Human LilrB2 is a β-amyloid receptor and its murine homolog PirB regulates synaptic plasticity in an Alzheimer's model - PubMed (original) (raw)

Human LilrB2 is a β-amyloid receptor and its murine homolog PirB regulates synaptic plasticity in an Alzheimer's model

Taeho Kim et al. Science. 2013.

Abstract

Soluble β-amyloid (Aβ) oligomers impair synaptic plasticity and cause synaptic loss associated with Alzheimer's disease (AD). We report that murine PirB (paired immunoglobulin-like receptor B) and its human ortholog LilrB2 (leukocyte immunoglobulin-like receptor B2), present in human brain, are receptors for Aβ oligomers, with nanomolar affinity. The first two extracellular immunoglobulin (Ig) domains of PirB and LilrB2 mediate this interaction, leading to enhanced cofilin signaling, also seen in human AD brains. In mice, the deleterious effect of Aβ oligomers on hippocampal long-term potentiation required PirB, and in a transgenic model of AD, PirB not only contributed to memory deficits present in adult mice, but also mediated loss of synaptic plasticity in juvenile visual cortex. These findings imply that LilrB2 contributes to human AD neuropathology and suggest therapeutic uses of blocking LilrB2 function.

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Figures

Fig. 1

Fig. 1. PirB is a receptor for oligomeric Aβ

(A) Monomeric (mono) or oligomerized (oligo) synthetic human Aβ42 peptides (fig. S1A) (21, 22) were analyzed by size exclusion column chromatography. Arrows indicate monomeric form; _V_0, void volume; absorbance at 215 nm is in arbitrary units. (B) The same peptides were analyzed by Western blotting with antibody to Aβ (4G8; detects Aβ17-24). (C) PirB-IRES-EGFP–transfected (top) or control IRES-EGFP–transfected (bottom) HEK293 cells (green) were treated with mono- or oligo-Aβ42 (100 nM total peptide, monomer equivalent), and bound Aβ42 (red) was visualized. See also fig. S1. DAPI, 4′,6-diamidino-2-phenylindole. (D) Quantification of Aβ42 binding represented in (C). AU denotes average signal per pixel (22); data are means ± SEM (PirB-IRES-EGFP, n = 5; IRES-EGFP, n = 4). (E) PirB-expressing cells were treated with oligo-Aβ42 (100 nM) and immunostained for Aβ and PirB. Colocalization is observed particularly at cell membrane (i.e., arrowheads). (F) Schematic of mouse PirB, the highly related mouse PirA1 and PirA4, and a rat PirB isoform (23). Amino acid sequence similarities to mouse PirB (% score, ClustalW) are indicated at bottom. Ig, immunoglobulin domain; ITIM, immunoreceptor tyrosine-based inhibitory motif. (G) Relative oligo-Aβ42 (200 nM) binding to HEK293 cells expressing mouse PirB, PirA1, or PirA4 or rat PirB; see also fig. S3. Data are means ± SEM (n = 4 or 5). (H) Dose-dependent binding of mono- or oligo-Aβ42 (squares and circles, respectively) to HEK293 cells expressing IRES-EGFP (green) or PirB-IRES-EGFP (red), assessed as a function of Aβ42 total concentration. (I) Binding curve of mono- or oligo-Aβ42 to PirB. Data (PirB-IRES-EGFP minus IRES-EGFP) are from (H) (22). (J) Scatchard plots of data from (I). Data are means ± SEM (n = 4). Calculated _K_d = 180 ± 52 nM; see also fig. S4. (K) Binding of oligo-Aβ42 to cultured cortical neurons (21 days in vitro) is diminished ~50% by deletion of PirB (PirB−/−), as assessed by alkaline phosphatase assay. Data are means ± SEM (n = 6). Estimated _K_d for neuronal PirB [dashed line: ΔPirB = wild type (WT) minus PirB−/−] is 110 nM.

Fig. 2

Fig. 2. LilrB2 is a PirB ortholog present in human brain and acts as a receptor for Aβ oligomers via the D1D2 domain

(A) Schematic of LilrB1, LilrB2, LilrB3, and Kir (3DL1), human homologs of mouse PirB. Amino acid sequence homologies (% score, ClustalW) to PirB or to LilrB2 are given at bottom. (B) Aβ42 oligomer (200 nM) selectively binds to LilrB2-expressing HEK293 cells but not to LilrB1-, LilrB3-, or Kir-expressing cells (arbitrary units); see fig. S5A. Similar LilrB1, LilrB2, and LilrB3 expression levels were verified by Western blotting (inset) with antibodies to Myc. Data are means ± SEM (n = 5). (C and D) Dose dependence of mono-Aβ42 (squares) or oligo-Aβ42 (circles) binding to LilrB2 expressed in HEK293 cells (22); data are means ± SEM (n = 4). _K_d = 206 ± 65 nM; see also fig. S5, B and C. (E) LilrB2 is expressed in frontal lobe of specimens from three adult humans (non-AD; C1 to C3) and from four Alzheimer’s patients (AD1 to AD4) (table S1). Protein extracts from fresh frozen frontal lobe were immunoprecipitated with control IgG or LilrB2-specific antibodies followed by Western blot analysis. (F) Quantitation of LilrB2 protein levels shown in (E). Data are means ± SEM. (G) Deletion of the D1D2 domain abrogates binding of Aβ42 oligomers to PirB and LilrB2. Top: Schematic of PirB and LilrB2 ectodomain constructs: full-length or truncated Ig domains fused to human IgG-Fc (hIgG-Fc). Bottom: Bar graphs of average band intensities ± SEM from experiments such as that shown in fig. S6C (n = 3). Note that sequence analysis using pairwise alignment indicates that the D1D2 domain of LilrB2 aligns closely with the D1D2 domain of PirB (28). (H) PirB-Fc or LilrB2-Fc binds predominantly to high-n oligomeric forms of Aβ42. Oligomerized Aβ42 (input; also contains low-n oligomers and monomeric Aβ42) was subjected to immunoprecipitation with full-length or truncated soluble PirB- or LilrB2-Fc proteins followed by Western blot analysis. Aβ oligomer binding domain–deficient PirB (D5D6)-Fc treatment was used as negative control (lane 1). Top right: Western blot with antibodies to Aβ specific for oligomeric forms (OMAB; see fig. S6D); Bottom left: quantification of Aβ42 binding. Data are normalized average band intensities ± SEM (n = 3).

Fig. 3

Fig. 3. PirB deletion rescues synaptic plasticity and behavioral deficits in AD models

(A) Acute application of oligo-Aβ42 inhibits LTP in WT hippocampal slices. fEPSPs were recorded from stratum radiatum in the CA1 region of hippocampal slices from 4- to 5-month-old WT mice with or without addition of oligo-Aβ42 (200 nM total peptide). Top panels show example fEPSP traces immediately before (light traces) and 45 min after (heavy traces) TBS; each is an average of five individual consecutive traces. Calibration bar = 0.5 mV/5 ms. The slope of the fEPSP after TBS, relative to baseline, is plotted as a function of time in the lower panel. Vehicle, n = 7 animals, 9 slices; Aβ42 oligomer, n = 6, 9 slices. (B) Aβ42 oligomer does not block LTP in PirB−/− slices. Vehicle, n = 5 animals, 8 slices; Aβ42 oligomer, n = 4, 6 slices. (C) Histograms of fEPSP slope measured 45 min after TBS. Each is a 2-min average of recordings taken from all slices of a given condition at time marked # in (A) and (B); all data are means ± SEM, ***P < 0.0001, t test. (D) Comparison of Aβ42 oligomer effects on hippocampal LTP from WT and PirB−/− mice; replotted from (A) and (B). (E) Novel object recognition memory of 9-month-old mice was evaluated by measuring percent of time mice spent exploring a novel versus a familiar object during a 10-min test session. (F) Novel place recognition memory (9-month-old) reflects percent time mice spent exploring familiar objects whose locations were or were not changed. Values are means ± SEM, *P < 0.05, paired t test. PirB+/−; APP/PS1 (PirB+/− Tg, n = 6), PirB−/−; APP/PS1 (PirB−/− Tg, n = 5). (G) Schematic of mouse visual system showing connections from eyes to lateral geniculate nucleus (LGN) to visual cortex. Cortical binocular zone (BZ) receives inputs from both eyes via the LGN. (H) In situ hybridization for Arc mRNA (digoxigenin-labeled antisense riboprobe) in visual cortex BZ of PirB;APP/PS1 littermates. At P22, one eye was removed; 10 days later (P32), induction of mRNA for the immediate early gene Arc at P32 was used to monitor width of territory receiving functional input from the open (ipsilateral) eye. Note that high Arc mRNA expression in layer 2/3 neurons within dashed lines, denoting domain of Arc induction in visual cortex. Scale bar, 500 μm. (I) Quantification of expansion in width of Arc mRNA signal in L2/3 visual cortex shown in (H). Data are means ± SEM; ***P < 0.001, t test; PirB+/− (n = 14 animals), PirB+/− Tg (n = 7), PirB−/− (n = 14), PirB−/− Tg (n = 10).

Fig. 4

Fig. 4. Cofilin is recruited and activated by PirB in an Aβ-dependent manner in vivo and in vitro and is altered in human AD frontal cortex

(A) PirB interacts with cofilin in vivo in PirB+/− Tg mice (P30, forebrain), assessed by immunoprecipitation for PirB. Other known PirB-proximal signaling and interactions such as tyrosine phosphorylation of PirB and SHP-2 recruitment to PirB are not altered in PirB+/− relative to PirB+/− Tg mice. Representative data are shown (n > 2). (B and C) Cofilin phosphorylation is reduced in both (B) juvenile (P30, forebrain) and (C) adult (P200, hippocampal synaptosomes) PirB+/−; APP/PS1 (PirB+/− Tg) mice relative to PirB+/− mice, and this reduction is rescued by PirB deletion (PirB−/− Tg). No significant alterations in LIM kinase (LIMK) 1/2 phosphorylation (Thr508/Thr505) were de tected. (D and E) Quantificaton of cofilin phosphorylation (left, expressed as pCofilin/total cofilin) and pLIMK levels (right) represented in (B) and (C). (D) Means ± SEM from four independent experiments (13 animals per genotype) (22) shown in (B). *P < 0.05, U test. (E) Means ± SEM (n = 3; **P < 0.01, t test) shown in (C). (F) Cortical neurons (DIV18 to 22) isolated from WT or PirB−/− embryos (E16) were treated with oligo-Aβ42 (100 nM) for 1 hour (top panels) or 24 hours (middle panels) and cofilin signaling or PSD-95 levels were analyzed by Western blotting. Anti-Tuj1 (βIII-tubulin) antibodies detect neuronal tubulin. Bottom panels: Expression of PirB in these neurons detected by PirB immunoprecipitation. (G) Summary of cofilin phosphorylation (left; *P < 0.05, U test, n = 7) or PSD-95 levels (right; *P < 0.05, U test, n = 6) represented in (F). (H) Increased cofilin activity and Tau phosphorylation (Ser396) in human frontal cortex specimens from Alzheimer’s patients (AD1 to AD4) relative to non-AD adults (C1 to C3) (table S1), assessed by Western blot analysis. (I) Summary of cofilin phosphorylation cases represented in (H). Means ± SEM, *P < 0.05, t test.

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