(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. …
Binding of ApoE Isoforms to 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 …
ApoE4 Impairs Recycling of the Reelin Receptor Apoer2.
(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 …
(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 …
The NHE inhibitor EMD87580 prevents the intracellular trapping of ApoE4 and its receptor Apoer2.
(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 …
A Specific Role for NHE6 in Apoer2 trafficking.
(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 …
NHE6 knockdown alleviates surface trafficking deficits induced by ApoE4.
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…
EMD87580 treatment differentially alters synaptic plasticity in ApoE3-KI and 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 …
NHE inhibition counteracts Aβ-induced LTP suppression in ApoE4-KI mice.
(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. …
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 …
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 …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (Homo sapiens) | HEK293 | Thermo Fisher | R70507, RRID: CVCL_0045 | Tested mycoplasma free annually, last test January 16,2018 |
Cell line (Homo sapiens) | HEK293T | ATCC | CRL-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 rat | Charles River | SC:400 | |
Antibody | goat anti- ApoE, pAb | EMD Millipore | 178479, RRID: AB_10682965 | 1:1000 (WB) |
Antibody | rabbit anti-Apoer2 | Herz Lab, #2561 | 1:1000 (WB) | |
Antibody | rabbit anti-Dab1 | Herz Lab, #5091 | 1:1000 (WB) | |
Antibody | mouse anti-FLAG M2 | Sigma-Aldrich | F3165, RRID: AB_259529 | 1:1000 (WB) |
Antibody | rabbit anti-GluA1 | Abcam | ab31232, RRID: AB_2113447 | 1:1000 (WB) |
Antibody | rabbit anti-GluA2/3 | EMD Millipore | 07–598, RRID: AB_310741 | 1:1000 (WB) |
Antibody | rabbit anti-GluN2B | Cell Signaling Technology | 4207S, RRID: AB_1264223 | 1:1000 (WB) |
Antibody | rabbit anti -Insulin Receptor B (4B8), mAb | Cell Signaling Technology | 3025, RRID: AB_ 2280448 | 1:1000 (WB) |
Antibody | rabbit anti-Lrp1 | Herz Lab | 1:5000 (WB) | |
Antibody | rabbit anti-Ldlr | Herz Lab | 1:1000 (WB) | |
Antibody | rabbit anti -NHE6 (C-terminus) | Herz Lab | 1:1000 (WB) | |
Antibody | mouse anti-phosphotyrosine (4G10) mAb | EMD Millipore | 05–321, RRID: AB_309678 | 1:1000 (WB) |
Antibody | rabbit anti-Transferrin receptor | Abcam | Ab61134, RRID: AB_943620 | 1:1000 (WB) |
Antibody | rabbit anti-B-Actin | Abcam | Ab8227, RRID: AB_2305186 | 1:3000 (WB) |
Peptide, recombinant protein | 6-cyano-7- nitroquinoxaline- 2,3-dione, CNQX | Sigma-Aldrich | C127 | |
Peptide, recombinant protein | ApoE3, human | Sigma-Aldrich | SRP4696 | |
Chemical compound, drug | B-27 Supplement (50X), serum free | Thermo Fisher | 17504044 | |
Chemical compound, drug | Bafilomycin A1 | Cayman Chemical | CAS88899-55-2 | |
Chemical compound, drug | DMEM | Sigma-Aldrich | D6046 | |
Chemical compound, drug | FuGENE | Promega | E2311 | |
Chemical compound, drug | HBSS (1X) | Gibco | 14175 | |
Chemical compound, drug | L-Glutamic acid (Glutamate) | Sigma-Aldrich | G1251 | |
Chemical compound, drug | Neurobasal Medium (1X) Liquid without Phenol Red | Thermo Fisher | 12348017 | |
Chemical compound, drug | NeutrAvidin Agarose | Thermo Fisher | 29201 | |
Chemical compound, drug | Nimodipine | Sigma- Aldrich | N3764 | |
Chemical compound, drug | NP-40 Alternative | EMD 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 | Corning | 30–002 CI | |
Chemical compound, drug | Phosphatase Inhibitor Cocktail | Thermo Fisher | 78420 | |
Chemical compound, drug | Poly-D -Lysine Solution | Sigma-Aldrich | A-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-Aldrich | P8340 | |
Chemical compound, drug | Sodium- hydrogen exchanger inhibitor | Merck KGaA | EMD87580 | |
Chemical compound, drug | Sulfo-NHS- SS-biotin | Pierce | 21331 | |
Chemical compound, drug | Tetrodotoxin | Sigma-Aldrich | T8024 | |
Chemical compound, drug | Triton X-100 | Sigma- Aldrich | CAS9002-93-1 | |
Chemical compound, drug | Vectashield with DAPI | Vector Labs | H-1200 | |
Recombinant DNA reagent | pcDNA3.1-Zeo | Invitrogen | V79020 | |
Recombinant DNA reagent | psPAX2 | Addgene | 12260 | |
Recombinant DNA reagent | pMD2.G | Addgene | 12259 | |
Recombinant DNA reagent | pLKO.1 | Addgene | 10878 | |
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/A | progenitor pcDNA3.1-Zeo |
Transfected construct (Homo sapiens) | pcDNA3.1-ApoE3 | (Chen et al., 2010) | N/A | progenitor pcDNA3.1-Zeo |
Transfected construct (Homo sapiens) | pcDNA3.1-ApoE4 | (Chen et al., 2010) | N/A | progenitor pcDNA3.1-Zeo |
Transfected construct (shRNA construct) | pLKO.1-shRNA scramble | this paper | N/A | progenitor pLKO.1 |
Transfected construct (shRNA construct) | pLKO.1-shNHE1 | Open Biosystem | TRCN0000044651 | progenitor pLKO.1 |
Transfected construct (shRNA construct) | pLKO.1-shNHE5 | this paper | N/A | progenitor pLKO.1 |
Transfected construct (shRNA construct) | pLKO.1-shNHE6 a | Open Biosystem | TRC N000 0068828 | progenitor pLKO.1 |
Transfected construct (shRNA construct) | pLKO.1-shNHE6 b | Open Biosystem | TRCN0000068830 | progenitor pLKO.1 |
Transfected construct (shRNA construct) | pLKO.1-shNHE6 c | Open Biosystem | TRCN0000068832 | progenitor pLKO.1 |
Transfected construct (shRNA construct) | pLKO.1-shNHE7 | Open Biosystem | TRCN0000068812 | progenitor pLKO.1 |
Transfected construct (shRNA construct) | pLKO.1-shNHE8 | this paper | N/A | progenitor pLKO.1 |
Transfected construct (shRNA construct) | pLKO.1-shNHE9 | Open Biosystem | TRCN0000068856 | progenitor pLKO.1 |
Transfected construct (Mus musculus) | pLVX-mCherry- Apoer2 | this paper | N/A | progenitor pLVXCMV100 |
Transfected construct (Homo sapiens) | pcDNA3.1- ApoE3-GFP | this paper | N/A | progenitor pcDNA3.1-Zeo |
Sequence-based reagent (oligo) | Scramble shRNA forward | IDT Inegrated DNA Technologies | N/A | 5’-CCGGCCTAAGGTTAAGTCGCCCT CGCTC-3' |
Sequence-based reagent (oligo) | Scramble shRNA reverse | IDT Inegrated DNA Technologies | N/A | 5'-GAGCGAGGGCGACTTAACCTTAGG TTTTTG-3’ |
Sequence-based reagent (oligo) | shRNA anti NHE1 (SLC9A1) forward | Open Biosystem | TRCN0000044651 | 5’-CCGCCATC GGATCTTCCCTTCCTTACTCG-3' |
Sequence-based reagent (oligo) | shRNA anti NHE1 (SLC9A1) reverse | Open Biosystem | TRCN0000044651 | 5'-AGTAAGGAAGGGAAGATCCGATGTTTTTG-3’ |
Sequence-based reagent (oligo) | shRNA anti NHE5 (SLC9A5) forward | IDT Inegrated DNA Technologies | N/A | 5’-CCGGAAGGACCACACTCATCTTAG TCTCG-3' |
Sequence based reagent (oligo) | shRNA anti NHE5 (SLC9A5) reverse | IDT Inegrated DNA Technologies | N/A | 5'-AGACTAAGATGAGTGTGGTCCTTT TTTTG-3’ |
Sequence-based reagent (oligo) | shRNA anti NHE6 (SLC9A6) -a forward | Open Biosystem | TRCN0000068828 | 5’-CCGGGCCGTTTATATGGCATAGGAACTC-3' |
Sequence-based reagent (oligo) | shRNA anti NHE6 (SLC9A6)- a reverse | Open Biosystem | TRCN0000068828 | 5'-GAGTTCCTATGCCATATAAACGGCTTTTTG-3’ |
Sequence-based reagent (oligo) | shRNA anti NHE6 (SLC9A6)-b forward | Open Biosystem | TRCN0000068830 | 5’-CCGGCCCTTGTCTCTCTTACTTAATCTCG-3' |
Sequence-based reagent (oligo) | shRNA anti NHE6 (SLC9A6)-b reverse | Open Biosystem | TRCN0000068830 | 5'-AGATTAAGTAAGAGAGACAAGGGTTTTTG-3’ |
Sequence-based reagent (oligo) | shRNA anti NHE6 (SLC9A6)-c forward | Open Biosystem | TRCN0000068832 | 5’-CCGGCCTTGGGTCTATCTTAGCATACTCG-3' |
Sequence-based reagent (oligo) | shRNA anti NHE6 (SLC9A6)-c reverse | Open Biosystem | TRCN0000068832 | 5'-AGTATGCTAAGATAGACCCAAGGTTTTTG-3’ |
Sequence-based reagent (oligo) | shRNA anti NHE7 (SLC9A7) forward | Open Biosystem | TRCN0000068812 | 5’-CCGGCCATTGTACT ATCCTCGTCTACTCG-3' |
Sequence-based reagent (oligo) | shRNA anti NHE7 (SLC9A7) reverse | Open Biosystem | TRCN0000068812 | 5'-AGTAGACGAGGATAGTACAATGGTTTTTG-3’ |
Sequence-based reagent (oligo) | shRNA anti NHE8 (SLC9A8) forward | IDT Inegrated DNA Technologies | N/A | 5’-CCGGAAGGCTTCATGTGGTTGGATGCTC-3' |
Sequence-based reagent (oligo) | shRNA anti NHE8 (SLC9A8) reverse | IDT Inegrated DNA Technologies | N/A | 5'-GAGCATCCAACCACAT GAAGCCTTTTTTTG-3’ |
Sequence-based reagent (oligo) | shRNA anti NHE9 (SLC9A9) forward | Open Biosystem | TRCN0000068856 | 5’-CCGGCTGGGCAGAAA GCAGAAGATTCTC-3' |
Sequence-based reagent (oligo) | shRNA anti NHE9 (SLC9A9) reverse | Open Biosystem | TRCN0000068856 | 5'-GAGAATCTTCTGCTTT CTGCCCAGTTTTTG-3’ |
Sequence-based reagent (oligo) | Apoer2 NT cloning site (sdm) forward | Inegrated DNA Technologies | N/A | 5'-TACAAATCTAGAGATCCG CTGCCGGGCGGCCAAG-3' |
Sequence-based reagent (oligo) | Apoer2 NT cloning site (sdm) reverese | Inegrated DNA Technologies | N/A | 5'-ACTCATGTCGACCGCTG CGGAGAGATG CTGAAGCTG-3' |
Sequence-based reagent (oligo) | mCherry (for mCherry-Apoer2) forward | Inegrated DNA Technologies | N/A | 5'-AAATTCGTCGACATGGTG AGCAAGGGCGA GGAGGATAAC-3' |
Sequence-based reagent (oligo) | mCherry (for mCherry-Apoer2) reverse | Inegrated DNA Technologies | N/A | 5'-GGGAACGTCTAGAG GACTTGTACAGCTC GTCCATG-3' |
Sequence-based reagent (oligo) | Apoer2 forward | Inegrated DNA Technologies | N/A | 5'-TGGAGCGCTAGCGC CACCATGGGCCGCCC AGAACTGG-3' |
Sequence-based reagent (oligo) | Apoer2 reverse | Inegrated DNA Technologies | N/A | 5'-AACCCGGAATTCTCA GGGCAGTCCAT CATCTTCAAGAC-3' |
Sequence-based reagent (oligo) | NheI-site removal (sdm) forward | Inegrated DNA Technologies | N/A | 5'-GTTTACCGTCGA CCTCTAGCTAG-3' |
Sequence-based reagent (oligo) | NheI-site removal (sdm) reverse | Inegrated DNA Technologies | N/A | 5'-AATGTCAAGGCCTCTCACTCTCTG-3' |
Sequence-based reagent (oligo) | CMVfull forward | Inegrated DNA Technologies | N/A | 5'-CAGTTTATCGATG GCCAGATATACGCG TTGACATTG-3' |
Sequence-based reagent (oligo) | CMVfull reverse | Inegrated DNA Technologies | N/A | 5'-TTTCCGCTAGCGGATCC CAGCTTGGGTCT CCCTATAGTGAGT-3' |
Sequence-based reagent (oligo) | ApoE3 (ApoE3-GFP) forward( | Inegrated DNA Technologies | N/A | 5'-ATCAGGGAATTCAAC CATGAAGGTTCTG TGGGCTGCG-3' |
Sequence-based reagent (oligo) | GFP (ApoE3-GFP) reverse | Inegrated DNA Technologies | N/A | 5'-ATTGGTGGATCCGCGT GATTGTCGCTG GGCACAG-3' |
Software, algorithm | Adobe Creative Cloud | Adobe | RRID: SCR_010279 | |
Software, algorithm | GraphPad Prism 7.0 | GraphPad Software | RRID: SCR_002798 | |
Software, algorithm | Fiji/ImageJ | NIH | RRID: SCR_002285 | |
Software, algorithm | LabView7.0 | National Instruments | RRID: SCR_014325 | |
Software, algorithm | Odyssey Imaging System | LI-COR | RRID: SCR_014579 | |
Software, algorithm | Clustal Omega | EMBL-EBI | RRID: SCR_001591 | |
Leica TCS SPE | Leica | RRID: SCR_002140 |