Prucalopride exerts neuroprotection in human enteric neurons

Abstract

Serotonin (5-hydroxytryptamine, 5-HT) and its transporters and receptors are involved in a wide array of digestive functions. In particular, 5-HT₄ receptors are known to mediate intestinal peristalsis and recent data in experimental animals have shown their role in neuronal maintenance and neurogenesis. This study has been designed to test whether prucalopride, a well-known full 5-HT₄ agonist, exerts protective effects on neurons, including enteric neurons, exposed to oxidative stress challenge. Sulforhodamine B assay was used to determine the survival of SH-SY5Y cells, human enteric neurospheres, and ex vivo submucosal neurons following H₂O₂ exposure in the presence or absence of prucalopride (1 nM). Specificity of 5-HT₄-mediated neuroprotection was established by experiments performed in the presence of GR113808, a 5-HT₄ antagonist. Prucalopride exhibited a significant neuroprotective effect. SH-SY5Y cells pretreated with prucalopride were protected from the injury elicited by H₂O₂ as shown by increased survival (73.5 ± 0.1% of neuronal survival vs. 33.3 ± 0.1%, respectively; P < 0.0001) and a significant reduction of proapoptotic caspase-3 and caspase-9 activation in all neurons tested. The protective effect of prucalopride was reversed by the specific 5-HT₄ antagonist GR113808. Prucalopride promotes a significant neuroprotection against oxidative-mediated proapoptotic mechanisms. Our data pave the way for novel therapeutic implications of full 5-HT₄ agonists in gut dysmotility characterized by neuronal degeneration, which go beyond the well-known enterokinetic effect.

the gastrointestinal tract serves as a major depot of the biogenic monoamine serotonin (5-hydroxytryptamine, 5-HT) with ∼95% of the body 5-HT localized in the gut (11). 5-HT is predominantly contained in enteroendocrine (i.e., enterochromaffin) cells (ECs) dispersed throughout the mucosal surface, but it is also synthesized and stored in a small proportion of neurons (2% of myenteric interneurons) of the enteric nervous system (ENS) (11, 12). The ENS expresses different types of 5-HT receptors (from 5-HT₁ to 5-HT₇) through which 5-HT modulates a remarkable number of physiological effects ranging from motility and secretion to mucosa growth and maintenance and neurogenesis (4, 19). For instance, 5-HT₄, which is mainly localized to the ascending excitatory neuronal reflex pathways, plays a prominent role in modulating propulsive patterns of gut motility (i.e., peristalsis), by releasing acetylcholine and other excitatory mediators, thus resulting in a prokinetic activity (11, 12, 19, 21). Various 5-HT₄ agonists have been developed to treat gastrointestinal disorders characterized by altered motility (25), including the 5-HT₄ full agonist prucalopride, which significantly improves symptoms and quality of life of patients with severe chronic constipation with excellent safety and tolerance profile (22). Interestingly, 5-HT₄ receptors have also been shown to modulate neuronal survival and neurogenesis (11). Indeed, in the central nervous system, 5-HT₄ has been shown to be involved in synaptic plasticity crucial to cognitive processes and memory consolidation (25). Recent findings also provide evidence for a role of 5-HT₄ in mediating neurogenesis and synaptic plasticity in the ENS (23). Furthermore, 5-HT₄^−/− mice showed loss of enteric neurons and reduced size of the surviving neurons at 1 mo in addition to delayed gastrointestinal transit (17). Finally, in vitro treatment with 5-HT₄ agonists increased the number of enteric neurons developing from precursor cells and/or surviving in culture, supporting neurogenetic properties mediated by this receptor subtype (17).¹

In this study, we tested the hypothesis that the 5-HT₄ full agonist prucalopride exerted a protective effect against neuronal injury using an established model of oxidative stress and different types of human neuronal cell lines, primary human enteric neurospheres (2), and human submucosal neuron whole-mount preparations obtained from colonic biopsies (16). We demonstrated that 5-HT₄ activation stimulated a specific protective response in the different neuronal cells analyzed, including human enteric neurons. Our findings open new avenues in the treatment of gastrointestinal disorders and homeostasis, especially in relation to neurodegeneration often underlying severe gut dysmotility.

METHODS

Neuronal cell lines and neurospheres culture.

Murine Neuro2A as well as human SH-SY5Y, SK-N-BE, S-K-NSH, LAN-1, and HEK293 (ATCC, Teddington, UK) cells were cultured in 95% air and 5% CO₂ at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Primary human enteric neurospheres were generated from human ENS stem cells containing neurosphere-like bodies according to a previously modified method (12). To produce oxidative stress, H₂O₂ was freshly prepared from 30% stock solution prior to each experiment. Cells were treated with 200 μM H₂O₂ in phosphate-buffered saline (PBS) for 30 min as previously indicated (3).

Western blotting.

Whole-cell lysates were obtained in immunoprecipitation (IP) buffer (Sigma-Aldrich, St. Louis, MO), protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), and phosphatase inhibitors (Sigma-Aldrich) on ice for 15 min. Protein concentration was evaluated by using the DC protein concentration assay kit (Bio-Rad, Hercules, CA). Fresh biopsies were sonicated on Bioruptor Pico Sonication System (V 1.1) (Diagenode, Liege, Belgium) in 100 μl of IP buffer and inhibitor cocktail (Sigma-Aldrich). Bands were visualized by the enhancer chemiluminescence method (GE Healthcare, Little Chalfont, UK). Primary antibodies used were anti-5-HT₄ (1:200; Abcam, Cambridge, UK), anti-HuC/D (1:200; Invitrogen, Carlsbad, CA), anti-p75 (1:200; Thermo Fisher Scientific, Waltham, MA), anti-γ-tubulin and anti-vinculin (1:10,000 and 1:50,000, respectively; Sigma-Aldrich), anti-caspase-3 and caspase-9 (both 1:200; Cell Signaling, Denver, CO), anti-neuronal nitric oxide synthase (nNOS) (1:50; Santa Cruz, Dallas, TX), and anti-peripheral choline acetyl transferase (pChAT) (1:100; Justus-Liebig University, Giessen, Germany). The experiments on neurospheres were performed on cells cultured for at least 14 days.

Cytotoxicity assay.

Sulforhodamine B colorimetric (SRB) assay was used for cell density determination. The method has been optimized for the toxicity screening of compounds to adherent cells in a 24-well plate (24). To determine the dose-response profiles of prucalopride, the SRB assay was performed by incubating cells for 0 (untreated), 30, and 60 min with prucalopride (Shire, Lexington, MA) at the following final concentrations: 10 mM, 10 μM, 10 nM, 1 nM, and 10 pM to establish a correct dose finding. Controls were exposed to 0.05% (vol/vol) dimethyl sulfoxide in culture medium while wells containing only culture medium served as blank. The selective antagonist GR113808 (10 nM) ([1-[2-[(methylsulfonyl)-amino]ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate, Sigma-Aldrich) was applied 30 min before prucalopride administration (Fig. 1A). Absorbance was measured at λ = 540 nm by spectrophotometry.

Fig. 1.

A: experimental design used in the present study. Arm-A: serum starvation (medium only) was applied at time 0 in cell cultures. Prucalopride (1 nM) was applied 30 min after challenge with H₂O₂ (200 μM). Arm-B: the 5-HT₄ antagonist GR113808 (10 nM) was applied 30 min before either prucalopride or H₂O₂. B: Western blot of total cell lysates with antibody to 5-HT₄ protein and with anti-vinculin in SH-SY5Y (lane 1), SK-N-BE (lane 2), SK-N-SH (lane 3), LAN-1 (lane 4), Neuro2A (N2A, lane 5), and HEK293 (lane 6). C: separate Western blot of total cell lysates with antibody to 5-HT₄ protein and with anti-vinculin in SH-SY5Y (lane 1) and human enteric neurospheres (lane 2). D: total cell lysates prepared from SH-SY5Y (a) and human enteric neurospheres (b) immunoblotted with anti-HuC/D, anti-p75, and anti-vinculin antibodies.

Cell proliferation assay.

SH-SY5Y cells (3×10⁵) were plated in duplicate and incubated with 10 μM bromodeoxyuridine (BrdU) (Millipore, Billerica, MA) for 1–48 h. BrdU-labeled cells adhered to the plate by centrifugation (300 g for 10 min) and then dried to the plate at room temperature for 1 h. Anti-mouse HRP-conjugated IgG antibody was used as secondary antibody. Absorbance measured at λ = 450 nm was used to calculate the BrdU labeling index.

Submucosal neuron whole-mount preparations from colonic biopsies.

A written, informed consent (protocol: PaBio-2011) was obtained from enrolled subjects (n = 3 female; age range: 30–55 yr) undergoing routine screening colonoscopy with biopsy sampling (n = 3 each subject from the descending colon). Subjects were recruited at the outpatient clinics of Internal Medicine and Gastroenterology Units of the Department of Medical and Surgical Sciences of University of Bologna. Biopsies were transferred to Sylgard-coated petri dish filled with ice-cold Hanks' solution and pinned flat, and the mucosa separated from the submucosa under a stereomicroscope (16). Fresh submucosal specimens were treated with prucalopride (1 nM) for 30 min in combination with H₂O₂ maintained in DMEM F12 medium. Biopsies were sonicated for eight cycles at 20–60 kHz.

Immunofluorescence.

Immunofluorescence was performed on neurospheres fixed in 4% paraformaldehyde, rinsed in PBS, and treated with blocking solution for 30 min. Previously described primary antibodies, i.e., rabbit anti-pChAT (1:100), mouse anti-nNOS (1:50), rabbit anti-5-HT₄ (1:200), and HuC/D (1:200), were applied to neurospheres overnight at 4°C. Specificity experiments included primary antibody omission and preincubation with the respective homologous antigen. After washing with PBS, a donkey anti-rabbit Cy3, anti-rabbit Alexa488, anti-goat Alexa488, and anti-mouse Alexa488 (1:200, Thermo Fisher Scientific) secondary antibodies were applied for 30 min at room temperature. Fluoroshield DAPI solution (Sigma-Aldrich) was used as mounting solution and to counterstain nuclei. Image capturing was performed on a Nikon microscope using DS-5M digital camera (Nikon Instruments, Düsseldorf, Germany).

Statistical analysis.

Statistical analysis was performed with GraphPad Prism software v.5.0 (San Diego, CA). Statistical significance was defined as P < 0.05 as evaluated by one-way ANOVA and Tukey's multiple-comparison test. Statistical analysis of densitometry was performed using Image Lab Software (Bio-Rad).

RESULTS

Evaluation of the 5-HT₄ expression in human enteric neurospheres and other neuronal cell lines.

The 5-HT₄ protein expression was studied by Western blot analysis using human neuroblastoma cell lines SH-SY5Y, SK-N-BE, S-K-NSH, and LAN-1; the human embryonic kidney cell line HEK293; a mouse neural crest-derived cell line, Neuro2A (used as a negative control for antibody specificity); and human enteric neurospheres (Fig. 1, B and C). All the human cell lines analyzed showed 5-HT₄ expression, whereas no signals were detected in the murine Neuro2A cells since the antibody used was specific for the human 5-HT₄ isoform. Western blot analysis of human enteric neurospheres and SH-SY5Y showed that these cells also expressed the neuronal marker HuC/D and the progenitor/stem cell marker p75 (Fig. 1D), thus indicating that these cells were suitable for studies on neuroprotection.

The 5-HT₄ agonist prucalopride protects SH-SY5Y cells from oxidative stress insult.

We tested different concentrations of the full 5-HT₄ agonist prucalopride on SH-SY5Y cells exposed to the oxidative stress agent H₂O₂. The oxidative stress evoked by 200 μM of H₂O₂ resulted in a significant reduction of cell survival (33.3 ± 0.1%) compared with untreated cells (P < 0.001). Prucalopride at 1 nM concentration showed the best protective effect, with a significantly higher cell survival compared with cells exposed to H₂O₂ (73.5 ± 0.1% of neuronal survival vs. 33.3 ± 0.1%, respectively; P < 0.0001) (Fig. 2A). To verify the specificity of the 5-HT₄-mediated protective effect, cells were exposed to the selective antagonist GR113808 (10 nM) applied 30 min before prucalopride administration (18). Pretreatment with GR113808 induced a significant reduction in cell survival, i.e., 23.3 ± 0.1 vs. the 73.5 ± 0.1% in the H₂O₂-treated cells exposed to prucalopride (P < 0.0001). These data are comparable to those obtained in presence of H₂O₂ alone, indicating that the observed protection was elicited via 5-HT₄ activation (Fig. 2A). Untreated cells exposed to GR113808 at 10 nM concentration showed a survival rate of 81.3 ± 0.1%, which was comparable to that of vehicle-treated cells, indicating lack of toxicity of the 5-HT₄ antagonist (Fig. 2A, P < 0.001). By contrast, prucalopride becomes toxic at high concentrations, with the highest toxic effect observed with 10 mM prucalopride as indicated by reduced survival (32 ± 1.2 and 29 ± 1.2% at 30 and 60 min, respectively) in cells unexposed to oxidative stress (cell survival being virtually 100%). Prucalopride at the lowest concentration tested (10 pM) failed to rescue neuronal cells from damage induced by oxidative stress (survival being 41 ± 0.8 vs. 32 ± 1.2%, respectively) (Fig. 2B).

Fig. 2.

A: percentage of SH-SY5Y survived after treatment with 1 nM prucalopride alone (C bar) or in combination with 200 μM H₂O₂ (prucalopride+H₂O₂ vs. H₂O₂ **P < 0.0001) for 30 min (D bar) and GR113808 either alone [GR113808 vs. untreated (NT); ns] (F bar) or with 200 μM H₂O₂ (prucalopride+H₂O₂ vs. prucalopride+GR113808+H₂O₂ ***P < 0.0001) (E bar). A bar, NT are considered as 100% cell survival; B bar, H₂O₂ alone. B: dose finding was established by treating SH-SY5Y cells for 30 or 60 min with prucalopride at different concentrations (10 mM, 10 μM, 10 nM, 1 nM, and 10 pM). Compared with 30 min, 10 nM prucalopride for 60 min resulted in a reduced neuroprotective effect (cell survival 80 vs. 60%, respectively, P < 0.0001). C: BrdU analysis in the SH-SY5Y cell line. Percentage of SH-SY5Y proliferation after treatment with 1 nM prucalopride, alone (C bar) and in combination with 200 μM H₂O₂ for 30 min (D bar). A bar, NT; B bar, 200 μM H₂O₂ for 30 min. ANOVA and Tukey's multiple comparison tests did not reached statistical significance (P = 0.384). D and E: Western blotting of SH-SY5Y total lysates did not show differences in procaspase-3 and -9 expression; lane 1: NT; lane 2: 1 nM prucalopride (Pruca) and H₂O₂; lane 3: H₂O₂ alone. F and G: Western blotting of SH-SY5Y showing significant differences in active caspase-3 and -9 expression (**P < 0.001 vs. NT); lane 1: NT; lane 2: 1 nM prucalopride and H₂O₂; lane 3: H₂O₂ alone.

Evaluation of prucalopride in neurogenesis in vitro.

To determine whether prucalopride induces neurogenesis in vitro, we evaluated the BrdU incorporation 24 h after the treatment with different concentrations of the 5-HT₄ agonist. Prucalopride did not evoke a significantly different incorporation of BrdU compared with untreated SH-SY5Y or H₂O₂-treated cells (P = 0.384) (Fig. 2C). This suggests that the effect of prucalopride treatment of SH-SY5Y cells exposed to oxidative stress is predominantly neuroprotective and not due to neuroproliferation.

Effect of prucalopride on caspase expression in neuronal cell lines and neurospheres.

To investigate the mechanisms underlying the neuroprotective effects mediated by prucalopride, we examined the expression of two apoptotic markers, i.e., caspase-3 and caspase-9, which are activated by oxidative stimuli. No changes in procaspase expression were found before and after treatments (Figs. 2, D and E, and 3, A and B), whereas a significant decrease of the active form of both oxidative stress-activated caspases-3 and caspase-9 was detected in SH-SY5Y cell lines (Fig. 2, F and G) and in human enteric neurospheres (Fig. 3, C and D) treated with 1 nM prucalopride.

Fig. 3.

A and B: Western blotting of human enteric neurospheres did not show differences in procaspase-3 and -9 expression; lane 1: NT; lane 2: 1 nM prucalopride and H₂O₂; lane 3: H₂O₂ alone. C and D: Western blotting showed significant differences in active caspase-3 and -9 expression (**P < 0.001 vs. NT); lane 1: NT; lane 2: 1 nM prucalopride and H₂O₂; lane 3: H₂O₂ alone. E and F: note nitrergic (nNOS) (E) and cholinergic (pChAT) (F) immunoreactive neurons (scale bar 100 μm). G and H: Western blotting for nNOS and pChAT showed rescue of these 2 neuronal subsets after prucalopride treatment; lane 1: NT; lane 2: H₂O₂ alone; lane 3: H₂O₂+prucalopride (*P < 0.05 pChAT/nNOS prucalopride+H₂O₂ vs. H₂O₂).

Effect of prucalopride in different subgroups of neurons.

To determine whether the neuroprotective effect evoked by prucalopride occurred in selective subpopulations of enteric neurons, we investigated the effect of prucalopride following the oxidative stress injury on the expression of two major enteric neuron transmitters nNOS (neuronal nitric oxide synthase, the enzyme that produces nitric oxide) and pChAT (peripheral cholinacetyltransferase, the enzyme that catalyzes the synthesis of acetylcholine) in human enteric neurospheres. Expression of both nNOS and pChAT was identified by immunofluorescence and Western blot (Figs. 3, E–G). H₂O₂ treatment induced a quantitative reduction of both nNOS and pChAT proteins in neurospheres compared with the total protein. In contrast, prucalopride treatment prevented the oxidative-induced reduction of each subset of transmitters compared with H₂O₂ treatment without prucalopride (Fig. 3, H and I, P < 0.05).

Western blot analysis confirmed the expression of 5-HT₄ in colonic biopsy-derived submucosae obtained during colonoscopy (Fig. 4A).

Fig. 4.

A: Western blot showing 5-HT₄ expression in submucosal neuron whole mounts (lane 1) compared with human SH-SY5Y (lane 2, positive control) and murine Neuro2A (lane 3, negative control). B and C: Western blotting did not show differences in procaspase-3 and -9 expression; lane 1: NT; lane 2: 1 nM prucalopride+H₂O₂; lane 3: H₂O₂ alone. D and E: Western blotting showed significant differences in active caspase-3 and -9 expression (**P < 0.001 vs. NT); lane 1: NT; lane 2: 1 nM prucalopride+H₂O₂; lane 3: H₂O₂ alone.

After treatment with prucalopride, submucosal whole-mount preparations were exposed to H₂O₂ and the levels of activated caspases were measured. Procaspase expression did not vary with any treatment (Figs. 4, B and C). However, activated caspase-3 and caspase-9 were significantly reduced in prucalopride-treated biopsies compared with H₂O₂ only (Fig. 4, D and E, P < 0.001).

DISCUSSION

The results of this study provide strong evidence for a neuroprotective role of 5-HT₄ subtype, which is expressed in human neuronal cell lines, neurospheres, and enteric neurons as confirmed by in vitro and ex vivo experiments. Stimulation of 5-HT₄ with the specific, high-affinity full agonist prucalopride induced significant protection against the oxidative stress injury elicited by H₂O₂.

The protective effect of prucalopride on human neurons, including enteric neurons, was demonstrated by 1) the increased survival of human neuronal cell lines exposed to oxidative stress and 2) reduction of apoptosis in neuronal cell lines, human enteric neurospheres obtained from perinatal gut biopsies, and whole-mount preparation of submucosal neurons from adult colonic tissues, following oxidative stress challenge. The abolition of all of these effects by pretreatment with the 5-HT₄ antagonist (GR113808) demonstrated that prucalopride-evoked protective effect was due to specific 5-HT₄ activation. Taken together these results expand previous data showing that other 5-HT₄ agonists (i.e., tegaserod and renzapride) protected primary cultures of mouse enteric neurons from apoptosis (17). Notably, our study indicated that the optimal neuroprotective effect was obtained with 1 nM prucalopride, which is at least 10 times lower compared with the doses used by Liu et al. (17) with either tegaserod or renzapride. The difference in terms of concentrations and related neuroprotective effect can be the result of a diverse efficiency in 5-HT₄ receptor activation exhibited by the different agonists used in our vs. Liu et al. study (17).

The neural network supplying the digestive system, including the ENS, exerts a prominent regulatory role on several digestive functions (6). A tight association links gut dysfunction to ENS abnormalities characterized by neuronal damage and/or loss (15). In addition to the many effects elicited by serotoninergic pathways, 5-HT₄ plays an important role in mediating 5-HT neuroprotective function as supported by the observation that mice lacking 5-HT₄ displayed a markedly reduced number of submucosal and myenteric neurons in an age-dependent manner (11, 17). Oxidative stress is well known to trigger neuronal loss and represents a key target for neuroprotection. A neuroprotective effect of 5-HT against oxidative stress-induced cell death has been supported by the results of a study showing that 5-HT treatment increased the activity of antioxidant enzyme and counteracted lipid peroxidation in the brain in a rat model of Parkinson's disease (1).

Our study extends the data reported in the brain, by showing that prucalopride, a specific 5-HT₄ receptor agonist, protects peripheral neurons against oxidative stress-mediated proapoptotic insults induced by exposure to H₂O₂, as indicated by the significant reduction in caspase-3 and caspase-9 activation, both key regulators of apoptosis in a variety of cells including the ENS (5, 7). The prucalopride-mediated neuroprotection in SH-SY5Y cell lines, human neurospheres, and ex vivo colonic submucosal neurons indicates that 5-HT₄ counteracts proapoptotic pathways by influencing the activation of both initiator and effector caspases. Notably, the finding that prucalopride rescued the neuronal expression of nNOS as well as pChAT that was reduced by oxidative stress challenge suggests that damage of functionally distinct neuronal populations, i.e., nitrergic (inhibitory) and cholinergic (excitatory) neurons (6, 9), can be prevented by 5-HT₄ stimulation. The 5-HT₄ activity is mediated by PKA/CREB signaling (17), a system crucial for neurogenesis, and neuronal connectivity in the brain and pharmacological modulation of PKA have been proposed as possible therapeutic targets in neuronal degeneration, e.g., Alzheimer's and Parkinson's diseases (14, 20). Ideally, 5-HT₄-mediated PKA activation might be exploited to restore neuronal circuitry also in enteric neuropathies.

Our results provide a pharmacological basis for prucalopride as a compound potentially useful in patients with enteric degeneration and loss of neurons, such as enteric neuropathies. These conditions are characterized by a severe abnormality of gastrointestinal motility and might represent the “tip of the iceberg” of functional digestive disorders (15). Enteric neuropathies can be either primary (i.e., of unknown etiology) or secondary to a variety of diseases including central neurodegenerative disorders. Patients with enteric neuropathies manifest a broad spectrum of digestive symptoms resulting from the severe abnormalities of gut motility. Typical examples of such conditions, which markedly impair the patients' quality of life and can be life threatening, include gastroparesis, chronic intestinal pseudo-obstruction, and severe slow-transit constipation/colonic inertia. The treatment of these conditions is mainly supportive and so far lacking a targeted, e.g., receptor-mediated, approach (9, 15).

Interestingly, a previous study reported that the 5-HT₄ partial agonist tegaserod increased the number of neurons and length of neurites of in vitro cultured neural crest-derived enteric neurons, indicating that activation of enteric neural 5-HT₄ receptors promotes neurogenesis (11, 17, 23). By contrast, our in vitro experiments did not show neurogenetic effects of prucalopride on human neuronal cell lines. This apparent discrepancy between our data and the previously reported results may be ascribed to a shorter period of analysis used in our investigation (24 h) compared with the Matsuyoshi et al. study (18) in which mosapride (another partial 5-HT₄ agonist) was used for 15 days in animal models. Whether a longer period of treatment with prucalopride can also induce an increase in cell proliferation and/or differentiation (i.e., neurite outgrowth) remains to be elucidated.

In conclusion, the present data highlight for the first time that the specific 5-HT₄ full agonist prucalopride evokes significant neuroprotective effects in neurons, including human enteric neurons, in vitro and ex vivo. Prucalopride is currently used for treatment of chronic constipation and has an excellent safety and tolerance profile. These findings widen the role of this compound beyond its well-defined enterokinetic property and support that 5-HT₄ activation can be a target for pharmacological intervention in neurodegenerative diseases of the gut. Ad hoc-designed clinical trials are awaited to test the efficacy of 5-HT₄ in stopping the progression of neuronal damage and loss in patients with severe gut dysmotility.

GRANTS

This work was supported by Ricerca Finalizzata RER2009 (Ita-MNGIE), Ministry of Health; the Italian Ministry of University and Research (PRIN/COFIN2009MFSXNZ_002); Telethon Grant GGP15171 (to R. De Giorgio); and NIH P30DK041301 grant “Imaging and Stem Cell Biology Core” (C. Sternini). R. De Giorgio is also the recipient of grants from “Fondazione del Monte di Bologna e Ravenna,” Bologna, Italy. N. Thapar is supported by Great Ormond Street Hospital Children's Charity (GOSHCC). D. Natarajan is funded through a GOSHCC grant (awarded to N. Thapar).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

F. Bianco, E. Bonora, N.T., U.V., C.S., and R.D.G. conception and design of research; F. Bianco, E. Bonora, D.N., M.V., F.T., F.G., E. Boschetti, and M.M. performed experiments; F. Bianco, E. Bonora, D.N., M.V., F.T., F.G., E. Boschetti, M.M., M.S., P.C., V.S., C.S., and R.D.G. analyzed data; F. Bianco, E. Bonora, D.N., M.V., N.T., F.T., F.G., E. Boschetti, U.V., F. Bazzoli, M.M., M.S., P.C., V.S., C.S., and R.D.G. interpreted results of experiments; F. Bianco, E. Bonora, and E. Boschetti prepared figures; F. Bianco, E. Bonora, U.V., and R.D.G. drafted manuscript; F. Bianco, E. Bonora, D.N., M.V., N.T., F.T., F.G., E. Boschetti, U.V., F. Bazzoli, M.M., M.S., P.C., V.S., C.S., and R.D.G. edited and revised manuscript; F. Bianco, E. Bonora, D.N., M.V., N.T., F.T., F.G., E. Boschetti, U.V., F. Bazzoli, M.M., M.S., P.C., V.S., C.S., and R.D.G. approved final version of manuscript.