

MAP4 within the microtubule binding repeat (MBR) domain in

the carboxyl (C)-terminus, and has been shown to regulate

microtubule assembly in vitro (Goedert et al., 1996; McDermott

et al., 1996). Immunohistochemistry and analysis of a ptl-1

transcriptional reporter line demonstrate that PTL-1 has a

neuronal expression pattern in adult worms (Goedert et al.,

1996; Gordon et al., 2008), and henceforth we refer to PTL-1 as

the tau/MAP2 homolog as these are the neuronal MAPs in

mammals (Dehmelt and Halpain, 2005). PTL-1 has also been

shown to have neuronal functions in worms, as it has been

implicated in the regulation of microtubule-based motility in

several neurons (Tien et al., 2011) and for the optimal

functioning of touch receptor neurons in the response to gentle

touch (Gordon et al., 2008). Therefore, C. elegans is a convenient

in vivo model in which to study the physiological roles of a tau/

MAP2-like protein without having to consider compensatory

effects produced by other closely-related MAPs.

Tau is implicated in several neurodegenerative conditions

called tauopathies, including Alzheimer’s disease, and these have

an increased incidence with age. Thus, it is of particular interest

to understand any age-dependent neuronal effects of perturbing

tau or tau-like proteins. A novel aging phenotype was recently

observed in C. elegans, where neurons display abnormal

structures such as branching or blebbing from the cell body or

axon, and these changes progressively accumulate as the

organism ages (Pan et al., 2011; Tank et al., 2011; Toth et al.,

2012). In a wild-type animal, these morphological changes occur

at a low frequency in early adulthood and gradually increase in

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frequency with age, however, the appearance of these aberrant

structures can be delayed or accelerated when mutations are

introduced in either tubulin genes (Pan et al., 2011), components

of the insulin signalling pathway (Pan et al., 2011; Tank et al.,

2011; Toth et al., 2012), or stress-response effectors (Pan et al.,

2011; Tank et al., 2011; Toth et al., 2012). We sought to examine

if loss of PTL-1 in C. elegans would affect the incidence of these

neuronal phenotypes.

We examined aging neurons in ptl-1 mutant strains and

observed that these animals show an increased incidence of

abnormal structures compared with wild-type animals, and

moreover, displayed a reduced lifespan. We were able to

rescue accelerated neuronal aging and shortened organismal

lifespan by re-expressing PTL-1 in a ptl-1 null mutant.

Interestingly, we found that gene dosage of PTL-1 is critical,

since either increasing or decreasing the number of copies of ptl-

1 is detrimental to the organism. Our findings demonstrate a key

role of PTL-1 in maintaining neuronal health with age and in

regulating whole organism lifespan.

Resultsptl-1(tm543) and ptl-1(ok621) mutant strains display allelic

differences in touch sensitivity

PTL-1 in C. elegans displays high sequence similarity to

mammalian tau/MAP2/MAP4 in the C-terminal MBR domain.

It is encoded by the gene ptl-1, which is composed of eight

coding exons, with exons 5–7 encoding the MBRs (Fig. 1A).

Two deletion alleles have been generated for ptl-1, both of which

were used in this study: the ok621 mutation generated by the

OMRF arm of the C. elegans Knockout Consortium (WormBase

ID: WBVar00091905), and the tm543 mutation by the National

Bioresource Project, Japan (WormBase ID: WBVar00249582)

(Fig. 1A). ptl-1(ok621) is a null mutation (Gordon et al., 2008),

whereas ptl-1(tm543) is a shorter deletion encompassing the

exons encoding the MBR region, and may result in a truncated

protein that has retained the N-terminal projection domain. There

are several putative isoforms of ptl-1, with transcripts labelled

F42G9.9a–d (http://www.wormbase.org). As a reference for our

experiments, we used the sequence of isoform a (F42G9.9a) as

this is a confirmed gene that generates a protein with five MBRs

(Goedert et al., 1996; Gordon et al., 2008), whereas the other

confirmed isoform of PTL-1 (isoform b) has only four putative

MBRs (Goedert et al., 1996; McDermott et al., 1996).

Mammalian MAPs such as tau and MAP2 are known to

execute a range of physiological functions in neurons (Drubin

and Kirschner, 1986; Avila et al., 2004; Dehmelt and Halpain,

2005; Ittner et al., 2010). As two alleles of ptl-1 are now

available, we investigated the role of PTL-1 in C. elegans

neurons by assaying both ptl-1(ok621) and ptl-1(tm543) mutant

strains. Although PTL-1 is expressed in several neuronal

subtypes in adult worms (Goedert et al., 1996; Gordon et al.,

2008), the highest levels are detected in the touch receptor

neurons, which regulate the response to gentle touch (Chalfie and

Sulston, 1981; Chalfie et al., 1985). Previously published data

indicate that ptl-1(ok621) mutants are mildly touch insensitive

compared with wild-type worms (Gordon et al., 2008). In line

with this, we found that ptl-1(ok621) worms were less touch

sensitive (touch sensitivity57462%) compared with wild-type

Fig. 1. ptl-1 alleles ok621 and tm543 display allelic differences in touch sensitivity. (A) Genomic locus of ptl-1 (transcript F42G9.9a) indicating regions

comprising the ok621 and tm543 deletions. Exons are shown as black boxes, introns as solid lines and untranslated regions at the 59 and 39 as white boxes; 59 is on

the left. Regions encoding putative microtubule binding repeats are marked 1–5 with arrowheads. (B) Data for sensitivity to gentle touch, shown for ptl-1(ok621)

and ptl-1(tm543) mutant strains and wild-type controls (n545 in total for two biological replicates). Assays were conducted on 1-day-old adults. Bars indicate

mean6s.e.m. One-way ANOVA, Bonferroni post-test; ns, no significance; *P,0.05.

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(8262%) (Fig. 1B). We also tested the ptl-1(tm543) C-terminaldeletion strain, and observed that there was no difference in touch

sensitivity between this mutant (8362%) and wild-type(Fig. 1B). These findings suggest that loss of full-length PTL-1, but not of the MBR domain alone, results in defective touchreceptor neurons.

ptl-1(ok621) and ptl-1(tm543) mutant strains show a highfrequency of abnormal neuronal structures in touchreceptor and GABAergic neurons in early adulthood

Recently, a novel age-related phenotype was reported in touchreceptor neurons where in wild-type animals, axons accumulated

branches and blebs, and cell bodies displayed branching as theanimals aged (Pan et al., 2011; Tank et al., 2011; Toth et al.,2012). The appearance of these abnormal neuronal structures wasaccelerated in the presence of mutations in mec-7 and mec-12

tubulin genes (Pan et al., 2011), both of which are highlyexpressed in touch neurons (Mitani et al., 1993; Savage et al.,1994; Fukushige et al., 1999). We investigated whether mutations

in ptl-1 would produce a similar effect. Initially, we studied theappearance of abnormal neuronal structures in wild-type and ptl-

1(ok621) animals by imaging individual worms until the animals

died (Fig. 2A,B; supplementary material Fig. S1). By assaying 15worms of each genotype every day over their entire lifespan, weobserved a progressive phenotype as previously reported, wherewild-type neurons accumulated branching and blebbing

structures with age. Interestingly, our data indicate that theaccumulation of morphological changes is significantlyaccelerated in the touch receptor neurons in ptl-1(ok621)

mutants. We observed that the proportion of ptl-1(ok621)

animals with neurons displaying abnormal structures reached50% at day 4, compared with wild-type animals where this level

was only reached at day 8 (Fig. 2C).

To complement this experiment, we used a transverseapproach, observing synchronised populations of wild-type and

ptl-1(ok621) animals on alternate days from day 1 to 15 ofadulthood. In this experiment, we recorded data for the touchreceptor neurons at the anterior and posterior halves of the animalseparately, as it became apparent that the posterior touch neurons

accumulate branches and blebs at a much faster rate comparedwith the anterior touch neurons, showing a high incidence ofthese abnormal structures even in early adulthood. Additionally,

since PTL-1 is not expressed in one of the three posterior touchneurons (PVM) (Goedert et al., 1996; Gordon et al., 2008), onlythe morphology of the two PLM neurons was considered in this

analysis. Although we were able to observe similar trends whencomparing the morphology of both posterior and anterior touchneurons of wild-type versus ptl-1 mutant strains, we observedhigh variability in the incidence of abnormal structures in the

posterior touch neurons in populations of wild-type animals andtherefore we restrict the discussion to findings obtained inanterior touch neurons. Data obtained from scoring posterior

touch neurons can be found in supplementary material Fig. S2.For both ptl-1 mutant strains, we separately scored for cell bodybranching, axon blebbing, and axon branching (Fig. 3A–C).

Consistent with our earlier findings, we observed a higherincidence of abnormal structures in the ptl-1(ok621) null mutantcompared with wild-type, and in addition we observed the same

phenotype in the MBR-deficient ptl-1(tm543) strain. In general,these structural abnormalities occur in a particular order, withcell body branching occurring first, followed by axon blebbing

and axon branching. Specifically, on day 5 of the assay we

observe cell body branching in 81% of ptl-1(ok621) (n537) and

73% of ptl-1(tm543) (n537) animals assayed compared with

36% of wild-type controls (n539) (Fig. 3A). At the same time

point, blebbing can be seen in 19% of ptl-1(ok621) and 22% of

ptl-1(tm543) animals compared with 5% in wild-type controls

(Fig. 3B), whereas axon branching is observed in 3% of both ptl-

1 mutant strains assayed compared with 0% of wild-type control

animals (Fig. 3C). Data obtained from scoring branching and

blebbing together in anterior touch neurons in ptl-1 mutant strains

can be found in supplementary material Fig. S2. Our results

suggest that the ptl-1(tm543) mutation could be hypomorphic,

such that the protein product in this mutant is able to sustain wild-

type levels of touch sensitivity, but is not sufficient to protect the

organism from susceptibility to age-dependent loss of structural

integrity. Taken together, these observations demonstrate that

PTL-1 is important in maintaining neuronal morphology in touch

receptor neurons, as a complete loss of this protein or expression

of a protein lacking the MBR domain results in a higher

incidence of abnormal neuronal structures in younger animals.

Fig. 2. ptl-1(ok621) mutant strain displays an accelerated onset of

appearance of abnormal neuronal structures in touch receptor neurons.

A representative animal is shown for each genotype (n515 total). Neurons

were visualised using the Pmec-4::gfp reporter. Worms were imaged every

day until death. Representative time points are shown, with arrowheads

indicating blebbing and arrows indicating branching phenotypes. For images

taken at all time points of a representative worm, see supplementary material

Fig. S1. Insets on day 10 show a close-up of the cell body for the respective

animal. (A) ALM neuron of a wild-type worm. The worm died on day 15.

(B) ALM neuron of a ptl-1(ok621) mutant worm. The worm died on day 14.

(C) Percentage of branching/blebbing observed in the complete data set of

wild-type and ptl-1(ok621) animals (n515). Values are mean6s.e.m. A

paired t-test was applied to the means of each strain at each time point and the

average of the entire curve compared; P,0.001. Scale bars: 50 mm.

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We next investigated if these effects are specific for selected

neuronal populations or whether they reflect a general

consequence of perturbing PTL-1 function. To this end, we

examined a second neuronal cell type in which PTL-1 is also

expressed (McKay et al., 2003; Gordon et al., 2008). Using a

Punc-47::gfp reporter line, we visualised the 25 GABAergic

neurons, which consist of neurons mainly in the ventral nerve

cord, as well as in the head and the tail (McIntire et al., 1997).

These GABAergic neurons were previously found to also display

an age-related phenotype, which is the incidence of branching

along commissures that extend to the dorsal side of the animal

(Tank et al., 2011). Representative images of the branching

phenotype in wild-type and ptl-1(ok621) animals are shown in

Fig. 3D. We assayed this phenotype in ptl-1 mutant animals and

observed an accelerated accumulation of branching structures in

both ptl-1(ok621) and ptl-1(tm543) animals compared with wild-

type (Fig. 3E). In particular at day 5, 68% of ptl-1(ok621) and

64% of ptl-1(tm543) animals displayed branching phenotypes

compared with 41% in wild-type controls. Data collected for each

ptl-1 mutant over 15 days can be seen in supplementary material

Fig. 3. ptl-1(ok621) and ptl-1(tm543) mutant strains show defects in maintaining neuronal integrity with age in touch receptor and GABAergic neurons.

Neurons were visualised using the Pmec-4::gfp reporter for touch receptor neurons or the Punc-47::gfp reporter for GABAergic neurons. Worms were imaged

every second day from day 1 to day 15. For imaging assays, the x2 test for independence was used to analyse differences between genotypes. (A–C) Anterior touch

neurons in ptl-1 mutants scored for (A) cell body branching, (B) blebbing along the axon and (C) axon branching. Sample sizes are indicated below graphs.

(D) Representative image of the phenotype scored in GABAergic neurons, showing healthy neurons and a branched commissure in wild-type, and representative

images of branched commissures in ptl-1(ok621) worms. Arrows indicate branching. Scale bars: 50 mm. (E) Data for the incidence of branching in GABAergic

neurons for ptl-1 mutant strains. (F) Assay for paralysis after levamisole exposure. Worms were scored for paralysis over 90 minutes on drug plates on day 1 and

day 7 of adulthood (n530). Statistical analysis, one-way ANOVA; ptl-1(ok621) and ptl-1(tm543) mutant strains, but not wild-type controls, show significant

decreases in drug sensitivity between day 1 and day 7. ns, no significance; *P,0.05.

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Fig. S2. We also found that ptl-1 mutant strains show

significantly lower sensitivity to the cholinergic agonist

levamisole compared with wild-type controls at early and mid

adulthood, with the ptl-1(tm543) strain appearing to have lower

sensitivity compared with ptl-1(ok621) (Fig. 3F). This suggests

that ptl-1 mutant animals are defective in cholinergic/GABAergic

transmission (Lewis et al., 1980). Additionally, we observed a

significant decrease in levamisole sensitivity for both ptl-1

mutant strains between day 1 and day 7, which correlates with the

higher incidence of branching commissures in GABAergic

neurons at later time points (Fig. 3E,F). In conclusion, ptl-1

mutant strains also display defects in a neuronal subtype other

than touch receptor neurons, suggesting that PTL-1 has a broader

role in the maintenance of neuronal structural integrity.

Lifespan reduction in ptl-1(ok621) and ptl-1(tm543) mutant

strains

Having found an age-related phenotype in strains carrying

mutations in ptl-1, we performed a lifespan assay to determine

if differences exist between wild-type and ptl-1 mutant strains.

We found that both ptl-1(ok621) (Fig. 4A) and ptl-1(tm543)

(Fig. 4B) mutants had a shorter lifespan compared with wild-

type. Analysis of the two survival curves indicates that this

difference is statistically significant; however, it appears that

lifespan reduction is more severe in the ptl-1(ok621) null mutant

strain that has a 37% shorter median lifespan compared with

wild-type, whereas the median lifespan in the ptl-1(tm543)

mutant strain is 10% shorter than wild-type.

Re-expressing PTL-1 under the control of the ptl-1

promoter rescues the age-dependent neuronal phenotype

and lifespan reduction in the ptl-1 null mutant line

To confirm that the neuronal aging and lifespan phenotypes

found in ptl-1 mutant worms are attributable to the loss of PTL-1

function, we generated a transgenic line expressing the ptl-1

cDNA under the control of the ptl-1 promoter and the PTL-1

39UTR control element. As no commercial antibody against PTL-

1 is available, we tagged PTL-1 at the C-terminus with the

short peptide V5 in order to enable detection of PTL-1 expressed

from the transgene. Transgenic worms were generated by

biolistic transformation, and an integrated line was obtained

after selection using the dual antibiotic method (Semple et al.,

2012). We confirmed that our transgene was expressed by

immunoblotting (Fig. 5A) and immunofluorescence (Fig. 5B).

The size of the band observed on the immunoblot is

consistent with previous experiments that have shown that

PTL-1 runs at ,75 kDa on an SDS-PAGE gel (Goedert et al.,

1996) (Fig. 5A). Furthermore, using a V5-specific antibody for

immunofluorescence staining, we detected PTL-1::V5

localising to axons and cell bodies in neurons, including

those comprising the nerve ring in the head, as well as in the

tail (Fig. 5B).

We crossed these PTL-1 transgenic worms with both the null

mutant ptl-1(ok621) and the Pmec-4::gfp reporter line, and

assayed for age-related morphological changes in touch receptor

neurons as described above. We confirmed expression of the

transgene in touch neurons by staining for PTL-1::V5 in

transgenic animals crossed with the Pmec-4::gfp reporter line

and observing co-localisation between signals from anti-V5

immunofluorescence and GFP (Fig. 5Biii). We also observed that

PTL-1 transgenic animals are touch sensitive both on a wild-type

and ptl-1(ok621) null mutant genetic background (Fig. 5C). For

assays on neuronal morphology, data are presented only for

anterior touch receptor neurons as we have shown that the

percentage of animals showing abnormal structures in these

neurons is low at early time points and that this is less variable

compared with the same phenotype in posterior touch receptor

neurons, making it easier to monitor changes in neuron

morphology with time. We found that re-expressing PTL-1 in

ptl-1(ok621) animals appeared to delay the accumulation of

abnormal neuronal structures such as branching and blebbing to

almost wild-type levels (Fig. 6Ai), such that when these animals

were assayed from day 5 onwards, the proportion of animals

showing these phenotypes in anterior touch neurons was not

significantly different from wild-type. For example, at day 5 of

this assay 20% of PTL-1 transgenic; ptl-1(ok621) worms (n540)

displayed abnormal structures in anterior touch neurons

compared with 32% in wild-type (n540). Thus we were able

to rescue the accelerated accumulation of abnormal structures in

touch neurons of ptl-1(ok621) mutants by re-expressing PTL-1 in

these animals. Somewhat surprisingly, we also observed that

animals expressing the PTL-1 transgene on a wild-type

background accumulated branches and blebbing structures with

increased frequency compared with non-transgenic wild-type

controls, and that these levels were comparable with that

observed in the strain carrying only the ptl-1(ok621) mutation

(Fig. 6Aii). For example, at day 5 of this assay, 55% of ‘PTL-1

transgenic; wild-type’ worms (n538) displayed abnormal

neuronal structures, compared with 32% in wild-type controls

(n540). It therefore appears that modulation of PTL-1 levels by

either a null mutation or increasing the gene copy number (by

introducing copy(ies) of a PTL-1 transgene in addition to the

endogenous gene locus) negatively affects neuronal integrity.

Furthermore, we conducted a lifespan assay on PTL-1

transgenic worms that were either wild-type at the genomic ptl-

1 locus or that carried the ptl-1(ok621) mutation (Fig. 6B). We

Fig. 4. ptl-1 mutant strains display a reduction in

lifespan. (A,B) Survival curves for (A) ptl-1(ok621) and

(B) ptl-1(tm543) mutant strains. Log rank and Gehan-

Breslow-Wilcoxon tests; P,0.05 for both ptl-1(ok621)

and ptl-1(tm543) compared with wild-type.

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found that re-expressing PTL-1 in a ptl-1 null mutant background

resulted in a survival curve that was very similar to non-

transgenic wild-type worms. Therefore, re-expressing PTL-1 in a

ptl-1 null mutant is able to rescue both the lifespan reduction and

the premature neuronal aging observed in ptl-1(ok621) animals

(Fig. 6A). Interestingly, expressing the PTL-1 transgene together

with endogenous PTL-1 led to a reduction in lifespan. This

reduction in lifespan for the ‘PTL-1 transgenic; wild-type’ strain

is statistically significant according to the Gehan-Breslow-

Wilcoxon test for significance. Increasing the number of copies

of ptl-1 thus appears to produce a detrimental effect both in terms

of neuronal health and whole organism lifespan, despite

displaying wild-type touch sensitivity.

Human tau does not robustly rescue for loss of PTL-1

As noted above, PTL-1 in C. elegans and tau in humans have

high sequence similarity in the C-terminal domain that is

important for microtubule binding, and also have a similar

overall charge distribution (Goedert et al., 1996; McDermott

et al., 1996; Gordon et al., 2008). To determine if there is

functional conservation between these two proteins, we generated

transgenic C. elegans strains expressing a cDNA of the longest

isoform of human tau (htau40, referred to here as htau). The htau

cDNA was expressed under the control of the ptl-1 promoter and

PTL-1 39 UTR as described above. htau transgenic animals were

similarly generated by biolistic transformation and an integrated

line was isolated. Immunoblotting analysis showed that htau is

expressed in the transgenic line but not in non-transgenic wild-

type worms (Fig. 7A).

Interestingly, we observed that htau expression in a wild-type

background resulted in touch insensitivity (Fig. 7B), and was also

detrimental to worms both in terms of neuronal aging and whole

organism lifespan (Fig. 8A,B). In particular, the transgenic line

expressing htau in a wild-type background displayed a higher

incidence of branching and blebbing phenotypes in anterior touch

receptor neurons compared with non-transgenic wild-type worms

at day 3, 5, 7 and 9 of the assay (Fig. 8Aii). In addition, this

transgenic line also showed a significant reduction in lifespan

compared with the wild-type control (Fig. 8B). Our data are

consistent with previous reports indicating that wild-type htau

expression under the control of a pan-neuronal promoter results

in defects in motility, cholinergic neuron transmission, and

lifespan (Kraemer et al., 2003). It is notable that htau transgenic

lines reported in the study conducted by Kraemer et al. were

Fig. 5. Re-expression of PTL-1 under the regulation of the ptl-1 promoter can be detected in neurons of transgenic animals and rescues touch sensitivity in a

null mutant. ‘PTL-1 Tg’ refers to the PTL-1::V5 transgene; the terms ‘wild-type’ and ‘ptl-1(ok621)’ following this refer to the genotype at the genomic ptl-1 locus,

whether wild-type or ok621 mutant, respectively. (A) Immunoblot showing the presence of a band corresponding to PTL-1::V5 expression from the transgene, probed

using an anti-V5 antibody. Non-Tg, non-transgenic wild-type animals. (B) Immunofluorescence micrographs showing the expression of the PTL-1::V5 transgene

(anti-V5 antibody) in neurons. Grayscale images on the left show the red channel only, the nerve ring is indicated by arrows, axons by arrowheads, and cell bodies by

asterisks. (Bi) The pharynx is shown and gfp expression can be observed from the Pmyo-2::gfp transformation reporter. (Bii) A tail neuron is shown with staining in

both the cell body and axon. (Biii) Co-localisation with a reporter line for touch receptor neurons (Pmec-4::gfp) is shown, demonstrating that the transgene is

expressed in touch neurons. Dotted lines indicate the outline of the animal as determined by phase-contrast microscopy. Ventral is down. Scale bars: 50 mm. (C) Data

for sensitivity to gentle touch, shown for PTL-1 transgenic animals (n.45 total for two biological replicates). Control strains are the same as those shown in Fig. 1B.

Assays were conducted on 1-day-old adults. Bars indicate mean6s.e.m. One-way ANOVA, Bonferroni post-test; ns, no significance; *P,0.05.

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generated by microinjection (Kraemer et al., 2003), whereas the

transgenic lines in our study were generated by biolistic

transformation, which we would expect to introduce a lower

transgene copy number than by microinjection (Praitis et al.,

2001). Therefore it appears that wild-type htau, even at a low

expression level, is detrimental to C. elegans when expressed in

addition to endogenous PTL-1.

We next investigated whether htau would rescue the defects

observed in the ptl-1 null mutant. When htau is expressed in

ptl-1(ok621) null mutant worms, touch sensitivity is rescued to

Fig. 6. Re-expression of PTL-1 rescues age-related abnormal neuron morphology and lifespan reduction in the ptl-1(ok621) mutant. ‘PTL-1 Tg’ refers to

the PTL-1::V5 transgene, and the terms ‘wild-type’ and ‘ptl-1(ok621)’ following this refer to the genotype at the genomic ptl-1 locus, whether wild-type or ok621

mutant, respectively. (A) Neuron imaging time course of PTL-1 transgenic worms, showing data for anterior touch receptor neurons visualised using the Pmec-

4::gfp reporter. (Ai) Control strains together with the PTL-1 Tg; ptl-1(ok621) rescue strain. (Aii) Effect of the PTL-1 Tg alone, compared with wild-type controls.

Worms were imaged every second day from day 1 to day 15. Sample sizes are indicated below the graph. The x2 test for independence was used to analyse

differences between genotypes; ns, no significance; *P,0.05. (B) Survival curves for PTL-1 transgenic worms. n5100 for each strain at the start of the assay.

Results of statistical analysis are shown in the table below.

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wild-type levels (Fig. 7B). However, the frequency of abnormal

structures observed in anterior touch receptor neurons is not

significantly different compared with the non-transgenic ptl-1

null mutant strain on days 3, 5, 7 and 9 of the assay (Fig. 8Ai).

This finding indicates that htau cannot compensate for loss of

PTL-1 in terms of protecting animals from premature neuronal

aging, but is able to rescue the functional defect in touch neurons.

Interestingly, we also observed that the transgenic line expressing

htau in a ptl-1 null mutant has a lifespan that is intermediate

between that of non-transgenic wild-type and ptl-1(ok621) null

mutant strains, and is not significantly different from these

control strains (Fig. 8B). While this latter observation suggests

that htau may compensate in part for the absence of PTL-1 in the

regulation of whole organism lifespan, overall our data indicate

that htau expression does not robustly rescue for the loss of

PTL-1.

Curiously, comparing htau transgenic lines that are either wild-

type or null for endogenous PTL-1 indicates that the presence of

the ptl-1(ok621) null mutation improved some of the detrimental

effects of htau expression. For example, in the touch assay, the

expression of htau resulted in reduced touch sensitivity in the

wild-type background but rescued touch responsiveness in the

null mutant (Fig. 7B). Furthermore, the htau transgenic line in a

wild-type background had a higher percentage of animals

displaying abnormal neuronal structures compared with the

htau transgenic line in a ptl-1 null mutant background at days 1,

3, 5 and 9 of the assay (Fig. 8A). In particular, on day 5, the

percentage of ‘htau transgenic; wild-type’ worms displaying

branching or blebbing was 60% (n540), compared with 40% in

‘htau transgenic; ptl-1(ok621)’ worms (n535) and 32% in non-

transgenic wild-type worms (n540). In terms of lifespan, the

strain carrying the htau transgene on a wild-type background has

a significantly shorter lifespan compared with non-transgenic

wild-type worms, but this effect is ameliorated in ‘htau

transgenic; ptl-1(ok621)’ strain (Fig. 8B). These data suggest

some degree of functional conservation between htau and PTL-1.

DiscussionPTL-1 is important for the maintenance of neuronal

integrity and lifespan in C. elegans

We have shown that PTL-1 in C. elegans is involved in the age-

associated preservation of neuronal structural integrity as well as

in the regulation of lifespan. Moreover, we observed that the

severity of phenotypes observed in htau transgenic worms is

dependent on the presence or absence of endogenous PTL-1,

suggesting some functional conservation between these proteins.

Our data indicate that targeting PTL-1 in C. elegans is a useful

model to study the physiological roles of neuronal tau/MAP2-like

MAPs in vivo.

Our focus in the study of PTL-1 is in the context of neuronal

integrity, where tau is understood to play a significant role. Tau is

pathologically important in several neurodegenerative disorders

in which the progressive formation of NFTs from aggregated

forms of tau is a histopathological hallmark of disease (reviewed

by Lee et al., 2001). It is increasingly appreciated that tau not

only assumes a toxic gain-of-function as its levels are elevated

and aggregated forms of tau accumulate (Gomez-Ramos et al.,

2006; Ittner et al., 2008; Clavaguera et al., 2009; Avila et al.,

2010), but rather also that the loss of normally functioning tau

results in disease (Gomez-Isla et al., 1997; Santacruz et al.,

2005). To clarify this issue, it is critical to acquire a thorough

understanding of the biological roles of tau in vivo in the absence

of pathology. As a microtubule-binding protein, tau plays many

important physiological functions (Weingarten et al., 1975;

Cleveland et al., 1977a; Baas et al., 1991; Chen et al., 1992;

Lee et al., 1998; Liao et al., 1998; Reynolds et al., 2008; Ittner

et al., 2010; Morris et al., 2011), however, investigating tau in

mammalian models is complicated by the presence of other

MAPs, which appear to share several biological roles (Dehmelt

and Halpain, 2005; Sontag et al., 2012). Due to the complex

functional redundancy between mammalian MAPs, many

studies aiming to address the biological functions of tau have

focussed on overexpression models, with few studies utilising

Fig. 7. Expression of human tau rescues touch sensitivity in the ptl-1(ok621) null mutant but not in a wild-type background. ‘htau Tg’ refers to the human

tau transgene, and the terms ‘wild-type’ and ‘ptl-1(ok621)’ immediately following this refer to the genotype at the ptl-1 locus, whether wild-type or ok621 mutant,

respectively. (A) Immunoblot showing the presence of a band corresponding to the htau40 transgene, probed using the tau 13 antibody. Non-Tg, non-transgenic

wild-type animals. (B) Data for sensitivity to gentle touch, shown for htau transgenic animals (n.45 total for two biological replicates). Control strains are the

same as those shown in Fig. 1B. Assays were conducted on 1-day-old adults. Bars indicate mean6s.e.m. One-way ANOVA, Bonferroni post-test; ns, no

significance; *P,0.05.

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gene-targeting approaches to investigate endogenous tau

(reviewed by Gotz and Ittner, 2008; Gotz et al., 2010). In

addition, three tau knockout mouse lines have been generated

(Harada et al., 1994; Dawson et al., 2001; Tucker et al., 2001)

and in general, these mice do not show defects in neuronal

development or function (reviewed by Ke et al., 2012), an

exception being in the case of an Alzheimer’s disease model,

where the loss of tau appeared to exacerbate effects of a mutated

b-amyloid precursor protein (Dawson et al., 2010). This general

lack of morphological defects is potentially due to compensatory

effects by MAP2 (Harada et al., 1994). Therefore, despite the

significant contribution of these studies, it would be useful to

have a model for tau where a gene-targeted approach can be

taken without also having to consider compensatory functions

Fig. 8. Expression of human tau does not robustly rescue defects observed in the ptl-1 null mutant, but phenotypes observed in tau transgenic lines are

dependent on endogenous PTL-1. ‘htau Tg’ refers to the human tau transgene, and the terms ‘wild-type’ and ‘ptl-1(ok621)’ immediately following this refer to

the genotype at the ptl-1 locus, whether wild-type or ok621 mutant, respectively. (A) Neuron imaging time course of htau transgenic worms showing data for

anterior touch receptor neurons visualised using the Pmec-4::gfp reporter. (Ai) Control strains together with the htau Tg; ptl-1(ok621) rescue strain. (Aii) Effect of

the htau Tg alone, compared with wild-type controls. Worms were imaged every second day from day 1 to day 15. Sample sizes are indicated below the graph.

Control strains, wild-type and ptl-1(ok621), are the same as those shown for the PTL-1 transgenics (Fig. 6A). The x2 test for independence was used to analyse

differences between genotypes; ns, no significance; *P,0.05. (B) Survival curves for htau transgenic worms. n5100 for each strain at the start of the assay.

Results of statistical analysis are shown in the table below. Control strains are the same as those shown for the PTL-1 transgenics (Fig. 6B).

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attributable to other MAPs. Investigating PTL-1 in C. elegans hasthe advantage of PTL-1 being the only homolog of tau/MAP2 in

the worm (McDermott et al., 1996; Gordon et al., 2008), meaningthat shared physiological functions between tau/MAP2 familymembers can be addressed.

There are two ptl-1 mutants available: ptl-1(ok621) is a null

mutant (Gordon et al., 2008), whereas the ptl-1(tm543) lineputatively generates a protein product containing only the N-terminal region (Fig. 1A). The N-terminus of PTL-1 is largely

conserved amongst nematodes, is proline-rich, highly acidic, andalso contains several serine/threonine-proline motifs that arepotential phosphorylation sites (Goedert et al., 1996; McDermott

et al., 1996; Gordon et al., 2008). Analysis of these mutant strainsallowed us not only to investigate the role of PTL-1 in vivo, butalso to dissociate functions of PTL-1 that are attributable solelyto microtubule-binding from functions of the full-length protein.

We found that both mutants of ptl-1 show a decreased capacity tomaintain neuronal integrity with age, as evidenced by a higherfrequency of abnormal morphological structures such as

branching and blebbing in touch receptor and GABAergicneurons in early adulthood compared with wild-type.Interestingly, this effect was also observed when we increased

the copy number of full-length PTL-1 by expression of a stabletransgene in addition to the endogenous locus. Therefore, correctgene dosage of PTL-1 is critical for the maintenance of neuronalstructures with age, as either increasing or decreasing this level is

detrimental to the organism. In addition when we assayed touchsensitivity, a response that requires functional touch receptorneurons, we found that the ptl-1(ok621) null mutant is less

responsive to gentle touch compared with wild-type, but thatthere was no difference in touch sensitivity from wild-type in theptl-1(tm543) mutant lacking the MBR domain, suggesting that

this allele may be hypomorphic, or that the N-terminal regionmay have functions that are sufficient to maintain touchsensitivity. Interestingly, we observed the opposite effect when

assaying for defects in cholinergic/GABAergic transmission,with ptl-1(tm543) animals displaying a more severe phenotypecompared with ptl-1(ok621). Importantly, our data indicate thatalthough the microtubule-binding functions of PTL-1 are not

necessary for wild-type touch sensitivity, the maintenance oftouch neuron structural integrity with age requires full lengthPTL-1. Our findings also demonstrate that wild-type functioning

of neurons may not be sufficient to preserve neuronal health withage. Another C. elegans MAP that is expressed in touch receptorneurons is the EMAP-like protein or ELP-1, which does not

display high sequence similarity to PTL-1 but also regulatestouch sensitivity (Hueston et al., 2008). In light of this, allelicdifferences in touch sensitivity observed between ptl-1 mutants

could be due to a requirement for both ELP-1 and the N-terminusof PTL-1 for wild-type touch responsiveness. A role of ELP-1 inneuronal aging, however, remains to be investigated.

Having found an age-associated neuronal phenotype in ptl-1

mutant lines, we investigated if these strains also displayeddefects in lifespan. We found that both mutants show a lifespanreduction compared with wild-type, and that furthermore,

increasing the number of copies of ptl-1 also results in ashortening of lifespan. This indicates that a tight regulation ofPTL-1 is required to maintain wild-type lifespan. As discussed

above, our observations also indicate a role for PTL-1 inmaintaining structural integrity in neurons, which may be relatedor additional to the role of this protein in regulating lifespan.

Previous reports suggest that the factors regulating wholeorganism lifespan and age-related neuronal integrity may be

separable. For example, C. elegans carrying mutations in the daf-

2 insulin receptor homolog are long-lived (Kenyon et al., 1993).

This lifespan extension requires the forkhead box O (FOXO)transcription factor daf-16, meaning that a daf-2;daf-16 doublemutant is not long-lived (Kenyon et al., 1993). Tank and

colleagues have shown that a daf-2 mutant displays significantlyless neuronal branching in early adulthood compared with wild-

type, and that this effect is ameliorated in daf-2;daf-16 doublemutants (Tank et al., 2011). Importantly, RNAi-mediated

knockdown of daf-16 in a daf-2 mutant in non-neuronal cellsresults in an animal with wild-type lifespan, but has no effect onthe daf-2-mediated delay of neuronal aging (Tank et al., 2011).

This and other evidence from previous reports (Pan et al., 2011;Tank et al., 2011) suggests that the processes that influence

neuronal aging and whole organism aging can be decoupled fromone another. Further investigations are required to determinewhether the function of PTL-1 in stabilising neuronal structures

with age influences lifespan, or vice versa.

If PTL-1 has biological functions in regulating both aging in

neurons and aging in the whole organism, where does this proteinexert its effects? In adult worms, it appears that PTL-1 is only

expressed in neurons (Goedert et al., 1996; Gordon et al., 2008).Previous studies have also established a role for PTL-1specifically in touch neurons, in particular with regards to

mechanosensation (Gordon et al., 2008) and microtubule-basedtransport (Tien et al., 2011). In the case of branching and bleb

formation in neurons, there is some evidence that thesemorphological changes are due to cell-autonomous effects(Tank et al., 2011; Toth et al., 2012). For example, expression

of DAF-16 only in neurons of a daf-2;daf-16 mutant delays theformation of abnormal neuron structures compared with a non-

transgenic daf-2;daf-16 control (Tank et al., 2011). In addition,reduced activity of the heat shock factor-1 transcription factor

(hsf-1) in the whole organism resulted in early onset branchingand blebbing in touch receptor neurons, but re-expressing hsf-1

only in this subset of neurons was able to rescue this effect (Toth

et al., 2012). With regards to the role of PTL-1 in regulatingorganismal lifespan, previous studies have suggested that

signalling events in neurons alone are sufficient to alterlifespan (Apfeld and Kenyon, 1999; Alcedo and Kenyon,2004). Therefore, it is possible that PTL-1 has a cell-

autonomous effect on neuronal integrity as well as wholeorganism aging. If neuronal functions of PTL-1 regulate whole

organism lifespan, it remains to be determined whether this refersto a role in all neurons or in a particular subset of neurons.

Ablation of sensory neurons such as thermosensory neurons (Leeand Kenyon, 2009) or gustatory and olfactory neurons (Alcedoand Kenyon, 2004) in C. elegans results in a shortening or

extension of lifespan, respectively. In addition, C. elegans

mutants defective in sensory cilia in some neurons have been

shown to be long-lived (Apfeld and Kenyon, 1999). Studiesperformed using several mechanosensory defective (mec)

mutants predominantly affecting touch receptor neurons, suchas mec-1, mec-8 and mec-12, have also demonstrated differencesin lifespan compared with wild-type (Apfeld and Kenyon, 1999;

Pan et al., 2011). As PTL-1 is expressed in most, if not all,neurons in the worm, the reduced lifespan of ptl-1 mutants may

be due to the loss of PTL-1 function in one or more of theseneuronal subsets.

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Human tau and C. elegans PTL-1 display somefunctional conservation

We generated tau transgenic worms that expressed the longestisoform of human tau under the control of the ptl-1 promoter, andobserved (1) that human tau expression is detrimental to worms,

(2) that human tau does not robustly rescue loss of PTL-1, and (3)that touch sensitivity, neuronal structural health and lifespanphenotypes of human tau transgenic lines are dependent on PTL-1. The negative effect of expressing human tau in C. elegans, as

also observed in (Kraemer et al., 2003; Miyasaka et al., 2005;Brandt et al., 2009), may either be due to overexpression of anyMAP having a detrimental effect, or due to a specific axonal role

of tau that evolved with the diversification of neuronal MAPsinto mainly axon-localised tau and mainly dendrite-localisedMAP2. In addition to some shared functions (Sontag et al., 2012),

tau and MAP2 play specific roles in their distinct subcellularcompartments (Kosik and Finch, 1987; Chen et al., 1992; Haradaet al., 1994; Hirokawa et al., 1996). In C. elegans, no such

diversification of neuronal MAPs exists, and as PTL-1 is ahomolog of both MAP2 and tau, it presumably has both axon-and dendrite-specific functions. Therefore, the axon-specificeffects of human tau could negatively affect the worm when tau

is present in addition to endogenous PTL-1, and could also beinsufficient to rescue for the loss of PTL-1 in a null mutant withregards to neuronal and whole organismal aging.

Our observation that human tau rescues touch insensitivity in aptl-1 null mutant, together with the finding that the detrimentaleffects of expressing tau are ameliorated in the absence of

endogenous PTL-1, suggests some functional conservationbetween tau and PTL-1. Although these proteins do not havehigh similarity over the entire sequence, the imperfect tandem

repeats in the C-terminal microtubule binding domain thatconstitute the only region of homology between these proteinsshow high amino acid identity. Therefore, these conservedfunctions may be those attributable to the microtubule-binding

capacity of tau and PTL-1.

Concluding remarks

We have found that C. elegans PTL-1 plays important functionsin maintaining neuronal health with age, and that these functionsmay be related or additional to a role in regulating whole

organism aging. This is consistent with the notion that at leastsome of the effects of tau pathology in neurodegenerativeconditions may be attributable to a loss of correctly functioning

tau, and not solely to a gain-of-toxic function due to aggregatedforms of tau. Furthermore, we have shown that PTL-1 is a usefulmodel for a gene-targeted approach to study the physiological

roles of a tau-like protein due to PTL-1 being the sole homolog oftau/MAP2 in C. elegans.

Materials and MethodsStrain information

C. elegans strains were cultured on NGM plates seeded with the Escherichia colistrain OP50. Hermaphrodite animals were used for all experiments. The wild-typestrain used for all experiments is N2 (Bristol). Strains N2 (Bristol), RB809 ptl-1(ok621), CZ10175 zdIs5[Pmec-4::gfp + lin-15(+)] I and EG1285 oxIs12[Punc-47::gfp + lin-15(+)] X were obtained from the Caenorhabditis Genetics Centre, andFX00543 ptl-1(tm543) was obtained from the National Bioresource Project, Japan(Dr S. Mitani). ptl-1 mutant lines RB809 and FX00543 were both outcrossed sixtimes to wild-type. For neuron imaging assays, strains involved were crossed withCZ10175 to visualise touch neurons and EG1285 to visualise GABAergic motorneurons.

The strains generated were as follows. APD004: ptl-1(ok621) outcrossed sixtimes to N2; APD009: ptl-1(ok621); oxIs12[Punc-47::gfp + lin-15(+)]; APD010:

ptl-1(ok621); zdIs5[Pmec-4::gfp + lin-15(+)]; APD015: ptl-1(tm543) outcrossed sixtimes to N2; APD016: ptl-1(tm543); zdIs5[Pmec-4::gfp + lin-15(+)]; APD017: ptl-

1(tm543); oxIs12[Punc-47::gfp + lin-15(+)]; APD025: apdIs4[Pptl-1:htau40:ptl-

1_39UTR; Pmyo-2:mCherry; Prpl-28::PuroR::rpl-16_outron::NeoR::let-858_39UTR]; APD026: apdIs5[Pptl-1:PTL-1::V5:ptl-1_39UTR; Pmyo-2:gfp; Prpl-28::PuroR::rpl-16_outron::NeoR::let-858_39UTR]; APD030: apdIs4[Pptl-

1:htau40:ptl-1_39UTR; Pmyo-2:mCherry; Prpl-28::PuroR::rpl-16_outron::NeoR::let-858_39UTR]; ptl-1(ok621); zdIs5 [Pmec-4::gfp + lin-15(+)]; APD031:apdIs4[Pptl-1:htau40:ptl-1_39UTR; Pmyo-2:mCherry; Prpl-28::PuroR::rpl-16_outron::NeoR::let-858_39UTR]; zdIs5[Pmec-4::gfp + lin-15(+)]; APD034: apdIs4

[Pptl-1:htau40:ptl-1_39UTR; Pmyo-2:mCherry; Prpl-28::PuroR::rpl-16_outron::NeoR::let-858_39UTR]; ptl-1(ok621); APD035: apdIs5[Pptl-1:PTL-1::V5:ptl-

1_39UTR; Pmyo-2:gfp; Prpl-28::PuroR::rpl-16_outron::NeoR::let-858_39UTR];ptl-1(ok621); zdIs5[Pmec-4::gfp + lin-15(+)]; APD036: apdIs5[Pptl-1:PTL-1::V5:ptl-1_39UTR; Pmyo-2:gfp; Prpl-28::PuroR::rpl-16_outron::NeoR::let-858_39UTR]; zdIs5[Pmec-4::gfp + lin-15(+)]; APD039: apdIs5[Pptl-1:PTL-1::V5:ptl-

1_39UTR; Pmyo-2:gfp; Prpl-28::PuroR::rpl-16_outron::NeoR::let-858_39UTR]; ptl-

1(ok621).

Plasmids

Cloning was performed using the Gateway (Invitrogen, Life Technologies) systemaccording to the manufacturer’s instructions. Dual antibiotic selection plasmidspBCN40 and pBCN41, encoding visual markers Pmyo-2::mCherry and Pmyo-

2::gfp, respectively, were generously provided by Drs J. Semple and B. Lehner(EMBL Centre for Genomic Regulation Systems Biology Unit, Barcelona). Theptl-1 promoter used is a 2.9 kb sequence upstream of ptl-1 and was cloned fromVancouver fosmid WRM0634bB04 (Source BioScience Lifesciences), using theprimers 59-GGGGACAACTTTGTATAGAAAAGTTGCATTCCGCATGGTTG-GAAAGAG-39 (forward primer including attB4 site) and 59-GGGGACTGCT-TTTTTGTACAAACTTGATTTTTCCTGAAAAATTGAAATTGGGAG-39 (reverseprimer including attB1r site). The ptl-1 cDNA was cloned from a C. elegans cDNAlibrary (RIKEN BioResource Centre, Japan; RDB No. 1864), and the V5 epitope taggedat the C-terminus using the forward primer 59-TCACGTAGAATCGAGACCG-AGGAGAGGGTTAGGGATAGGCTTACCCGCCGCGCGATTGAATATAAAAT-CAGG-39. Site attB1 was added using 59-GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGTCAACCCCTCAATCAGAG-39, and attB2 using 59-GGGGACC-ACTTTGTACAAGAAAGCTGGGTTTACGTAGAATCGAGACCGAGGAG-39. ThePTL-1 39 UTR was cloned from fosmid WRM0634bB04 using forward primer 59-GGGGACAGCTTTCTTGTACAAAGTGGGATAACAATCGCTGATGTATACCG-CGC-39 (incorporating attB2r) and reverse primer 59-GGGGACAACTTTGTAT-AATAAAGTTGACACTTTTAATTACCACTTTATTGAAGAG-39 (incorporatingattB3). The 59 entry clone was generated using a BP reaction into pDONRP4P1R, themiddle entry clone using a BP reaction into pDONR221, and the 39 entry clone using aBP reaction into pDONRP2RP3 (pDONR vectors from Invitrogen). The htau40 entryclone (in pENTR-SD-D-Topo) was kindly provided by Dr L. Ittner (Brain and MindResearch Institute, University of Sydney). The Multisite LR reaction (Invitrogen) wasused to combine entry clones into destination vector pBCN40 or pBCN41.

Generation of transgenic lines

Transgenic worms for rescue experiments were generated by biolistic transformationusing the PDS-1000/HeTM particle delivery system (BioRad) according to themanufacturer’s instructions. Wild-type worms were bombarded with 7 mg oflinearised plasmid DNA using previously established methods (Praitis et al., 2001).Selection post-bombardment was undertaken using the dual antibiotic selectionprotocol (Semple et al., 2012). Integrated lines expressing htau40 cDNA and PTL-1cDNA tagged at the carboxyl (C)-terminus with V5 under the control of the ptl-1

promoter and PTL-1 39UTR were obtained and outcrossed six times to wild-type.

Touch sensitivity assay

Animals were synchronised by hypochlorite treatment and cultured at 25 C, with1-day-old adults used for all assays. Touch assays were performed according toestablished methods (Chalfie and Sulston, 1981). A positive response wasdetermined as acceleration of the animal away from touch. The number of positiveresponses was then expressed as a percentage of total touches (ten) for eachanimal. Assays were conducted blind to the genotype of the worms.

Neuron imaging assay

Age-matched animals synchronised by egg-laying were cultured on plates at 20 C.Starting animals were 1-day-old adults in all cases. For longitudinal assays whereindividual animals were monitored every day during their lifetimes, these animalswere individually mounted into 0.2% tetramisole (Sigma) on 3% agarose padsprepared on standard microscope slides. These animals were rescued by pickingthem onto a drop of M9 buffer, and recovered well if incubated in tetramisole forunder two minutes. For transverse assays where populations of animals weremonitored every second day for 15 days, surviving adult worms on a plate weremounted as described, but were not recovered post-imaging. To separate adultworms from their progeny, adult worms were moved to new NGM plates every

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second day until the assay was completed. Assays were conducted blind to thegenotype of the worms. To score the incidence of aberrant neuronal structures intouch receptor neurons, worms were scored as positive if the neuron displayedbranching or blebbing at the cell body or axon. For scoring in GABAergic neurons,worms were scored as positive if at least one of the observed commisures displayedbranching. For all transverse assays, the proportion of animals scored as positive wasexpressed then as a percentage of the sample size observed at that time point.

Lifespan assay

Age-matched animals synchronised by egg-laying were cultured on plates at 25 Cand the number of surviving animals recorded every day until death. 100 1-day-oldadults per strain were plated at the start of each assay. Animals that were lost ordisplayed internal hatching or bursting were censored. To separate adult wormsfrom their progeny, adult worms were moved to new NGM plates every secondday until the assay was completed. Survival curves were generated using GraphPadPrism 5 (GraphPad Software Inc.).

Immunofluorescence

One-day-old adults synchronised by egg-laying and cultured at 23 C were used forall experiments. Animals were permeabilised using the freeze-crack protocol aspreviously described (Crittenden and Kimble, 1999). Samples were immediatelyfixed in ice-cold 4% paraformaldehyde at 4 C for .12 hours. The primary antibodyused was mouse monoclonal anti-V5 (R960-25, Life Technologies) [1:200]. Thesecondary antibody used was goat anti-mouse Alexa Fluor-568 (Sigma) [1:500]. Allantibody dilutions were made in 30% (v/v) normal goat serum (Life Technologies).Samples were mounted onto microscope slides using VectaShield mounting medium(Vector Labs) containing 49,6-diamidino-2-phenylindole (DAPI) where applicable.Samples were imaged using a BX51 Microscope (Olympus). Micrographs werecaptured using AnalySIS software (Olympus).

Immunoblot

Samples for immunoblot were prepared by washing worms off plates in M9 buffer,followed by repeated washes in M9 and a final wash in distilled water. Samplesresuspended in Laemmli buffer underwent three freeze-thaw cycles in liquid nitrogenbefore boiling, and were immediately loaded onto 10% acrylamide gels. Primaryantibodies used are mouse monoclonal tau 13 antibody (Abcam) [1:5000] that bindshuman tau, mouse monoclonal anti-V5 antibody conjugated to HRP (R961-25, LifeTechnologies) [1:1500], and mouse monoclonal anti-acetyl alpha-tubulin (T7451,Sigma) [1:2000]. The secondary antibody used was goat anti-mouse conjugated tohorseradish peroxidase (NA931, GE Healthcare) [1:10,000]. Blots were developedwith Immobilon Western chemiluminescent substrate (Millipore) on film.

Pharmacological assays

Pharmacological assays were performed on unseeded plates spread with levamisole(Sigma) at a final concentration of 1 mM. Levamisole was allowed to equilibrate onthe plates for 1–2 hours at room temperature prior to scoring. Animals to be assayedwere synchronised by egg-laying and were cultivated at 20 C. Animals were pickedonto drug plates and scored at room temperature for paralysis (defined as noresponse to tapping with a platinum wire) at 15-minute intervals for 90 minutes.Scoring was conducted blind to the genotype of the worms.

Statistical analysis

All statistical analysis was performed using the GraphPad Prism 5/6 software(GraphPad Software Inc.) or Microsoft Excel (Microsoft).

AcknowledgementsThe authors gratefully acknowledge Dr M. Hilliard for providingreagents, Drs J. Semple and B. Lehner for dual antibiotic selectionvectors, and Dr L. Ittner for the htau40 construct. The authors thankmembers of the Nicholas lab and Gotz lab for helpful discussions. Somestrains were provided by the Caenorhabditis Genetics Center (CGC),which is funded by the National Institutes of Health (NIH) Office ofResearch Infrastructure Programs [grant number P40 OD010440].Deposited in PMC for release after 12 months.

Author contributionsY.L.C., J.G. and H.R.N. designed research, Y.L.C. and X.F.performed research, Y.L.C., X.F., J.G. and H.R.N. analysed data,and Y.L.C., J.G., and H.R.N. wrote the paper.

FundingH.R.N. is supported by a University of Sydney Re-entry Fellowship,J.G. by the Estate of Dr Clem Jones AO, and grants from the

Australian Research Council and the National Health and MedicalResearch Council of Australia.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.jcs124404/-/DC1

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