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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.

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The N-terminal histidine-rich region of β-neurexin1/2/3 and S4 inserts of α/β-neurexin1/2 are responsible for Aβ42 oligomer interaction.(a) Representative images showing the binding of biotin-Aβ42 oligomers (250 nM, monomer equivalent) to COS-7 cells expressing the indicated isoform of extracellularly HA-tagged NRX. S4(−) and S4(+) indicate without and with an insert at splicing site 4, respectively. HA fluorescent signals correspond to surface HA. Note that in assays with S4-negative isoforms, there is no bound biotin-Aβ42 oligomer signal on cells expressing α-NRX1, 2, or 3 whereas cells expressing β-NRX1, 2, and 3 all have significant bound biotin-Aβ42 oligomer signal. Cells expressing β-NRX1, 2, and 3 lacking the histidine-rich domain (∆HRD) have no biotin-Aβ42 oligomer binding signal. Cells expressing S4-positive isoforms of α-NRX1 or 2 or β-NRX1, 2, or 3 have significant bound biotin-Aβ42 oligomers. Scale bar represents 30 μm. (b) Quantification of bound biotin-Aβ42 oligomers for each NRX construct. n = 30 cells for each construct from three independent experiments, one-way ANOVA, P < 0001. *P < 0.01 and ***P < 0.0001 compared with HA-CD4 and §P < 0.0001 between S4(−) and S4(+) by Bonferroni multiple comparisons tests. Data are presented as mean ± SEM.
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f3: The N-terminal histidine-rich region of β-neurexin1/2/3 and S4 inserts of α/β-neurexin1/2 are responsible for Aβ42 oligomer interaction.(a) Representative images showing the binding of biotin-Aβ42 oligomers (250 nM, monomer equivalent) to COS-7 cells expressing the indicated isoform of extracellularly HA-tagged NRX. S4(−) and S4(+) indicate without and with an insert at splicing site 4, respectively. HA fluorescent signals correspond to surface HA. Note that in assays with S4-negative isoforms, there is no bound biotin-Aβ42 oligomer signal on cells expressing α-NRX1, 2, or 3 whereas cells expressing β-NRX1, 2, and 3 all have significant bound biotin-Aβ42 oligomer signal. Cells expressing β-NRX1, 2, and 3 lacking the histidine-rich domain (∆HRD) have no biotin-Aβ42 oligomer binding signal. Cells expressing S4-positive isoforms of α-NRX1 or 2 or β-NRX1, 2, or 3 have significant bound biotin-Aβ42 oligomers. Scale bar represents 30 μm. (b) Quantification of bound biotin-Aβ42 oligomers for each NRX construct. n = 30 cells for each construct from three independent experiments, one-way ANOVA, P < 0001. *P < 0.01 and ***P < 0.0001 compared with HA-CD4 and §P < 0.0001 between S4(−) and S4(+) by Bonferroni multiple comparisons tests. Data are presented as mean ± SEM.

Mentions: Given that the NRX family is composed of many different isoforms such as α/β-isoforms and alternative splicing site 4 (S4)-positive or S4-negative isoforms18, which have differing binding affinity and selectivity for NRX-interacting proteins, we next tested which NRX isoforms interact with AβOs (Fig. 3). In the case of S4-negative isoforms, biotin–Aβ42 oligomers interacted with NRX1β, 2β and 3β at a similar level but not with NRX1α, 2α or 3α, indicating that Aβ42 oligomers interact with β-NRX-specific domains in the absence of an insert at S4. Given that the N-terminal histidine-rich domain (HRD; amino acids 50–83 in NRX1β) is unique to the β-isoforms48, we next tested the binding of biotin–Aβ42 oligomers to COS-7 cells expressing NRX1β lacking the HRD (HA-NRX1β∆HRD) and detected no binding (Fig. 3a,b). Further, COS-7 cells expressing HA-NRX2β∆HRD or HA-NRX3β∆HRD also displayed no binding signal (Fig. 3a,b). These results indicate that the HRD of β-NRX1/2/3 is one of the domains responsible for Aβ42 oligomer binding. Next, we investigated Aβ42 oligomer-binding to S4-positive NRX isoforms. Biotin–Aβ42 oligomers interacted with S4-positive NRX1α and 2α but not 3α (Fig. 3a,b), indicating that the inserts at the S4 site of NRX1 and NRX2 interact with Aβ42 oligomers. Indeed, S4-positive NRX1β and 2β displayed stronger binding of Aβ42 oligomers than S4-negative NRX1β and 2β, respectively (Fig. 3a,b). The enhancement of binding by the S4 insert is similar to the difference in the binding signals of S4-negative and S4-positive NRX1α and 2α (Fig. 3b), suggesting that Aβ42 oligomer-binding to the S4 insert additively increases the binding of Aβ42 to NRX1β and 2β. Together, these data indicate that the HRDs of NRX1β, 2β and 3β and the S4 inserts of NRX1α/β and 2α/β are responsible for Aβ42 oligomer interaction.


Amyloid- β Oligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization
The N-terminal histidine-rich region of β-neurexin1/2/3 and S4 inserts of α/β-neurexin1/2 are responsible for Aβ42 oligomer interaction.(a) Representative images showing the binding of biotin-Aβ42 oligomers (250 nM, monomer equivalent) to COS-7 cells expressing the indicated isoform of extracellularly HA-tagged NRX. S4(−) and S4(+) indicate without and with an insert at splicing site 4, respectively. HA fluorescent signals correspond to surface HA. Note that in assays with S4-negative isoforms, there is no bound biotin-Aβ42 oligomer signal on cells expressing α-NRX1, 2, or 3 whereas cells expressing β-NRX1, 2, and 3 all have significant bound biotin-Aβ42 oligomer signal. Cells expressing β-NRX1, 2, and 3 lacking the histidine-rich domain (∆HRD) have no biotin-Aβ42 oligomer binding signal. Cells expressing S4-positive isoforms of α-NRX1 or 2 or β-NRX1, 2, or 3 have significant bound biotin-Aβ42 oligomers. Scale bar represents 30 μm. (b) Quantification of bound biotin-Aβ42 oligomers for each NRX construct. n = 30 cells for each construct from three independent experiments, one-way ANOVA, P < 0001. *P < 0.01 and ***P < 0.0001 compared with HA-CD4 and §P < 0.0001 between S4(−) and S4(+) by Bonferroni multiple comparisons tests. Data are presented as mean ± SEM.
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f3: The N-terminal histidine-rich region of β-neurexin1/2/3 and S4 inserts of α/β-neurexin1/2 are responsible for Aβ42 oligomer interaction.(a) Representative images showing the binding of biotin-Aβ42 oligomers (250 nM, monomer equivalent) to COS-7 cells expressing the indicated isoform of extracellularly HA-tagged NRX. S4(−) and S4(+) indicate without and with an insert at splicing site 4, respectively. HA fluorescent signals correspond to surface HA. Note that in assays with S4-negative isoforms, there is no bound biotin-Aβ42 oligomer signal on cells expressing α-NRX1, 2, or 3 whereas cells expressing β-NRX1, 2, and 3 all have significant bound biotin-Aβ42 oligomer signal. Cells expressing β-NRX1, 2, and 3 lacking the histidine-rich domain (∆HRD) have no biotin-Aβ42 oligomer binding signal. Cells expressing S4-positive isoforms of α-NRX1 or 2 or β-NRX1, 2, or 3 have significant bound biotin-Aβ42 oligomers. Scale bar represents 30 μm. (b) Quantification of bound biotin-Aβ42 oligomers for each NRX construct. n = 30 cells for each construct from three independent experiments, one-way ANOVA, P < 0001. *P < 0.01 and ***P < 0.0001 compared with HA-CD4 and §P < 0.0001 between S4(−) and S4(+) by Bonferroni multiple comparisons tests. Data are presented as mean ± SEM.
Mentions: Given that the NRX family is composed of many different isoforms such as α/β-isoforms and alternative splicing site 4 (S4)-positive or S4-negative isoforms18, which have differing binding affinity and selectivity for NRX-interacting proteins, we next tested which NRX isoforms interact with AβOs (Fig. 3). In the case of S4-negative isoforms, biotin–Aβ42 oligomers interacted with NRX1β, 2β and 3β at a similar level but not with NRX1α, 2α or 3α, indicating that Aβ42 oligomers interact with β-NRX-specific domains in the absence of an insert at S4. Given that the N-terminal histidine-rich domain (HRD; amino acids 50–83 in NRX1β) is unique to the β-isoforms48, we next tested the binding of biotin–Aβ42 oligomers to COS-7 cells expressing NRX1β lacking the HRD (HA-NRX1β∆HRD) and detected no binding (Fig. 3a,b). Further, COS-7 cells expressing HA-NRX2β∆HRD or HA-NRX3β∆HRD also displayed no binding signal (Fig. 3a,b). These results indicate that the HRD of β-NRX1/2/3 is one of the domains responsible for Aβ42 oligomer binding. Next, we investigated Aβ42 oligomer-binding to S4-positive NRX isoforms. Biotin–Aβ42 oligomers interacted with S4-positive NRX1α and 2α but not 3α (Fig. 3a,b), indicating that the inserts at the S4 site of NRX1 and NRX2 interact with Aβ42 oligomers. Indeed, S4-positive NRX1β and 2β displayed stronger binding of Aβ42 oligomers than S4-negative NRX1β and 2β, respectively (Fig. 3a,b). The enhancement of binding by the S4 insert is similar to the difference in the binding signals of S4-negative and S4-positive NRX1α and 2α (Fig. 3b), suggesting that Aβ42 oligomer-binding to the S4 insert additively increases the binding of Aβ42 to NRX1β and 2β. Together, these data indicate that the HRDs of NRX1β, 2β and 3β and the S4 inserts of NRX1α/β and 2α/β are responsible for Aβ42 oligomer interaction.

View Article: PubMed Central - PubMed

ABSTRACT

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

No MeSH data available.


Related in: MedlinePlus