Defining the role of glia in the
enteric nervous system
Meenakshi Rao, MD, PhD
Columbia University
October 9, 2015
Disclosures
No relevant conflicts to disclose.
Enteric nervous
system (ENS)
• Can function
independently of CNS
• Regulates:
Gastrointestinal motility
Secretion
Mucosal maintenance
Mucosal immunity
Hormonal secretion
Neurons.Glia
1
Enteric glia are essential for GI function
• Chemical ablation (HSV‐Tk + ganciclovir)
–
–
–
–
Severe jejunoileitis
Altered epithelial proliferation
Altered epithelial barrier
Neuronal degeneration
Bush et. al., Cell, 1998.
• Immune‐mediated targeting of enteric glia
These methods all targeted GFAP‐expressing cells.
Proteolipid protein 1 (PLP1) is a new marker
of enteric glia
Rao, M., et al. Glia, 2015.
Genetic model for conditional ablation of
enteric glia
Rosa26
LoxP
STOP
LoxP
DTA
Rosa26
Cre recombinase
Rosa26
LoxP
DTA
Rosa26
PLPCreERT: Expresses tamoxifen‐inducible Cre
recombinase within PLP1‐expressing cells
Experimental groups:
Cre+, Tamoxifen
Cre‐, Tamoxifen
2
Cre ‐
Cre +
Crypts
S100+ cells/0.1mm2
p < 0.001
300
200
100
0
Cre-
Cre+
Villi
Ablation of PLP1+ glia does NOT result in
intestinal inflammation
How does loss of enteric glia affect
gastrointestinal function?
• Intestinal‐epithelial barrier: No effect
• Epithelial proliferation & repair: No effect
• Enteric neurons
Survival: No effect
Function: ?
3
GI Transit Time (min)
Enteric glial loss alters neuronal function
250
p = 0.07
200
150
100
50
0
15
15
Cre-
Cre+
Females
n.s.
GI Transit Time (min)
GI Transit Time (min)
Males
250
200
150
100
50
0
8
8
Cre-
Cre+
250
p = 0.02
200
150
100
50
0
7
7
Cre-
Cre+
Enteric glial loss does not alter UGI transit
n.s.
80
60
40
20
0
Cre-
Females
Gastric Emptying (%)
Gastric
Emptying
Gastric Emptying (%)
Males
100
Cre+
8
6
n.s.
4
2
0
80
60
40
20
0
Cre-
Cre+
10
Cre-
Geometric Center
Small Intestinal
Transit
Geometric Center
10
n.s.
100
8
6
2
0
Cre+
n.s.
4
Cre-
Cre+
Analyzing colonic motility ex vivo
Females
CMMC
15
10
5
0
Cre-
p = 0.03
Cre-
Cre+
Cre+
4
Summary
• Enteric glia widely express PLP1
• Conditional expression of DTA in PLP1‐
expressing cells is a robust, non‐inflammatory
model of enteric glial ablation
• Enteric glia play a sexually dimorphic role in
colonic motility
Does this underlie sex differences in
functional disorders?
Thanks
Michael Gershon, M.D.
NASPGHAN Foundation
Gabriel Corfas, Ph.D.
NIH (NIDDK)
Svetlana Sabel, M.D.
Daniella Rastelli
Lauren Dong
Sophia Chiu
Milton Fund
Glaxo Smith Kline
CUMC Dept. of Pediatrics
BCH Div. of Pediatric GI
Wanda Setlik
Bradlee Nelms
Michael Rutlin, Ph.D.
5
Glial ablation alters colonic motility in a
sex‐dependent manner
Males
Females
15
15
p = 0.03
5
0
Speed (mm/sec)
2.5
Contraction
Speed
CMMC
10
5
Cre+
p = n.s.
2.0
1.5
1.0
0.5
0.0
10
0
Cre-
Cre-
Cre+
Cre-
Cre+
2.5
Speed (mm/sec)
Contraction
Frequency
CMMC
p = n.s.
2.0
p = n.s.
1.5
1.0
0.5
0.0
Cre-
Cre+
Immuno‐EM confirms loss of S100β+ enteric
glia
Cre ‐
Cre +
S100β
PLP1
S100β.PLP1
GFAP
PLP1
GFAP.PLP1
PLP1 only
GFAP only
PLP1 & GFAP
6
GFAP HSV‐Tk mice express Tk protein
in epithelial cells
GFAP
HSV-Tk
GFAP.HSV-Tk
Enteric glial ablation does not alter
epithelial barrier penetration
15000
Fluorescein (ng/mL)
15000
FITC (ng/mL)
10000
5000
10000
5000
n.s.
n.s.
0
0
DSS
Cre-
Cre+
M.W. 4400 Da
Cre-
Cre+
M.W. 478 Da
Enteric glial ablation does not alter
bacterial penetration
n.s.
10
16S qPCR
Mesenteric
Lymph Nodes
CT
8
6
4
2
0
Cre-
Cre+
7
n.s.
10
6
n.s.
4
2
0
Cre-
Cre+
Duodenum
n.s.
30
8
Cre-
Cre+
Ileum
Ki67+ Cells/Crypt
Villus to Crypt Ratio
Glial ablation does not affect epithelial
proliferation
n.s.
20
10
0
Cre-
Cre+
Duodenum
Cre-
Cre+
Ileum
8