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Neuron, Volume 85
Supplemental Information
NFB-Activated Astroglial Release
of Complement C3 Compromises Neuronal Morphology
and Function Associated with Alzheimer’s Disease
Hong Lian, Li Yang, Allysa Cole, Lu Sun, Angie C.-A. Chiang, Stephanie W. Fowler,
David J. Shim, Jennifer Rodriguez-Rivera, Giulio Taglialatela, Joanna L. Jankowsky,
Hui-Chen Lu, and Hui Zheng
Lian et al.
Inventory of Supplementary Information
•
Supplemental Figures, Figure Legends, Table and Videos
o Figure S1, related to Figure 1
o Figure S2, related to Figure 2
o Figure S3, related to Figure 3
o Figure S4, related to Figure 4
o Figure S5, related to Figure 6
o Table S1, related to Experimental Procedures and Figure 7
o Video S1, related to Experimental Procedures and Figure 3
o Video S2, related to Figure 5
•
Supplemental Experimental Procedures
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Figure S1, related to Figure 1. Characterization of the NFκB and complement pathway in IκBα mutants
(A) Microarray analysis of 3 month-old brain IκBα conditional knockout (NcKO) mice and littermate
controls. Data was processed by Bio-conductor. Green indicates downregulated genes and red
indicates upregulated genes. Threshold was set as 1.5 fold difference and P < 0.0001 (t-test). (B)
Comparison of IκBα mRNA expression in primary neuronal or astroglial cultures under basal (TNFα -) or
TNFα stimulated (TNFα +) conditions. IκBα knockout (KO) cells were used as a negative control. (C)
Breeding scheme for generating brain (NcKO), forebrain neurons (CcKO) or astrocyte (GcKO) IκBα
conditional knockout mice using Nestin-Cre, CamKIIα-Cre or GFAP-Cre lines respectively. (D) qPCR
analysis of IκBα mRNA levels in NcKO and GcKO mice relative to wild-type controls (Ctrl). (E) Western
blot analysis of IκBα protein expression in GcKO mice. (F) Representative Nissl images of GcKO and Ctrl
brain slices showing indistinguishable brain morphology between the two groups. Scale bar: 1 mm. (G)
Location of the two putative κB sites (κB-1, κB-2) in the C3 promoter. (H) ChIP with anti-p65 antibody
(p65) followed by PCR of the two κB sites in the presence (+) or absence (-) of TNFα. Anti-IgG (IgG) and
anti-H3 (H3) were used as negative and positive controls respectively. (I and J) qPCR results of neuronal
or astroglial C3 mRNA expression normalized to hypoxanthine guanine phosphoribosyl transferase 1
(Hprt1, panel I) and phosphoglycerate kinase 1 (Pgk1, panel J). (K and L) mRNA expression of
complement proteins C1q, C4, Cfh and Cfb in primary astroglia (K) or in mouse hippocampi (L). C1q and
C4 are required for the classical complement activation pathway; Cfh and Cfb are involved in the
alternative complement pathway. All assays were done using 2- to 3-month-old mice. N = 4/group for
(A); N=3 per group per experiment for others. Data is shown as mean ± SEM. B: Three-way ANOVA
followed by planned means comparison. D: One-way ANOVA followed by Bonferroni post-hoc analysis.
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I and J: Two-way ANOVA followed by pairwise comparison. K and L: Student’s t-test; *P < 0.05; **P <
0.01; ***P < 0.001; NS: non-significant.
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Figure S2, related to Figure 2. Characterization of neuron-astroglia co-cultures and C3 effect
(A) Representative staining of the microglia marker Iba1 and neuronal marker NeuN in co-cultured
neurons. (B) Double staining of the microglia marker Iba1 and astroglia marker GFAP in co-cultured
astroglia grown in insert. (C) Quantification of the percentage of microglia in neurons or astroglia. N=16
and 15 random fields for neuronal and astroglial cultures respectively. (D) Double-immunostaining of
wild-type or IκBα KO astroglia (WTA or KOA) co-cultured neurons with anti-VGAT and anti-MAP2
antibodies. Images underneath each panel are enlarged view of the bracketed areas. Quantification of
density of VGAT+MAP2+ inhibitory synapses is shown in Figure 2C. (E) Representative dendritic
structures of wild-type neuronal monocultures (WTN) incubated with conditioned media derived from
wild-type (WTCM) or IκBα KO (KOCM) astroglial cultures. (F) Quantification of dendritic complexity of
WTCM or KOCM treated WT neurons. NWTCM= 34; NKOCM=31. (G) C3 levels in WTCM or KOCM astroglial
conditioned media following immunodepletion with anti-GFP or anti-C3 antibodies. N=4 per group. (H)
Representative dendritic structures of wild-type neurons incubated with conditioned media derived
from WTCM or KOCM astroglial cultures with GFP or C3 immunodepletion; (I) Quantification of
dendritic complexity of (H). NWTCM GFP=37; NWTCM C3=40; NKOCM GFP=35; NKOCM C3=33. Scale bar: 100 μm in
(A, B), 10 μm in (D), and 20 μm in (E, H). Data is displayed as mean ± SEM. F: Two-way ANOVA. G: Twoway ANOVA followed by planned means comparison. I: Three-way ANOVA followed by Bonferroni
post-hoc analysis. *P < 0.05; **P < 0.01.
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Figure S3, related to Figure 3. Reduced spine density in GcKO mice
(A) Total spine density in Ctrl and GcKO mouse brains. (B) Density of each of the four spine types in Ctrl
and GcKO mouse brains. (C) Frequency of each spine types. Data is displayed as mean ± SEM. Student’s
t-test; *P < 0.05; **P < 0.01. This figure is the same as Figure 3 except that N represents the number of
mice instead of dendritic segments (N=3). Means for each mouse were generated and used for
statistical analysis.
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Figure S4, related to Figure 4. Neuronal C3aR mediates the effect of astroglia NFκB activation
(A) Representative dendritic morphology of wild-type neurons co-cultured with IκBα KO astroglia (KOA)
and treated with either vehicle (DMSO), 1μM C3aRA, or 1μM C5aRA. (B) Sholl analysis of KOA neurons
treated with DMSO or C3aRA at 1, 2, or 5 μM. NDMSO=31; N1μm=28; N2μM=31; N5μM=30. (C) Sholl analysis
of KOA neurons treated with DMSO or C5aRA at dosages of 0.1, 0.5, or 1 μM. NDMSO=30; N0.1μm=30;
N0.5μM=34; N1μM=30. (D) Representative VGluT1- and MAP2-double staining images of wild-type or IκBα
KO astroglia (WTA or KOA) co-cultured neurons treated with DMSO or C3aRA. Images underneath each
panel are enlarged view of the bracketed areas. (E) Quantification of number of VGluT1+MAP2+
synaptic puncta per 10 μm of dendrite in WTA and KOA co-cultured neurons treated with DMSO or
C3aRA. NWTA DMSO=64; NKOA DMSO=60; NWTA C3aRA=61; NKOA C3aRA=57. (F) Relative spine density of Ctrl or
GcKO mice injected with DMSO or C3aRA. Panel (F) is the same as Figure 4F except that N represents
the number of mice instead of dendritic segments (N=3). Scale bar: 20 μm in (A) and 10 μm in (D). B
and C: Two-way ANOVA followed by Bonferroni post-hoc analysis. E and F: Two-way ANOVA followed
by pairwise comparisons. *P < 0.05; **P < 0.01.
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Figure S5, related to Figure 6. Activated NFκB/C3 pathway does not affect total GluR1 expression
(A) Representative images of WT or IκBα KO astroglia (WTA or KOA) co-cultured neurons stained
against total GluR1 and Syn. Inset: Enlarged images of the bracketed areas. (B) Quantification of
relative fluorescence intensity of total GluR1 in Syn+ puncta. NWTA=53 000; NKOA=34 000. (C) Western
blots of GluR1 in Ctrl and GcKO cortical protein lysates. γ-tubulin was used as a loading control. (D)
Quantification of (C). (E) Blots of total GluR1 in WT or IκBα KO astroglia co-cultured neurons. (F)
Quantification of (E). (G) Kinetic changes of surface GluR1 in response to C3aRA treatment. The bar
graphs are relative surface GluR1 fluorescence intensity of wild-type neurons co-cultured with either
WTA or KOA) and treated with 1 μM of C3aRA for 15 min, 30 min, 1 hour or 2 hours. DMSO: DMSO
treatment 15 min. NDMSO WTA=44; NDMSO KOA=41; NC3aRA WTA 15min=41; NC3aRA KOA 15min=43; NC3aRA WTA 30min=36;
NC3aRA KOA 30min=39; NC3aRA WTA 1hr=37; NC3aRA KOA 1hr=39; NC3aRA WTA 2hr=35; NC3aRA KOA 2hr=40. B, D, and F: ttest; G: Three-way ANOVA followed by planned means comparison. ***P < 0.001; NS: non-significant.
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Table S1. Demographics of AD patients and control subjects
Case #
986
1013
767
785
1008
1029
1229
Diagnosis
Control
Control
Control
Control
Control
Control
Control
Brain Area
Frontal Cortex
Frontal Cortex
Frontal Cortex
Frontal Cortex
Frontal Cortex
Frontal Cortex
Frontal Cortex
Age
83.1
>89
86
82.7
77.4
73
>89
PMI
2
6
8
13
12
4
12
Braak
1
1
2
1
0
0
2
Plaque
1
4
4
4
4
4
3
1673
1811
1816
1756
1749
1776
1777
1827
AD
AD
AD
AD
AD
AD
AD
AD
Frontal Cortex
Frontal Cortex
Frontal Cortex
Frontal Cortex
Frontal Cortex
Frontal Cortex
Frontal Cortex
Frontal Cortex
81
>89
75
68
79
>89
67
>89
5.5
18
22
11.5
6
6.25
20.5
5
6
6
6
6
6
6
6
6
2
2
1
1
1
2
1
2
Table S1, related to Figure 7. Braak scores of 0 to 6 indicate the severity of tangle pathology from none
to most severe. Plaque load is scored from 1 to 4 with 1 representing the highest plaque load. PMI:
post-mortem interval.
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Video S1, related to Experimental Procedures and Figure 3. 3D view of dendritic spines from a GFPpositive neuron in the cortical region of AAV-GFP injected mice.
Video S2, related to Figure 5. A 2 min recording with 10 sec intervals of AAV-GCaMP6s-infected
hippocampal slice cultures from Ctrl and GcKO mice showing enhanced GCaMP baseline fluorescence
in the GcKO slice compared to the controls.
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Supplemental Experimental Procedures
Mouse breeding
Mice were housed 2–5 per cage with ad libitum access to food and water in a room with a 12 hr
light/dark cycle in a specific pathogen free mouse facility. All procedures were performed in
accordance with NIH guidelines and with the approval of the Baylor College of Medicine Institutional
Animal Care and Use Committee. The Nestin-Cre (Tronche et al., 1999), C3aR-/- (Humbles et al., 2000),
IκBα+/- (Beg et al., 1995), the tet-activator (TTA) line CamKIIα-TTA (Mayford et al., 1996), and the tetresponsive APP transgenic line tetO-APPswe/ind line 102 (Jankowsky et al., 2005) are available from
the Jackson Laboratory. The GFAP-Cre transgenic mice (Bajenaru et al., 2002) were obtained from
National Cancer Institute Mouse repository. The CamKIIα-Cre mice (Dragatsis and Zeitlin, 2000) were
provided by Dr. Mauro Costa-Mattioli of Baylor College of Medicine, and the IκBαfl/+ mice were
obtained from Dr. Rudolf Rupec (University of Munich, Germany) (Rupec et al., 2005). The C3aR
knockout mice are on 129S4 background. All other lines have been backcrossed to C57BL/6J
background for a minimum of 10 generations. The double transgenic APP/TTA males were outcrossed
to FVB to generate F1 offspring for behavioral testing. Genotyping was performed by PCR of tail DNA
one week before weaning.
Primary cell culture and neuron-astroglia coculture
Cortices and hippocampi were isolated from new born pups in dissection medium (HBSS with 10 mM
HEPES, 0.6% glucose, 1% v/v Pen/Strep) and cut into small pieces. Tissue was digested with 2.5%
trypsin at 37°C for 15 min before trypsin inhibitor (1 mg/ml) was added. Tissue was collected after 5
min 1000 rpm centrifugation, triturated and resuspended in culture media (Neuronal medium:
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Neurobasal medium supplemented with 2% B27, 0.5 mM L-glutamine; Astroglia medium: DMEM
supplemented with 10% fetal bovine serum). For neuronal culture, cells were plated onto poly-D-lysine
(PDL)-coated glass coverslips at 50,000 cells/cm2 (low density for morphological measurement) or 2 X
105 cells/cm2 (high density for RNA and protein assays) and incubated at 37°C with 5% CO2 for 14 days
before experiments. For astroglia culture, cells were plated onto PDL-coated T-75 flasks at 50,000
cells/cm2. When confluent, the cultures were shaken at 220 rpm overnight at 37°C to remove
unwanted cell types (microglia, oligodendrocytes, neurons and fibroblasts), trypsinized with 0.5%
trypsin in EDTA, and plated onto PDL-coated culture vessels. For neuron-glia co-culture, purified
astroglia were seeded onto matrigel-coated cell culture inserts (Costar, #3450, #3470) and cultured in
astroglial media for 3-7 days followed by 24 hr preconditioning in neuronal media. Co-cultured neurons
were prepared following the protocol of primary neuronal culture. Astroglial inserts were transferred
onto DIV1 neuronal cultures and media was replaced. 5 μM AraC was supplemented to curb glia
proliferation. Co-cultures were maintained for 2 weeks before morphological characterization or
electrophysiological recording.
Protein extraction and Western blotting
Adult mice were anesthetized with isoflurane and euthanized by cervical dislocation. Brain tissues were
collected and immediately used for protein extraction or stored long-term at -80˚C after liquid nitrogen
snap freeze. To extract protein, tissue or cells were homogenized in RIPA buffer supplemented with
protease inhibitors (Roche, #04693116001) and phosphatase inhibitors (Roche, #04906845001) and
centrifuged at 14,000 rpm for 20 min to collect the supernatant. For nuclear protein extraction, brain
tissue was homogenized in extraction buffer (10 mM HEPES, 1 mM EDTA, 2 mM EGTA, 1 mM DTT, 0.5
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mM PMSF supplemented with protease and phosphatase inhibitors) and centrifuged at 1000 x g for 10
min at 4°C to pellet nuclear fraction. Pellet was then rinsed with extraction buffer, centrifuged, and
resuspended in buffer (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5% NP-40
supplemented with protease and phosphatase inhibitors). Lysates were used for ELISA after protein
concentration was determined or mixed with 5X Laemmli buffer and boiled at 95°C for 5 min for
Westerns. For Western blotting, 15 μg of protein samples were loaded onto 10% SDS-polyacrylamide
gels, run at 100 V for 2 hr, then transferred onto nitrocellulose membranes at 90 V for 2 hr at 4°C in
transfer buffer (50 mM Tris, 40 mM glycine, 20 % methanol). The membrane was then blocked with 5%
milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and probed with diluted primary antibody
(Mouse anti-GluR1, N-terminal, Millipore, #MAB2263, 1:2000; Rabbit anti-IκBα, Santa Cruz, #SC371,
1:1000; Mouse anti-γ-tubulin, Sigma, #T9026, 1:20,000; Rabbit anti-NSE, Cell Signaling, #8171)
overnight at 4°C. Membranes were washed 3 x 10 min in TBST and blotted with secondary antibody
(Horse anti-Rabbit-HRP, Vector Labs, 1:5000; Horse anti-Mouse-HRP, Vector Labs, 1:5000) for 1 hr at
room temperature. The membranes were again washed 3 x 10 min in TBST, incubated in ECL solution
(GE Healthcare Life Sciences), and exposed to film. After developing, the films were digitized on a
flatbed scanner.
qRT-PCR
cDNA was synthesized after reverse transcription of total RNA and analyzed as described previously
(Yang et al., 2009). Briefly, total RNA was extracted from mouse hippocampi or primary cells using
RNeasy Mini kit (Qiagen, #74106). Reverse transcription was carried out using Superscript III First
Strand synthesis system (Invitrogen, #18080-051). qPCR recipe includes 12.5 μl 2 X SYBR Green PCR
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master mix (Roche, #04673484001), 1.5 μl each of sense and anti-sense primer at 5 μM, 5 μl 50 X
diluted cDNA and 4.5 μl H2O. The primer sequences are as follows:
5’- AAG CAT CAA CAC ACC CAA CA-3’ (C3 Fwd)
5’- CTT GAG CTC CAT TCG TGA CA-3’ (C3 Rev)
5’-AAT GTG TCC GTC GTG GAT CTG A-3’ (GAPDH Fwd)
5’-GATGCCTGCTTCACCACCTTCT-3’ (GAPDH Rev)
5’-GGCTGGAGCATCCAGTTTGA-3’ (C1q Fwd)
5’-GTCATGGTCAGCACACAGGC-3’ (C1q Rev)
5’-GATGACAAGAACGTGAGTGTCC-3’ (C4 Fwd)
5’-CCCTTTAGCCACCAATTTCAGG-3’ (C4 Rev)
5’-GAGCGCAACTCCAGTGCTT-3’ (Cfb Fwd)
5’-GAGGGACATAGGTACTCCAGG-3’ (Cfb Rev)
5’-AGGCTCGTGGTCAGAACAAC-3’ (Cfh Fwd)
5’-GTTAGACGCCACCCATTTTCC-3’ (Cfh Rev)
5’-CCTAAGATGAGCGCAAGTTGAA-3’ (Hprt1 Fwd)
5’-CCACAGGACTAGAACACCTGCTAA-3’ (Hprt1 Rev)
5’-CTCCGCTTTCATGTAGAGGAAG-3’ (Pgk1 Fwd)
5’-GACATCTCCTAGTTTGGACAGTG-3’ (Pgk1 Rev)
Enzyme-linked immunosorbent assay (ELISA) and chromatin immunoprecipitation (ChIP)
C3 levels in brain lysates were determined using mouse C3 ELISA kit (Genway, #GWB-7555C7). 100 μl
of concentration-determined protein samples were used for detection according to the manufacturer’s
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instruction. The absorbance was read on a spectrophotometer with a wavelength of 450 nm.
Concentrations were quantified as absolute amount, determined by the kit, divided by total protein
amount loaded and presented as ng/mg of total protein. Relative quantification of nuclear p65 level
was determined using Cayman NFκB (p65) Transcription Factor Assay kit (#10007889) following
manufacturer’s instruction. ChIP assay was performed using the Active Motif ChIP-IT Express Enzymatic
Shearing kit (#53035). Antibodies used in ChIP included the negative control rabbit IgG (Santa Cruz,
#SC-2027), the positive control rabbit anti-Histone3 (Active Motif, #39163), and the NFκB p65 antibody
(Cell signaling, #8242).
Conditioned media preparation and immunodepletion
Primary astroglial culture media in T25 flasks were replaced by fresh neuronal media for 2 days for
preconditioning. Astroglial conditioned neuronal media was prepared by incubating astroglia cultures
with fresh neuronal media for 2 days after the preconditioning. For immunodepletion, control antibody
(goat anti-GFP, Santa Cruz, #33856) and C3 antibody (goat anti-C3, MP Biomedicals, #55444) were
added to conditioned media at 1 μg/ml and incubated at 4˚C overnight on an end-to-end rotator. The
next day, Protein G agarose beads (Invitrogen, # 15920-010) were added to conditioned media at 40
μl/ml and incubated for 1 hr at 4˚C to pull down antigen-antibody complexes. Media were then filtered
through 0.22 μm filters. To use the conditioned media, 2/5 of the media of the WT neurons cultured
for 10 days were replaced with the prepared conditioned media, and cultures were maintained for 4
days before fixation.
Pharmacological treatment and viral infection of primary cultures
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Primary astroglia were treated with 50 ng/ml TNFα (Sigma, #T0157) for 20 hrs to induce C3 expression.
JSH23 (Calbiochem, #481408) inhibits NFκB activity through blocking NFκB cytoplasmic-nuclear
translocation. 20 μM JSH23 was added to astroglia cultures along with TNFα to test the dependency of
C3 upregulation on NFκB. To chelate intracellular calcium or antagonize C3aR or C5aR activity in cocultured neurons, BAPTA/AM (Calbiochem, #196419), C3aRA (Calbiochem, #SB290157), C5aRA
(Calbiochem, #W54011), or C3 (Millipore, #204885) at different dosages (specified in figure legend)
were added to co-cultures at DIV 10 for long-term incubation or at DIV 14 for short-term incubation.
Neurons were fixed, lysed, or tested for synaptic activity. For adeno-associated virus (AAV) infection of
neuronal culture, viral particles encoding calcium indicator GCaMP6s under human synaptophysin
promoter, purchased from UPenn Vector Core (#AV-1-PV2824), were added to neuronal cultures at
MOI of 2.41 X 106 immediately after neurons were seeded. Virus was removed after 24 hrs when
media was replaced.
Synthetic Aβ42 (Invitrogen, #03-111) and rAβ42 (Sigma #SCP0048) peptides were prepared
following the protocol previously described (Stine et al., 2003). Briefly, lyophilized peptide was
dissolved in Hexafluoroisopropanol (HFIP) and incubated at RT for 2 hr. HFIP was then dried down by
SpeedVac allowing the formation of Aβ films. The film was then resuspended in DMSO, sonicated in
water bath for 10 min, diluted in 10 mM HCl, and incubated at 37˚C for 24 hrs. Primary astroglia
cultures were treated with 100 nM Aβ for 20 min and were fixed for staining. Cultures treated for 20
hrs were harvested for qPCR analysis.
Histology and immunostaining
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For mouse brain sections, deeply anaesthetized mice were perfused with 4% PFA. Mouse brains were
further fixed in 4% PFA overnight at 4°C then dehydrated in 30% sucrose. 30 μm coronal sections were
cut with a microtome and stored in cryoprotectant at -20˚C. Sections were then washed in PBS and
mounted in fluoromount before confocal microscopy. Cultured neurons on coverslips were fixed in 4%
PFA for 20 min at room temperature before extensive washing with PBS (5 min, 3X). Cells were then
blocked with 3% BSA, 2% goat serum in PBST for 30 min and incubated in diluted primary antibody
solution (Mouse anti-MAP2, Millipore, #MAB3418, 1:2000; Rabbit anti-Synaptophysin, Synaptic
systems, #101 002, 1:2000; Mouse anti-GluR1, Millipore, #MAB2263, 1:500; Rabbit anti-VGluT1,
Synaptic systems, #135 302, 1:1000; Mouse anti-VGAT, Synaptic systems, #131 011, 1:1000; Mouse
anti-GFAP, Millipore, #MAB3402, 1:2000; Rabbit anti-p65, Cell signaling, #8242, 1:100) overnight at 4
˚C. Cells were washed the next day in PBS and incubated in diluted secondary antibodies for 1-2 hrs
before the final washing. Coverslips were mounted in DAPI solution before imaging. For
immunostaining of surface GluR1 in co-cultured neurons, glial inserts were removed and neurons were
incubated with diluted anti-GluR1 antibody (1:100) at 37 ˚C for 5 min before fixation.
Image processing and quantification
Brain sections of AAV-GFP injected mice and stained neuronal cultures were imaged using a Leica laser
confocal microscope. For neuronal culture, synapses were recognized as puncta positive for synaptic
markers (Syn, VGluT1 or VGAT) in proximity to the dendritic marker MAP2. Random dendrites were
selected and synaptic density was calculated as number of puncta divided by length of the dendrite.
NeuronJ (Meijering, 2004) and Advanced Sholl analysis plugins of Fiji software were used to process
MAP2-positive images and to calculate total dendritic length and dendritic complexity. Random fields
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were chosen for quantification of microglia in co-cultures. The number of Iba1-positive cells was
divided by the number of NeuN-positive cells for neuronal cultures and Iba1-positive fluorescent area
was divided by GFAP-positive area for astroglia cultures. For measurement of surface and total GluR1
fluorescence intensity, gain and exposure values of confocal microscope were kept the same during
imaging, and Z-stack imaging was taken at 63 x magnification using 0.17 μm Z-steps. Adobe Photoshop
was used to measure GluR1 expression in Syn+ puncta using Z-stack images positive for both GluR1 and
Syn channels. Bright puncta were chosen from the Syn channel and gray value of the overlapped
puncta area in the GluR1 channel was measured. GluR1 fluorescence intensity over the whole cell
surface and blots of surface GluR1 were quantified using ImageJ. The threshold was adjusted to allow
ImageJ ROI manager to acquire all neuronal surfaces, and the average fluorescence intensity was
measured. All the quantifications were done using at least two sister wells from 2 independent culture
experiments. Nuclear/Cytoplasmic p65 ratio was measured by ImageJ as the mean grey value of
nuclear area defined by DAPI divided by the cytoplasmic area of the same size.
For AAV-GFP injected mouse brain sections, dendritic spines were 3D reconstructed by
Neurolucida (MBF Bioscience). Number of spines along fluorescent dendrites was counted manually
and divided by the length of the dendrite to calculate spine density. Classification and quantification of
frequency and density of different spine types (stubby, mushroom, long-thin and filophodia) were
performed using the IMARIS software (Bitplane). Criteria for the classification included: (1) stubby:
spine length < 1 μm; (2) Mushroom: Max (spine head width) > spine neck length; (3) Long-thin: spine
neck length > Max (spine head width), Mean (head width) >= Mean (neck width); (4) Filophodia: Max
(spine head width) <= Mean (neck width). Genotypes of the mice were blind to investigators during
quantification.
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Surface biotinylation
Neuron-glia co-cultures grown in 6-well plates were used for surface receptor biotinylation assay. At
DIV 14 of co-cultures, astroglia inserts were removed and neurons were rinsed gently with PBS++
buffer (PBS plus 1 mM MgCl2 and 0.1 mM CaCl2) followed by 15 min incubation in 1 mg/ml sulfo-NHSSS-biotin (Pierce #21331) in PBS++ solution at 4˚C. Biotinylation reaction was then quenched by 100
mM glycine in PBS++ for 15 min, and neurons were harvested and lysed for 30 min in buffer (1 %
CHAPS, 150 mM NaCl, 50 mM Tris pH 7.4, 2 mM EDTA, 2 mM EGTA, plus phosphatase inhibitors and
protease inhibitors) before centrifugation at 14,000 rpm for 20 min. Biotinylated surface proteins in
the supernatant were pulled down by pre-washed 1:1 slurry Ultralink-neutravidin beads (Pierce,
#53114) after 1 hr rotation at 4˚C, washed with lysis buffer 4 times, and eventually eluted in Laemmli
buffer for 15 min.
Slice culture and calcium imaging
Slice cultures were prepared following the protocol described in (Gogolla et al., 2006). Brains were
collected from 6-9 day-old pups and submerged in dissection media (MEM containing Hank’s Salts and
25 mM HEPES, 2% v/v penicillin/streptomycin, 1.2 mg/ml Tris base) where they were then sectioned
transversely at 350 µm with a Leica vibratome. The hippocampus was dissected from each section and
3-4 slices plated on each 0.4 µm Millicell culture insert, 30 mm in diameter, within a 6-well culture
plate. 1 ml of culture media (MEM containing 2% v/v penicillin/streptomycin, 0.6 mg/ml Tris base, 25%
heat-inactivated horse serum, 25% 1X HBSS, 3.4 % NaHCO3) was added to each well outside of the
insert and changed every 3 days. 1 μl AAV-GCaMP6s virus at a titer of 2.41 X 1013 TU/ml was added to
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the media immediately before the slices were plated and removed the next day when media was
replaced.
AAV-GCaMP6s-infected neurons and slices were imaged using EVOS fluorescence microscope
(AMG) at 10x magnification. Images were processed by Adobe Photoshop to measure GFP fluorescence.
For co-cultures, each neuronal cell body was chosen as a region of interest (ROI). For each cultured
slice, 15 random regions were chosen as ROIs. Background was measured as the gray value of blank
area. Gray values of ROIs were subtracted with background values to indicate GFP fluorescence from
GCaMP expression. Basal GFP fluorescent intensity was averaged from the measurement of 13 random
images of neuronal cultures or slice cultures.
Electrophysiology
Whole-cell patch-clamp recordings in hippocampal neuronal cultures were performed at 32±0.5°C on
DIV 11–15 neurons using low-resistance pipettes (3–6 MΩ). Neurons were visualized with an upright
microscope (BW51X, Olympus). The extracellular solution contained (in mM) 25 HEPES, 129 NaCl, 5 KCl,
2 CaCl2, 1 MgCl2 and 30 glucose; 1 μM TTX and 100 μM picrotoxin were added to the extracellular
solution for mEPSC recordings. The intercellular solution contained (in mM) 40 HEPES, pH 7.2, 110 KGluconic acid, 10 phosphocreatine, 10 EGTA, 2 MgATP, 2 Na2ATP, 0.3 mM Na2GTP. Neurons were
voltage clamped at -70 mV. Signals were amplified using a MultiClamp 700B amplifier (Molecular
Devices), filtered at 2 kHz and digitized at 10 kHz via a DigiData 1322A interface (Molecular Devices).
Data were collected using pClamp 9 software (Molecular Devices). Cumulative distributions were
generated using consecutive mEPSCs of 3 minutes. Cellular input and series resistances were
monitored through the patch-clamp electrode and cells were excluded from data analysis if more than
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a 15% change occurred during the course of the experiment. Analysis of mEPSCs was performed using
Mini Analysis Program (Synaptosoft).
Field recordings of Schaffer collateral LTP was performed as described (Peethumnongsin et al.,
2010; Polito et al., 2014). Brains were isolated from control and astroglial IκBα knockout (GcKO) mice
following behavioral testing. Stimulation of Schaffer collaterals from the CA3 region was performed
with bipolar electrodes, while borosilicate glass capillary pipettes filled with recording ACSF
(resistances of 2 to 3.5 MΩ) were used to record field excitatory postsynaptic potentials (fEPSPs) in the
CA1 region. Signals were amplified using a MultiClamp 700 B amplifier (Axon), digitized using a Digidata
1322A (Axon) with a 2 kHz low pass filter and a 3 Hz high pass filter, and then captured and stored
using Clampex 9 software (Axon) for offline data analysis.
Behavioral testing
Conditioned fear was carried out using training and context protocols over the course of two days. On
both days mice were initially transferred in their home cages to a holding room for 30 minutes to
acclimate. The mice were then transferred individually in a clean cage to the testing room and placed
in a testing chamber (VFC-008, Med Associates, St. Albans, VT, USA) within a sound attenuated cubicle.
On the training day, the mice were subjected to white noise for 2 minutes, followed by an auditory cue
for 30 seconds, then a mild foot shock (0.7 mA) for 2 seconds. This pattern was repeated once more for
a total of two foot shocks. The next day, mice were placed back in the same chamber for 5 minutes of
white noise for the context test. The movements of the mice were tracked and recorded by a video
camera, and the freezing frequency was scored by automated software (FreezeFrame, Version 2.0, San
Diego Instruments).
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Lian et al.
Morris water maze (MWM) testing was performed according to Fowler et al. (2014). Briefly,
animals were handled for 3 days prior to testing. Animals received 1 day of training in a straight swim
channel, followed by up to 8 days of acquisition training and short-term memory probe in a circular
water maze apparatus. Trials were recorded and tracked using ANY-maze Video Tracking System.
Acquisition training in the MWM consisted of 4 trials/day, during which animals were placed into the
water facing the wall at one of the four cardinal points (N, E, S, W) of the tank and given 60 sec to
locate the submerged platform. If the animal failed to locate the platform in the allotted time, it was
guided there by the experimenter. At the end of each training session, the platform was removed for a
probe test. Animals were placed in the tank at positions halfway between the cardinal points (NE, NW,
SE, and SW) and allowed 45 sec to navigate the maze. Short term memory performance was evaluated
by two standards: 1) at least 35% of their swim path in the correct quadrant and 2) ≥40% of their
platform crossings over the correct location compared to the other three equivalent platform sites
within the pool. Animals were retired from MWM training when they reached criterion performance.
Days to criterion, proximity to target and the number of times the animal crossed the platform location
was recorded for subsequent analyses.
The radial arm water maze (RAWM) was created by installing clear Plexiglass triangular inserts
into the water tank maze, creating 6 arms (20 cm wide x 34 cm long) connected at the center. An
escape platform was located at the end of one arm and submerged 1 cm below the surface. The
hidden platform location was constant on all trials, but mice were placed into a different arm at the
beginning of each trial, with the starting position randomly selected. Each animal received 1 day of
RAWM training consisting of eight trials. If a mouse did not locate the platform in 60 sec, it was guided
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there by the experimenter. Working memory errors (re-entries into incorrect arms) were calculated
for each trial, and trials 2-6 were averaged to provide a performance index for RAWM learning.
Statistics
All data was presented as mean ± SEM. Outliers were identified using Grubbs’ method with α=.05.
Pairwise comparisons were analyzed using a two-tailed Student’s t-test, while a one-way, two-way, or
three-way ANOVA followed by Bonferroni post-hoc analysis, pairwise comparison, or planned means
comparison was used for multiple comparisons. P values less than or equal to 0.05 were considered
statistically significant and marked as “*”. P values less or equal to 0.01 and 0001 were marked as “**”
and “***” respectively.
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