Neurogastroenterology & Motility Neurogastroenterol Motil (2015) 27, 1156–1161
doi: 10.1111/nmo.12605
MeCP2 in the enteric nervous system G. WAHBA ,*
,#
S. C. SCHOCK ,†
,#
E. CLARIDGE ,* M. BETTOLLI ,‡ D. GRYNSPAN ,‡ P. HUMPHREYS ‡
& W. A. STAINES *
*Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada †Research Institute, Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada ‡Departments of Surgery, Pathology and Paediatrics (Neurology), Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada
Key Messages
• MeCP2 • • •
is expressed throughout development of the GI tract with appearance at or before E11.5 in mice, specifically in the enteric nervous tissue. This study aimed to investigate if MeCP2 is expressed in the human and murine ENS and at which stages of development. Immunofluorescence was performed on frozen sections of human intestinal tissue and dissociated murine enteric neuronal cultures. Western blots on intestinal dissections of developmental and adult mice confirmed MeCP2 expression. This study opens the possibility for treatment of the GI dysfunction in Rett syndrome with peripheral acting drugs and growth factors.
Abstract Background Rett syndrome (RTT) is an intellectual deficit and movement disorder that develops during early childhood in girls. Affected children are normal until 6–18 months of age, after which symptoms begin to appear. Most cases of RTT are due to mutations in the MeCP2 gene leading to disruption of neuronal communication in the central nervous system. In addition, RTT patients show peripheral ailments such as gastrointestinal (GI), respiratory, and cardiac dysfunction. The etiology of intestinal dysfunction in RTT is not well-understood. Reports on the presence of MeCP2 in the peripheral nervous system are scant. As such we examined the levels of MeCP2 in human and murine GI tissue and assessed MeCP2 expression
at various developmental stages. Methods Immunohistochemistry for MeCP2, HuC/D, juvenile beta tubulin, and GFAP was performed on human and murine intestine. Western blots of these same tissues were probed with MeCP2, vAChT, nNOS, and betaactin antibodies. Key Results MeCP2 is expressed throughout the GI tract. MeCP2 is expressed specifically in the enteric nervous system of the GI tract. MeCP2 is expressed in the GI tract throughout development with appearance beginning at or before E11.5 in the murine intestine. Conclusions & Inferences The proof of MeCP2 expression in enteric neurons suggests that the GI dysmotility in Rett may arise from enteric network dysfunction secondary to MeCP2 mutation. Keywords development, enteric nervous system, gastrointestinal dysmotility, gastrointestinal tract, MeCP2, myenteric plexus, Rett syndrome.
Address for Correspondence William A Staines, Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Rd, Ottawa ON, Canada K1H 8M5. Tel: +1-613-562-5800 (8188); fax: +1-613-562-5434; e-mail: [email protected] # These authors contributed equally and should be considered co-first author. Received: 29 January 2015 Accepted for publication: 29 April 2015
Abbreviations: ENS, enteric nervous system; GFAP, glial fibrillary acidic protein; GI, gastrointestinal; KO, knockout; LM, longitudinal muscle; MeCP2, methyl binding protein 2; MP, myenteric plexus; nNOS, neuronal nitric oxide synthase; PBS, phosphate buffered saline; RTT, Rett syndrome; SDS, sodium dodecyl sulphate; SI, small intestine; Tuj, juvenile beta tubulin; vAChT, vesicular acetylcholine transporter.
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MeCP2 in the ENS
INTRODUCTION
with special attention to the ENS cells considering MeCP2’s well-established role in neuronal maturation elsewhere.
Rett syndrome (RTT) is a neurological and developmental disorder affecting 1 in 13 000 girls.1 It is characterized by normal development until 6– 18 months preceding the onset of symptoms which include motor difficulties, severe mental retardation, and seizures.2 Rett syndrome is caused by de novo mutations in the gene coding for the methyl binding protein-2 (MeCP2),3 a mediator of synaptic development and plasticity.4 Methyl binding protein-2 levels are highest in the brain, where it is expressed in all regions at different developmental stages.5,6 During embryonic cortical development, MeCP2 protein is barely detectable but increases greatly postnatally.6 Only mature postmigratory neurons express MeCP2 and silencing the gene has no effect on migration or differentiation of neuroblasts.7 Increased expression of MeCP2 coincides with an important period of synaptogenesis in the brain.4 It has been suggested that MeCP2’s role resides in synaptogenesis and lifelong maintenance of synapses and neuronal networks.8 Rett syndrome patients do not show signs of neurodegeneration; however, neurons from MeCP2 knockout (KO) mice have fewer dendrites, abnormal dendritic spines, disorganized axons, and altered neurotransmitter levels.9–11 These findings all suggest that RTT is a disease of synaptic function. Apart from the well-studied neurological pathologies in RTT, general eating and nutrition problems also contribute significantly to its morbidity. In a large nationwide American study, 92% (n = 983) of parents of RTT patients reported that their child demonstrated some sort of GI dysmotility and 80% specifically reported constipation.12,13 Apart from constipation, dysmotility in the upper GI tract is also seen. Gastroesophageal dysmotility and gastrointestinal reflux disease are common as well.12,14 Thus, dysmotility is prevalent in RTT affecting the entire length of the GI tract. We speculate that MeCP2 associated enteric neuronal dysfunction may underlie some of the GI symptoms in RTT. To our knowledge no one has yet investigated the mechanistic basis of the GI symptoms in RTT. A previous study reported that protein levels of MeCP2 were high in the brain but present at negligible levels in the stomach and the intestines.5 We surmised that this might be due to a false negative due to sampling error because of the small proportion of enteric nervous system (ENS) cells relative to muscularis. As a result, we more closely examined the presence of MeCP2 in human and murine GI tissue
© 2015 John Wiley & Sons Ltd
METHODS Immunofluorescence on human frozen tissue Frozen formalin fixed human colonic, intestinal, and appendiceal sections were obtained from the Children’s Hospital of Eastern Ontario with REB approval. Small intestine (SI), colon, and appendix were sampled from a 16 year old female with ulcerative colitis, a 16 year old male with ulcerative colitis and a 13 year old female with appendicitis respectively. Sections were incubated with mouse anti-HuC/D (1 : 100; Invitrogen, Carlsbad, CA, USA) and rabbit anti-MeCP2 (1 : 200; Millipore, Darmstadt, Bundesland, Germany) overnight at 4 °C, and then rinsed with 10 mM PBS. Sections were then incubated with the appropriate secondary antibodies (1 : 200; Invitrogen) for 5 h at 37 °C and then rinsed with 10 mM PBS. All antibodies were diluted in 10 mM PBS containing 0.3% Triton-X. Sections were then imaged with a Zeiss Axio Observer D1 microscope (New York, NY, USA). The MeCP2 antibody is specific for this protein as evidenced by its adsorption by the immunizing peptide.15 We and others have shown staining to be abolished in complete MeCP2 KO mice.15 Furthermore, no staining was seen with any of the secondary antibodies in the absence of primary antibody.
Immunofluorescence on dissociated cultures All animal procedures were approved by the animal care facility at the University of Ottawa. Dissociated enteric neurons were obtained from 4 to 6 month old CD-1 mice by dissecting the myenteric plexus (MP) and the longitudinal muscle (LM) from the SI. Tissue was enzymatically dissociated by incubation at 37 °C in 1 mg/mL collagenase followed by 0.05% trypsin. Cultures were plated onto 10% matrigel (BD Biosciences, San Jose, CA, USA) and 33 lg/mL collagen (Enzo Life Sciences, Farmingdale, NY, USA) coated 96 well plates and grown in vitro for 4 days. Cells were then fixed in 4% paraformaldehyde for 20 min at room temperature. Anti-MeCP2 staining procedure was followed as described above. Cultures were then stained with mouse anti-juvenile beta tubulin (Tuj) antibody (1 : 25; gift from Dr. A. Frankfurter) following this same procedure but secondary incubation time was reduced to 30 min.
MeCP2 Western Blot Whole SIs from adult CD-1 mice (4–6 month old) were extracted and separated from the MP and LM as described above. The remaining SI wall and the myenteric/muscle strip were sonicated for 20 s in radioimmunoprecipitation assay buffer containing protease inhibitors. Sonicated tissue was incubated for 30 min on ice vortexing every 5 min. Tissue was centrifuged at 12 000 g at room temperature for 5 min to pellet cell debris. The protein concentration was assessed using a Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Following this, 15 lg of protein was added to 5 lL 49 SDS protein sample buffer and water and boiled for 5 min at 100 °C. The samples were electrophoresed on a 4% polyacrylamide gel and transferred onto a nitrocellulose membrane using an iBlotâ Gel Transfer Device (Invitrogen). The immunoblot was blocked overnight in 10% skim milk in
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(Fig. 1). Methyl binding protein-2 immunoreactivity was not expressed in all cell types present in the gut sections (Fig. 2) but rather showed selectivity. The MeCP2-positive cells were grouped together in discrete clusters corresponding to the myenteric and submucosa ganglia of the ENS. Methyl binding protein-2positive cells co-labeled with HuC/D, a neuronal marker, confirming the impression of selective expression of MeCP2 in the ENS. Dissociated mouse enteric neurons in culture also express MeCP2 (Fig. 3A). Methyl binding protein-2 immunoreactivity is strongest in Tuj-1 positive neurons.
Tris-based buffer saline with Tween (TBST). The immunoblot was then incubated for 1.5 h at room temperature with anti-beta actin antibody bound to horse radish peroxidase (1 : 10 000; Abcam, Cambridge, MA, USA), and then rinsed with TBST for 30 min. The immunoblot was exposed to a chemiluminescence reagent (Millipore) and visualized with X-ray film (Diamed, Mississauga, ON, Canada). The immunoblot was then stripped using Reblot Solution (Millipore) for 20 min at room temperature. Anti-MeCP2 antibody (1 : 1000; Millipore; expected band size of ~75 kDa) was applied overnight with shaking at 4 °C. Afterwards, the immunoblot was rinsed with TBST and horse radish peroxidase-bound anti-rabbit secondary antibody (1 : 5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied for 2 h with shaking at room temperature. The immunoblot was rinsed again and then exposed to a chemiluminescence reagent and visualized with Xray film. All antibodies were diluted in 10% non-fat milk in TBST. The blot was also probed with anti-nitric oxide synthase (NOS; 1 : 1000; Santa Cruz Biotechnology; expected band size of 155 kDa) and anti-vesicular acetylcholine transporter (VAChT; 1 : 1000; Synaptic Systems, Goettingen, Germany; expected band size of 70 kDa) as described above.
MeCP2 levels are proportional to neuronal content in lysates To confirm that MeCP2 is expressed in the neuronal component of the intestinal tract, western blot was performed on protein lysates from the SI after removal of the MP and on the MP itself. The MP lysate contains a higher concentration of neuronal protein than the SI in which the MP was removed. This lysate still has some neuronal content due to the presence of the submucosal plexus, a small neuronal layer, which was not removed during the dissection process. The western blot revealed a single band for MeCP2 at the expected protein weight of ~75 kDa. The lysate from the isolated MP showed higher levels of MeCP2 than the SI without the MP (but still containing the submucous neuronal plexus); this result shows that MeCP2 is in fact present in neurons and/or other nerve plexus cells in the GI. Methyl binding protein-2 levels correlated with neurochemical markers (nNOS and
MeCP2 protein levels change with age The western blotting method described above was repeated for protein extractions from the entire SI of embryonic and postnatal mice at ages indicated in the figure. Exposure time for MeCP2 antibody immunoreactivity was increased from 2 to 45 min in order to detect MeCP2 in whole GI lysates.
RESULTS MeCP2 is expressed throughout the GI tract, exclusively within the ENS The staining of human frozen gut tissue showed positive immunoreactivity for MeCP2 in all sections of the GI similar to that seen in the cerebral cortex
A
B
Figure 1 Localization of MeCP2 to mouse cerebral cortex. Image of the cingulate region of neocortical sections from MeCP2 knockout (A) and wild type (B) mice. MeCP2 (green) DAPI (blue).
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Figure 2 Localization of MeCP2 to human enteric neurons throughout the distal gastrointestinal tract. Inverted immunofluorescence on frozen human tissue sections from colon, small intestine and appendix, stained with anti-HuC/D, a neuronal marker, and anti-MeCP2; scale bar: 50 lm, magnification: 2009.
same was true for MeCP2 presence in the ENS. We dissected GI tissue from embryonic and postnatal mice and assessed MeCP2 protein levels by western blot. Methyl binding protein-2 was present and levels remained constant from the earliest embryonic time point investigated (Fig. 5).
VAChT) present in high levels in enteric neuronal tissue (Fig. 4). It should be noted that the MP lysate in Fig. 4 was not purely neuronal as during dissection the longitudinal muscle strongly adheres to the MP and is difficult to detach.
MeCP2 protein levels remain constant with age DISCUSSION
Since MeCP2 protein levels have been shown to increase throughout development in the brain in an area-specific manner, we sought to determine if the
A
Our main findings are: (i) MeCP2 is expressed throughout the GI tract, (ii) MeCP2 is expressed specifically in
B
C
Figure 3 Nuclear localization of MeCP2 to cultured neurons from the mouse small intestine. (A) MeCP2 plus Tuj-1 double immunofluorescence in dissociated ENS cultures fixed at DIV4. (B) single anti-Tuj-1 (C) single anti-MeCP2. Scale bar: 50 lm.
© 2015 John Wiley & Sons Ltd
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Previously, it has been reported that at E11.5, MeCP2 is only detectable in the spinal cord and hindbrain5 but not in other brain structures such as the cortex and hippocampus. It is thus important to note that along with these brain areas, the ENS develops at an early embryonic age. Neuroblasts migrate to the foregut at E9.5, demonstrate electric activity at E11.5, and form synapses as early as E12.5.17–19 Considering that MeCP2 expression in the brain coincides with important stages of synaptogenesis and maturity,4 it follows that MeCP2 protein can be found early in the ENS. It would be interesting to determine whether GI symptoms, which are prominent in RTT, are observed earlier than the onset of CNS symptoms, as MeCP2 may become functionally important in the ENS earlier than the CNS. We were unable to determine whether the ENS is MeCP2 positive prior to E11.5 due to impractical surgical access to the GI in embryos earlier than this date. These findings reveal the potential relevance of peripheral nervous MeCP2 distribution in the autonomic pathophysiology of RTT, and suggest that the GI pathology in RTT may be mediated through primary dysfunction of the ENS and not only due to dysfunction in descending CNS pathways or as a non-specific association of global development delay mediated through factors such as immobility and aerophagia. Further studies into the GI pathology of RTT will be required to elucidate the function of MeCP2 in these neurons and to determine the mechanisms by which its abrogation causes GI dysmotility. Our findings open the possibility of treatment of the GI dysfunction in this disorder with peripheral acting drugs and growth factors.
Figure 4 MeCP2 immunoreactivity coisolates with neuronal markers and is depleted in GI tissue upon removal of enteric neurons. Western blot analysis of MeCP2 protein levels in comparison to neurochemical markers (nNOS and VAChT) in lysates from mouse small intestine from which the MP has been removed and the isolated MP strip.
Figure 5 MeCP2 is expressed very early in enteric tissue E11.5 and maintains a constant level of expression through to P14. Western blot analysis of MeCP2 protein levels in GI lysates extracted from mice at different developmental points. The MeCP2 (75 kDa) bands for the lysates is shown on top with the beta-actin (41.7 kDa) bands shown on the bottom. This same pattern was repeated in five mice at each age.
the enteric nervous tissue, and (iii) MeCP2 is expressed throughout development in the GI tract with appearance at or before E11.5 in mice. Until now there has been little attention paid to MeCP2 expression in the gut. A past study indicated that MeCP2 protein levels are high in the brain but present at insignificant levels in the stomach and the intestines.5 However, in that study, western blots were performed on whole lysates of GI tissue. We found that expression of MeCP2 protein is limited to neuronal cells which are a small minority in the GI cell population. We speculate that perhaps in the previous study MeCP2 expressing neurons were too dilute to be detected in western blots. We investigated protein levels of MeCP2 and not RNA levels as MeCP2 has been shown to be strongly posttranscriptionally regulated; RNA levels have been reported to be equal in different tissues as opposed to protein levels which are tissue-specific.5 We have now demonstrated that MeCP2 protein is present within human and murine enteric neurons at levels that persist throughout postnatal development.
FUNDING This work was funded by the Jessica Carr Found and the CHEO Research Foundation.
DISCLOSURE No competing interests declared.
AUTHOR CONTRIBUTION Performed research: GW, SS; Designed research study: WS, SS, GW; Contributed essential reagents: MB, DG; Analyzed data: GW, WS, SS; Drafting of manuscript: GW, SS, PH, WS, DG; Supervised the study: PH and WS.
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REFERENCES 1 Percy AK. Rett syndrome: current status and new vistas. Neurol Clin 2002; 20: 1125–41. 2 Hagberg B, Witt-Engerstr€ om I. Rett syndrome: a suggested staging system for describing impairment profile with increasing age towards adolescence. Am J Med Genet 1986; 25: 47–59. 3 Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is caused by mutations in X-linked MECP2, encoding methylCpG-binding protein 2. Nat Genet 1999; 23: 185–8. 4 Guy J, Cheval H, Selfridge J, Bird A. The role of MeCP2 in the brain. Annu Rev Cell Dev Biol 2011; 27: 631–52. 5 Shahbazian MD, Antalffy B, Armstrong DL, Zoghbi HY. Insight into Rett syndrome: MeCP2 levels display tissue-and cell-specific differences and correlate with neuronal maturation. Hum Mol Genet 2002; 11: 115–24. 6 Kishi N, Macklis JD. MECP2 is progressively expressed in post-migratory neurons and is involved in neuronal maturation rather than cell fate decisions. Mol Cell Neurosci 2004; 27: 306–21. 7 Smrt RD, Eaves-Egenes J, Barkho BZ, Santistevan NJ, Zhao C, Aimone JB, Gage FH, Zhao X. Mecp2 deficiency leads to delayed maturation and altered gene expression in hippocampal neurons. Neurobiol Dis 2007; 27: 77–89.
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8 Nguyen M, Du F, Felice CA, Shan X, Nigam A, Mandel G, Robinson JK, Ballas N. MeCP2 is critical for maintaining mature neuronal networks and global brain anatomy during late stages of postnatal brain development and in the mature adult brain. J Neurosci 2012; 32: 10021–34. 9 Belichenko PV, Wright EE, Belichenko NP, Masliah E, Li HH, Mobley WC, Francke U. Widespread changes in dendritic and axonal morphology in Mecp2-mutant mouse models of rett syndrome: evidence for disruption of neuronal networks. J Comp Neurol 2009; 514: 240–58. 10 Ide S, Itoh M, Goto Y. Defect in normal developmental increase of the brain biogenic amine concentrations in the mecp2-null mouse. Neurosci Lett 2005; 386: 14–7. 11 Lappalainen R, Riikonen RS. High levels of cerebrospinal fluid glutamate in Rett syndrome. Pediatr Neurol 1996; 15: 213–6. 12 Motil KJ, Caeg E, Barrish JO, Geerts S, Lane JB, Percy AK, Annese F, McNair L et al. Gastrointestinal and nutritional problems occur frequently throughout life in girls and women with Rett Syndrome. J Pediatr Gastroenterol Nutr 2012; 55: 292. 13 Schwartzman F, Vıtolo MR, Schwartzman JS, Morais MB. Eating practices, nutritional status and constipation in patients with Rett syndrome. Arquivos de Gastroenterolgia 2008; 45: 284–9.
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14 Motil KJ, Schultz RJ, Browning K, Trautwein L, Glaze DG. Oropharyngeal dysfunction and gastroesophageal dysmotility are present in girls and women with Rett syndrome. J Pediatr Gastroenterol Nutr 1999; 29: 31–7. 15 Yazdani M, Deogracias R, Guy J, Poot RA, Bird A, Barde Y-A. Disease modeling using embryonic stem cells: MeCP2 regulates nuclear size and RNA synthesis in neurons. Stem Cells 2012; 30: 2128–39. 16 Nan X, Tate P, Li E, Bird A. DNA methylation specifies chromosomal localization of MeCP2. Mol Cell Biol 1996; 16: 414–21. 17 Kapur RP, Yost C, Palmiter RD. A transgenic model for studying development of the enteric nervous system in normal and aganglionic mice. Development 1992; 116: 167– 75. 18 Hao MM, Boesmans W, Van den Abbeel V, Jennings EA, Bornstein JC, Young HM, Vanden Berghe P. Early emergence of neural activity in the developing mouse enteric nervous system. J Neurosci 2011; 31: 15352– 61. 19 Vannucchi MG, Faussone-Pellegrini MS. Synapse formation during neuron differentiation: an in situ study of the myenteric plexus during murine embryonic life. J Comp Neurol 2000; 425: 369–81.
doi: 10.1111/nmo.12605
MeCP2 in the enteric nervous system G. WAHBA ,*
,#
S. C. SCHOCK ,†
,#
E. CLARIDGE ,* M. BETTOLLI ,‡ D. GRYNSPAN ,‡ P. HUMPHREYS ‡
& W. A. STAINES *
*Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada †Research Institute, Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada ‡Departments of Surgery, Pathology and Paediatrics (Neurology), Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada
Key Messages
• MeCP2 • • •
is expressed throughout development of the GI tract with appearance at or before E11.5 in mice, specifically in the enteric nervous tissue. This study aimed to investigate if MeCP2 is expressed in the human and murine ENS and at which stages of development. Immunofluorescence was performed on frozen sections of human intestinal tissue and dissociated murine enteric neuronal cultures. Western blots on intestinal dissections of developmental and adult mice confirmed MeCP2 expression. This study opens the possibility for treatment of the GI dysfunction in Rett syndrome with peripheral acting drugs and growth factors.
Abstract Background Rett syndrome (RTT) is an intellectual deficit and movement disorder that develops during early childhood in girls. Affected children are normal until 6–18 months of age, after which symptoms begin to appear. Most cases of RTT are due to mutations in the MeCP2 gene leading to disruption of neuronal communication in the central nervous system. In addition, RTT patients show peripheral ailments such as gastrointestinal (GI), respiratory, and cardiac dysfunction. The etiology of intestinal dysfunction in RTT is not well-understood. Reports on the presence of MeCP2 in the peripheral nervous system are scant. As such we examined the levels of MeCP2 in human and murine GI tissue and assessed MeCP2 expression
at various developmental stages. Methods Immunohistochemistry for MeCP2, HuC/D, juvenile beta tubulin, and GFAP was performed on human and murine intestine. Western blots of these same tissues were probed with MeCP2, vAChT, nNOS, and betaactin antibodies. Key Results MeCP2 is expressed throughout the GI tract. MeCP2 is expressed specifically in the enteric nervous system of the GI tract. MeCP2 is expressed in the GI tract throughout development with appearance beginning at or before E11.5 in the murine intestine. Conclusions & Inferences The proof of MeCP2 expression in enteric neurons suggests that the GI dysmotility in Rett may arise from enteric network dysfunction secondary to MeCP2 mutation. Keywords development, enteric nervous system, gastrointestinal dysmotility, gastrointestinal tract, MeCP2, myenteric plexus, Rett syndrome.
Address for Correspondence William A Staines, Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Rd, Ottawa ON, Canada K1H 8M5. Tel: +1-613-562-5800 (8188); fax: +1-613-562-5434; e-mail: [email protected] # These authors contributed equally and should be considered co-first author. Received: 29 January 2015 Accepted for publication: 29 April 2015
Abbreviations: ENS, enteric nervous system; GFAP, glial fibrillary acidic protein; GI, gastrointestinal; KO, knockout; LM, longitudinal muscle; MeCP2, methyl binding protein 2; MP, myenteric plexus; nNOS, neuronal nitric oxide synthase; PBS, phosphate buffered saline; RTT, Rett syndrome; SDS, sodium dodecyl sulphate; SI, small intestine; Tuj, juvenile beta tubulin; vAChT, vesicular acetylcholine transporter.
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INTRODUCTION
with special attention to the ENS cells considering MeCP2’s well-established role in neuronal maturation elsewhere.
Rett syndrome (RTT) is a neurological and developmental disorder affecting 1 in 13 000 girls.1 It is characterized by normal development until 6– 18 months preceding the onset of symptoms which include motor difficulties, severe mental retardation, and seizures.2 Rett syndrome is caused by de novo mutations in the gene coding for the methyl binding protein-2 (MeCP2),3 a mediator of synaptic development and plasticity.4 Methyl binding protein-2 levels are highest in the brain, where it is expressed in all regions at different developmental stages.5,6 During embryonic cortical development, MeCP2 protein is barely detectable but increases greatly postnatally.6 Only mature postmigratory neurons express MeCP2 and silencing the gene has no effect on migration or differentiation of neuroblasts.7 Increased expression of MeCP2 coincides with an important period of synaptogenesis in the brain.4 It has been suggested that MeCP2’s role resides in synaptogenesis and lifelong maintenance of synapses and neuronal networks.8 Rett syndrome patients do not show signs of neurodegeneration; however, neurons from MeCP2 knockout (KO) mice have fewer dendrites, abnormal dendritic spines, disorganized axons, and altered neurotransmitter levels.9–11 These findings all suggest that RTT is a disease of synaptic function. Apart from the well-studied neurological pathologies in RTT, general eating and nutrition problems also contribute significantly to its morbidity. In a large nationwide American study, 92% (n = 983) of parents of RTT patients reported that their child demonstrated some sort of GI dysmotility and 80% specifically reported constipation.12,13 Apart from constipation, dysmotility in the upper GI tract is also seen. Gastroesophageal dysmotility and gastrointestinal reflux disease are common as well.12,14 Thus, dysmotility is prevalent in RTT affecting the entire length of the GI tract. We speculate that MeCP2 associated enteric neuronal dysfunction may underlie some of the GI symptoms in RTT. To our knowledge no one has yet investigated the mechanistic basis of the GI symptoms in RTT. A previous study reported that protein levels of MeCP2 were high in the brain but present at negligible levels in the stomach and the intestines.5 We surmised that this might be due to a false negative due to sampling error because of the small proportion of enteric nervous system (ENS) cells relative to muscularis. As a result, we more closely examined the presence of MeCP2 in human and murine GI tissue
© 2015 John Wiley & Sons Ltd
METHODS Immunofluorescence on human frozen tissue Frozen formalin fixed human colonic, intestinal, and appendiceal sections were obtained from the Children’s Hospital of Eastern Ontario with REB approval. Small intestine (SI), colon, and appendix were sampled from a 16 year old female with ulcerative colitis, a 16 year old male with ulcerative colitis and a 13 year old female with appendicitis respectively. Sections were incubated with mouse anti-HuC/D (1 : 100; Invitrogen, Carlsbad, CA, USA) and rabbit anti-MeCP2 (1 : 200; Millipore, Darmstadt, Bundesland, Germany) overnight at 4 °C, and then rinsed with 10 mM PBS. Sections were then incubated with the appropriate secondary antibodies (1 : 200; Invitrogen) for 5 h at 37 °C and then rinsed with 10 mM PBS. All antibodies were diluted in 10 mM PBS containing 0.3% Triton-X. Sections were then imaged with a Zeiss Axio Observer D1 microscope (New York, NY, USA). The MeCP2 antibody is specific for this protein as evidenced by its adsorption by the immunizing peptide.15 We and others have shown staining to be abolished in complete MeCP2 KO mice.15 Furthermore, no staining was seen with any of the secondary antibodies in the absence of primary antibody.
Immunofluorescence on dissociated cultures All animal procedures were approved by the animal care facility at the University of Ottawa. Dissociated enteric neurons were obtained from 4 to 6 month old CD-1 mice by dissecting the myenteric plexus (MP) and the longitudinal muscle (LM) from the SI. Tissue was enzymatically dissociated by incubation at 37 °C in 1 mg/mL collagenase followed by 0.05% trypsin. Cultures were plated onto 10% matrigel (BD Biosciences, San Jose, CA, USA) and 33 lg/mL collagen (Enzo Life Sciences, Farmingdale, NY, USA) coated 96 well plates and grown in vitro for 4 days. Cells were then fixed in 4% paraformaldehyde for 20 min at room temperature. Anti-MeCP2 staining procedure was followed as described above. Cultures were then stained with mouse anti-juvenile beta tubulin (Tuj) antibody (1 : 25; gift from Dr. A. Frankfurter) following this same procedure but secondary incubation time was reduced to 30 min.
MeCP2 Western Blot Whole SIs from adult CD-1 mice (4–6 month old) were extracted and separated from the MP and LM as described above. The remaining SI wall and the myenteric/muscle strip were sonicated for 20 s in radioimmunoprecipitation assay buffer containing protease inhibitors. Sonicated tissue was incubated for 30 min on ice vortexing every 5 min. Tissue was centrifuged at 12 000 g at room temperature for 5 min to pellet cell debris. The protein concentration was assessed using a Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Following this, 15 lg of protein was added to 5 lL 49 SDS protein sample buffer and water and boiled for 5 min at 100 °C. The samples were electrophoresed on a 4% polyacrylamide gel and transferred onto a nitrocellulose membrane using an iBlotâ Gel Transfer Device (Invitrogen). The immunoblot was blocked overnight in 10% skim milk in
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(Fig. 1). Methyl binding protein-2 immunoreactivity was not expressed in all cell types present in the gut sections (Fig. 2) but rather showed selectivity. The MeCP2-positive cells were grouped together in discrete clusters corresponding to the myenteric and submucosa ganglia of the ENS. Methyl binding protein-2positive cells co-labeled with HuC/D, a neuronal marker, confirming the impression of selective expression of MeCP2 in the ENS. Dissociated mouse enteric neurons in culture also express MeCP2 (Fig. 3A). Methyl binding protein-2 immunoreactivity is strongest in Tuj-1 positive neurons.
Tris-based buffer saline with Tween (TBST). The immunoblot was then incubated for 1.5 h at room temperature with anti-beta actin antibody bound to horse radish peroxidase (1 : 10 000; Abcam, Cambridge, MA, USA), and then rinsed with TBST for 30 min. The immunoblot was exposed to a chemiluminescence reagent (Millipore) and visualized with X-ray film (Diamed, Mississauga, ON, Canada). The immunoblot was then stripped using Reblot Solution (Millipore) for 20 min at room temperature. Anti-MeCP2 antibody (1 : 1000; Millipore; expected band size of ~75 kDa) was applied overnight with shaking at 4 °C. Afterwards, the immunoblot was rinsed with TBST and horse radish peroxidase-bound anti-rabbit secondary antibody (1 : 5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied for 2 h with shaking at room temperature. The immunoblot was rinsed again and then exposed to a chemiluminescence reagent and visualized with Xray film. All antibodies were diluted in 10% non-fat milk in TBST. The blot was also probed with anti-nitric oxide synthase (NOS; 1 : 1000; Santa Cruz Biotechnology; expected band size of 155 kDa) and anti-vesicular acetylcholine transporter (VAChT; 1 : 1000; Synaptic Systems, Goettingen, Germany; expected band size of 70 kDa) as described above.
MeCP2 levels are proportional to neuronal content in lysates To confirm that MeCP2 is expressed in the neuronal component of the intestinal tract, western blot was performed on protein lysates from the SI after removal of the MP and on the MP itself. The MP lysate contains a higher concentration of neuronal protein than the SI in which the MP was removed. This lysate still has some neuronal content due to the presence of the submucosal plexus, a small neuronal layer, which was not removed during the dissection process. The western blot revealed a single band for MeCP2 at the expected protein weight of ~75 kDa. The lysate from the isolated MP showed higher levels of MeCP2 than the SI without the MP (but still containing the submucous neuronal plexus); this result shows that MeCP2 is in fact present in neurons and/or other nerve plexus cells in the GI. Methyl binding protein-2 levels correlated with neurochemical markers (nNOS and
MeCP2 protein levels change with age The western blotting method described above was repeated for protein extractions from the entire SI of embryonic and postnatal mice at ages indicated in the figure. Exposure time for MeCP2 antibody immunoreactivity was increased from 2 to 45 min in order to detect MeCP2 in whole GI lysates.
RESULTS MeCP2 is expressed throughout the GI tract, exclusively within the ENS The staining of human frozen gut tissue showed positive immunoreactivity for MeCP2 in all sections of the GI similar to that seen in the cerebral cortex
A
B
Figure 1 Localization of MeCP2 to mouse cerebral cortex. Image of the cingulate region of neocortical sections from MeCP2 knockout (A) and wild type (B) mice. MeCP2 (green) DAPI (blue).
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Figure 2 Localization of MeCP2 to human enteric neurons throughout the distal gastrointestinal tract. Inverted immunofluorescence on frozen human tissue sections from colon, small intestine and appendix, stained with anti-HuC/D, a neuronal marker, and anti-MeCP2; scale bar: 50 lm, magnification: 2009.
same was true for MeCP2 presence in the ENS. We dissected GI tissue from embryonic and postnatal mice and assessed MeCP2 protein levels by western blot. Methyl binding protein-2 was present and levels remained constant from the earliest embryonic time point investigated (Fig. 5).
VAChT) present in high levels in enteric neuronal tissue (Fig. 4). It should be noted that the MP lysate in Fig. 4 was not purely neuronal as during dissection the longitudinal muscle strongly adheres to the MP and is difficult to detach.
MeCP2 protein levels remain constant with age DISCUSSION
Since MeCP2 protein levels have been shown to increase throughout development in the brain in an area-specific manner, we sought to determine if the
A
Our main findings are: (i) MeCP2 is expressed throughout the GI tract, (ii) MeCP2 is expressed specifically in
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C
Figure 3 Nuclear localization of MeCP2 to cultured neurons from the mouse small intestine. (A) MeCP2 plus Tuj-1 double immunofluorescence in dissociated ENS cultures fixed at DIV4. (B) single anti-Tuj-1 (C) single anti-MeCP2. Scale bar: 50 lm.
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Previously, it has been reported that at E11.5, MeCP2 is only detectable in the spinal cord and hindbrain5 but not in other brain structures such as the cortex and hippocampus. It is thus important to note that along with these brain areas, the ENS develops at an early embryonic age. Neuroblasts migrate to the foregut at E9.5, demonstrate electric activity at E11.5, and form synapses as early as E12.5.17–19 Considering that MeCP2 expression in the brain coincides with important stages of synaptogenesis and maturity,4 it follows that MeCP2 protein can be found early in the ENS. It would be interesting to determine whether GI symptoms, which are prominent in RTT, are observed earlier than the onset of CNS symptoms, as MeCP2 may become functionally important in the ENS earlier than the CNS. We were unable to determine whether the ENS is MeCP2 positive prior to E11.5 due to impractical surgical access to the GI in embryos earlier than this date. These findings reveal the potential relevance of peripheral nervous MeCP2 distribution in the autonomic pathophysiology of RTT, and suggest that the GI pathology in RTT may be mediated through primary dysfunction of the ENS and not only due to dysfunction in descending CNS pathways or as a non-specific association of global development delay mediated through factors such as immobility and aerophagia. Further studies into the GI pathology of RTT will be required to elucidate the function of MeCP2 in these neurons and to determine the mechanisms by which its abrogation causes GI dysmotility. Our findings open the possibility of treatment of the GI dysfunction in this disorder with peripheral acting drugs and growth factors.
Figure 4 MeCP2 immunoreactivity coisolates with neuronal markers and is depleted in GI tissue upon removal of enteric neurons. Western blot analysis of MeCP2 protein levels in comparison to neurochemical markers (nNOS and VAChT) in lysates from mouse small intestine from which the MP has been removed and the isolated MP strip.
Figure 5 MeCP2 is expressed very early in enteric tissue E11.5 and maintains a constant level of expression through to P14. Western blot analysis of MeCP2 protein levels in GI lysates extracted from mice at different developmental points. The MeCP2 (75 kDa) bands for the lysates is shown on top with the beta-actin (41.7 kDa) bands shown on the bottom. This same pattern was repeated in five mice at each age.
the enteric nervous tissue, and (iii) MeCP2 is expressed throughout development in the GI tract with appearance at or before E11.5 in mice. Until now there has been little attention paid to MeCP2 expression in the gut. A past study indicated that MeCP2 protein levels are high in the brain but present at insignificant levels in the stomach and the intestines.5 However, in that study, western blots were performed on whole lysates of GI tissue. We found that expression of MeCP2 protein is limited to neuronal cells which are a small minority in the GI cell population. We speculate that perhaps in the previous study MeCP2 expressing neurons were too dilute to be detected in western blots. We investigated protein levels of MeCP2 and not RNA levels as MeCP2 has been shown to be strongly posttranscriptionally regulated; RNA levels have been reported to be equal in different tissues as opposed to protein levels which are tissue-specific.5 We have now demonstrated that MeCP2 protein is present within human and murine enteric neurons at levels that persist throughout postnatal development.
FUNDING This work was funded by the Jessica Carr Found and the CHEO Research Foundation.
DISCLOSURE No competing interests declared.
AUTHOR CONTRIBUTION Performed research: GW, SS; Designed research study: WS, SS, GW; Contributed essential reagents: MB, DG; Analyzed data: GW, WS, SS; Drafting of manuscript: GW, SS, PH, WS, DG; Supervised the study: PH and WS.
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