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Amyloid- β Oligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization

View Article: PubMed Central - PubMed

ABSTRACT

Alzheimer’s disease (AD) is characterized by excessive production and deposition of amyloid-beta (Aβ) proteins as well as synapse dysfunction and loss. While soluble Aβ oligomers (AβOs) have deleterious effects on synapse function and reduce synapse number, the underlying molecular mechanisms are not well understood. Here we screened synaptic organizer proteins for cell-surface interaction with AβOs and identified a novel interaction between neurexins (NRXs) and AβOs. AβOs bind to NRXs via the N-terminal histidine-rich domain (HRD) of β-NRX1/2/3 and alternatively-spliced inserts at splicing site 4 of NRX1/2. In artificial synapse-formation assays, AβOs diminish excitatory presynaptic differentiation induced by NRX-interacting proteins including neuroligin1/2 (NLG1/2) and the leucine-rich repeat transmembrane protein LRRTM2. Although AβOs do not interfere with the binding of NRX1β to NLG1 or LRRTM2, time-lapse imaging revealed that AβO treatment reduces surface expression of NRX1β on axons and that this reduction depends on the NRX1β HRD. In transgenic mice expressing mutated human amyloid precursor protein, synaptic expression of β-NRXs, but not α-NRXs, decreases. Thus our data indicate that AβOs interact with NRXs and that this interaction inhibits NRX-mediated presynaptic differentiation by reducing surface expression of axonal β-NRXs, providing molecular and mechanistic insights into how AβOs lead to synaptic pathology in AD.

No MeSH data available.


Related in: MedlinePlus

A candidate screen isolates neurexin1β as an Aβ42 oligomer-interacting protein.(a) Immunoblotting with an anti-β-Amyloid 1–16 antibody (6E10) confirms the formation of soluble oligomers of untagged amyloid-β (1–42) peptide (Aβ) and of biotin-tagged Aβ42 peptides (biotin-Aβ). The preparations include both low and high molecular weight (HMW) oligomers. The preparation without an oligomerization incubation step (Fresh) does not include HMW oligomers. Full gel blots for the cropped blots (a) are shown in the Supplementary Fig. 4. (b) The biotin-Aβ42 oligomers bind to COS-7 cells expressing the N-terminal extracellular HA-tagged known Aβ42 oligomer receptors, paired immunoglobulin-like receptor B (HA-PirB) and prion protein (HA-PrPc), but not those expressing HA-CD4 as a negative control. Surface HA was immunostained to verify expression of these constructs on the COS-7 cell surface. (c) Representative images showing cell surface binding assays testing for interaction between biotin-Aβ42 oligomers (250 nM, monomer equivalent) and known synaptic organizers. Biotin-Aβ42 oligomers were added to COS-7 cells expressing the indicated construct. Note that biotin-Aβ42 oligomers bind to COS-7 cells expressing HA-neurexin (NRX)1βS4(−), but not to those expressing any of the other organizers including HA-neuroligin1 (HA-NLG1). For the N-terminal extracellular HA-tagged constructs, surface HA was immunostained to verify expression of the construct on the COS-7 cell surface. Scale bars represent 30 μm (b,c).
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f1: A candidate screen isolates neurexin1β as an Aβ42 oligomer-interacting protein.(a) Immunoblotting with an anti-β-Amyloid 1–16 antibody (6E10) confirms the formation of soluble oligomers of untagged amyloid-β (1–42) peptide (Aβ) and of biotin-tagged Aβ42 peptides (biotin-Aβ). The preparations include both low and high molecular weight (HMW) oligomers. The preparation without an oligomerization incubation step (Fresh) does not include HMW oligomers. Full gel blots for the cropped blots (a) are shown in the Supplementary Fig. 4. (b) The biotin-Aβ42 oligomers bind to COS-7 cells expressing the N-terminal extracellular HA-tagged known Aβ42 oligomer receptors, paired immunoglobulin-like receptor B (HA-PirB) and prion protein (HA-PrPc), but not those expressing HA-CD4 as a negative control. Surface HA was immunostained to verify expression of these constructs on the COS-7 cell surface. (c) Representative images showing cell surface binding assays testing for interaction between biotin-Aβ42 oligomers (250 nM, monomer equivalent) and known synaptic organizers. Biotin-Aβ42 oligomers were added to COS-7 cells expressing the indicated construct. Note that biotin-Aβ42 oligomers bind to COS-7 cells expressing HA-neurexin (NRX)1βS4(−), but not to those expressing any of the other organizers including HA-neuroligin1 (HA-NLG1). For the N-terminal extracellular HA-tagged constructs, surface HA was immunostained to verify expression of the construct on the COS-7 cell surface. Scale bars represent 30 μm (b,c).

Mentions: To test whether there are any synaptic organizers that interact with Aβ oligomers, we performed cell surface binding assays in which soluble oligomers of amyloid-β (1–42) peptide conjugated with biotin (biotin–Aβ42) were added onto COS-7 cells expressing each synaptic organizer. We first confirmed that the biotin–Aβ42 peptides were properly oligomerized into low and high molecular weight oligomers by western blot analysis (Fig. 1a) and also that the biotin–Aβ42 oligomers bound COS-7 cells expressing the known Aβ42 oligomer receptors, the paired immunoglobulin-like receptor B (PirB)46 and the cellular prion protein (PrP c)47 (Fig. 1b). We screened a total of 22 synaptic organizers. We found no significant binding of biotin–Aβ42 oligomers on COS-7 cells expressing NLG1 or NLG2 (Fig. 1c) although a previous study reported an interaction between AβOs and NLG145. Instead, we detected significant binding of biotin–Aβ42 oligomers on COS-7 cells expressing (NRX1βS4(−)) (Fig. 1c). Interestingly, we did not detect any binding signals on COS-7 cells expressing any of the other synaptic organizers that we tested including type IIa receptor-type protein tyrosine phosphatases (RPTPs: PTPσ, PTPδ, and LAR), LRRTM2, TrkC, and Slitrk family members (Fig. 1c).


Amyloid- β Oligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization
A candidate screen isolates neurexin1β as an Aβ42 oligomer-interacting protein.(a) Immunoblotting with an anti-β-Amyloid 1–16 antibody (6E10) confirms the formation of soluble oligomers of untagged amyloid-β (1–42) peptide (Aβ) and of biotin-tagged Aβ42 peptides (biotin-Aβ). The preparations include both low and high molecular weight (HMW) oligomers. The preparation without an oligomerization incubation step (Fresh) does not include HMW oligomers. Full gel blots for the cropped blots (a) are shown in the Supplementary Fig. 4. (b) The biotin-Aβ42 oligomers bind to COS-7 cells expressing the N-terminal extracellular HA-tagged known Aβ42 oligomer receptors, paired immunoglobulin-like receptor B (HA-PirB) and prion protein (HA-PrPc), but not those expressing HA-CD4 as a negative control. Surface HA was immunostained to verify expression of these constructs on the COS-7 cell surface. (c) Representative images showing cell surface binding assays testing for interaction between biotin-Aβ42 oligomers (250 nM, monomer equivalent) and known synaptic organizers. Biotin-Aβ42 oligomers were added to COS-7 cells expressing the indicated construct. Note that biotin-Aβ42 oligomers bind to COS-7 cells expressing HA-neurexin (NRX)1βS4(−), but not to those expressing any of the other organizers including HA-neuroligin1 (HA-NLG1). For the N-terminal extracellular HA-tagged constructs, surface HA was immunostained to verify expression of the construct on the COS-7 cell surface. Scale bars represent 30 μm (b,c).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5304201&req=5

f1: A candidate screen isolates neurexin1β as an Aβ42 oligomer-interacting protein.(a) Immunoblotting with an anti-β-Amyloid 1–16 antibody (6E10) confirms the formation of soluble oligomers of untagged amyloid-β (1–42) peptide (Aβ) and of biotin-tagged Aβ42 peptides (biotin-Aβ). The preparations include both low and high molecular weight (HMW) oligomers. The preparation without an oligomerization incubation step (Fresh) does not include HMW oligomers. Full gel blots for the cropped blots (a) are shown in the Supplementary Fig. 4. (b) The biotin-Aβ42 oligomers bind to COS-7 cells expressing the N-terminal extracellular HA-tagged known Aβ42 oligomer receptors, paired immunoglobulin-like receptor B (HA-PirB) and prion protein (HA-PrPc), but not those expressing HA-CD4 as a negative control. Surface HA was immunostained to verify expression of these constructs on the COS-7 cell surface. (c) Representative images showing cell surface binding assays testing for interaction between biotin-Aβ42 oligomers (250 nM, monomer equivalent) and known synaptic organizers. Biotin-Aβ42 oligomers were added to COS-7 cells expressing the indicated construct. Note that biotin-Aβ42 oligomers bind to COS-7 cells expressing HA-neurexin (NRX)1βS4(−), but not to those expressing any of the other organizers including HA-neuroligin1 (HA-NLG1). For the N-terminal extracellular HA-tagged constructs, surface HA was immunostained to verify expression of the construct on the COS-7 cell surface. Scale bars represent 30 μm (b,c).
Mentions: To test whether there are any synaptic organizers that interact with Aβ oligomers, we performed cell surface binding assays in which soluble oligomers of amyloid-β (1–42) peptide conjugated with biotin (biotin–Aβ42) were added onto COS-7 cells expressing each synaptic organizer. We first confirmed that the biotin–Aβ42 peptides were properly oligomerized into low and high molecular weight oligomers by western blot analysis (Fig. 1a) and also that the biotin–Aβ42 oligomers bound COS-7 cells expressing the known Aβ42 oligomer receptors, the paired immunoglobulin-like receptor B (PirB)46 and the cellular prion protein (PrP c)47 (Fig. 1b). We screened a total of 22 synaptic organizers. We found no significant binding of biotin–Aβ42 oligomers on COS-7 cells expressing NLG1 or NLG2 (Fig. 1c) although a previous study reported an interaction between AβOs and NLG145. Instead, we detected significant binding of biotin–Aβ42 oligomers on COS-7 cells expressing (NRX1βS4(−)) (Fig. 1c). Interestingly, we did not detect any binding signals on COS-7 cells expressing any of the other synaptic organizers that we tested including type IIa receptor-type protein tyrosine phosphatases (RPTPs: PTPσ, PTPδ, and LAR), LRRTM2, TrkC, and Slitrk family members (Fig. 1c).

View Article: PubMed Central - PubMed

ABSTRACT

Alzheimer’s disease (AD) is characterized by excessive production and deposition of amyloid-beta (Aβ) proteins as well as synapse dysfunction and loss. While soluble Aβ oligomers (AβOs) have deleterious effects on synapse function and reduce synapse number, the underlying molecular mechanisms are not well understood. Here we screened synaptic organizer proteins for cell-surface interaction with AβOs and identified a novel interaction between neurexins (NRXs) and AβOs. AβOs bind to NRXs via the N-terminal histidine-rich domain (HRD) of β-NRX1/2/3 and alternatively-spliced inserts at splicing site 4 of NRX1/2. In artificial synapse-formation assays, AβOs diminish excitatory presynaptic differentiation induced by NRX-interacting proteins including neuroligin1/2 (NLG1/2) and the leucine-rich repeat transmembrane protein LRRTM2. Although AβOs do not interfere with the binding of NRX1β to NLG1 or LRRTM2, time-lapse imaging revealed that AβO treatment reduces surface expression of NRX1β on axons and that this reduction depends on the NRX1β HRD. In transgenic mice expressing mutated human amyloid precursor protein, synaptic expression of β-NRXs, but not α-NRXs, decreases. Thus our data indicate that AβOs interact with NRXs and that this interaction inhibits NRX-mediated presynaptic differentiation by reducing surface expression of axonal β-NRXs, providing molecular and mechanistic insights into how AβOs lead to synaptic pathology in AD.

No MeSH data available.


Related in: MedlinePlus