Sera of Patients With Celiac Disease and Neurologic Disorders Evoke a Mitochondrial-Dependent Apoptosis In Vitro

GASTROENTEROLOGY 2007;133:195–206

Sera of Patients With Celiac Disease and Neurologic Disorders Evoke a Mitochondrial-Dependent Apoptosis In Vitro ELISABETTA CERVIO,* UMBERTO VOLTA,‡,§ MANUELA VERRI,* FEDERICA BOSCHI,* ORNELLA PASTORIS,* ALESSANDRO GRANITO,‡,§ GIOVANNI BARBARA,§,储 CLAUDIA PARISI,‡ CRISTINA FELICANI,储 MARCELLO TONINI,* and ROBERTO DE GIORGIO§,储

Background & Aims: The mechanisms underlying neurologic impairment in celiac disease remain unknown. We tested whether antineuronal antibody– positive sera of patients with celiac disease evoke neurodegeneration via apoptosis in vitro. Methods: SH-Sy5Y cells were exposed to crude sera, isolated immunoglobulin (Ig) G and IgG-depleted sera of patients with and without celiac disease with and without neurologic disorders, and antineuronal antibodies. Adsorption studies with gliadin and tissue transglutaminase (tTG) were performed in celiac disease sera. Apoptosis activated caspase-3, apaf-1, Bax, cytochrome c, cleaved caspase-8 and caspase-9 and mitochondrial respiratory chain complexes were evaluated with different methods. Results: SH-Sy5Y cells exposed to antineuronal antibody–positive sera and isolated IgG from the same sera exhibited a greater percentage of TUNEL-positive nuclei than that of antineuronal antibody–negative sera. Neuroblasts exposed to antineuronal antibody–negative celiac disease sera also showed greater TUNEL positivity and apaf-1 immunolabeled cells than controls. Antigliadin- and anti-tTG– depleted celiac disease sera had an apoptotic effect similar to controls. Anti– caspase-3 immunostained cells were greater than controls when exposed to positive sera. The mitochondrial respiratory chain complex was reduced by positive sera. Western blot demonstrated only caspase-9 cleavage in positive sera. Cytochrome c and Bax showed reciprocal translocation (from mitochondria to cytoplasm and vice versa) after treatment with positive sera. Conclusions: Antineuronal antibodies and, to a lower extent, combined antigliadin and anti-tTG antibodies in celiac disease sera contribute to neurologic impairment via apoptosis. Apaf-1 activation with Bax and cytochrome c translocation suggest a mitochondrial-dependent apoptosis.

C

eliac disease is an autoimmune chronic inflammatory intestinal disease resulting from sensitivity to ingested gluten-containing foods.1– 4 Although the disease primarily affects the gastrointestinal tract, celiac

disease is a classic example of a systemic disorder involving many organs, such as skin, thyroid, pancreas, liver, and heart, as well as joints, muscles, bones, the central nervous system (CNS), and the peripheral nervous system.5 Approximately 10% of patients with celiac disease display neurologic symptoms related to a wide array of disorders, such as epilepsy, myoclonus, cerebellar ataxia, multifocal leukoencephalopathy, dementia, chorea, migraine, multiple sclerosis, memory/attention impairment, and peripheral axonal and demyelinating neuropathies.3,6 – 8 The nature of this association is unclear, and whether a specific neurologic complication occurs in celiac disease remains unsettled. Malabsorption may lead to vitamin and trace element deficiencies. Therefore, patients who develop neurologic dysfunction should be carefully screened for these alterations. However, malabsorption alone does not explain the pathophysiology and clinical course of many of the associated neurologic disorders. Other mechanisms proposed include gluten toxicity,9 genetic factors,10 and autoimmunity.11 The concept that autoimmunity can act as a mechanism triggering neurologic dysfunction is strengthened by the identification of lymphocytic infiltration in the central and peripheral nervous systems12,13 as well as circulating antineuronal antibodies.14,15 These antibodies bind to CNS and enteric nervous system (ENS) neurons. Using appropriate tissue sections (eg, cerebellum, brain cortex, and intestine), sera of patients with antineuronal antibodies provide labeling of either CNS or ENS neurons at indirect immunofluorescence. Two different patterns have been so far recognized: one characterized by a predominant neuronal nuclear labeling (ie, “Hu-like pattern”) and the other with a cytoplasmic staining of CNS Abbreviations used in this paper: BSA-PBST, bovine serum albumin in phosphate-buffered saline with Triton X-100; DMEM, Dulbecco’s modified Eagle medium; ENS, enteric nervous system; FCS, fetal calf serum; NADH, nicotinamide adenine dinucleotide; tTG, tissue transglutaminase; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling. © 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.04.070

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*Department of Physiological & Pharmacological Sciences, University of Pavia, Pavia, Italy; and Departments of ‡Internal Medicine, Cardioangiology, and Hepatology and 储Internal Medicine & Gastroenterology, §Centro Unificato di Ricerca BioMedica Applicata, University of Bologna, Bologna, Italy

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neurons such as Purkinje cells (“Yo-like pattern”).11,14,15 A high prevalence of these antibodies has been shown to correlate with cerebellar ataxia, epilepsy, and peripheral neuropathy related to celiac disease.11 However, whether antineuronal antibodies elicit neurologic impairment still remains unclear. The present study was designed to gain insights into the pathogenetic mechanisms of patients with neurologic celiac disease by testing the hypothesis that sera containing antineuronal antibodies present in a subset of such patients may evoke neuronal damage. For this purpose, we elected to use a neuroblastoma cell line of human origin (ie, SH-Sy5Y) because these cells provide a reliable model for analyzing molecular pathways involved in different neurodegenerative disorders. In this report, we specifically focused on neuronal apoptosis and related pathways as indicators of neuronal damage/degeneration resulting from sera of patients with celiac disease with and without neurologic manifestations.

din, antiendomysial, and anti–tissue transglutaminase [anti-tTG]) tested positive in all patients.11,16 Diagnosis of celiac disease was confirmed by endoscopic duodenal biopsy. Histologic findings were graded according to Marsh’s revised criteria.17,18 A formal neurologic assessment was routinely performed in all patients with celiac disease on presentation, including analysis of the time of onset of any neurologic symptoms with respect to diagnosis of celiac disease. In addition, we included 4 non– celiac disease patients with neurologic disorders (2 patients with cerebellar ataxia and 2 with epilepsy; 1 male and 3 females; age range, 5–37 years) without (n ⫽ 2) and with antineuronal antibodies (n ⫽ 2, titer 1:200). Control sera, including 10 sex- and age-matched blood donors, tested negative to all of the aforementioned antibodies as expected. All patients gave their informed consent to participate in the present study, which was approved by the St Orsola-Malpighi University Hospital Ethics Committee (reference no. 1804/2006).

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Antineuronal Antibodies

Materials and Methods Patients This study included 9 selected patients (6 women and 3 men; age range, 21– 61 years) with celiac disease and concomitant CNS or peripheral nervous system dysfunction associated with the presence of antineuronal antibodies (Table 1). The study included also 6 patients with celiac disease (5 women and 1 man; age range, 30 – 46 years) without neurologic abnormalities and without antineuronal antibodies. In 2 patients, one from the group with celiac disease and antineuronal antibodies and the second from the group with celiac disease without neurologic abnormalities and without antineuronal antibodies, sera were collected and immunoglobulin (Ig) G purified as indicated in the following text. Patients with celiac disease were diagnosed at the Departments of Internal Medicine, Cardioangiology, Hepatology, and Internal Medicine and Gastroenterology of the University of Bologna. Celiac disease–related antibodies (antiglia-

The presence of antineuronal antibodies to the CNS and ENS was detected by indirect immunofluorescence on 5-␮m cryostat sections of monkey and rat cerebellum as well as rat ileum and colon (Medic, Turin, Italy). Sera were tested at the initial dilution of 1:10 (in phosphate-buffered saline [PBS]) and, when positive, were titrated up to the end point. Rabbit anti-human IgG and IgA (Dako, Copenhagen, Denmark) were used as secondary antibody at the appropriate working dilution (1:60 and 1:100 on rat and monkey tissue, respectively).11

Purification of IgG Antibodies Sera of 2 patients with celiac disease with neurologic disorders and with or without antineuronal antibodies, respectively, were used. IgG were purified from sera using a protein G agarose column according to the manufacturer’s instructions (KPL, Gaithersburg, MD). An automatic immunoturbidimetric assay (Modular, Roche Diagnostics, Basel, Switzerland) showed that the

Table 1. List of Patients With Celiac Disease With Concomitant Neurologic Disorders and Antineuronal Antibodies Patients with celiac disease

Age (y)

1 2 3 4 5 6 7 8 9

37 24 61 28 42 45 61 59 21

Sex

Antigliadin antibodies IgA (titer)

Anti-tTG IgA (AU)

Antineuronal antibodies IgG (titer)

Associated neurologic disorder

F F F M F F F M M

1:40 1:160 1:10 1:80 1:20 1:80 1:40 1:20 1:80

15 ⬎20 8 18 ⬎20 ⬎20 11 12 14

CNS (1:200) CNS (1:100) CNS (1:200) CNS (1:50) ENS (1:1600) ENS (1:200) CNS (1:200) ENS (1:100) ENS (1:100)

Multiple sclerosis Cerebellar ataxia Cerebellar ataxia Epilepsy Cognitive disorders Memory/attention impairment Cerebellar ataxia Epilepsy Moyamoya disease

NOTE. Antigliadin antibodies of the IgA class were detected by immunofluorescence with a cutoff value ⱖ1:10; anti-tTG of the IgA class were detected by enzyme-linked immunosorbent assay with a cutoff value of ⬎7 AU.

IgG aliquots obtained at the end of the procedure contained more than 95% of total IgGs and no IgA or IgM Igs, whereas the IgG-depleted samples contained only IgA and IgM Igs. Commercially available human pure IgG and IgA used in this study were purchased by Bethyl Laboratories Inc (Montgomery, TX).

Adsorption Studies Six sera obtained from previously examined patients with celiac disease without neurologic disorders and antineuronal antibodies, positive for anti-tTG and antigliadin antibodies, were incubated overnight at 4°C, under continuous shaking, with guinea pig liver transglutaminase (Sigma Chemical Co, St Louis, MO) or crude gliadin (Sigma Chemical Co) at a concentration of 10 mg of protein/mL of undiluted serum. Antibody-antigen complexes were then separated from sera by ultracentrifugation at 100,000g for 30 minutes.19 An enzyme-linked immunosorbent assay for the detection of anti-tTG and antigliadin antibodies was performed before and after adsorption of sera.

Cell Culture Procedures and Experimental Treatments The human neuroblastoma cell clone SH-Sy5Y was maintained in 100-cm2 dishes in a 1:1 mixture of Dulbecco’s modified Eagle medium (DMEM) and Ham’s F-12 containing 15% fetal calf serum (FCS), penicillin (100 IU/mL), streptomycin (100 ␮g/mL), and nonessential amino acids (100 ␮g/mL) in an incubator at 37°C and gassed continuously with a mixture of 5% CO2 and 95% O2. Cells were detached every 2 days with trypsinEDTA (0.05% trypsin and 0.53 mmol/L EDTA), followed by centrifugation, resuspension, and finally seeding in new plates. All reagents were obtained from Gibco BRL Laboratories (Gaithersburg, MD). Passages never exceeded 30. For experimental analyses, cells were grown in either cell culture dishes or on poly-L-lysine– coated (Sigma Immunochemicals, St Louis, MO) (50 ␮g/mL) glass coverslips. SH-Sy5Y neuroblasts were exposed to sera of patients with celiac disease and neurologic disorders with or without antineuronal antibodies, sera of non– celiac disease patients with or without neurologic disorders, and control sera/FCS. Each of the above sera (5% or 1:20) was diluted in DMEM and Ham’s F-12. Furthermore, neuroblasts were exposed to commercially available human pure IgG and IgA (both diluted in DMEM and Ham’s F-12 to 1:1000 at 0.001 mg/mL concentration) and to isolated IgG from patients with celiac disease with neurologic disorders with or without antineuronal antibodies (both diluted 1:20 at 0.063 and 0.064 mg/mL concentrations, respectively) and related sera without IgG (diluted to 5%). Neuroblastoma cells were exposed to these sera for 12 hours (ie, activated apaf-1, caspase-3, cleaved

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caspase-8 and caspase-9) and 24 hours (ie, to identify apoptosis by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling [TUNEL] technique). Mitochondrial fraction from neuroblasts, cultured for 6 or 12 hours with antineuronal antibody–positive sera (from patients with celiac disease with neurologic impairment) and antineuronal antibody–negative sera (from patients with celiac disease without neurologic abnormalities and blood donors), was processed for respiratory chain complexes (including nicotinamide adenine dinucleotide [NADH]-ubiquinone oxidoreductase, succinate dehydrogenase, and cytochrome oxidase) and citrate synthase specific activities. Finally, an exposition time of 16 hours was used to evaluate Bax translocation and release of cytochrome c from mitochondria.

Nuclear Staining With Hoechst 33258 Dye This method was applied to prove the occurrence of nuclear condensation as a result of the apoptosis induced by sera of patients with celiac disease positive for antineuronal antibodies. Briefly, cells were cultured for 24 hours in DMEM containing 5% of antineuronal antibody–positive sera of patients with neurologic celiac disease, antineuronal antibody–negative sera of patients with celiac disease without neurologic abnormalities, or control sera, fixed in 3.7% buffered formaldehyde (10 minutes) and in methanol (20 minutes) at room temperature, washed in PBS, and stained with Hoechst 33258 dye (Sigma Immunochemicals) (10 ␮g/mL) for 15 minutes at room temperature, rinsed with PBS, and finally mounted upside down on glass slides in a drop of Mowiol (Calbiochem, Darmstadt, Germany). SH-Sy5Y cells were examined with a fluorescent microscope. Further control experiments were performed in parallel using cells cultured for 24 hours in DMEM containing 15% FCS.

TUNEL Method SH-Sy5Y neuronal cells were grown for 24 hours on poly-L-lysine– coated coverslips and then incubated for 24 hours in DMEM and Ham’s F-12 containing 5% of celiac disease sera of patients with and without neurologic disorders and with and without antineuronal antibodies, sera of non– celiac disease patients with neurologic disorders with and without antineuronal antibodies, isolated IgG from patients with celiac disease with neurologic disorders with or without antineuronal antibodies (and related sera without IgG), commercially available human pure IgG and IgA (1:1000), and control sera. Additional control experiments were performed in parallel using cells cultured for 24 hours in DMEM containing 15% FCS. All experiments were performed in duplicate. Fixed neuroblastoma cells were processed with either an in situ apoptosis detection kit (Neurotacs II, Trevigen, Gaithersburg, MD) or the dead-end fluorometric TUNEL system (Promega, Madison, WI) according to the manu-

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facturer’s instructions.20,21 With the Neurotacs II method, the apoptotic nuclei appeared dark brown and could be visualized with phase-contrast light transmission microscopy (Leica, Westlar, Germany); with the TUNEL system, which allowed identification of fluorescein isothiocyanate–labeled DNA fragments, apoptotic cells were visualized with a microscope equipped with confocal laser scanning microscopy (Leica TCS-SP). As a positive control for apoptosis, cells were incubated for 12 hours with DMEM and dopamine (100 mmol/L), a wellestablished proapoptotic substance for this cell line.22

Immunocytochemistry

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SH-Sy5Y cells were grown for 24 hours on polyL-lysine– coated coverslips, cultured for 12 hours in DMEM containing 15% of FCS or 5% of celiac disease sera of patients with and without neurologic disorders and with and without antineuronal antibodies, sera of non– celiac disease patients with neurologic disorders with and without antineuronal antibodies, isolated IgG from patients with celiac disease with neurologic disorders with or without antineuronal antibodies (and related sera without IgG), commercially available human pure IgG and IgA (1:1000), and control sera. Cells were fixed as previously described and then incubated for 30 minutes in buffer containing 3% bovine serum albumin in PBS, pH 7.4, with 0.1% Triton X-100 (BSA-PBST). Primary rabbit antibodies against the active fragment of caspase-3 (1:1000; Trevigen) and anti–apaf-1 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) were used in experiments performed incubating the SH-Sy5Y cells with sera and IgG/IgA as indicated previously, whereas primary mouse anti– cytochrome c (1:7500; BD PharMingen, Franklin Lakes, NJ) and rabbit anti-human Bax (1:1000; BD PharMingen) were used in experiments with either sera of patients with celiac disease with neurologic disorders and with antineuronal antibodies or control sera (blood donors/FCS). Cells were incubated in a humid chamber at 4°C overnight, rinsed with PBS, and incubated for 60 minutes at room temperature with the respective secondary antibodies (Molecular Probes Inc, Eugene, OR) diluted 1:500 in 3% BSA-PBST. Coverslips were finally rinsed with PBS and mounted upside down on glass slides in a drop of Mowiol. For double-labeling studies with cytochrome c and Bax in mitochondria, SH-Sy5Y cells were grown for 24 hours on poly-L-lysine– coated coverslips and then cultured for 12 hours in DMEM containing 15% of FCS or 5% of either antineuronal antibody– containing sera or control sera. Then, cells were incubated with the mitochondrial marker MitoTracker Deep Red 633 probe (Molecular Probes Inc), 500 nmol/L in growth medium, at 37°C for 30 minutes. Cells were then fixed with growth medium containing 3.7% formaldehyde at 37°C for 15 minutes. After fixation, cells were permeabilized with PBS containing 0.2% Triton X-100 (Sigma Chemical Co) at room

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temperature for 5 minutes. Mouse anti– cytochrome c monoclonal antibody (1:7500; BD Biosciences, Franklin Lakes, NJ) or rabbit anti-human Bax (1:1000; BD PharMingen) was applied for 1 hour at room temperature. Cells were then washed with PBS and incubated for 1 hour at room temperature with an Alexa 488 – conjugated goat anti-mouse or goat anti-rabbit IgG antibody (Molecular Probes Inc) diluted 1:500 in 3% BSA-PBST. Samples were finally counterstained with Hoechst 33258. Controls for double immunolabeling technique were performed to determine that the primary antibodies do not cross-react when mixed together and that the secondary antibodies recognize the appropriate antigen-antibody complexes.

Specific Enzymatic Activities To determine enzyme activities, neuroblasts cultured for 6 or 12 hours in DMEM containing 15% of FCS or 5% of antineuronal antibody– containing sera were weighed and subsequently homogenized in 0.25 mol/L sucrose in a precooled Potter-Braun S homogenizer. The homogenate was diluted with 0.25 mol/L sucrose (ie, 1 g of cells in 10 mL of sucrose solution). This homogenate was then centrifuged at 800g for 15 minutes in a refrigerated centrifuge (Beckman J2-21, rotor JA-20, Beckman Coulter, Fullerton, CA), and the supernatant was stored in ice. The sediment was rehomogenized in 0.25 mol/L sucrose and centrifuged at 800g for 15 minutes. The 2 supernatants obtained were centrifuged at 14,000g for 20 minutes. The mitochondrial sediment was gently resuspended in sucrose solution at a final dilution of 100 mg/mL. An aliquot of this solution (60 ␮L) was used to assess the protein content, whereas the remaining portion was used to evaluate enzyme activities. The maximum rates of the following enzyme activities were evaluated in the mitochondrial fraction: citrate synthase for the tricarboxylic acid cycle and NADH-ubiquinone oxidoreductase, succinate dehydrogenase, and cytochrome oxidase for the electron transfer chain. Enzyme activities were recorded graphically for at least 3 minutes with a double recorder spectrophotometer (Shimadzu 1601; Shimadzu Biotech, Milan, Italy), and each value was calculated from 2 blind determinations on the same sample. Enzyme specific activities were expressed as nanomolar of substrate transformed per minute per milligram of protein.23

Western Blot Analysis After treatment with sera, neuroblastoma cells were first rinsed twice with ice-cold PBS and then lysed for 10 minutes on ice in 200 ␮L of ice-cold lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 140 mmol/L NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mmol/L sodium orthovanadate, 2 g/mL aprotinin, 2 g/mL pepstatin, 2 g/mL leupeptin, and 1 ␮mol/L microcystin-LR and Triton X-100 1%). The lysates were sonicated for 10

seconds and then centrifuged at 9660g for 5 minutes at 4°C, and aliquots were taken for protein quantitation using the Pierce BCA protein assay kit (Rockford, IL). For the evaluation of the presence of cytochrome c and Bax in the cytoplasm and mitochondria, treated cells were first rinsed twice with ice-cold PBS and then lysed for 10 minutes on ice in 200 ␮L of ice-cold lysis buffer without Triton X-100 (50 mmol/L Tris-HCl, pH 7.5, 140 mmol/L NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mmol/L sodium orthovanadate, 2 g/mL aprotinin, 2 g/mL pepstatin, 2 g/mL leupeptin, and 1 ␮mol/L microcystin-LR). The lysates were sonicated for 10 seconds and then ultracentrifuged at 15,000g for 15 minutes at 4°C. Supernatant containing cytosol proteins was used to determine cytochrome c release from mitochondria. Sediment, containing membrane proteins, was rehomogenized in lysis buffer with Triton X-100 1% and ultracentrifuged at 15,000g for 15 minutes at 4°C, and aliquots of all samples were taken for protein determination using the Pierce BCA protein assay kit. Samples containing equal protein amounts (55 ␮g) were mixed with Laemmli’s loading buffer, boiled for 5 minutes, and electrophoresed in a 9% or 15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis minigel at 100 V. Resolved proteins were then electrophoretically transferred onto a nitrocellulose membrane (BioRad Laboratories, Hercules, CA) for 1 hour at 4°C under a constant current of 100 V. Membranes were saturated with 5% low-fat dry milk in Tris-buffered saline (TBST) (25 mmol/L Tris-HCl, pH 7.5, 140 mmol/L NaCl, and 0.05% Tween 20) for 1 hour at room temperature and then incubated overnight at 4°C with the following antibodies: 2 rabbit polyclonal antibodies raised against the peptides of human origin corresponding to amino acids 217–350 mapping within the caspase-8 p20 subunit and amino acids 315–397 mapping within the carboxy terminus of caspase-9 (both diluted 1:1000; Santa Cruz Biotechnology), a mouse anti– cytochrome c monoclonal antibody, and finally a rabbit anti-human Bax antibody (1:800 and 1:1000, respectively; BD PharMingen). After several washes with TBST, membranes were incubated for 60 minutes at room temperature with horseradish peroxidase– conjugated secondary antibodies (1:1500; Santa Cruz Biotechnology). Following incubation with secondary antibodies, membranes were rinsed and the specific signal was detected by enhanced chemiluminescence with the Immuno-Star HRP Substrate Kit (Bio-Rad Laboratories). As a positive control for the activation of the 2 caspases, SH-Sy5Y cells were cultured for 48 hours in DMEM containing tumor necrosis factor–related apoptosis-inducing ligand, also known as Apo2 ligand (0.1 ␮g/mL; PeproTech House, London, England). This ligand is known to induce apoptosis through a membrane receptor in tumor cell lines but not in normal cells.24,25 To verify that the cytochrome c in the cytosolic fraction was not due to mitochondrial contamination, an

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antibody to cytochrome oxidase IV (0.5 ␮g/mL; Molecular Probes Inc) was used. While present in the mitochondrial fraction, cytochrome oxidase IV was absent from the cytosolic fraction.

Quantification of Apoptosis The number of TUNEL-positive and activated caspase-3 and apaf-1 immunolabeled neurons was counted in microscopic fields and expressed as a percentage of detected neurons/field. At least 3 randomly selected fields per dish were blindly counted by 2 expert investigators. In each experiment with various sera (including isolated IgG, IgG-depleted, and antigliadin- and anti-tTG– depleted sera) and commercially available human pure IgG/IgA (at different dilutions/incubation times), one dish per experimental condition was counted and experiments were repeated 3 times.

Confocal Microscopy Coverslips were observed with a confocal laser scanning microscopy (Leica TCS-SP system mounted on a Leica DMIRBE inverted microscope). An Ar/Vis laser at 300/385 nm, 500/530 nm, and 650/720 nm was used to excite Hoechst 33258, Alexa 488, and Alexa 633 fluorescence, respectively. Optical sections (0.5 ␮m) were recorded using a 63⫻ oil-immersion objective. Images were processed and labeled using Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA) and transferred to dedicated software for construction of figure sets.

Statistical Analysis Data are expressed as mean ⫾ SEM. The statistical significance of the mean difference of the percentage of cells positive for TUNEL, caspase-3, and apaf-1 immunoreactivities exposed to antineuronal antibody–positive or –negative celiac disease sera, celiac disease sera without neurologic disorders, and antineuronal antibodies adsorbed with either gliadin and tTG proteins and control sera/FCS was assessed by one-way analysis of variance with Fisher’s protected least significant difference test for multiple comparisons. The same test was applied for specific enzymatic activities. Differences were considered statistically significant at P ⬍ .05.

Results The results obtained pointed to the evidence that sera containing antineuronal antibodies from patients with celiac disease and neurologic disease have the ability to promote apoptosis by activating the mitochondrial pathway in a human neuroblastoma cell line. Of particular interest, even sera of patients with celiac disease without neurologic disorders and antineuronal antibodies caused a substantial degree of apoptosis. The following are the data that emerged in different types of experiments performed in this study.

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Antineuronal Antibodies The immunofluorescent pattern observed with sera containing antineuronal antibodies was characterized by a bright staining in the cytoplasm or nuclei of Purkinje cells and granular layer of the cerebellar cortex (ie, antineuronal antibodies to the CNS, identified in 5 of the investigated patients). A positive immunofluorescent staining was also observed in virtually all enteric neurons of the myenteric and submucosal plexuses of the rat ileum and colon (antineuronal antibodies to the ENS, identified in 4 patients).11 The identified antineuronal antibodies were confined to the IgG subclass. Relative titers are listed in Table 1.

Induction of TUNEL and Hoechst 33258 Positivity

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Incubation of SH-Sy5Y cells with antineuronal antibody–positive sera induced TUNEL positivity characterized by an intense nuclear fluorescence. Furthermore, all the TUNEL-positive nuclei (Figure 1A) showed chromatin condensation, as indicated by the DNA staining Hoechst 33258. Quantitative analysis showed that after 24 hours of exposure, the number of TUNEL-positive nuclei induced by antineuronal antibody–positive sera of patients with neurologic celiac disease (42.7% ⫾ 3.8%) was significantly greater than that evoked by antineuronal antibody–negative sera (21.8% ⫾ 3.1%; P ⬍ .001), control sera (6.7% ⫾ 0.7%; P ⬍ .001), and FCS (3.4% ⫾ 0.8%; P ⬍ .001) (Figure 1B). A time course analysis showed that the number of apoptotic neuroblastoma cells evoked by antineuronal antibody–positive and –negative sera at 12 and 24 hours was similar and significantly greater as compared with that induced by control sera or FCS (P ⬍ .05). Conversely, after 6 hours of exposure, no significant difference was found between the number of TUNEL-positive nuclei induced by antineuronal anti-

Figure 1. Neuronal apoptosis induced by antineuronal antibody–positive sera of patients with celiac disease and neurologic impairment (celiac disease with neurologic disorder and antineuronal antibodies, CD/ND/NA) in SH-Sy5Y cells. (A) Representative microphotographs showing TUNEL (green fluorochrome) (arrows). Calibration bar ⫽ 15 ␮m. (B) Quantitative analysis of neuroblastoma cells exposed to antineuronal antibody– positive sera showing a significantly greater percentage of TUNELpositive nuclei compared with antineuronal antibody–negative celiac disease sera (celiac disease without neurologic disorder and without antineuronal antibodies, CD/WND/WNA), control sera (blood donors), and FCS (n ⫽ 4 experiments). Data are expressed as mean ⫾ SEM (n ⫽ 6 –10 subjects); *P ⬍ .05, **P ⬍ .001.

Figure 2. Time course analysis showing a significantly greater number of apoptotic SH-Sy5Y cells evoked by antineuronal antibody–positive celiac disease sera (celiac disease with neurologic disorder and antineuronal antibodies, CD/ND/NA) at 12 and 24 hours, but not at 6 hours, as compared with antineuronal antibody–negative celiac disease sera (celiac disease without neurologic disorder and without antineuronal antibodies, CD/WND/WNA), control sera, and FCS (n ⫽ 4 experiments). Data are expressed as mean ⫾ SEM (n ⫽ 6 –10 subjects); *P ⬍ .05, **P ⬍ .001.

body–positive sera of patients with neurologic celiac disease and antineuronal antibody–negative sera of patients with celiac disease without neurologic abnormalities, control sera, and FCS (Figure 2).

Activation of Caspase-3, Apaf-1, and Caspase-9 SH-Sy5Y cells exposed to antineuronal antibody– positive sera showed a markedly activated caspase-3 and apaf-1 immunoreactivity detectable in the cytoplasm of cells (Figure 3). Very few cells exposed to sera without antineuronal antibodies, control sera, or FCS displayed activated caspase-3 and apaf-1 immunoreactivity (Figure 3). These findings were confirmed by quantitative analysis showing that antineuronal antibody–positive sera were able to evoke a significantly greater number of activated caspase-3 cells (32.6% ⫾ 2.4%; P ⬍ .001) compared with antineuronal antibody–negative sera (13.9% ⫾ 1.3%), control sera (11.6% ⫾ 1.6%), and FCS (9.4% ⫾ 1.3%). Both antineuronal antibody –positive and –negative sera were able to evoke a significantly greater number of activated apaf-1 cells (49.0% ⫾ 2.4% and 27.4% ⫾ 1%, respectively; P ⬍ .001) compared with control sera (9.8% ⫾ 1.1%) and FCS (8.1% ⫾ 1.4%). However, the number of activated apaf-1 cells induced by antineuronal antibody–positive sera was significantly higher (P ⬍ .001) than that induced by antineuronal antibody–negative sera (Figure 3). The involvement of the caspase-9 pathway was demonstrated by Western blot analysis, which detected the active (cleaved) p10 subunit of caspase-9 in SH-Sy5Y cells treated with antineuronal antibody–positive sera. In contrast, active caspase-9 was virtually undetectable in neuroblastoma cells treated with antineuronal antibody–negative sera and was absent in cells treated with control sera or FCS (Figure 4A). Caspase-8 p20 subunit, the active form of caspase-8, was not detectable in all samples (Figure 4B).

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Experiments Aimed at Strengthening the Contribution of Antineuronal Antibodies to Apoptosis Sera of non– celiac disease patients with neurologic disorders and with antineuronal antibodies (n ⫽ 2) evoked a considerable degree of TUNEL positivity (27.5% ⫾ 0.8%), activated caspase-3 (27.9% ⫾ 3.6%), and apaf-1 (32% ⫾ 1.9%), values that were greater than those of sera of non– celiac disease patients (n ⫽ 2) with neurologic disorders but without antineuronal antibodies (10.9% ⫾ 0.6%, 7.2% ⫾ 0.7%, and 10% ⫾ 1.2%, respectively). Sera of patients with celiac disease without neurologic disorders but with antineuronal antibodies (n ⫽ 2) induced TUNEL positivity (42% ⫾ 1%), activated caspase-3 (36.1% ⫾ 1.5%), and apaf-1 (39% ⫾ 2.2%) in a comparable fashion to those of celiac disease sera with neurologic disorders and antineuronal antibodies (42.7% ⫾ 3.8%, 32.6% ⫾ 2.4%, and 49.0% ⫾ 2.4%, respectively). The values obtained with sera of patients with celiac disease with neurologic disorders and without an-

tineuronal antibodies (n ⫽ 2) showed the following results for the 3 investigated apoptotic markers: 10.5% ⫾ 0.7%, 10.8% ⫾ 1.1%, and 14% ⫾ 1% for TUNEL, caspase-3, and apaf-1, respectively. These results were comparable to control sera. IgG isolated from the serum of 1 patient with celiac disease with a neurologic disorder and antineuronal antibodies evoked a marked TUNEL positivity (39% ⫾ 1.3%), activated caspase-3 (33.2% ⫾ 1.2%), and apaf-1 (37.8% ⫾ 1.2%), values that were similar or slightly lower than those of original sera (42.7% ⫾ 3.8%, 32.6% ⫾ 2.4%, and 49.0% ⫾ 2.4%, respectively). Conversely, IgG isolated from the serum of 1 patient with celiac disease with neurologic disorder but without antineuronal antibodies behaved as control sera with regard to the 3 aforementioned apoptotic parameters (11.8% ⫾ 0.9%, 9.7% ⫾ 1.1%, and 10.3% ⫾ 1.5%, respectively). Similar results were obtained with IgG-depleted sera of 1 patient with celiac disease with antineuronal antibodies (13.3% ⫾ 0.9%, 10.7% ⫾ 0.7%, and 12.4% ⫾ 1.1%) and 1 patient without

Figure 4. Western blot of total proteins of SH-Sy5Y cells showing (A) caspase-9 but not (B) caspase-8 activation after 12 hours of exposure to FCS (n ⫽ 4 experiments), tumor necrosis factor–related apoptosis-inducing ligand (Trail), control sera, antineuronal antibody–positive (CD/ND/ NA), and antineuronal antibody– negative (CD/WND/WNA) celiac disease sera.

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Figure 3. (Top panel) A representative example of caspase-3 intense cytoplasmic staining in cells exposed to antineuronal antibody–positive celiac disease sera (CD/ND/NA) compared with controls. Quantitative data show a significantly greater number of caspase-3–positive neuroblastoma cells exposed to antineuronal antibody–positive celiac disease sera compared with antineuronal antibody–negative celiac disease sera (CD/WND/WNA), control sera, and FCS (n ⫽ 4 experiments). (Bottom panel) Similar results with apaf-1 immunolabeling and related quantitative results. Data are expressed as mean ⫾ SEM (n ⫽ 6 –10 subjects); **P ⬍ .001. Calibration bars ⫽ 10 ␮m.

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antineuronal antibodies (13% ⫾ 0.9%, 9% ⫾ 1.1%, and 12% ⫾ 1.9%, respectively), which were comparable to controls. Finally, commercially available human pure IgG and IgA evoked TUNEL positivity, activated caspase-3, and apaf-1 (IgG: 4.2% ⫾ 0.9%, 5.5% ⫾ 1%, and 6.2% ⫾ 0.9%; IgA: 5.2% ⫾ 1%, 5% ⫾ 0.7%, and 4.4% ⫾ 0.9%) comparable to FCS.

Adsorption Studies

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Enzyme-linked immunosorbent assay confirmed the lack of both antigliadin and anti-tTG antibodies in sera of patients with celiac disease without neurologic disorders and antineuronal antibodies adsorbed with the respective antigen. Compared with crude celiac disease sera, the incubation of adsorbed sera with SH-Sy5Y cells evoked a significant reduction of the 3 apoptotic markers, that is, TUNEL, activated caspase-3, and apaf-1 (4.8% ⫾ 1.1%, 5.8% ⫾ 0.4%, and 5.5% ⫾ 0.5% for antigliadin-depleted sera, P ⬍ .05; 4.8% ⫾ 1%, 6.1% ⫾ 0.4%, and 5.9% ⫾ 0.5% for anti-tTG– depleted sera, P ⬍ .05). These values were comparable to those obtained with FCS.

Increased Citrate Synthase and Decreased NADH-Ubiquinone Oxidoreductase Activities Because data of caspases and apaf-1 activation suggested an involvement of mitochondrial-dependent apoptosis mechanisms, we verified whether this abnormality involved the mitochondrial respiratory chain enzyme activity of SH-Sy5Y cells. Neuroblastoma cells exposed to antineuronal antibody–positive sera showed that the specific activity of citrate synthase at 6 and 12 hours (95 ⫾ 13 and 97 ⫾ 8 nmol · min⫺1 · mg protein⫺1, respectively) was significantly higher than that found in cells treated with FCS (67 ⫾ 4 nmol · min⫺1 · mg protein⫺1; P ⬍ .05). The NADH-ubiquinone oxidoreductase specific activity was significantly lower in SH-Sy5Y cells treated for 12 hours with sera containing antineuronal antibodies (9 ⫾ 1 nmol · min⫺1 · mg protein⫺1) than that found in cells treated with FCS (19 ⫾ 3 nmol · min⫺1 · mg protein⫺1; P ⬍ .05). There were no significant changes in other specific enzymatic activities measured (Figure 5).

Figure 5. The specific activity of citrate synthase (CS) after 6 or 12 hours of exposure to antineuronal antibody–positive celiac disease sera (CD/ND/NA) was significantly higher than controls. In contrast, the specific activity of NADH-ubiquinone oxidoreductase after 12 hours of exposure to antineuronal antibody–positive celiac disease sera (CD/ND/ NA) was significantly lower than controls. No significant differences in specific enzymatic activities of succinate dehydrogenase (SDH) and cytochrome oxidase (COX) were observed after 6 and 12 hours of exposure to either FCS or antineuronal antibody–positive celiac disease sera (CD/ND/NA).

Bax and mitochondria revealed that SH-Sy5Y cells treated with FCS or control sera displayed a diffuse fluorescence, indicating the presence of Bax throughout the cytosol under normal conditions (Figure 7A). Cells treated with antineuronal antibody–positive sera exhibited dot- or rod-like patterns, as visualized with the MitoTracker Deep Red 633 probe, indicating Bax translocation from cytosol into mitochondria (Figure 7B). To further strengthen these findings, we performed a fractionation of treated SH-Sy5Y cells into cytosolic and membrane fractions. By Western blot analysis, we could detect cytochrome c in the cytosolic fraction (Figure 6C) and Bax in the membrane fraction of mitochondria (Figure 7C) after 16 hours of exposure to antineuronal antibody–positive sera. These data confirm the immunocytochemical results on cytochrome c release and show that Bax is likely to be involved in this process. Neither translocation of Bax nor release of cytochrome c could be seen in the fractions from neuroblastoma cells treated with antineuronal antibody–negative sera, control sera, or FCS (Figures 6C–7C).

Cytochrome c and Bax Double Labeling Double-labeling immunocytochemistry experiments for cytochrome c and mitochondria revealed that SH-Sy5Y cells treated with FCS or control sera exhibited clear dot- or rod-like patterns distributed evenly throughout the cytoplasm, indicating that cytochrome c was localized in mitochondria under normal conditions (Figure 6A). Cells treated with antineuronal antibody–positive sera revealed a diffuse fluorescence, indicating the presence of cytochrome c throughout the cytosol due to its release from mitochondria (Figure 6B). Furthermore, double-labeling immunocytochemistry experiments for

Discussion Growing evidence indicates that neurologic diseases can be associated with celiac disease.3,6 – 8 In our experience, about 50% of patients with neurologic celiac disease with different disorders (such as cerebellar ataxia, epilepsy, peripheral neuropathy, and cognitive disorders) showed the presence of circulating antineuronal antibodies labeling CNS and ENS neurons (as indicated in Table 1). Although the possible molecular target(s) of these autoantibodies is still undefined, several groups, including ours, have postulated that its presence can

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Figure 6. Cytochrome c release from mitochondria induced by exposure of SH-Sy5Y cells to antineuronal antibody–positive celiac disease sera (CD/ND/NA). (A) Cells treated with control sera showing cytochrome c immunoreactive dot-like structures. (B) Cells treated with antineuronal antibody–positive celiac disease sera (CD/ND/NA) for 16 hours showed a diffuse cytoplasmic cytochrome c localization as exemplified in the merge. Calibration bar for A and B ⫽ 16 ␮m. (C) Western blot of cytosolic protein (CP) and membrane protein (MP) fractions of SH-Sy5Y cells showed a clear cytochrome c release into the cytosol after 16 hours of exposure to antineuronal antibody–positive celiac disease sera (CD/ND/NA), as compared with FCS, control sera, and antineuronal antibody–negative celiac disease sera (CD/WND/WNA). The absence of cytochrome oxidase IV (COX IV) from cytosolic fraction excludes a mitochondrial contamination.

contribute to the pathogenesis of neural impairment.5,7,11,12,26 Thus, autoimmunity directed against central or peripheral (including enteric) neurons may be exploited in vitro to test whether sera of patients with celiac disease with neurologic diseases and antineuronal antibodies may lead to neurodegenerative changes. In this study, we used a human neuroblastoma cell line (ie,

SH-Sy5Y) because these cells have been previously established as a valuable model to investigate mechanisms involved in several neurotoxic/neurodegenerative diseases. We focused on apoptosis as a hallmark of neuronal damage occurring in different neurologic disorders, including those related to celiac disease. In particular, our results showed that antineuronal antibody–positive sera

Figure 7. Translocation of Bax from cytosol to mitochondria after 16 hours of exposure of neuroblastoma cells to control sera (sequence of pictures in A) or antineuronal antibody–positive celiac disease sera (CD/ND/NA) (sequence of pictures in B). Arrows in pictures in B indicate Bax translocation from cytosol to mitochondria as exemplified in the merge. Calibration bar for A ⫽ 16 ␮m; calibration bar for B ⫽ 20 ␮m. (C) Western blot of cytosolic protein (CP) and membrane protein (MP) fractions of SH-Sy5Y cells showed Bax translocation from cytosol to mitochondria after 16 hours of exposure to antineuronal antibody–positive celiac disease sera (CD/ND/NA) compared with FCS, control sera, and antineuronal antibody–negative celiac disease sera (CD/WND/WNA). The absence of cytochrome oxidase IV (COX IV) from cytosolic fraction excludes a mitochondrial contamination.

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evoked nuclear changes (ie, chromatin condensation as identified by Hoechst 33258) accompanied by TUNEL positivity (ie, DNA fragmentation) and activation of caspase-3 and apaf-1. This notion has been strengthened by several lines of evidence indicating that sera of non– celiac disease patients with neurologic disorders and antineuronal antibodies, sera of patients with celiac disease without neurologic disorders and with antineuronal antibodies, as well as IgG isolated from a patient with celiac disease with a neurologic disorder and antineuronal antibodies induced a remarkable degree of apoptosis with changes in TUNEL, caspase-3, and apaf-1 parameters. On the same line, IgG isolated from a patient with celiac disease with a neurologic disorder but lacking antineuronal antibodies, as well as sera depleted of IgG from patients with celiac disease with and without antineuronal antibodies, evoked a degree of apoptosis similar to controls. Taken together, these results show that antineuronal antibodies alter neuronal survival in vitro, thus contributing to neurologic diseases irrespective of whether they are from celiac disease or not. These effects were also observed treating SH-Sy5Y cells with antineuronal antibody–negative sera of patients with celiac disease without neurologic disorders, although less pronounced than with antineuronal antibody–positive sera. Compared with crude sera of patients with celiac disease without neurologic disorders and without antineuronal antibodies, antigliadin- and anti-tTG– depleted sera showed less apoptotic potential. The lack of caspase-8 activation, along with the positive involvement of caspase-9 and apaf-1, suggested that the apoptotic pathway was dependent on mitochondria impairment. Also, the alteration of respiratory chain enzymes, cytochrome c and Bax translocation, is additional proof of involvement of a mitochondrial-dependent process. The results showing that neuronal apoptosis is also caused by antineuronal antibody–negative celiac disease sera suggest that even these sera may bear a neurotoxic potential. This concept has been strengthened by data obtained with the same antineuronal antibody–negative celiac disease sera adsorbed with either gliadin and tTG. Indeed, adsorbed celiac disease sera evoked a lower degree of apoptosis as compared with crude sera. Thus, apoptosis mediated by celiac disease sera without antineuronal antibodies may be ascribed to several factors such as the wide array of other autoantibodies contained in the sera, namely the combination of both antigliadin and antitTG antibodies. Although these agents caused neural damage per se, they may contribute to overt clinical manifestations by acting in concert with antineuronal antibodies present in the sera of patients with celiac disease with neurologic disorders. The results that emerged in this study also provide evidence that proapoptotic events in the neuroblastoma cell line were predominantly evoked by celiac disease sera containing antineuronal antibodies. Preliminary data

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from our group showed that adsorption of celiac disease sera containing antineuronal antibodies with crude gliadin and tTG did not block central and enteric neuron immunolabeling, thus indicating lack of cross-reactivity between antineuronal antibodies and serologic markers of celiac disease.27 This finding further strengthens the peculiar identity of antineuronal antibodies present in neurologic celiac disease sera as potential effectors of neurodegenerative abnormalities, such as mitochondrialdependent neuronal apoptosis. The occurrence of this degenerative mechanism confirms and expands previous data indicating that sera of patients with type 128 –30 and type 231 diabetes and peripheral neuropathy evoked toxic or apoptotic effects through autoimmune-mediated pathways. In addition, neuronal apoptosis has been induced by exposing neuroblastoma cells and isolated myenteric neurons to sera of patients with paraneoplastic syndrome and high titers of circulating antineuronal antibodies targeting the Hu molecules (ie, anti-Hu antibodies), a group of RNA-binding proteins involved in neuronal maintenance and survival.32 Compared with anti-Hu antibodies, antineuronal antibodies found in patients with neurologic celiac disease usually have a much lower titer (range, 1:50 –1600) at immunofluorescence and do not recognize specific molecular antigen(s), as shown by Western blot and immunoblotting (Volta and De Giorgio, unpublished data, April, 2002). Nonetheless, the fact that apoptosis can be evoked by sera with anti-Hu antibodies and by sera containing undetermined antineuronal antibodies of patients with celiac disease provides further evidence that immune-mediated pathways are involved in neuronal damage. Dysregulation of the apoptotic processes can contribute to the pathogenetic mechanisms underlying different conditions, including cancer, autoimmunity, and neurodegeneration. In this context, our data indicated that proapoptotic effects predominantly triggered by celiac disease sera carrying antineuronal antibodies involve mitochondrial dysfunction. This conclusion is based on the finding showing activated apaf-1, a cytochrome c– dependent messenger. This enzyme, which is normally present in the cytoplasm in an inactive form, binds to cytochrome c, with subsequent recruitment of procaspase-9,33–35 activation of caspase-9, and cleavage of procaspase-3 to caspase-3. In the mitochondrial pathway of apoptosis, the complex of cytochrome c, apaf-1, and caspase-9, referred to as “apoptosome,” is a critical activator of the effector caspases. In line with these results, pointing toward a mitochondrial-dependent mechanism of apoptosis, immunocytochemical and Western blot analysis showed Bax translocation into mitochondria in the neuroblastoma cell line exposed to celiac disease sera containing antineuronal antibodies. Recent reports showed that Bax, a proapoptotic member of the Bcl-2 family, can directly induce cytochrome c release from mitochondria by interacting with Bcl-2.36 –38 Bax

protein resides in the cytoplasm in living cells and translocates to mitochondria when cells receive an apoptotic signal.39 – 41 Using a specific antibody, we identified an intense and diffuse cytoplasmic cytochrome c immunoreactive pattern in SH-Sy5Y cells treated with celiac disease sera containing antineuronal antibodies. Western blot experiments confirmed these results. In this study, the possibility that apoptosis evoked by celiac disease sera containing antineuronal antibodies could be dependent on a pathway other than mitochondria impairment was tested by evaluating caspase-8 activation. This enzyme results from death receptor activation at the plasma membrane level.42 Western blot results showed caspase-9, but not caspase-8, activation in protein extracts from SH-Sy5Y cells treated with celiac disease sera containing antineuronal antibodies. Thus, the lack of caspase-8 activation strengthened the concept that neuroblastoma cells exposed to celiac disease sera with antineuronal antibodies are altered by apoptotic mechanisms involving a mitochondrial-dependent pathway. Furthermore, measurement of specific enzymatic activities provides further evidence for this concept. We found that the specific activity of citrate synthase and NADH-ubiquinone oxidoreductase was increased or decreased, respectively, in SH-Sy5Y cells exposed to celiac disease sera containing antineuronal antibodies. In contrast, no significant changes were observed for other specific enzymatic activities such as succinate dehydrogenase and cytochrome oxidase. The abnormalities to citrate synthase and NADH-ubiquinone oxidoreductase, along with the lack of changes to other specific enzymatic activities, suggest a possible uncoupling between the Krebs cycle and the mitochondrial respiratory chain leading to cellular sufferance and apoptosis.43,44 In conclusion, the present study shows that antineuronal antibodies in sera of patients with celiac disease with neurologic disorders evoked apoptosis in a human neuroblastoma cell line. This neuronal damage was caused by the presence of antineuronal antibodies of the IgG class, because their deprivation from sera was associated with an apoptotic degree similar to controls. When examining celiac disease sera without neurologic disorders and antineuronal antibodies, a certain degree of apoptosis, although to a lower extent, was observed, suggesting a neurotoxic potential intrinsic to celiac disease. This proapoptotic mechanism is likely to depend on the combination of antigliadin and anti-tTG antibodies. Based on our findings, it is possible to speculate that overt neurologic manifestations in patients with celiac disease originate from a combination of subthreshold neurotoxic effects due to humoral markers of celiac disease with those triggered predominantly by antineuronal antibodies.

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Received August 28, 2006. Accepted April 12, 2007. Address requests for reprints to: Roberto De Giorgio, MD, PhD, Department of Internal Medicine & Gastroenterology, St Orsola-Malpighi Hospital, Via Massarenti, 9, 40138 Bologna, Italy. e-mail: [email protected]; fax: (39) 051-34-58-64. This work was supported by grants from the Italian Ministry of University and Research (COFIN Projects n° 2003064378_003, 2004062155_003) to G.B. and R. De G. and F.A.R. (to M.T.) and R.F.O. (to G.B. and R. De G.) funds from University of Pavia and Bologna. R. De G. is a recipient of a grant from “Fondazione Del Monte di Bologna e Ravenna”, Bologna, Italy. The authors thank Dr Catia Sternini (CURE Digestive Diseases Research Center, Division of Digestive Diseases, Departments of Medicine and Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA) for a critical reading of the manuscript and insightful comments, as well as Dr Patrizia Vaghi for her excellent assistance on confocal microscopy at the Centro Grandi Strumenti of the University of Pavia.