1. Neuroscience

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
https://doi.org/10.7554/eLife.40048
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Cite this article
  1. Xunde Xian
  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
(2018)
Reversal of ApoE4-induced recycling block as a novel prevention approach for Alzheimer’s disease
eLife 7:e40048.
https://doi.org/10.7554/eLife.40048
  2. Download BibTeX
  3. Download .RIS
9 figures, 2 videos, 1 table and 1 additional file

Figures

Figure 1
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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. …

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
Figure 2
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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 …

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
Figure 3
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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 …

https://doi.org/10.7554/eLife.40048.009
Figure 4
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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 …

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
Figure 5
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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 …

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
Figure 6
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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 …

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
Figure 7
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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…

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
Figure 8
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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 …

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
Figure 9
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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. …

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

Videos

Video 1
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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 …

https://doi.org/10.7554/eLife.40048.005
Video 2
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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 …

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

Additional files

Transparent reporting form
https://doi.org/10.7554/eLife.40048.021

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