ORIGINAL PAPER

HLA-DR3-DQ2 Mice Do Not Develop Ataxia in the Presenceof High Titre Anti-gliadin Antibodies

Volga Tarlac & Louise Kelly & Nupur Nag &

Judy Allen-Graham & Robert P. Anderson &

Elsdon Storey

Published online: 20 October 2012# Springer Science+Business Media New York 2012

Abstract Recently, it has been suggested that anti-gliadinantibodies (αGAb) may produce “gluten ataxia”, even in theabsence of celiac disease enteropathy. αGAb are reportedlypresent in 12–50% of patients with sporadic ataxia, but also in12 % of the general population, such that the importance ofαGAb as a cause of sporadic ataxia is not conclusively settled.We aimed to determine whether mice transgenic for HLA-DR3-DQ2 and immunised with gliadin to achieve high titresof αGAb would develop ataxia and/or cerebellar damage.From 6 weeks of age, HLA-DR3-DQ2 transgenic mice wereimmunised fortnightly with gliadin (n010) or a saline control(n06) in adjuvant. Serum titres were measured by αGAbenzyme-linked immunosorbent assay. At 24 weeks of age,mice were tested for locomotor function using the acceleratingrotarod, ledged beam, ink-paw gait, and several neurologicalseverity score subtests. Brains were then collected and pro-cessed for immunohistochemistry. Sections were analysed forlymphocytic infiltration, changes in morphology and Purkinjecell (PC) dendritic volume and the number of PCs counted viaunbiased stereology. Gliadin-immunised mice developed highαGAb titres while controls did not. There was no statistically

significant difference between the gliadin and sham-immunised HLA-DR3-DQ2 mice on any of the tests of motorcoordination, in lymphocytic infiltration, PC number or indendritic volume. High levels of αGAb are not sufficient toproduce ataxia or cerebellar damage in HLA-DR3-DQ2 trans-genic mice.

Keywords Gluten ataxia . Celiac disease . Ataxia . Mouse .

Cerebellum

Introduction

Celiac disease (CD) is a common enteropathy caused by animmune-mediated reaction to ingested gluten in geneticallysusceptible individuals. Gluten consists of a combination ofgliadin and glutenin proteins found in the endosperm of wheatand other related cereal grains. Presence of CD4 T cellsspecific for gliadin peptides selectively deamidated by tissuetransglutaminase is central to the pathogenesis of celiac dis-ease. CD is strongly associated (>90 %) with HLA-DQ2,typically encoded by the DR3-DQ2 haplotype [1]. It is diag-nosed on the basis of positive anti-tissue transglutaminase andendomysial antibodies, small bowel histopathology and re-sponse to a gluten-free diet. CD classically manifests as mal-absorption, weight loss, fatigue and steatorrhea. However,many more patients present with non-specific symptomsand/or are asymptomatic—a concept known as the “celiaciceberg” [2]. Celiac disease may also result in systemic man-ifestations, such as dermatitis herpetiformis.

In 1966, it was postulated that CD may be associatedwith sensory ataxia, based on a case study of 16 biopsy-proven CD patients [3]. Since then, many other neurologicalsyndromes have been linked with CD, including epilepsywith occipital calcification, migraine, cerebellar ataxia, atax-ia–myoclonus syndrome, chorea, myelopathy, peripheral neu-ropathy, myopathy, depression, attention deficit-hyperactivity

V. Tarlac : L. Kelly :N. Nag : J. Allen-Graham : E. StoreyDepartment of Medicine and Van Cleef Roet Centre for NervousDisease, Monash University (Alfred Hospital Campus),Commercial Road,Melbourne, VIC 3004, Australia

R. P. AndersonDivision of Immunology,Walter & Eliza Hall Institute of Medical Research,Royal Parade,Parkville, VIC 3052, Australia

E. Storey (*)Department of Neuroscience, The Alfred Hospital,Commercial Road,Melbourne, VIC 3004, Australiae-mail: elsdon.storey@monash.edu

Cerebellum (2013) 12:370–376DOI 10.1007/s12311-012-0425-z

disorder, and white matter lesions onMRI [3–5]. Neurologicaldisorders in aggregate have been estimated to occur in 10% ofCD patients [5].

Clinically silent gluten sensitivity (associated with anti-gliadin antibodies (αGAb), but not with CD enteropathy)has been proposed to be causal in a high proportion ofcases of sporadic ataxia. In 1996, the Sheffield groupreported that 57 % of patients with various forms ofneurological dysfunction of unknown cause had αGAbpresent, compared with 12 % of healthy blood donors[6]. In follow-up papers, the Sheffield group described28 patients with otherwise unexplained ataxia, 82 % ofwhom carried HLA-DQ2, and in 15 of whom small bowelbiopsy was entirely normal, and 221 patients with sporad-ic ataxia, of whom 41 % had αGAb, compared with 12 %in controls [7]. Other groups have reported concordantresults: Pellecchia et al. [8] found that 3/24 patients withidiopathic cerebellar ataxia (ICA) had positive αGAb andbiopsy proven CD. In another large study of 104 ICApatients, it was found that 11.5 % had αGAb, seven ofwhom had small bowel histopathological changes [9].However, this rate of serological positivity is the sameas found in the controls of the Sheffield group [6] and inanother series from the UK [10]. A study of both sporadicand dominantly inherited ataxias found αGAb in 7/26(27 %) and 9/24 (37 %) of patients, respectively [11].While these workers interpreted the presence of αGAb asdemonstrating a role for gluten in dominantly inheritedataxias, an alternative interpretation of their data is thatcerebellar damage may result in the development of anti-cerebellar antibodies that can cross-react with gliadinepitopes. Several other groups have failed to find evi-dence of CD in ICA [12] or an association of ataxia,epilepsy, cognitive impairment, or neuropathy in patientswith dermatitis herpetiformis [13]. More recently, a largeFinnish study has failed to find an association betweenαGAb positivity and neurological symptoms, althoughthere was an association with rheumatoid arthritis anddepression [14].

In this present study, we utilised mice transgenic forhuman HLA-DR3-DQ2 on a complete mouse MHC classII knockout background and expressing human CD4 [15] todetermine whether they would develop ataxia and/or cere-bellar damage after immunisation with gliadin to producehigh-titre αGAb.

Subjects/Materials and Methods

Animals All experiments were conducted on HLA-DR3-DQ2transgenic mice, with all procedures approved by the institu-tional Animal Ethics Committee. HLA-DR3-DQ2 mice weregenerated as described previously [15]. Male and female

HLA-DR3-DQ2 founder mice were obtained (a gift from J.McCluskey, University of Melbourne, Australia) and used toestablish a colony. Mice were housed in a pathogen-freefacility in cages with up to six same-sex littermates on a12 h light/dark cycle with lights on at 06:00. The mice weremaintained on gluten-free food pellets (Specialty Feeds, Aus-tralia) and water was provided ad libitum.

Immunisation At 6–8 weeks of age, HAL-DR3-DQ2 trans-genic mice were immunised intraperitoneally with 20 μg ofgliadin (n011, five males and six females; Vital Diagnostics,Australia) or saline (n09; two died, leaving three males andfour females) in complete Freund’s adjuvant. A further threebooster injections of 20 μg gliadin or saline control in incom-plete Freund’s adjuvant were administered at fortnightly inter-vals (Table 1). One mouse from each group received anincorrect booster injection, leaving 10 gliadin and six saline-immunised mice, respectively.

ELISA An in-house enzyme-linked immunosorbent assay(ELISA) was used to measure αGAb titres in immunisedmice. Briefly, blood samples were collected via submandib-ular bleeds pre- and 1 week post-each booster immunisation,with a final bleed at 25 weeks of age following locomotortesting. Table 1 shows the timeline of the immunisationschedule. Blood was centrifuged at 1,000 rpm and serumcollected and frozen at −20 °C for subsequent analysis ofαGAb by ELISA. High-bind ELISA plates (Greiner, Ger-many) were coated with 500 ng of gliadin in 0.1 MNaHCO3, pH9.0 overnight at room temperature (RT).Plates were washed four times with phosphate bufferedsaline with 0.05 % Tween 20. The plates were then blockedwith 1 % bovine serum albumin (BSA; Sigma, Australia) inPBS. The mouse serum was serially diluted in 0.5 % BSA inPBS, added to plates in duplicates and incubated for 1 h atRT. The plates were washed as previously before the addi-tion of rabbit anti-mouse HRP Ab (DAKO, Australia) dilut-ed 1:1,000 in 0.5 % BSA and incubated for 1 h at RT.Following washing, the bound antibodies were detected bySigmaFAST OPD substrate (Sigma, Australia) and the

Table 1 Experimental timeline

Week of age Immunisation/testing

5 Prebleed

6–7 Gliadin/saline in complete Freund’s adjuvant

8–9 Gliadin/saline in incomplete Freund’s adjuvant1

10–11 Gliadin/saline in incomplete Freund’s adjuvant2

12–13 Bleed1

13–14 Gliadin/saline in incomplete Freund’s adjuvant3

15–16 Bleed2

24 Final bleed and locomotor testing

Cerebellum (2013) 12:370–376 371

reaction stopped by the addition of 2 M H2SO4. All plateswere read at 492 nm in a multi-well plate reader.

Locomotor Tests Motor function was assessed with the fol-lowing behavioural tests at 24 weeks of age: (1) NeurologicalSeverity Score (NSS) subtests [16]: Mice were tested once onlyand scored pass/fail in: (a) beam walking, (b) ability to walkstraight immediately when placed on a flat surface, (c) abilityto balance on a 0.5-cm width square section beam for 10 s, (4)ability to balance on a 0.5-cm diameter round-section beam for10 s, (5) ability to traverse 3, 2, and 1 cm wide×30 cm longflat beams. (2) Ink-paw test: This assesses gait, stride andmovement deficits [17]. The fore- and hind-paws of the mousewere marked with different-coloured non-toxic inks and themice then ran through a narrow open-bottomed tunnel on asheet of paper (50×25 cm), away from a light source. Testingconsisted of three test runs, and was conducted after threeconsecutive days of training at three practice runs per day. Pawprints were measured for (a) average stride length, (b) averageinter-hind-paw base width, (c) average inter-fore–paw basewidth, and (d) overlap of fore-paw/hind-paw placement. Theaverage of five values from the middle section of each of thethree test runs was calculated. (3) Ledged-beam test [18]: Thisassesses gait ataxia (accurate paw placement) on a progres-sively narrowing (3.5→0.5 cm) upward-sloping beam, with asafety ledge on either side 0.5 cm wide and 1 cm lower thanthe central beam surface. The mice underwent training, con-sisting of three practice trials per day for three consecutivedays, followed by three recorded test trials on the fourth day.The trials were filmed, and the mean latency to first foot fault(contact with the safety ledge) and the total number of footslips were recorded. (4) Accelerating rotarod test: Thisassesses motor coordination and learning [19]. The rod accel-erated over a 5-min period from 4 to 40 rpm. Each mouse wastrained over three consecutive days (four practice trials perday) followed by a fourth day of testing consisting of four testtrials with 5-min rest between each trial. The mean latency tofall was calculated across the four test trials.

Tissue Processing All mice were euthanized at 25 weeks ofage with sodium pentobarbital (100 mg/kg) and were thentranscardially perfused with 10 mL of PBS, followed by10 mL of 4 % paraformaldehyde (PFA) in PBS. The cere-bellum was divided into left and right hemispheres and post-fixed in fresh 4 % PFA in PBS for the right hemisphere andin Bouin’s fixative (9 % PFA, 0.9 % picric acid and 5 %acetic acid) in PBS for the left hemisphere.

Immunohistochemistry The right cerebellar hemisphere wasembedded in paraffin and cut into 7-μm thick sections. Thesections were deparaffinised in xylene and then rehydratedthrough graded ethanol to PBS. Antigens were un-masked byheating in a microwave with 0.1 M citrate buffer, pH6. The

sections were pretreated in 0.3 % hydrogen peroxide in PBSfor 30 min to remove endogenous peroxidase activity, fol-lowed by blocking in 5 % BSA in PBS for 1 h at RT, and thenstained for either calbindin (mouse anti-calbindin; Cell Sig-naling, Australia; dilution 1:400), CD19 (rat anti-mouseCD19; AbD Serotec, U.K.; dilution 1:150) or CD3 (mouseanti-mouse CD3; BD Biosciences, Australia; dilution 1:50)overnight at 4 °C. The sections were washed three times inPBS before applying the LSAB kit (Dako, Australia). Rinsedsections were then developed using diaminobenzidine,according to the manufacturer’s protocol (Dako, Australia).The sections were mounted in DPX (Merck, Germany). Con-trol sections underwent the same staining procedure, exceptfor the omission of the primary antibody.

Stereology The left halves of the dissected cerebelli wereplaced in Bouin’s fixative and processed through graded eth-anol into glycol methacrylate resin (Technovit 7100; HeraeusKulzer, Germany). Embedded cerebelli were then seriallysectioned at 20 μm, and every 15th pair was then mountedonto slides and stained with hematoxylin and eosin (HE) andthe sections mounted using DPX. Cell counts were performedon an upright Leica DMLB2 microscope (Leica, Germany)fitted with a Ludl motorised microscope stage (Ludl Electron-ic Products, USA). The sections were viewed (OptronicsCamera; Optronics, USA) and analysed using Stereo-Investigator software (version 8.0; MBF Bioscience, USA).Contour lines were traced around the Purkinje cell (PC) layerat ×5 magnification to isolate the area of interest and three-dimensional counting frames were then placed onto the area ina systematic random manner with a random starting point andthen 200 μm step lengths between subsequent frames. The x,y, z dimensions of the counting frames were 60×50×15 μm,respectively. All PC nuclei that were either partially or com-pletely inside the counting frame were counted, except thosethat intersected either of the two exclusion boundaries (lowerand left sides). Based on the following formula, an estimate oftotal PC number was calculated:

PC Chð Þ ¼ 2 Q�ð Þ 1 f1=ð Þ 1 f2=ð Þ 1 f3=ð Þð Þ

Raw counts of PCs (Q−) were multiplied by the recipro-cals of the sampling fractions to estimate total number ofPCs in the cerebellum (this then multiplied by two as onlyhalf the cerebellum was used for stereological analysis). Theco-efficient of error was estimated for each contour usingthe Gunderson method (M01) and satisfied the requirementthat there was little variance between counts.

Molecular Layer Volume and Purkinje Cell Dendritic DensityEstimation Images of anti-calbindin-immunostained sectionswere analysed using Image J software (NIH, USA) to assess

372 Cerebellum (2013) 12:370–376

the relative density of the PC dendrites within the molecularlayer—a measure of the extent of PC dendritic arborisation iftotal PC numbers are unchanged. Randomly placed rectangularselections were used tomeasure the density of immunoreactivityof both the granular and molecular layers. The density of im-munoreactivity of the PC dendrites in the molecular layer wascalculated for each section by subtracting the density of thegranular layer (to account for any variations in backgroundstaining intensity between sections). The relative density of themolecular layer was calculated by dividing the total intensity bythe area sampled. Molecular layer volume can also be used toassess the extent of PC dendritic arborisation indirectly. TheH&E-stained slides used for PC number stereology were alsoused to measure the volume of the molecular layer. Using theCellSense software, V1.4 (Olympus, Japan), the entire molecu-lar layer was traced on every 15th slide for at least eight sectionsper cerebellar hemisphere and the area calculated. Using theformula: V ¼ ISF�P

A� tð Þ, where V is volume, ISF is theinverse of the sampling fraction, ΣA is the sum of areas fromeach slide and t is the section thickness in millimetres, thevolume of the molecular layer was calculated in cubic milli-metres and then doubled to obtain the volume per cerebellum.

Statistical Analysis Analysis was performed using the Stu-dents t test for all data. All calculations and statistics wereperformed using Microsoft Excel and GraphPad PRISM (ver-sion 5.04; GraphPad Software, USA). With the exception ofthe ELISA results, all other data is reported as mean±SD.

Results

HLA-DR3-DQ 2 Transgenic Mice Develop a High TitreαGAb Response

Figure 1 shows that gliadin—but not saline—immunised HLA-DR3-DQ2mice developed highαGAb titres after gliadin immu-nisation with mean titres of one in 2.15×105 at bleed1, one in2.62×105 at bleed2 and one in 1.03×105 at the final bleed. Forcomparison, anti-gliadin titres in newly diagnosed celiac diseasepatients (treated for <2 months) were (median 1 in 3.2×104;range <1 in 8×103−1 in 2.5×105). The high αGAb titres weremaintained until the end of the experiment at week25.

Locomotor Ability is not Affected in Gliadin-ImmunisedHLA-DR3-DQ2 Mice

At 24 weeks of age, 10 weeks after the final gliadin injection,there was no difference in the average number of ledged beampaw-slips of the left, right, fore and hind paws of saline-treated(n06) compared with gliadin-treated (n010) HLA-DR3-DQ2mice (Fig. 2a). Total paw-slips on the ledged beam were alsounaffected by gliadin treatment (Fig. 2a). There was no

significant difference on either length of hind stride, hind-base, fore-base or the amount of overlap of the fore and hindpaw placement between the saline-treated (n06) and gliadin-treated (n010) mice on the ink-paw test (Fig. 2b). The NSSalso failed to show a significant difference between the controland gliadin-treated mice (data not shown). The mean latencyto fall on the accelerating rotarod for saline-treatedHLA-DR3-DQ2mice (n05) was 102±24.1 s compared with 105±15.9 sfor the gliadin-treated group (n09); these results were notsignificant (p00.92; Fig. 2c). One mouse from each treatmentgroup failed to perform the rotarod task and was excludedfrom the rotarod analysis. As male mice were heavier, whichmay impair rotarod performance, linear regression analysiswas performed to determine whether the greater weight ofmale mice would affect their latency to fall on the acceleratingrotarod (Fig. 2d). The weight of the mice did not significantlyimpact on their latency to fall on this test.

Purkinje Cell Number, Dendritic Density and Molecular LayerVolume is Unaffected in Gliadin-Immunised HLA-DR3-DQ2Mice

The mean number of PCs in gliadin-immunised mice (n010)was 2.15×105±3.66 m×104, compared with 1.81×105±4.47×104 in saline-treated mice (n06; Fig. 3a); this differencewas not statistically significant (p00.13). The total numbers ofPCs are in agreement with previously published data [20].Densitometry analysis of calbindin-immunostained cerebellarsections revealed no difference in the relative density of PCdendritic arborisation between the control and gliadin-treatedHLA-DR3-DQ2 mice (p00.08; Fig. 3b). Figure 3c–d showrepresentative calbindin-stained cerebellar sections fromsaline- and gliadin-treated mice, illustrating the lack of grossmorphological changes. The mean volume of the molecularlayer, a surrogate marker of PC cell dendritic density, was notsignificantly different between the two groups (p00.82). In the

Fig. 1 Anti-gliadin antibody titre in saline (n06) and gliadin (n08)injected mice before and after immunisation was measured by ELISA.Gliadin immunised mice maintained a high titre of anti-gliadin anti-bodies until the final bleed taken at the end of the experiment at25 weeks. Bleed1, p00.015; bleed2, p00.005; and final bleed,p00.02. Error bars represent SD

Cerebellum (2013) 12:370–376 373

Fig. 2 At 24 weeks of age, mice were tested for gait disturbances.Gliadin treated HLA-DR3-DQ2 (n010) compared to saline (n06)-treatedmice had a similar number of paw slips with the right fore and hind (R-Fore, R-Hind) and left fore and hind (L-Fore, L-Hind) paws as measuredon the ledged beam. The total of all limb paw slips was also similarbetween the saline (7.56±5.37)- and gliadin (8.44±5.39)-treated mice(p00.76; a). The ink paw test was unable to detect a difference in thelength of the hind stride (H-Stride; p00.34), hind base (H-Base; p00.92),

fore-base (F-Base; p00.5) and overlap of the fore and hind placement(p00.37) in saline (n06)- and gliadin (n010)-treated mice (b). Motorcoordination was measured by the latency to fall off an acceleratingrotarod (c); there was no difference in the latency to fall between thesaline (n05) and gliadin (n09) treated mice (p00.9). The effect of weighton latency to fall of the rotarod was calculated using linear regression (d).Weight did not affect the latency to fall in gliadin (R200.3973)- or saline(R200.4929)-treated mice. Error bars represent SD

Fig. 3 Purkinje cell number was counted in 25-week-old mice usingstereology of every 15th pair of HE stained 20 μm thick cerebellarsections. The total number of Purkinje cells was unaffected by gliadintreatment (p00.16) when compared to saline-treated mice (a). Densi-tometry of calbindin-stained cerebellar sections of saline (n05) andgliadin (n06) immunised HLA-DQ2-DR3 mice was quantified and nodifference in Purkinje cell arbour density was detected (p00.08; b).

Calbindin-stained cerebellar sections of saline (c) and gliadin (d)immunised mice showing healthy PCs and normal dendritic arbourisa-tion (scale bar 100 μm). The volume of the molecular layer wasdetermined from the HE-stained stereology sections of saline (n06)-and gliadin (n010)-treated HLA-DR3-DQ2 by measuring the size ofthe molecular layer; no difference was found between the two groups(p00.82). Error bars represent SD

374 Cerebellum (2013) 12:370–376

saline-treated mice (n06), the mean molecular layer volumewas 9.74±1.183 mm3 and in gliadin-treated mice (n010) 9.62±0.861 mm3 (Fig. 3e). The mean ratio of molecular layervolume to total cerebellar volume was also similar betweensaline (0.49±0.02) and gliadin-treated groups (0.48±0.02; p00.32). Immunohistochemistry for CD3 and CD19 cells, repre-senting T-cells and B-cells, respectively, was negative in bothsaline (n06)- and gliadin (n010)-treated mice when comparedwith positive control lymph node sections (data not shown).

Discussion

The prevalence, and indeed even the existence, of “glutenataxia” is the subject of continued debate [21]. Unfortunate-ly, no widely available and accepted animal model exists inwhich to explore this controversy. We hypothesised that, ifanti-gliadin antibodies are responsible for gluten ataxia,induction of very high antibody titres in mice expressingthe necessary permissive HLA-DQ2 human transgene, andtheir persistence across a number of months, might result ina murine model of “gluten ataxia”. In this regard, the factthat such mice do not develop characteristic celiac diseaseenteropathy [15] does not invalidate the model, as onlyminorities of patients with “gluten ataxia” are reported todevelop enteropathy [22]. However, the immunised micefailed to develop either motor deficits or pathological cere-bellar involvement. This does not disprove the existence of“gluten ataxia”, of course, but does at least indicate thatsustained high titres of anti-gliadin antibodies are not suffi-cient for its development.

Our study has several strengths. Firstly, we were able toachieve persistently high anti-gliadin titres across the immu-nisation period. Secondly, we employed a number of objec-tive tests of murine ataxia. This is important becausedifferent strains, and presumably also different causes ofataxia, may result in differential impairment even acrosstests as superficially similar as constant speed and acceler-ating rotarods [23, 24]. Thirdly, we conducted quantitativeneuropathology on the mice after death and were able toshow that there was no loss of Purkinje cells, and no pruningof molecular layer volume or calbindin immunoreactivity(which, taken together, indicate that there was no loss ofPurkinje cell dendritic arbours). Purkinje cell loss has pre-viously been shown to be a feature of gluten ataxia [7].Qualitatively, we demonstrated complete absence of cere-bellar lymphocytic infiltration—a prominent pathologicalfeature of gluten ataxia [7, 25].

This study also has a number of potential drawbacks.Firstly, the numbers of mice studied were small. As againstthis, the genetic homogeneity offered by the immunised andcontrol mice, their matched ages, and the standardized motorassessments all reduce variability and therefore numbers

required to show an effect. Secondly, the duration of theexperiment from first immunisation to death was relativelyshort at 18 weeks. Even with the high titres achieved, this mayhave been insufficient time for cerebellar disease to develop.Against this, Boscolo et al. [26] showed that passive intrace-rebral (ventricular) immunisation of mice with human anti-gliadin antibodies impaired rotarod performance at 3 and 6 h.In a follow-up study, they found that intraventricular injectionof human anti-transglutaminase 2/3/6 scFv IgA into miceproduced immediate transient ataxia [27], suggesting that ashort period of exposure to the pathogenic antibody/ies maybe sufficient. Thirdly, the relevant cerebellar autoantigen(s)epitope(s) might not be conserved between humans and mice.However, two postulated autoantigens—synapsin 1 [28] andtransglutaminase 6 [27]—seem to be recognised in mice byhuman antibodies, implying epitopic cross-reactivity. Fourth-ly, we did not measure anti-transglutaminase 6 levels, al-though this possible cross-reactive epitope for neurological(as distinct from enteropathic) manifestations of gluten sensi-tivity is involved in neurogenesis and neuronal differentiation[29]. This has recently received indirect support through theidentification of mutations in the transglutaminase 6 gene ascausative in one type of dominant cerebellar ataxia [30],although antibodies to transglutaminase 6 are only found inabout half of patients diagnosed with gluten ataxia, and arealso present in a substantial proportion of non-ataxic patientswith celiac disease [31]. Lastly, given that about 10 % of thepopulation has anti-gliadin antibodies, but only a small pro-portion of even those with manifest enteropathy have evensubtle evidence of cerebellar dysfunction [32], we cannotexclude that other genetic variability beyond that at theMHC loci, not present in our genetically homogeneous mice,may be important determinants of susceptibility.

Acknowledgments This work was supported by grant (436791)from the National Health and Research Council (NH & MRC). Wethank Dr J. McCluskey and Dr Kate Keech (Department of Immunol-ogy, Melbourne University, Australia) for kindly providing the HLA-DR3-DQ2 transgenic mice.

Conflict of Interest The authors declare no conflict of interest forthis submission.

References

1. Sollid LM, Thorsby E. HLA susceptibility genes in celiac disease:genetic mapping and role in pathogenesis. Gastroenterology.1993;105:910–22.

2. Catassi C, Rätsch IM, Fabiani E, Rossini M, Bordicchia F, CandelaF, et al. Coeliac disease in the year 2000: exploring the iceberg.Lancet. 1994;343:200–3.

3. Cooke WT, Smith WT. Neurological disorders associated withadult coeliac disease. Brain. 1966;89:683–722.

Cerebellum (2013) 12:370–376 375

4. Vaknin A, Eliakim R, Ackerman Z, Steiner I. Neurological abnor-malities associated with celiac disease. J Neurol. 2004;251:1393–7.

5. Finelli PF, McEntee WJ, Ambler M, Kestenbaum D. Adult celiacdisease presenting as cerebellar syndrome. Neurology. 1980;30:245–9.

6. Hadjivassiliou M, Gibson A, Davies-Jones GA, Lobo AJ, Stephen-son TJ, Milford-Ward A. Does cryptic gluten sensitivity play a partin neurological illness? Lancet. 1996;347:369–71.

7. Hadjivassiliou M, Grünewald RA, Chattopadhyay AK, Davies-Jones GA, Gibson A, Jarratt JA, et al. Clinical, radiological,neurophysiological, and neuropathological characteristics of glu-ten ataxia. Lancet. 1998;352:1582–5.

8. Pellecchia MT, Scala R, Filla A, De Michele G, Ciacci C, BaroneP. Idiopathic cerebellar ataxia associated with celiac disease: lackof distinctive neurological features. JNNP. 1999;66:32–5.

9. Bürk K, Bösch S, Müller CA, Melms A, Zühlke C, Stern M, et al.Sporadic cerebellar ataxia associated with gluten sensitivity. Brain.2001;124:1013–9.

10. Unsworth DJ, Brown DL. Serological screening suggests that adultcoeliac disease is underdiagnosed in the UK and increases theincidence by up to 12 %. Gut. 1994;35:61–4.

11. Bushara K, Hallett M. Prevalence of antigliadin antibodies inataxia patients. Neurology. 2004;62:1237–8.

12. Combarros O, Infante J, López-Hoyos M, Bartolomé MJ, BercianoJ, Corral J, et al. Celiac disease and idiopathic cerebellar ataxia.Neurology. 2000;54:2346.

13. Wills AJ, Turner B, Lock RJ, Johnston SL, Unsworth DJ, Fry L.Dermatitis herpetiformis and neurological dysfunction. J NeurolNeurosurg Psychiatr. 2002;72:259–61.

14. Ruuskanen A, Kaukinen K, Collin P, Huhtala H, Valve R, Mäki M,et al. Positive serum antigliadin antibodies without celiac diseasein the elderly population: does it matter? Scand J Gastroenterol.2010;45:1197–202.

15. de Kauwe AL, Chen Z, Anderson RP, Keech CL, Price JD, WijburgO, et al. Resistance to celiac disease in humanized HLA-DR3-DQ2-transgenic mice expressing specific anti-gliadin CD4+ T cells. JImmunol. 2009;182:7440–50.

16. Chen Y, Constantini S, Trembovler V, Weinstock M, Shohami E. Anexperimental model of closed head injury in mice: pathophysiology,histopathology, and cognitive deficits. J Neurotrauma. 1996;13:557–68.

17. Klapdor K, Dulfer BG, Hammann A, Van der Staay FJ. A low-cost method to analyse footprint patterns. J Neurosci Meth.1997;75:49–54.

18. Bye N, Habgood MD, Callaway JK, Malakooti N, Potter A,Kossmann T, et al. Transient neuroprotection by minocycline

following traumatic brain injury is associated with attenuatedmicroglial activation but no changes in cell apoptosis or neutrophilinfiltration. Exp Neurol. 2007;204:220–33.

19. Jones BJ, Roberts DJ. The quantitative measurement of motorinco–ordination in naive mice using an accelerating rotarod. JPharma Pharmacol. 1968;20:302–4.

20. Woodruff-Pak DS. Stereological estimation of Purkinje neuronnumber in C57BL/6 mice and its relation to associative learning.Neuroscience. 2006;141:233–43.

21. Grossman G. Neurological complications of coeliac disease: whatis the evidence? Pract Neurol. 2008;8:77–89.

22. HadjivassiliouM, Sanders D,WoodroofeN,WilliamsonC,GrünewaldR. Gluten ataxia. Cerebellum. 2008;7:494–8.

23. Kamens HM, Crabbe JC. The parallel rod floor test: a measure ofataxia in mice. Nat Protoc. 2007;2:277–81.

24. Rustay NR, Wahlsten D, Crabbe JC. Assessment of genetic suscep-tibility to ethanol intoxication in mice. PNAS. 2003;100:2917–22.

25. Mittelbronn M, Schittenhelm J, Bakos G, de Vos RA, WehrmannM, Meyermann R, et al. CD8(+)/perforin/granzyme B(+) effectorcells infiltrating cerebellum and inferior olives in gluten ataxia.Neuropathology. 2010;30:92–6.

26. Boscolo S, Sarich A, Lorenzon A, Passoni M, Rui V, Stebel M, etal. Gluten ataxia: passive transfer in a mouse model. Ann NYAcadSci. 2007;1107:319–28.

27. Boscolo S, Lorenzon A, Sblattero D, Florian F, Stebel M, MarzariR, et al. Anti transglutaminase antibodies cause ataxia in mice.PLoS One. 2010;5:e9698.

28. Alaedini A, Okamoto H, Briani C, Wollenberg K, Shill HA,Bushara KO, et al. Immune cross-reactivity in celiac disease:anti-gliadin antibodies bind to neuronal synapsin I. J Immunol.2007;178:6590–5.

29. Thomas H, Beck K, Adamczyk M, Aeschlimann P, Langley M,Oita RC et al (2011) Transglutaminase 6: a protein associated withcentral nervous system development and motor function. AminoAcids. Available from: http://www.springerlink.com/content/j032glrt53077845/

30. Wang JL, Yang X, Xia K, Hu ZM, Weng L, Jin X, et al. TGM6identified as a novel causative gene of spinocerebellar ataxiasusing exome sequencing. Brain. 2010;133:3510–8.

31. Hadjivassiliou M, Aeschlimann P, Strigun A, Sanders DS, WoodrofeN, Aeschlimann D. Autoantibodies in gluten ataxia recognise a novelneuronal transglutaminase. Ann Neurol. 2008;64:332–43.

32. Versino M, Franciotta D, Colnaghi S, Biagi F, Zardini E, Bianchi PI,et al. Cerebellar signs in celiac disease. Neurology. 2009;72:2046–8.

376 Cerebellum (2013) 12:370–376