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Oligophrenin-1 a Rho GTPase-activating protein (RhoGAP) involved in X-linked mental retardation is expressed in the enteric nervous system.

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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: j.furness@unimelb.edu.au
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. This finding suggests that oligophrenin-1 and Rho family members have a
role in the modulation of neuronal connectivity within the
enteric nervous system, and in long-term changes in gastrointestinal function.
ACKNOWLEDGMENTS
We thank Daniel Poole for assistance in preparing images for publication, and Heather Robbins for assistance
with microscopy.
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