Oligophrenin-1 a Rho GTPase-activating protein (RhoGAP) involved in X-linked mental retardation is expressed in the enteric nervous system.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 273A:671– 676 (2003) Oligophrenin-1, a Rho GTPaseActivating Protein (RhoGAP) Involved in X-Linked Mental Retardation, Is Expressed in the Enteric Nervous System JUNHUA XIAO, CRAIG B. NEYLON, BILLIE HUNNE, AND JOHN B. FURNESS* Department of Anatomy and Cell Biology, and Centre for Neuroscience, University of Melbourne, Parkville, Australia ABSTRACT Oligophrenin-1 is a RhoGTPase-activating protein (RhoGAP) that is involved in the regulation of shape changes in dendritic spines, and outgrowth of axons and dendrites in the brain. These changes in neuronal morphology are central to the mechanisms of plasticity, learning, and memory. Although the enteric nervous system also exhibits long-term changes in neuronal function, the expression and involvement of oligophrenin-1 has not previously been investigated. We show by RT-PCR analysis that oligophrenin-1 mRNA is expressed in the myenteric plexus (MP) of the guinea pig ileum. Sequencing of RT-PCR products showed that guinea pig oligophrenin-1 mRNA is 98% and 87% homologous to human and mouse oligophrenin-1, respectively, except that a 42 bp sequence is absent from the guinea pig mRNA. This 42 bp sequence codes for a sequence of 14 amino acids located near the carboxy-terminal end of the RhoGAP domain in the human sequence. An antibody that recognizes human oligophrenin-1 identiﬁed a 91 kDa protein band in rat and mouse brain lysates and in guinea pig sciatic nerve, and a 36 kDa protein band in both puriﬁed enteric ganglion cell and brain lysate from guinea pig. Oligophrenin-1 is localized speciﬁcally to neurons and varicose axons in the MPs and submucosal plexuses (SMPs) of the guinea pig and rat, but is not detectable in glial cells, smooth muscle, or other cell types. These ﬁndings indicate that oligophrenin-1 is expressed in the enteric nervous system, where it may regulate morphological changes in axons and dendrites, and thus modulate neuronal connectivity. Anat Rec Part A 273A:671– 676, 2003. © 2003 Wiley-Liss, Inc. Key words: oliogophrenin-1; enteric nervous system; neuronal plasticity; gene sequence; immunohistochemistry The enteric nervous system has structural and organizational features in common with the central nervous system (CNS). However, enteric neurons demonstrate considerably greater plasticity compared to central neurons (Gabella, 1990; Giaroni et al., 1999). The plasticity of the enteric nervous system is revealed after neuronal injury; for example, regrowth and reconnection of axons occur after transection and reanastomosis of the intestine (Sarna et al., 1983; Galligan et al., 1989). Enteric neuron plasticity can also be observed when there is no direct injury to neurons; for example, subsequent to a stenosis of the small intestine, enteric neurons double their crosssectional area in the following 3–5 weeks (Gabella, 1984). Uninjured enteric neurons can also produce collaterals that innervate sites vacated following section of the axons © 2003 WILEY-LISS, INC. of other neurons (Galligan et al., 1990). Changes in enteric neurons also occur in pathology; for example, hypertrophy of enteric neurons innervating the inﬂamed mucosa occurs in Crohn’s disease (Sharkey and Lomax, 2001). Altered *Correspondence to: Prof. John B. Furness, Dept. of Anatomy and Cell Biology, University of Melbourne, Parkville, VIC 3010, Australia. Fax: ⫹61-3-93475219. E-mail: firstname.lastname@example.org Received 13 January 2003; Accepted 10 March 2003 DOI 10.1002/ar.a.10072 672 XIAO ET AL. physiological properties of neurons have been reported in diseases of the human gastrointestinal tract and in animal models of gastrointestinal pathology (Holzer, 1998). Although changes in enteric neurons are well documented, particularly in development, after injury and in gastrointestinal diseases, the molecular mechanisms involved in remodeling of enteric neurons are not known. Oligophrenin-1 was ﬁrst discovered as one of a small number of genes in which a mutation causes nonsyndromic mental retardation (MR) in humans (Ramakers, 2000). It functions within the dendritic synapse to orchestrate changes in the actin cytoskeleton that are essential for directed neurite outgrowth and regulation of synaptic connectivity (Ramakers, 2000). Abnormalities in oligophrenin-1 are associated with a wide range of clinical neuropathies, including enlarged cerebral ventricles, cerebellar hypoplasia, seizures, MR, and ataxia (Tentler et al., 1999). It is also important in neuronal development (Billuart et al., 1998), and is up-regulated in brain tumors and tumor-adjacent tissue (Ljubimova et al., 2001). Oligophrenin-1 is a GTPase-activating protein (RhoGAP) that stimulates GTP hydrolysis of Rho family members (Billuart et al., 1998). Rho proteins are key signaling intermediates that link extracellular stimuli to dynamic changes in neuronal morphology (Luo et al., 1996; Luo, 2000). Oligophrenin-1 regulates the interaction of Rho proteins with the actin cytoskeleton (Hall, 1998). The clinical manifestations of abnormalities in oligophrenin-1 expression are well known in the CNS; however, its proposed involvement in synaptic organization suggests that it may have a crucial role in other neuronal systems. Molecules that regulate neuronal connectivity in the enteric nervous system are of considerable interest to investigators, because certain forms of MR are also associated with visceral control abnormalities (Lubs et al., 1996; Billuart et al., 2000). Moreover, modulation of excitability, caused by changes in the activity of neural pathways within the enteric nervous system, underlies gastrointestinal diseases and pathologies, including irritable bowel syndrome (IBS) and inﬂammatory bowel disease (IBD) (Giaroni et al., 1999). Thus, molecules that regulate synaptic transmission and plasticity in the CNS are also likely to play important and similar roles in the enteric nervous system. In the present study, we demonstrate that oligophrenin-1 is expressed in the enteric nervous system. Preparation of Enriched Enteric Ganglia We prepared the specimens by a method based on that of Jeitner et al. (1991). Segments of ileum were placed in ice-cold, sterile Dulbecco’s modiﬁed Eagle’s medium (DMEM; JRH Biosciences, Melbourne, Australia) containing 100 U/ml penicillin, 100 g/ml streptomycin, and 1 M nicardipine. External muscle (EM), including the myenteric plexus (MP), was dissected from the segments at cold temperature, and collected in sterile medium on ice. EM/MP preparations were digested at 37°C in collagenase type 1A (1 mg/ml, C-9891; Sigma) in a shaking water bath. After 15 min, the solution was vortexed and ﬁltered through a 400-m nylon mesh ﬁlter. The dissociated ganglia were then collected by passing the ﬁltrate through a 40-m nylon mesh ﬁlter membrane. Material that did not pass through the initial ﬁlter was subjected to further collagenase digestion cycles (15 min). Ganglia present on the 40-m ﬁlter were washed into Krebs or PBS solution and collected by centrifugation at 250 g for 5 min. RT-PCR of Oligophrenin-1 mRNA The longitudinal muscle (LM) and MP of the guinea pigs were separated by dissection, and total RNA was isolated from this tissue and the brain using the RNeasy Mini Kit (Qiagen, Sydney, Australia). RNA was reverse-transcribed using random oligonucleotide primers. One forward and two reverse primers based on the human oligophrenin-1 nucleotide sequence (Genbank accession number NM_002547) 5⬘ TG ATA AGA CAC TTG GTC AA 3⬘ (2151–2170 bp), 5⬘ GTC CTT TCT CGG AGG AA 3⬘ (2441– 2425 bp), and 5⬘ TGT CAG CAT CCC CCT CCT TG 3⬘ (2812–2793 bp) were used to amplify LM/MP cDNA. Samples were incubated at 95°C for 1 min, followed by 30 cycles of 30 sec at 95°C, 30 sec at 55°C, and 3 min at 72°C. A ﬁnal extension was performed for 10 min at 72°C. PCR fragments were puriﬁed with UltrafreeR-MC ﬁlters (Millipore, Sydney, Australia) and then cloned into pCR 4Blunt-TOPO vector (Invitrogen, Melbourne, Australia). The identity of clones was conﬁrmed by DNA sequencing using Big Dye terminator (Applied Biosystems, Foster City, CA). The sequences were analyzed using BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast). The predicted secondary structure of human oligophrenin-1 protein was determined using SMART (http://smart.embl-heidelberg. de/), BLAST-Conserved domain database (http://www.ncbi. nlm.nih.gov/Structure/cdd/wrpsb.cgi), and Prosite (http:// au.expasy.org/prosite/). Sequence alignment was performed using Pileup (GCG). MATERIALS AND METHODS Tissue and Animals Western Blotting The current experiments utilized male and female guinea pigs (200 – 400 g) and rats (240 g). Tissue samples were taken from animals that were killed by a blow to the head and cutting the carotid arteries and the spinal cord. All procedures were approved by the University of Melbourne Animal Experimentation Ethics Committee. Fresh segments of ileum were removed from each animal and placed in phosphate-buffered saline (PBS: 0.15 M NaCl in 0.01 M sodium phosphate buffer, pH 7.2) containing the L-type calcium channel blocker nicardipine (10– 6 M; Sigma, Sydney, Australia) to inhibit tissue contraction, at room temperature (RT). The pieces were opened along the mesenteric border and cleaned of contents by a PBS wash. Protein was extracted from isolated myenteric ganglia; guinea pig sciatic nerve; and rat, mouse, and guinea pig brain. Isolated ganglia were thawed and then sonicated on ice in lysis buffer (0.04% Triton X-100, 1 mM EGTA, 5 g/ml leupeptin, 30 mM Tris HCl pH 7.4, 50 M PMSF), and the lysates were frozen at –20°C until used. To prepare brain lysate, the dissected brains were placed in cold 1 mM PBS buffer and then transferred into modiﬁed RIPA buffer (50 mM Tris HCl at pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml pepstatin, 1 mM Na3VO4, 1 mM NaF), which was followed by homogenization on ice using a power control unit (Littau-Luzern, Switzerland) at setting 4 for 15 sec. The homogenate was centrifuged at OLIGOPHRENIN-1 IN THE ENTERIC NERVOUS SYSTEM 673 TABLE 1. Amino acid sequence alignment of predicted guinea pig (gp) oligophrenin-1 and human (hu) oligophrenin-1 (NP_002538) sequences* gp 1 hu 513 gp 51 hu 563 HSKENLMTPSNMGVIFGPTLMRAQEDTVAAMMNIKFQNIVVEILIEHFDK 50 : HSKENLMTPSNMGVIFGPTLMRAQEDTVAAMMNIKFQNIVVEILIEHFGK 562 䡠 䡠 䡠 IYLGPPEES-----------------------------HKPITISKRLLRERT 74 : : : : : : : : : : : : : : IYLGPPEESAAPPVPPPRVTARRHKPITISKRLLRERT 600 *The region covered includes the C-terminal end of the Rho-GTPase-activating domain (human RhoGAP ⫽ aa513-600). Dashes in the guinea pig sequence represent amino acids present in the human sequence but not in guinea pig. 12,000 g at 4°C for 5 min, and the supernatant was taken for immunoblotting. Protein concentration was measured using the BioRad (Sydney, Australia) protein assay. Equal amounts of proteins (10 g) were separated on a 10% SDS-polyacrylamide gel. Molecular weights were determined using prestained protein standards (BioRad). Proteins were transferred overnight at RT onto polyvinylidene diﬂuoride-based membranes (Hydrond-P; Amersham, Australia) followed by 1 hr of blocking (5% skim milk in PBS plus 0.1% Tween20 (PBS-T)). Blots were then incubated with anti-human oligophrenin-1 C-terminus antibody (diluted 1:1000; Santa Cruz, CA) for 1 hr at RT, followed by extensive washing in PBS-T. After incubation with secondary antibody (HRP-conjugated rabbit antisheep antibody, 1:1000 dilution; DAKO, Carpinteria, CA) and washing in PBS-T, immunoreactive bands were detected with enhanced chemiluminescence (ECL, Amersham, Melbourne, Australia). Immunohistochemistry Segments of ileum were pinned tautly, mucosa side down, onto a balsa-wood board, and ﬁxed overnight at 4°C in 2% formaldehyde plus 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.0. Tissue was cleared of ﬁxative with 3 ⫻ 10-min washes in dimethylsulphoxide followed by 3 ⫻ 10-min washes in PBS. Tissue was stored at 4°C in PBS containing sodium azide (0.1%). Fixed tissue was dissected into LM/MP and submucosal plexus (SMP) wholemounts. Wholemount preparations were preincubated in 10% normal horse serum in PBS containing 1% Triton X-100 for 30 min at room temperature. Oligophrenin-1 antiserum (Santa Cruz) was diluted (1:100) in 1.8% NaCl in 0.01 M sodium phosphate buffer, pH 7.2, containing 0.1% sodium azide. Double-labeling was achieved using a combination of anti-oligophrenin-1 and anti-Hu antibodies. Following incubation in primary antibodies for 2–3 nights at 4°C in a humid chamber, the preparations were rinsed in PBS and then incubated for 1 hr at RT with the appropriate secondary antibody. Tissue was mounted in glycerol buffered with 0.5 M sodium carbonate (pH 8.6). The Hu antibody was used as a marker of all enteric neurons (Lin et al., 2002). Preparations were examined on a Zeiss Axioplan microscope equipped with the appropriate ﬁlters, and images were taken with a SpotRT cooled charge-coupled device camera and SpotRT 3.2 software (Diagnostic Instruments, Sterling Heights, MI). The proportions of neurons in which antigen immunoreactivity was colocalized were determined by examining ﬂuorescently-labeled, double-stained preparations. The percentages of anti-Hu-immunoreactive neurons that were also oligophrenin-immunoreactive were calculated. RESULTS To determine whether oligophrenin-1 mRNA was expressed in the enteric nervous system, we performed RTPCR on total RNA prepared from guinea pig LM/MP. Using primers based on the human oligophrenin-1 sequence (Billuart et al., 1998), we ampliﬁed two PCR fragments (300 bp and 660 bp). Identically-sized bands were ampliﬁed from brain RNA, indicating that they corresponded to human oligophrenin-1. The identity of the ampliﬁed fragments was conﬁrmed by cloning into pCR 4Blunt-TOPO vector and DNA sequencing, which was followed by determination of the predicted amino acid sequence (Table 1). The nucleotide sequence was 98% identical to the corresponding human oligophrenin-1 sequence (accession number NM_002547) and 87% identical to mouse oligophrenin-1 (accession number NM_052976), except that a 42 bp sequence is absent from the guinea pig mRNA. The ampliﬁed sequence is contained within the coding region and corresponds to residues 513– 600 of human oligophrenin-1. Interestingly, guinea pig oligophrenin-1 protein lacks a sequence of 14 amino acids (AAPPVPPPRVTARR), which is located near the carboxy end of the RhoGAP domain in the human sequence (Table 1). This 14 amino acid sequence is within an exon encoding 49 amino acids of the human oligophrenin gene, which suggests that the omission is not due to exon skipping. To examine whether oligophrenin-1 protein is expressed in the enteric nervous system, we performed Western analysis on brain and myenteric ganglion lysates. Antibody raised against the C-terminal 19 amino acids of the human sequence identiﬁed a single band of molecular weight 91 kDa in mouse and rat brain cell lysates, conﬁrming the speciﬁcity of the antibody. The antibody recognized a single band in guinea pig brain and myenteric ganglion lysates, which was of lower molecular weight (36 kDa) than that in the mouse and rat brains (Fig. 1B). However, an immunoreactive 91 kDa protein was found in extracts of guinea-pig sciatic nerve, in which immunohistochemistry revealed oligophrenin-1 immunoreactivity in myelin-forming glial cells. Immunohistochemical analysis using the anti-human oligophrenin-1 antibody revealed immunoreactivity in nerve cell bodies in the ileum of guinea-pigs and rats (Fig. 1C), but detectable immunoreactivity was absent from glial cells, smooth muscle, and other cell types. In guineapig tissue dissected at room temperature, oligophrenin-1 immunoreactivity was observed in both the nucleus and cytoplasm of myenteric neurons. Oligophrenin-1 immunoreactivity was also found in a small proportion of varicose axons of the MP (Fig. 1Ca). The origins of these varicose 674 XIAO ET AL. Fig. 1. Evidence for oligophrenin-1 expression in the enteric nervous system. A: RT-PCR products from guinea pig brain and LM/MP of guinea pig ileum. Products of 300 bp (band 1) and 660 bp (band 2) were obtained using combinations of one forward primer and two reverse primers. No bands were observed when reverse transcriptase was omitted or when the RNA was replaced by water (not shown). B: Western blots of protein extracts from rat, mouse, and guinea pig brain lysates and guinea pig enteric ganglion protein. Protein (10 g) was boiled and then run on SDS-PAGE under reduced conditions. Blots were probed with anti-human oligophrenin-1 (C-terminus) antibody. A protein with molecular weight of 91 kDa was detected in rat and mouse brain lysates, whereas a protein of lower molecular weight (⬃36 kDa) was observed in guinea pig brain and ganglion lysates. C: Oligophrenin-1 immunoreactivity in guinea pig and rat ileum. Immunoreactivity was observed in neurons and axons of MP and SMP ganglia. a: Oligophrenin-1 in guinea pig MP; arrow indicates axon. b: Oligophrenin-1 in rat MP. c and d: Double staining of oligophrenin-1 (c) and anti Hu antibody (d) in guinea pig SMP. Bars: 50 m. ﬁbres have not been determined. In rats, immunoreactivity was detected in the cytoplasm of neurons with no evidence for nuclear immunoreactivity (Fig. 1Cb). Oligophrenin has a nuclear localization signal, but why it was detectable in the nuclei of guinea-pig but not rat enteric neurons is not known. To estimate the proportion of neurons containing oligophrenin-1 immunoreactivity in guinea-pig MP and SMP ganglia, double-labeling studies were performed using anti-Hu antibody, which labels all neurons in the enteric plexuses. Oligophrenin-1 immunoreactivity was found in about 78% and 82% of neurons in the guinea pig MPs and SMPs, respectively (Fig. 1Cc and d). Oligophrenin-1 is an important signaling molecule that regulates dendritic shape changes at synapses in the brain. However, its expression in neurons outside the CNS has not been previously described. In the present study, we report that oligophrenin-1 is expressed in the guineapig and rat enteric nervous systems. A ⬃660 bp RT-PCR product ampliﬁed from guinea-pig MP is almost identical to the human brain cDNA sequence, except for the absence of a region coding for a 14 amino acid proline-rich sequence near the carboxy-terminal end of the RhoGAP DISCUSSION OLIGOPHRENIN-1 IN THE ENTERIC NERVOUS SYSTEM domain. Antibody raised against the C-terminus of the human protein detects a band of lower molecular weight in the guinea-pig enteric nervous system and brain, indicating the possibility of a different isoform in this species. We also show that oligophrenin-1 is expressed in most neurons of the guinea-pig and rat MP. Given the putative role of oligophrenin-1 in learning and memory in the brain, these results indicate that oligophrenin-1 may play an important role in plasticity of the enteric nervous system, and perhaps in other peripheral neurons. Some indication of its role might be obtained from investigation of human enteric nervous system disorders and from genetically engineered mice. Oligophrenin-1 was ﬁrst identiﬁed as one of seven genes that cause nonspeciﬁc X-linked MR (Billuart et al., 1998; Tentler et al., 1999; Ramakers, 2000). This form of MR is associated with abnormalities in dendrites and dendritic spines resulting in impairments in synaptic transmission and information processing. This is consistent with the identiﬁcation of oligophrenin-1 as a RhoGAP. Oligophrenin-1 stimulates the GTPase activity of the Rho family members RhoA, Rac1, and Cdc42, in vitro (Billuart et al., 1998). Rho proteins are signaling molecules that regulate the actin cytoskeleton and are therefore involved in neuronal migration, dendrite outgrowth, and axon guidance (Luo et al., 1994; Threadgill et al., 1997). Rho-GTPases are also key regulators of synaptic connectivity. Our ﬁnding that oligophrenin-1 is found in nerve cell bodies in the guinea-pig and rat small intestine, but is undetectable in glial cells, smooth muscle, and other cell types, indicates that it is a speciﬁc neuronal protein present in both central and peripheral neurons. It is likely, therefore, that oligophrenin-1 is involved in modulating dendritic shape and altering connectivity at both central and peripheral synapses. The results also raise the possibility that aberrant Rho signaling may underlie some forms of gastrointestinal dysfunction. It is interesting that oligophrenin-1 expression is high in areas of the CNS surrounding glioblastomas—regions in which considerable remodeling can be expected (Ljubimova et al., 2001). Oligophrenin-1 is also overexpressed in colorectal tumors, where there is also extensive tissue reorganization (Pinheiro et al., 2001). The axonal expression of oligophrenin-1 in the MP is consistent with a role of RhoGAPs in axonal outgrowth and remodeling (Luo et al., 1994, 1996; Zipkin et al., 1997). Such processes occur during development, in gastrointestinal (GI) diseases and in recovery after GI injury (Sharkey and Lomax, 2001), although the molecular processes that underlie remodeling in the enteric nervous system are not well understood (Giaroni et al., 1999). Given its critical role in development and in the CNS, oligophrenin-1 may be involved in axon remodeling in the enteric nervous system. The existence of different isoforms of the oligophrenin-1 protein is suggested by the different molecular weights observed in Western blots: 91 kDa in rat and mouse brain and guinea-pig sciatic nerve, and 36 kDa in guinea-pig brain and enteric nervous system. A search of the sequence databases (BLAST) indicated that there is no other mammalian gene that includes the sequence against which the antiserum was raised. Thus, the 36 kDa protein appears to be a genuine isoform of oligophrenin-1. The antibody used in Western analysis was raised against the carboxy-terminus of human oligophrenin-1; our data indicate that it also recognizes guinea pig, rat, and mouse 675 homologues. The omission of a 14 amino acid proline-rich segment from the guinea-pig sequence may have functional consequences with respect to the ability of oligophrenin-1 to interact with other factors. Despite the observed differences, the oligophrenin-1 sequence appears to be highly conserved across species (human, rat, mouse, and guinea-pig), consistent with its important role in neuronal function. In conclusion, our results demonstrate that oligophrenin-1, a gene thought to be involved in structural development and modulation of neuronal shape in the CNS, is expressed in the enteric nervous system. 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