1. Neuroscience
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Reversal of ApoE4-induced recycling block as a novel prevention approach for Alzheimer’s disease

  1. Xunde Xian  Is a corresponding author
  2. Theresa Pohlkamp
  3. Murat S Durakoglugil
  4. Connie H Wong
  5. Jürgen K Beck
  6. Courtney Lane-Donovan
  7. Florian Plattner
  8. Joachim Herz  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
  2. Jkb Consult Inc SPRL, Belgium
Research Article
Cite this article as: eLife 2018;7:e40048 doi: 10.7554/eLife.40048
9 figures, 2 videos, 1 table and 1 additional file

Figures

Binding of ApoE Isoforms to Apoer2.

(A and B) ApoE isoforms interact with ApoE receptor 2 (Apoer2) as tested by co-immunoprecipitation. ApoE3 and ApoE4 bind Apoer2 with similar affinity, whereas ApoE2 binding to Apoer2 is poor. ApoE-conditioned media (0, 0.1, 0.3, 1, 3 and 10 µg/ml ApoE) were incubated with Apoer2-Fc (secreted Apoer2 ectodomain fused to Fc) bound to protein-G beads and pulled down to perform immunoblotting for ApoE. Representative immunoblot images (A) and quantification (B) are shown. (C) Apoer2 co-localizes with ApoE in primary neurons. Primary cortical neurons were infected with lentiviral mCherry-Apoer2 (red) and subsequently treated with ApoE3-GFP-conditioned media (green). A single plane of a z-stack is shown with the orthogonal xz- and yz-views as indicated. White lines indicate the vertical and horizontal cuts. Boxed vesicles are shown enlarged in the panels below labeled ApoE, Apoer2 and Merge. Additionally 3D movies of the cells are provided online (Videos 1 and 2).

https://doi.org/10.7554/eLife.40048.003
Figure 1—source data 1

Binding of ApoE Isoforms to Apoer2.

https://doi.org/10.7554/eLife.40048.004
ApoE4 Impairs Recycling of the Reelin Receptor Apoer2.

(A) Timeline for experiment shown in B and C. (B and C) Apolipoprotein E (ApoE) isoforms reduce surface expression of Apoer2. ApoE-conditioned media treatment reduces the surface expression of Apoer2 in presence of Reelin in primary neurons. Apoer2 surface levels show a higher reduction with ApoE4 than ApoE3. Other ApoE receptors, such as low-density lipoprotein receptor-related protein 1 (Lrp1) and low-density lipoprotein receptor (Ldlr), as well as the endocytic receptor for transferrin (TfR) and insulin receptor (IR) exhibit comparable surface levels in the presence of ApoE3 or ApoE4. Levels of surface proteins and total proteins were analyzed by immunoblotting using antibodies raised against Apoer2, Lrp1, Ldlr, IR and TfR. Quantitative analysis of the ratio of surface and total receptor levels is shown (C). (D and E) Proteins from primary neurons incubated with ApoE-conditioned media were immunoprecipitated with anti-Apoer2 or control rabbit IgG and immunoblotted with anti-ApoE antibody. Input is shown in the right panel of (D) and quantification in (E). (F and G) ApoE4, but not ApoE3, induces phosphorylation of Dab1 independent of Reelin. Primary neurons were incubated with ApoE-conditioned media or Reelin and tested for phospho-Dab1 and total Dab1. Quantitative analysis is shown (G). All data are expressed as mean ± SEM from three independent experiments. *p<0.05, **p<0.01, ***p<0.001. Statistical analysis was performed using one-way ANOVA and Dunnett’s post-hoc test (C and G) or Student’s t-test (E).

https://doi.org/10.7554/eLife.40048.007
Figure 2—source data 1

ApoE4 Impairs Recycling of the Reelin Receptor Apoer2.

https://doi.org/10.7554/eLife.40048.008
Working model illustrating the hypthetical mechanism of the vesicular trafficking defect incurred by human Apoe4.

(A) Cysteines/arginines at residues 112 and 158 account for the difference in relative charge and isoelectric point (IEP) of human ApoE isoforms. (B) Endosomal ApoE4/Apoer2 aggregates form upon acidification. Endosomal pH is regulated by the vacuolar-type H+-ATPase (vATPase, proton pump) and organellar Na+/H+ exchangers (NHEs, proton leak). After binding to ApoE4, Apoer2 undergoes endocytosis, is sequestered in endosomes and recycling is delayed. (C) Endosomal ApoE4/Apoer2 resolve when the pH is lowered further. Accelerated acidification through NHE6 inhibition activity promotes dissociation of ApoE4 and Apoer2, resulting in the efficient recycling of Apoer2 back to cell plasma membrane.

https://doi.org/10.7554/eLife.40048.009
The NHE inhibitor EMD87580 prevents the intracellular trapping of ApoE4 and its receptor Apoer2.

(A) Chemical structure of the NHE inhibitor EMD87580. (B and C) EMD87580 increases the Reelin-induced surface expression of Apoer2 in ApoE4-treated neurons in a dose-dependent manner. Primary neurons were pre-treated with EMD87580 at the indicated concentrations and then incubated with Reelin with or without cell-derived ApoE4. Surface and total Apoer2 levels were analyzed by immunoblotting. (D and E) The effect of EMD87580 on Reelin-induced Apoer2 trafficking in the presence of ApoE3 or ApoE4. Primary neuronal cells were treated with EMD87580, Reelin and either ApoE3- or ApoE4-conditioned media. (F and G) Bafilomycin, a proton pump inhibitor, counteracts the effect of EMD87580 on Apoer2 recycling in a dose-dependent manner. Primary neurons were pre-treated with or without bafilomycin in the presence or absence of EMD87580 and subsequently incubated with ApoE4 and Reelin. Surface and total Apoer2 levels were analyzed by immunoblotting. (H and I) Bafilomycin shifts the EMD87580 dose response curve of Apoer2 surface expression. All values are expressed as mean ±SEM from three independent experiments. *p<0.05, **p<0.01, ***p<0.001. Statistical analysis was performed using one-way ANOVA and Dunnett’s post-hoc test (C, E and G) or Student’s t-test (I).

https://doi.org/10.7554/eLife.40048.010
Figure 4—source data 1

The NHE inhibitor EMD87580 prevents the intracellular trapping of ApoE4 and its receptor Apoer2.

https://doi.org/10.7554/eLife.40048.011
A Specific Role for NHE6 in Apoer2 trafficking.

(A and C) shRNA knockdown of NHE6, but not other NHEs (NHE1, 5, 7, 8, 9) restores ApoE4-impaired Apoer2 recycling. Lentivirus-mediated shRNAs targeting NHE1, 5, 6, 7, 8, or 9 were applied to primary neurons. Cells were then treated with ApoE4-conditioned media and Reelin, and cell surface and total Apoer2 were determined by immunoblotting. Arrows indicate conditions with restored Apoer2 surface levels. Three different shRNA constructs against NHE6 showed significant attenuation of Apoer2 cell surface levels (shNHE6 a, b, c). (B and D) Quantitative analysis of (A) and (C). All data are expressed as mean ±SEM of three independent experiments. ***p<0.001. Statistical analysis was performed using Student’s t-test (B and D).

https://doi.org/10.7554/eLife.40048.012
Figure 5—source data 1

A Specific Role for NHE6 in Apoer2 trafficking.

https://doi.org/10.7554/eLife.40048.013
NHE6 knockdown alleviates surface trafficking deficits induced by ApoE4.

(A) Lentiviral shRNA knockdown efficiency of NHE6 protein expression in primary rat cortical neurons. (B) Lentiviral shRNA directed against NHE6 restores the ApoE4-induced trafficking deficits of surface receptors. Primary cortical neurons were infected with three different lentiviral shRNAs directed against NHE6 (shNHE6 a, b and c; lanes 6–7) or scrambled shRNA control (lanes 4, 5). Infected cultures were treated without (lanes 1 and 4) or with (lanes 2, 3, 5–8) cell-derived ApoE4 and Reelin (all lanes) and the cell surface biotinylation assay was performed for Apoer2, GluN2B, GluA1 and GluA2/3. (D–F) Quantitative analysis of immunoblot signal from (B). All data are expressed as mean ±SEM from three independent experiments. *p<0.05, **p<0.01, ***p<0.001. Statistical analysis was performed using one-way ANOVA and Dunnett’s post-hoc test (C–F).

https://doi.org/10.7554/eLife.40048.014
Figure 6—source data 1

NHE6 knockdown alleviates surface trafficking deficits induced by ApoE4.

https://doi.org/10.7554/eLife.40048.015
EMD87580 treatment differentially alters synaptic plasticity in ApoE3-KI and ApoE4-KI mice.

Mice were pre-treated with EMD87580 in vivo and acute hippocampal slices were subsequently analyzed by recording extracellular field potentials (A and B) Input-output curves are shown for ApoE3-KI (A) and ApoE4-KI (B). (A) ApoE3-KI slices treated with Reelin or treated with EMD87580 and Reelin exhibited increased I/O slopes compared to control (Ctrl: 1.127 ± 0.18; EMD87580: 1.653 ± 0.15; Reelin: 1.97 ± 0.14; Reelin and EMD87580: 2.23 ± 0.16; F = 9.567, p<0.05). (B) I/O curves were increased in ApoE4-KI slices at baseline (1.86 ± 0.16) compared to ApoE3-KI control (1.127 ± 0.18). Neither EMD87580 nor Reelin significantly affected the I/O slopes in ApoE4-KI slices (EMD: 1.705 ± 0.10; Reelin: 1.43 ± 0.09; Reelin and EMD: 1.67 ± 0.09; F = 1.8, p=0.14). (C and D) Results from LTP recordings in ApoE3-KI (C) and ApoE4-KI (D). Representative traces before (solid line) and 40 min after (dashed line) theta-burst stimulation (TBS) for each treatment paradigm are shown in the top panels. Bottom panels depict LTP recordings and quantification of average LTP responses between 40 and 60 min after TBS (bar graphs). (C) ApoE3-KI slices treated with Reelin (152.4% ± 21.69, n = 5) and EMD87580 (149.30 ± 25.29, n = 7) had increased LTP compared to control slices (109.7% ± 9.7, n = 6). Combined Reelin treatment with EMD87580 increased LTP (129.4 ± 17.6, n = 7) compared to control. (D) Untreated ApoE4-KI slices showed enhanced LTP (140.80% ± 10.5, n = 12) when compared to untreated ApoE3-KI slices (109.7% ± 9.7, n = 6). ApoE4-KI slices treated with Reelin did not further potentiate LTP (134.70% ± 9.63, n = 14), whereas ApoE4-KI slices treated with EMD87580 exhibited reduced LTP (102 ± 11.9, n = 14). ApoE4-KI slices with EMD87580 show increased LTP when treated with Reelin (136.30 ± 11.87, n = 15) as compared to EMD87580 treatment alone. Open circles: no additions; Open squares: Reelin alone; Gray diamonds: EMD87580 alone; Filled triangles: Reelin and EMD87580 treated.

https://doi.org/10.7554/eLife.40048.016
Figure 7—source data 1

EMD87580 treatment differentially alters synaptic plasticity in ApoE3-KI and ApoE4-KI mice.

https://doi.org/10.7554/eLife.40048.017
NHE inhibition counteracts Aβ-induced LTP suppression in ApoE4-KI mice.

(A–D) Treatment of hippocampal slices with AD brain extracts impairs long-term potentiation (LTP) in ApoE3-KI and ApoE4-KI mice. Reelin can attenuate the LTP deficits induced by AD extracts in ApoE3-KI, but not ApoE4-KI mice. Inhibition of NHE counteracts the LTP deficits induced by AD extract in ApoE4-KI mice. Hippocampal slices were prepared from 2- to 3-month-old ApoE3-KI and ApoE4-KI mice. Extracellular field recordings were performed in slices treated with AD brain extract, Reelin and/or EMD87580. Control slices were treated with control brain extract. Theta burst stimulation (TBS) was performed 20 min after stable baseline was attained. Representative traces are shown in each panel, before TBS induction (black) and 40 min after TBS (grey). (C, D) Quantitative analysis of normalized fEPSP slopes at 40–60 min post TBS for (A), respectively (B). All data are expressed as mean ±SEM. *p<0.05, **p<0.01. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test (C, D).

https://doi.org/10.7554/eLife.40048.018
Figure 8—source data 1

NHE inhibition counteracts Aβ-induced LTP suppression in ApoE4-KI mice.

https://doi.org/10.7554/eLife.40048.019
Restoration of vesicular trafficking and synaptic homeostasis by NHE6 inhibition in the presence of ApoE4.

(A) Effect of ApoE isoforms on ApoE receptor signaling at the synapse. Apoer2 induces NMDAR tyrosine phosphorylation by activating SFKs in response to Reelin in the postsynaptic neuron. Astrocyte-derived ApoE3 (green ovals) or ApoE4 (gray ovals) bind to Apoer2 and are constitutively but slowly internalized. Apoer2 undergoes accelerated endocytosis in response to Reelin signaling. ApoE4 sequesters Apoer2 in intracellular compartments thereby reducing the ability of the postsynaptic neuron to phosphorylate (activate) NMDA receptors in response to Reelin (shown on the right), whereas ApoE2 or ApoE3 efficiently recycle back to the cell surface and thus deplete surface Apoer2 levels to a lesser extent (illustrated on the left for ApoE3). Aβ oligomers interfere with NMDAR tyrosine phosphorylation by activating tyrosine phosphatases. This panel has been reproduced from Chen et al. (2010). (B) When NHE6 (red) is functioning normally, early endosomal pH is maintained at ~6.5 by the action of the proton pump and proton leakage through NHE6. This pH level is close to the isoelectric point (pI) of ApoE4, which is particularly sensitive to structural unfolding into a molten globule state upon entering acidic compartments (Morrow et al., 2002). The resulting reduced solubility may hamper efficient release of ApoE4 from its receptors (such as Apoer2 in neurons). Upon NHE6 inhibition, endosomal pH is further reduced, resulting in the release of ApoE4 from its receptors through improved solubility and the repulsion forces proteins exert upon each other when the pH is below their pI. Thus, trafficking and recycling of Apoer2 and the neurotransmitter receptors it associates with is restored. It should be noted that this property of ApoE4 to undergo impaired trafficking through endosomal compartments is universal and not restricted to neurons. Therefore, NHE6 inhibition may also aid in the clearance of amyloid-β in the brain and LDL removal in the liver.

© 2010 PNAS. All rights reserved. Subpanel A in Figure 9 is reproduced from Chen et al. (2010), PNAS with permission.

https://doi.org/10.7554/eLife.40048.020

Videos

Video 1
Supporting material for Figure 1C. 3D View of Apoer2 co-localizes with Apoe in primary neurons.

N-terminal mCherry-labeled Apoer2 (red) and C-terminal GFP-labeled ApoE3 (green) co-localize intracellularly in primary neurons. Rat primary cortical neurons were infected with lentiviral mCherry-Apoer2 and subsequently exposed to ApoE3-GFP-conditioned media. Confocal microscopy was performed as described in the Materials and methods section.

https://doi.org/10.7554/eLife.40048.005
Video 2
Supporting material for Figure 1C.

3D View of Apoer2 co-localizes with Apoe in primary neurons. N-terminal mCherry-labeled Apoer2 (red) and C-terminal GFP-labeled ApoE3 (green) co-localize intracellularly in primary neurons. Rat primary cortical neurons were infected with lentiviral mCherry-Apoer2 and subsequently exposed to ApoE3-GFP-conditioned media. Confocal microscopy was performed as described in the Materials and methods section.

https://doi.org/10.7554/eLife.40048.006

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Cell line
(Homo sapiens)
HEK293Thermo FisherR70507,
RRID: CVCL_0045
Tested
mycoplasma
free annually, last test January
16,2018
Cell line
(Homo sapiens)
HEK293TATCCCRL-3216,
RRID: CVCL_0063
Tested
mycoplasma
free annually,
last test January
16,2018
Strain, strain
background
(Mus musculus)
Mouse/ApoE3ki
(B6.129P2- Apoetm2
(APOE*3)Mae N8)
(Knouff et al., 1999;
Sullivan et al., 1997)
Originally
provided
by Dr. Nobuyo
Maeda
Strain,
strain background
(Mus musculus)
Mouse/ApoE4ki
(B6.129P2- Apoetm2
(APOE*4)Mae N8)
(Knouff et al., 1999;Sullivan et al., 1997)Originally
provided by
Dr. Nobuyo
Maeda
Strain, strain
background
(Rattus norvegicus)
SD ratCharles RiverSC:400
Antibodygoat anti-
ApoE, pAb
EMD
Millipore
178479,
RRID: AB_10682965
1:1000 (WB)
Antibodyrabbit
anti-Apoer2
Herz Lab, #25611:1000 (WB)
Antibodyrabbit
anti-Dab1
Herz Lab, #50911:1000 (WB)
Antibodymouse
anti-FLAG M2
Sigma-AldrichF3165,
RRID: AB_259529
1:1000 (WB)
Antibodyrabbit
anti-GluA1
Abcamab31232,
RRID: AB_2113447
1:1000 (WB)
Antibodyrabbit
anti-GluA2/3
EMD Millipore07–598,
RRID: AB_310741
1:1000 (WB)
Antibodyrabbit
anti-GluN2B
Cell Signaling
Technology
4207S, RRID: AB_12642231:1000 (WB)
Antibodyrabbit anti
-Insulin Receptor
B (4B8), mAb
Cell Signaling
Technology
3025, RRID: AB_
2280448
1:1000 (WB)
Antibodyrabbit anti-Lrp1Herz Lab1:5000 (WB)
Antibodyrabbit anti-LdlrHerz Lab1:1000 (WB)
Antibodyrabbit anti
-NHE6
(C-terminus)
Herz Lab1:1000 (WB)
Antibodymouse
anti-phosphotyrosine
(4G10) mAb
EMD
Millipore
05–321, RRID: AB_3096781:1000 (WB)
Antibodyrabbit
anti-Transferrin
receptor
AbcamAb61134,
RRID: AB_943620
1:1000 (WB)
Antibodyrabbit
anti-B-Actin
AbcamAb8227,
RRID:
AB_2305186
1:3000 (WB)
Peptide,
recombinant protein
6-cyano-7-
nitroquinoxaline-
2,3-dione, CNQX
Sigma-AldrichC127
Peptide,
recombinant protein
ApoE3, humanSigma-AldrichSRP4696
Chemical
compound, drug
B-27 Supplement
(50X), serum free
Thermo Fisher17504044
Chemical
compound, drug
Bafilomycin A1Cayman
Chemical
CAS88899-55-2
Chemical
compound, drug
DMEMSigma-AldrichD6046
Chemical
compound, drug
FuGENEPromegaE2311
Chemical
compound, drug
HBSS (1X)Gibco14175
Chemical
compound, drug
L-Glutamic acid
(Glutamate)
Sigma-AldrichG1251
Chemical
compound, drug
Neurobasal
Medium (1X) Liquid
without Phenol Red
Thermo Fisher12348017
Chemical
compound,
drug
NeutrAvidin
Agarose
Thermo Fisher29201
Chemical
compound,
drug
NimodipineSigma-
Aldrich
N3764
Chemical
compound,
drug
NP-40 AlternativeEMD
Millipore
492016
Chemical
compound,
drug
32%
Paraformaldehyde
AQ solution
Fisher
Scientific
15714S
Chemical
compound,
drug
PBS (1X)Sigma-
Aldrich
D8537
Chemical
compound,
drug
Penicillin-
Streptomycin
Solution, 100X
Corning30–002 CI
Chemical
compound,
drug
Phosphatase
Inhibitor
Cocktail
Thermo
Fisher
78420
Chemical
compound,
drug
Poly-D
-Lysine Solution
Sigma-AldrichA-003-M
Chemical
compound,
drug
Protein A-
Sepharose 4B
Thermo
Fisher
101042
Chemical
compound,
drug
Protein G-
Sepharose 4B
Thermo
Fisher
101142
Chemical
compound,
drug
Proteinase
Inhibitor
Cocktail
Sigma-AldrichP8340
Chemical
compound, drug
Sodium-
hydrogen
exchanger
inhibitor
Merck KGaAEMD87580
Chemical
compound, drug
Sulfo-NHS-
SS-biotin
Pierce21331
Chemical
compound, drug
TetrodotoxinSigma-AldrichT8024
Chemical
compound, drug
Triton X-100Sigma-
Aldrich
CAS9002-93-1
Chemical
compound, drug
Vectashield
with DAPI
Vector LabsH-1200
Recombinant
DNA reagent
pcDNA3.1-ZeoInvitrogenV79020
Recombinant
DNA reagent
psPAX2Addgene12260
Recombinant
DNA reagent
pMD2.GAddgene12259
Recombinant
DNA reagent
pLKO.1Addgene10878
Recombinant
DNA reagent
pLVXCMV100(Dean et al., 2017)N/A

Transfected
construct
(Mus musculus)
pCrl, Reelin
expression vector
(D'Arcangelo et al., 1997)N/A
Transfected
construct
(Mus musculus)
pcDNA3.1-Apoer2-Fc(Hiesberger et al., 1999)N/A
Transfected
construct
(Homo sapiens)
pcDNA3.1-ApoE2(Chen et al., 2010)N/Aprogenitor
pcDNA3.1-Zeo
Transfected
construct
(Homo sapiens)
pcDNA3.1-ApoE3(Chen et al., 2010)N/Aprogenitor
pcDNA3.1-Zeo
Transfected
construct
(Homo sapiens)
pcDNA3.1-ApoE4(Chen et al., 2010)N/Aprogenitor
pcDNA3.1-Zeo
Transfected
construct
(shRNA construct)
pLKO.1-shRNA
scramble
this paperN/Aprogenitor pLKO.1
Transfected
construct
(shRNA construct)
pLKO.1-shNHE1Open BiosystemTRCN0000044651progenitor pLKO.1
Transfected
construct
(shRNA construct)
pLKO.1-shNHE5this paperN/Aprogenitor
pLKO.1
Transfected
construct
(shRNA construct)
pLKO.1-shNHE6 aOpen
Biosystem
TRC
N000
0068828
progenitor
pLKO.1
Transfected
construct
(shRNA construct)
pLKO.1-shNHE6 bOpen
Biosystem
TRCN0000068830progenitor
pLKO.1
Transfected
construct
(shRNA construct)
pLKO.1-shNHE6 cOpen
Biosystem
TRCN0000068832progenitor
pLKO.1
Transfected
construct
(shRNA construct)
pLKO.1-shNHE7Open BiosystemTRCN0000068812progenitor
pLKO.1
Transfected
construct
(shRNA construct)
pLKO.1-shNHE8this paperN/Aprogenitor
pLKO.1
Transfected
construct
(shRNA construct)
pLKO.1-shNHE9Open BiosystemTRCN0000068856progenitor
pLKO.1
Transfected
construct
(Mus musculus)
pLVX-mCherry-
Apoer2
this paperN/Aprogenitor
pLVXCMV100
Transfected
construct
(Homo sapiens)
pcDNA3.1-
ApoE3-GFP
this paperN/Aprogenitor
pcDNA3.1-Zeo
Sequence-based
reagent (oligo)
Scramble
shRNA forward
IDT Inegrated
DNA Technologies
N/A5’-CCGGCCTAAGGTTAAGTCGCCCT
CGCTC-3'
Sequence-based
reagent (oligo)
Scramble
shRNA reverse
IDT Inegrated
DNA Technologies
N/A5'-GAGCGAGGGCGACTTAACCTTAGG
TTTTTG-3’
Sequence-based
reagent (oligo)
shRNA anti NHE1
(SLC9A1) forward
Open
Biosystem
TRCN00000446515’-CCGCCATC
GGATCTTCCCTTCCTTACTCG-3'
Sequence-based
reagent (oligo)
shRNA anti NHE1
(SLC9A1) reverse
Open
Biosystem
TRCN00000446515'-AGTAAGGAAGGGAAGATCCGATGTTTTTG-3’
Sequence-based
reagent (oligo)
shRNA anti
NHE5 (SLC9A5)
forward
IDT Inegrated DNA TechnologiesN/A5’-CCGGAAGGACCACACTCATCTTAG
TCTCG-3'
Sequence based
reagent (oligo)
shRNA anti
NHE5 (SLC9A5)
reverse
IDT Inegrated
DNA
Technologies
N/A5'-AGACTAAGATGAGTGTGGTCCTTT
TTTTG-3’
Sequence-based

reagent (oligo)
shRNA anti
NHE6 (SLC9A6)
-a forward
Open
Biosystem
TRCN00000688285’-CCGGGCCGTTTATATGGCATAGGAACTC-3'
Sequence-based
reagent (oligo)
shRNA anti
NHE6 (SLC9A6)-
a reverse
Open
Biosystem
TRCN00000688285'-GAGTTCCTATGCCATATAAACGGCTTTTTG-3’
Sequence-based
reagent (oligo)
shRNA anti NHE6
(SLC9A6)-b forward
Open
Biosystem
TRCN00000688305’-CCGGCCCTTGTCTCTCTTACTTAATCTCG-3'
Sequence-based
reagent
(oligo)
shRNA anti NHE6
(SLC9A6)-b reverse
Open
Biosystem
TRCN00000688305'-AGATTAAGTAAGAGAGACAAGGGTTTTTG-3’
Sequence-based
reagent (oligo)
shRNA anti NHE6
(SLC9A6)-c forward
Open
Biosystem
TRCN00000688325’-CCGGCCTTGGGTCTATCTTAGCATACTCG-3'
Sequence-based
reagent (oligo)
shRNA anti NHE6
(SLC9A6)-c reverse
Open
Biosystem
TRCN00000688325'-AGTATGCTAAGATAGACCCAAGGTTTTTG-3’
Sequence-based
reagent (oligo)
shRNA anti NHE7
(SLC9A7) forward
Open
Biosystem
TRCN00000688125’-CCGGCCATTGTACT
ATCCTCGTCTACTCG-3'
Sequence-based
reagent (oligo)
shRNA anti NHE7
(SLC9A7) reverse
Open
Biosystem
TRCN00000688125'-AGTAGACGAGGATAGTACAATGGTTTTTG-3’
Sequence-based
reagent (oligo)
shRNA anti NHE8
(SLC9A8) forward
IDT Inegrated
DNA Technologies
N/A5’-CCGGAAGGCTTCATGTGGTTGGATGCTC-3'
Sequence-based
reagent (oligo)
shRNA anti NHE8
(SLC9A8) reverse
IDT Inegrated
DNA Technologies
N/A5'-GAGCATCCAACCACAT
GAAGCCTTTTTTTG-3’
Sequence-based
reagent (oligo)
shRNA anti NHE9
(SLC9A9) forward
Open BiosystemTRCN00000688565’-CCGGCTGGGCAGAAA
GCAGAAGATTCTC-3'
Sequence-based
reagent (oligo)
shRNA anti NHE9
(SLC9A9) reverse
Open BiosystemTRCN00000688565'-GAGAATCTTCTGCTTT
CTGCCCAGTTTTTG-3’
Sequence-based
reagent (oligo)
Apoer2 NT cloning
site (sdm) forward
Inegrated DNA
Technologies
N/A5'-TACAAATCTAGAGATCCG
CTGCCGGGCGGCCAAG-3'
Sequence-based
reagent (oligo)
Apoer2 NT cloning
site (sdm) reverese
Inegrated
DNA
Technologies
N/A5'-ACTCATGTCGACCGCTG
CGGAGAGATG
CTGAAGCTG-3'
Sequence-based
reagent (oligo)
mCherry (for
mCherry-Apoer2)
forward
Inegrated
DNA
Technologies
N/A5'-AAATTCGTCGACATGGTG
AGCAAGGGCGA
GGAGGATAAC-3'
Sequence-based
reagent (oligo)
mCherry (for
mCherry-Apoer2)
reverse
Inegrated
DNA
Technologies
N/A5'-GGGAACGTCTAGAG
GACTTGTACAGCTC
GTCCATG-3'
Sequence-based
reagent (oligo)
Apoer2 forwardInegrated
DNA
Technologies
N/A5'-TGGAGCGCTAGCGC
CACCATGGGCCGCCC
AGAACTGG-3'
Sequence-based
reagent (oligo)
Apoer2 reverseInegrated
DNA
Technologies
N/A5'-AACCCGGAATTCTCA
GGGCAGTCCAT
CATCTTCAAGAC-3'
Sequence-based
reagent (oligo)
NheI-site removal
(sdm) forward
Inegrated
DNA
Technologies
N/A5'-GTTTACCGTCGA
CCTCTAGCTAG-3'
Sequence-based
reagent (oligo)
NheI-site removal
(sdm) reverse
Inegrated
DNA
Technologies
N/A5'-AATGTCAAGGCCTCTCACTCTCTG-3'
Sequence-based
reagent (oligo)
CMVfull forwardInegrated
DNA
Technologies
N/A5'-CAGTTTATCGATG
GCCAGATATACGCG
TTGACATTG-3'
Sequence-based
reagent (oligo)
CMVfull reverseInegrated
DNA Technologies
N/A5'-TTTCCGCTAGCGGATCC
CAGCTTGGGTCT
CCCTATAGTGAGT-3'
Sequence-based
reagent (oligo)
ApoE3 (ApoE3-GFP) forward(Inegrated
DNA
Technologies
N/A5'-ATCAGGGAATTCAAC
CATGAAGGTTCTG
TGGGCTGCG-3'
Sequence-based
reagent (oligo)
GFP (ApoE3-GFP) reverseInegrated
DNA
Technologies
N/A5'-ATTGGTGGATCCGCGT
GATTGTCGCTG
GGCACAG-3'
Software,
algorithm
Adobe Creative CloudAdobeRRID: SCR_010279
Software,
algorithm
GraphPad Prism 7.0GraphPad
Software
RRID: SCR_002798
Software,
algorithm
Fiji/ImageJNIHRRID: SCR_002285
Software,
algorithm
LabView7.0National
Instruments
RRID: SCR_014325
Software,
algorithm
Odyssey Imaging
System
LI-CORRRID: SCR_014579
Software,
algorithm
Clustal OmegaEMBL-EBIRRID: SCR_001591
Leica TCS SPELeicaRRID: SCR_002140

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