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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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Aβ42 oligomers reduce surface expression of neurexin1β on axons.(a–c) Representative time-lapse images of axons expressing extracellularly super-ecliptic pHluorin (SEP)-tagged NRX1βS4(+) (a), SEP-NRX1βS4(−) (b) and SEP-NRX1βS4(−) lacking the histidine-rich domain (SEP-NRX1βS4(−)∆HRD) (c) treated with Aβ42 oligomers (500 nM, monomer equivalent) at t = 0 min. Scale bars represent 5 μm. (d) Quantification of SEP intensity signals at 5 min before and 10, 30 and 60 min after Aβ42 oligomer treatment. n = 29 for SEP-NRX1βS4(+), n = 26 for SEP-NRX1βS4(−), and n = 33 for SEP-NRX1βS4(−)ΔHRD puncta from 9 cells for each condition from three independent experiments, two-way repeated measures ANOVA, F(3, 255) = 42.94, P < 0.0001 for time and F(2, 85) = 18.95, P < 0.0001 for construct. **P < 0.001, and ***P < 0.0001 compared with SEP signal at 5 min before the treatment, and ***P < 0.0001 compared with SEP-NRX1βS4(−)ΔHRD by Bonferroni multiple comparisons tests. n.s., not significant. Data are presented as mean ± SEM.
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f6: Aβ42 oligomers reduce surface expression of neurexin1β on axons.(a–c) Representative time-lapse images of axons expressing extracellularly super-ecliptic pHluorin (SEP)-tagged NRX1βS4(+) (a), SEP-NRX1βS4(−) (b) and SEP-NRX1βS4(−) lacking the histidine-rich domain (SEP-NRX1βS4(−)∆HRD) (c) treated with Aβ42 oligomers (500 nM, monomer equivalent) at t = 0 min. Scale bars represent 5 μm. (d) Quantification of SEP intensity signals at 5 min before and 10, 30 and 60 min after Aβ42 oligomer treatment. n = 29 for SEP-NRX1βS4(+), n = 26 for SEP-NRX1βS4(−), and n = 33 for SEP-NRX1βS4(−)ΔHRD puncta from 9 cells for each condition from three independent experiments, two-way repeated measures ANOVA, F(3, 255) = 42.94, P < 0.0001 for time and F(2, 85) = 18.95, P < 0.0001 for construct. **P < 0.001, and ***P < 0.0001 compared with SEP signal at 5 min before the treatment, and ***P < 0.0001 compared with SEP-NRX1βS4(−)ΔHRD by Bonferroni multiple comparisons tests. n.s., not significant. Data are presented as mean ± SEM.

Mentions: We next tested another possible mechanism: Aβ42 oligomers could decrease the surface expression of NRXs on axons and thereby diminish NRX-mediated presynaptic differentiation. We cotransfected hippocampal neurons with mCherry (for imaging neuronal morphology) and NRX1β extracellularly tagged with super-ecliptic pHluorin (SEP) (SEP-NRX1β), and then performed time-lapse imaging of SEP-NRX1β expressed on mCherry-positive axons (Fig. 6). SEP is a pH-sensitive GFP variant that yields fluorescence at neutral pH (e.g. on the cell surface) but is quenched at low pH (e.g. inside cytoplasmic vesicles), thus it allows for monitoring the surface expression level of tagged proteins50. In mCherry-expressing axons, cotransfected SEP-NRX1βS4(+), SEP-NRX1βS4(−), or SEP-NRX1βS4(−) lacking the HRD (SEP-NRX1βS4(−)∆HRD) had punctate distributions (Fig. 6a–c). Our immunocytochemistry further confirmed that these SEP-NRX1β puncta colocalized with VGLUT1 puncta (Supplementary Fig. 2), suggesting that the SEP-NRX1β proteins likely accumulated at presynaptic boutons. The application of Aβ42 oligomers into the extracellular solution decreased the SEP fluorescent signal of SEP-NRX1βS4(+) and SEP-NRX1βS4(−) with similar decay (Fig. 6d). The similarity of the decay suggests that the binding of Aβ42 oligomers to the S4 insert is not essential for AβO-induced reduction of NRX surface expression. On the other hand, the application of Aβ42 oligomers did not affect the SEP signal of SEP-NRX1βS4(−)∆HRD (Fig. 6d), which does not bind Aβ42 oligomers (Supplementary Fig. 3). Thus, the binding of Aβ42 oligomers to the HRD is essential for AβO-induced reduction of NRX surface expression. Together with the results of our coculture assays (Fig. 4), these data suggest that Aβ42 oligomers diminish NRX-mediated presynaptic differentiation by decreasing surface expression of β-NRXs on axons.


Amyloid- β Oligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization
Aβ42 oligomers reduce surface expression of neurexin1β on axons.(a–c) Representative time-lapse images of axons expressing extracellularly super-ecliptic pHluorin (SEP)-tagged NRX1βS4(+) (a), SEP-NRX1βS4(−) (b) and SEP-NRX1βS4(−) lacking the histidine-rich domain (SEP-NRX1βS4(−)∆HRD) (c) treated with Aβ42 oligomers (500 nM, monomer equivalent) at t = 0 min. Scale bars represent 5 μm. (d) Quantification of SEP intensity signals at 5 min before and 10, 30 and 60 min after Aβ42 oligomer treatment. n = 29 for SEP-NRX1βS4(+), n = 26 for SEP-NRX1βS4(−), and n = 33 for SEP-NRX1βS4(−)ΔHRD puncta from 9 cells for each condition from three independent experiments, two-way repeated measures ANOVA, F(3, 255) = 42.94, P < 0.0001 for time and F(2, 85) = 18.95, P < 0.0001 for construct. **P < 0.001, and ***P < 0.0001 compared with SEP signal at 5 min before the treatment, and ***P < 0.0001 compared with SEP-NRX1βS4(−)ΔHRD by Bonferroni multiple comparisons tests. n.s., not significant. Data are presented as mean ± SEM.
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f6: Aβ42 oligomers reduce surface expression of neurexin1β on axons.(a–c) Representative time-lapse images of axons expressing extracellularly super-ecliptic pHluorin (SEP)-tagged NRX1βS4(+) (a), SEP-NRX1βS4(−) (b) and SEP-NRX1βS4(−) lacking the histidine-rich domain (SEP-NRX1βS4(−)∆HRD) (c) treated with Aβ42 oligomers (500 nM, monomer equivalent) at t = 0 min. Scale bars represent 5 μm. (d) Quantification of SEP intensity signals at 5 min before and 10, 30 and 60 min after Aβ42 oligomer treatment. n = 29 for SEP-NRX1βS4(+), n = 26 for SEP-NRX1βS4(−), and n = 33 for SEP-NRX1βS4(−)ΔHRD puncta from 9 cells for each condition from three independent experiments, two-way repeated measures ANOVA, F(3, 255) = 42.94, P < 0.0001 for time and F(2, 85) = 18.95, P < 0.0001 for construct. **P < 0.001, and ***P < 0.0001 compared with SEP signal at 5 min before the treatment, and ***P < 0.0001 compared with SEP-NRX1βS4(−)ΔHRD by Bonferroni multiple comparisons tests. n.s., not significant. Data are presented as mean ± SEM.
Mentions: We next tested another possible mechanism: Aβ42 oligomers could decrease the surface expression of NRXs on axons and thereby diminish NRX-mediated presynaptic differentiation. We cotransfected hippocampal neurons with mCherry (for imaging neuronal morphology) and NRX1β extracellularly tagged with super-ecliptic pHluorin (SEP) (SEP-NRX1β), and then performed time-lapse imaging of SEP-NRX1β expressed on mCherry-positive axons (Fig. 6). SEP is a pH-sensitive GFP variant that yields fluorescence at neutral pH (e.g. on the cell surface) but is quenched at low pH (e.g. inside cytoplasmic vesicles), thus it allows for monitoring the surface expression level of tagged proteins50. In mCherry-expressing axons, cotransfected SEP-NRX1βS4(+), SEP-NRX1βS4(−), or SEP-NRX1βS4(−) lacking the HRD (SEP-NRX1βS4(−)∆HRD) had punctate distributions (Fig. 6a–c). Our immunocytochemistry further confirmed that these SEP-NRX1β puncta colocalized with VGLUT1 puncta (Supplementary Fig. 2), suggesting that the SEP-NRX1β proteins likely accumulated at presynaptic boutons. The application of Aβ42 oligomers into the extracellular solution decreased the SEP fluorescent signal of SEP-NRX1βS4(+) and SEP-NRX1βS4(−) with similar decay (Fig. 6d). The similarity of the decay suggests that the binding of Aβ42 oligomers to the S4 insert is not essential for AβO-induced reduction of NRX surface expression. On the other hand, the application of Aβ42 oligomers did not affect the SEP signal of SEP-NRX1βS4(−)∆HRD (Fig. 6d), which does not bind Aβ42 oligomers (Supplementary Fig. 3). Thus, the binding of Aβ42 oligomers to the HRD is essential for AβO-induced reduction of NRX surface expression. Together with the results of our coculture assays (Fig. 4), these data suggest that Aβ42 oligomers diminish NRX-mediated presynaptic differentiation by decreasing surface expression of β-NRXs on axons.

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