nat. neurosci.; doi:10.1038/nn.3725 leptin signaling in astrocytes … · 2014-06-25 · nature...
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nature neuroscience
co r r e c t i o n n ot i c e
Nat. Neurosci.; doi:10.1038/nn.3725
Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feedingJae Geun Kim, Shigetomo Suyama, Marco Koch, Sungho Jin, Pilar Argente-Arizon, Jesús Argente, Zhong-Wu Liu, Marcelo R Zimmer, Jin Kwon Jeong, Klara Szigeti-Buck, Yuanqing Gao, Cristina Garcia-Caceres, Chun-Xia Yi, Natalina Salmaso, Flora M Vaccarino, Julie Chowen, Sabrina Diano, Marcelo O Dietrich, Matthias H Tschöp & Tamas L Horvath
In the PDF version of the supplementary information initially published, Supplementary Figures 1a,b and 5a and the corresponding legends were missing. In the HTML version, the legends were present but the figure panels were missing. The errors have been corrected in the HTML and PDF versions of the supplementary information as of 10 June 2014.
Supplementary information Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feeding Jae Geun Kim1, Shigetomo Suyama1, Marco Koch1, 2, Sungho Jin1, Pilar Argente-‐Arizon3, Jesús Argente3 Zhong-‐Wu Liu1, Marcelo R. Zimmer1, Jin Kwon Jeong1,4, Klara Szigeti-‐Buck1, Yuanqing Gao5, Cristina Garcia-‐Caceres5, Chun-‐Xia Yi5, Natalina Salmaso6, Flora M. Vaccarino6,7, Julie Chowen3, Sabrina Diano1,4,7, Marcelo O Dietrich1, Matthias H. Tschöp5 and Tamas L. Horvath1,4,7 1Program in Integrative Cell Signaling and Neurobiology of Metabolism, Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut, USA. 2Institute of Anatomy, University of Leipzig, 04103 Leipzig, Germany. 3Hospital Infantil Universitario Niño Jesús, Department of Endocrinology, Instituto de Investigación La Princesa and Centro de Investigación Biomédica en Red de la Fistiopatología (CIBER) de Fisiopatología de Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain. 4Department of Obstetrics, Gynecology, and Reproductive Sciences, New Haven, Connecticut, USA. 5Institute for Diabetes and Obesity, Helmholtz Zentrum München & Technische Universität München, Germany. 6Child Study Center, Yale University School of Medicine, New Haven, Connecticut, USA. 7Department of Neurobiology, Yale University School of Medicine, Connecticut, USA.
Nature Neuroscience: doi:10.1038/nn.3725
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NeuNLepR
s100 β
Astrocytes in hypothalamus
AgRPLepR
s100 β
AgRP neuronsin hypothalamus
Enr
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a
MergedGFAP LepR
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+//+
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Supplementary figure 1. Presence of leptin receptors in hypothalamic astrocytes. (a) Double
fluorescence labeling of the astrocyte marker GFAP (red) and leptin receptors (leptin receptor-driven
expression of EGFP, green) shows co-localization of GFAP-immunolabeling with EGFP-tagged
leptin receptor-containing profiles (white arrows) in the arcuate nucleus (Arc). Scale bar = 100 μm.
(b) The truncated leptin receptor (exon 17) allele was confirmed by in situ hybridization combined
with immunohistochemistry. Red fluorescence indicates GFAP-positive astrocyte and green
fluorescence indicates POMC neurons. White dots indicate mRNA signals of the leptin receptor
containing exon 17. White arrows indicate cells expressing mRNA of leptin receptors. White
arrowheads indicate leptin receptor-negative cells. Scale bar = 30 μm. (c) To further confirm
astrocyte-specific expression of leptin receptors, transcript of leptin receptor b (LepRb) was
amplified from RNA bound to the ribosomes selectively in astrocytes isolated from AldH-EGFP-
L10a mice and in AgRP neurons isolated from AgRP-cre:floxed Rpl22HA mice. To check purification
and contamination of the samples, we analyzed enrichment of AgRP (marker for AgRP purification),
NeuN (Marker for neuronal cell contamination) and s100β (marker for astrocyte contamination)
compared to that of input samples. Purple bar graphs represent enrichment of LepRb in the AgRP
neurons or astrocytes of the hypothalamus compared to that of input controls.
Nature Neuroscience: doi:10.1038/nn.3725
loxPloxP
Exon 17Exon 16 18b18aLepr flox/flox
Cre ER Tam
Tamoxifen
Cre ERT2GFAP promoter Cre ERT2 Cre ERT2
off on
a
GFAP creERT2
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LadderLepR LepR +
GFAP cre
Tamoxifen
WTExon 17 Deleted
f/f f/f
Supplementary figure 2. Strategy of transgenic mice with selective impairment of leptin receptor
signaling in astrocyte. (a) Schematic drawing of floxed leptin receptor construct, which was used to
generate a mouse line allowing cell and time-specific knockout of leptin receptors in astrocytes. (b) RT-
PCR shows a deletion of exon 17 of the leptin receptor in primary astrocytes from GFAP-LepR KO mice.
Representative images were selected from at least 3 time repeated experiments.
Nature Neuroscience: doi:10.1038/nn.3725
Tamoxifen
Cre ERT2GFAP promoter Cre ERT2 Cre ERT2
off on
loxPloxP
STOP tdTomatoAi14 tdTomato
Cre ER Tam
GFAP creERT2
50 μm
GFAP tdTomato Merged
Iba-1 tdTomato Merged
NeuN tdTomato Merged
b
c
ROSA26
a
Supplementary figure 3. Verification of astrocyte-specific Cre-recombination. (a) Schematic
drawing of human GFAP-driven Cre promoter construct under control of estrogen receptors. (b) GFAP-
CreERT2 mice were crossed with tdTomato-loxP reporter mice to confirm successful Cre-mediated
recombination in GFAP-positive cells. (c) Ai14 tdTomato mice express red fluorescence in GFAP-
positive cells following cre-mediated recombination. tdTomato fluorescences were not co-localized with
signals of Iba-1, a biomarker for microglia or NeuN, a biomarker for neuron. White arrows indicate
double-labeled cells. Representative images were selected from at least 3 time repeated experiments.
Scale bar = 50 μm.
Nature Neuroscience: doi:10.1038/nn.3725
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Supplementary figure 4. Effect of astrocyte-specific deletion of leptin receptors on morphology of
astrocytes in the hippocampal CA3. (a) To confirm expression of leptin receptor in hippocampal
astrocytes, we performed ribosome profiling to isolated RNA bound to the ribosomes selectively from
hippocampal astrocytes and analyzed enrichment of LepRb. Purple bar graph represents enrichment of
LepRb in astrocytes of the hippocampus compared to that of input control. (b) Representative image of
GFAP-immunolabeling in the hippocampal CA3 of GFAP-LepR+/+ (+/+) or GFAP-LepR–/– (–/–) mice. (c
The number of GFAP-positive cells (n=5 slices for GFAP-LepR+/+ ; n=4 slices for GFAP-LepR–/–,
p=0.7150, t(7)=0.3803) and (d) length (n=8 cells for GFAP-LepR+/+; n=8 cells for GFAP-LepR–/–,
p>0.9999, t(14)=0.0) and (e) number (n=8 cells for GFAP-LepR+/+; n=8 cells for GFAP-LepR–/–, p=0.456
t(14)=0.7667) of astrocyte primary projections did not differ between GFAP-LepR+/+ and GFAP-LepR–/–
mice. Results are means ± the s.e.m. P values for unpaired comparisons were analyzed by two-tailed
Student’s t-test.
Nature Neuroscience: doi:10.1038/nn.3725
a
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_ / _+ /+
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***
***
c
Supplementary figure 5. Leptin receptor signaling in astrocytes affects contact formation
between astrocytes and melanocortin cells. (a) Representative electron micrograph showing
astrocyte coverage (green pseudo-color and blue arrows) onto POMC-labeled cells. POMC cells of
GFAP-LepR–/– mice had less coverage of their perikaryal membranes by astrocytic processes
compared to controls. Scale bar = 1 μm. Coronal sections labeled with GFP in AgRP or POMC cells
were incubated with GFAP antibody. (b, d) Representative pictures of double-labeled AgRP or
POMC-GFP and GFAP-positive cells in the Arc of GFAP-LepR+/+ or GFAP-LepR–/– mice.
Percentage of (c) POMC (n=7 slices for GFAP-LepR+/+; n=6 slices for GFAP-LepR–/–, p<0.0001,
t(11)=8.226) or (e) AgRP cells (n=8 slices for GFAP-LepR+/+; n=8 slices for GFAP-LepR–/–,
p=0.0004, t(14)=4.672) contacted with GFAP-fiber signals was reduced in GFAP-LepR–/– mice.
White arrows indicate cells interacted with astrocyte fibers. ***, p<0.001 versus GFAP-LepR+/+
mice. Results are means ± the s.e.m. P values for unpaired comparisons were analyzed by two-tailed
Student’s t-test. Scale bar = 100 μm.
+/+ _/_
Nature Neuroscience: doi:10.1038/nn.3725
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Supplementary figure 6. The probability distribution and average peak amplitude of miniature
postsynaptic currents on AgRP or POMC neurons. (a, b) Cummulative probability distribution and
average peak amplitude (inserts) of mIPSC or mEPSC onto AgRP neurons (Fig. 6a: n=9 cells for GFAP-
LepR+/+; n=9 cells for GFAP-LepR–/–, p=0.7715, t(798)=0.2906; Fig. 6b: n=9 cells for GFAP-LepR+/+;
n=9 cells for GFAP-LepR–/–, p=0.2788, t(798)=1.084). No-differences were observed between control
and GFAP-LepR–/– mice. (c) GFAP-LepR–/– mice showed a significantly increase in probability
distribution and average peak amplitude (inserts) of mIPSC onto POMC neurons (n=9 cells for GFAP-
LepR+/+; n=9 cells for GFAP-LepR–/–, p<0.0001, t(896)=7.604). (d) GFAP-LepR–/– mice revealed an
increase in probability distribution of mEPSC amplitude onto POMC neurons (n=23 cells for GFAP-
LepR+/+; n=25 cells for GFAP-LepR–/–, p<0.0001, t(3998)=5.255). **, p<0.01; ***, p<0.001 versus
GFAP-LepR+/+. Results are means ± the s.e.m. P values for unpaired comparisons were analyzed by two-
tailed Student’s t-test.
Nature Neuroscience: doi:10.1038/nn.3725
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Supplementary figure 7. Metabolic phenotypes of GFAP-LepR+/+ and GFAP-LepR–/– mice. Five-
week-old male mice were administrated tamoxifen (Tx) or vehicle (Veh) and then analyzed metabolic
phenotype at three-month-old age. GFAP-LepR–/– mice did not show significant changes of (a) body
weight (n=6 mice for GFAP-LepR+/+-veh; n=8 mice for GFAP-LepR+/+-tx; n=9 mice for GFAP-LepR–/–-
tx, p=0.3720, t(12)=0.9274 for GFAP-LepR+/+-veh versus GFAP-LepR+/+-tx; p=0.08, t(15)=1.874 for
GFAP-LepR+/+-tx versus GFAP-LepR–/–-tx), (b) fat mass (n=6 mice for GFAP-LepR+/+-veh; n=6 mice
for GFAP-LepR+/+-tx; n=5 mice for GFAP-LepR–/–-tx, p=0.588, t(10)=0.5630 for GFAP-LepR+/+-veh
versus GFAP-LepR+/+-tx; p=0.1974, t(9)=1.392 for GFAP-LepR+/+-tx versus GFAP-LepR–/–-tx) (c) lean
mass (n=6 mice for GFAP-LepR+/+-veh; n=6 mice for GFAP-LepR+/+-tx; n=5 mice for GFAP-LepR–/–-tx,
p=0.405, t(10)=0.8686 for GFAP-LepR+/+-veh versus GFAP-LepR+/+-tx; p=0.1410, t(9)=1.614 for
GFAP-LepR+/+-tx versus GFAP-LepR–/–-tx) (d) food intake (n=5 mice for GFAP-LepR+/+-veh; n=7 mice
for GFAP-LepR+/+-tx; n=7 mice for GFAP-LepR–/–-tx, p=0.8471, t(10)=0.1979 for GFAP-LepR+/+-veh
versus GFAP-LepR+/+-tx; p=0.6162, t(12)=0.05146 for GFAP-LepR+/+-tx versus GFAP-LepR–/–-tx) (e, f)
energy expenditure (n=8 mice for GFAP-LepR+/+; n=8 mice for GFAP-LepR–/–, p=0.8378, t(14)=0.2086
for light period; p=0.8641, t(14)=0.1743 for dark period; p=0.9966, t(14)=0.004295 for total) and (g, h)
physical activity (n=8 mice for GFAP-LepR+/+; n=8 mice for GFAP-LepR–/–, p=0.1184, t(14)=1.663 for
light period; p=0.2703, t(14)=1.148 for dark period; p=0.1692, t(14)=1.149 for total) when compared to
littermate control mice. Results are means ± the s.e.m. P values for unpaired comparisons were analyzed
by two-tailed Student’s t-test. Nature Neuroscience: doi:10.1038/nn.3725
a:leptin+/+
Merged
POMC-GFPFos
:leptin/_ _
b:fasted+/+
Merged
AgRP-GFPFos
:fasted/_ _
Supplementary fig. 8. Representative images of Fos-positive POMC or AgRP neurons in the arcuate of GFAP-LepR+/+ and GFAP-LepR- /- mice. (a) Representative
images show double labeled POMC-GFP and Fos cells in the Arc of GFAP-LepR+/+ and
GFAP-LepR- /- mice. Scale bar = 100 μm. (b) Representative images show double
labeled AgRP-GFP and Fos cells in the Arc of GFAP-LepR+/+ and GFAP-LepR- /- mice.
Scale bar = 100 μm.
Nature Neuroscience: doi:10.1038/nn.3725