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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.


Aβ42 oligomers diminish neurexin-mediated excitatory presynaptic differentiation.(a) Representative images of triple immunolabeling for VGLUT1, VGAT and surface HA in HEK293 cells expressing the indicated extracellularly HA-tagged construct cocultured with cultured hippocampal neurons and treated with Aβ42 oligomers (AβOs, 500 nM, monomer equivalent) or vehicle. HA-CD4 is used as a negative control protein as it lacks synaptogenic activity. AβOs seem not to affect VGLUT1 accumulation induced by an HA-TrkC non-catalytic isoform (HA-TrkC) or VGLUT1 or VGAT accumulation induced by HA-Slitrk2. In contrast, AβOs seem to decrease VGLUT1 accumulation induced by HA-NLG1, HA-NLG2, or HA-LRRTM2. AβOs seem not to affect VGAT accumulation induced by HA-NLG1 or HA-NLG2. Scale bar represents 20 μm. (b,c) Quantification of the total intensity of VGLUT1 (b) and VGAT (c) puncta on HEK293 cells expressing the indicated HA-tagged proteins divided by HEK293 cell area. n = 30 cells for each construct from three independent experiments, one-way ANOVA, P < 0.0001. #P < 0.05 and *P < 0.01 for the indicated comparisons between vehicle control and AβO treatment by Bonferroni multiple comparisons tests. n.s., not significant. Data are presented as mean ± SEM.
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f4: Aβ42 oligomers diminish neurexin-mediated excitatory presynaptic differentiation.(a) Representative images of triple immunolabeling for VGLUT1, VGAT and surface HA in HEK293 cells expressing the indicated extracellularly HA-tagged construct cocultured with cultured hippocampal neurons and treated with Aβ42 oligomers (AβOs, 500 nM, monomer equivalent) or vehicle. HA-CD4 is used as a negative control protein as it lacks synaptogenic activity. AβOs seem not to affect VGLUT1 accumulation induced by an HA-TrkC non-catalytic isoform (HA-TrkC) or VGLUT1 or VGAT accumulation induced by HA-Slitrk2. In contrast, AβOs seem to decrease VGLUT1 accumulation induced by HA-NLG1, HA-NLG2, or HA-LRRTM2. AβOs seem not to affect VGAT accumulation induced by HA-NLG1 or HA-NLG2. Scale bar represents 20 μm. (b,c) Quantification of the total intensity of VGLUT1 (b) and VGAT (c) puncta on HEK293 cells expressing the indicated HA-tagged proteins divided by HEK293 cell area. n = 30 cells for each construct from three independent experiments, one-way ANOVA, P < 0.0001. #P < 0.05 and *P < 0.01 for the indicated comparisons between vehicle control and AβO treatment by Bonferroni multiple comparisons tests. n.s., not significant. Data are presented as mean ± SEM.

Mentions: NRXs mediate the presynaptic induction activity of NLGs and LRRTM1/2/3 in hippocampal neurons181920212227282930. We thus tested whether Aβ42 oligomers affect NRX-mediated presynaptic differentiation in coculture-based artificial synapse formation assays (Fig. 4). As reported previously, HEK293 (hereinafter HEK) cells expressing NLG1, NLG2, or LRRTM2 induced the accumulation of the excitatory presynaptic marker VGLUT1. NLG1- or NLG2-expressing HEK cells further induced the accumulation of the inhibitory presynaptic marker VGAT in cocultured hippocampal neurons. Treatment with Aβ42 oligomers significantly decreased VGLUT1 accumulation induced by NLG1, NLG2, or LRRTM2 (Fig. 4a,b). Interestingly, treatment with Aβ42 oligomers had no effect on VGAT accumulation induced by NLG1 or NLG2 (Fig. 4a,c). These data indicate that Aβ treatment diminishes excitatory, but not inhibitory, presynaptic differentiation induced by NRX-interacting synaptic organizers. Notably, treatment with Aβ42 oligomers did not affect TrkC-induced or Slitrk2-induced VGLUT1 accumulation (Fig. 4a,b), which is mediated by RPTPs in cocultured hippocampal neurons22232425, indicating that Aβ treatment diminishes NRX-mediated, but not RPTP-mediated, presynaptic differentiation. Aβ42 oligomers did not affect VGAT accumulation induced by HEK cells expressing Slitrk2 (Fig. 4a,c). The observed phenotypes induced by Aβ42 oligomers are not due to the reduction of surface expression levels of the tested organizers on HEK cells because Aβ42 oligomers did not alter their surface expression (Supplementary Fig. 1). Together these results indicate that Aβ42 oligomers selectively diminish NRX-mediated excitatory presynaptic differentiation and also that RPTP-mediated presynaptic differentiation is insensitive to Aβ42 oligomers.


Amyloid- β Oligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization
Aβ42 oligomers diminish neurexin-mediated excitatory presynaptic differentiation.(a) Representative images of triple immunolabeling for VGLUT1, VGAT and surface HA in HEK293 cells expressing the indicated extracellularly HA-tagged construct cocultured with cultured hippocampal neurons and treated with Aβ42 oligomers (AβOs, 500 nM, monomer equivalent) or vehicle. HA-CD4 is used as a negative control protein as it lacks synaptogenic activity. AβOs seem not to affect VGLUT1 accumulation induced by an HA-TrkC non-catalytic isoform (HA-TrkC) or VGLUT1 or VGAT accumulation induced by HA-Slitrk2. In contrast, AβOs seem to decrease VGLUT1 accumulation induced by HA-NLG1, HA-NLG2, or HA-LRRTM2. AβOs seem not to affect VGAT accumulation induced by HA-NLG1 or HA-NLG2. Scale bar represents 20 μm. (b,c) Quantification of the total intensity of VGLUT1 (b) and VGAT (c) puncta on HEK293 cells expressing the indicated HA-tagged proteins divided by HEK293 cell area. n = 30 cells for each construct from three independent experiments, one-way ANOVA, P < 0.0001. #P < 0.05 and *P < 0.01 for the indicated comparisons between vehicle control and AβO treatment by Bonferroni multiple comparisons tests. n.s., not significant. Data are presented as mean ± SEM.
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f4: Aβ42 oligomers diminish neurexin-mediated excitatory presynaptic differentiation.(a) Representative images of triple immunolabeling for VGLUT1, VGAT and surface HA in HEK293 cells expressing the indicated extracellularly HA-tagged construct cocultured with cultured hippocampal neurons and treated with Aβ42 oligomers (AβOs, 500 nM, monomer equivalent) or vehicle. HA-CD4 is used as a negative control protein as it lacks synaptogenic activity. AβOs seem not to affect VGLUT1 accumulation induced by an HA-TrkC non-catalytic isoform (HA-TrkC) or VGLUT1 or VGAT accumulation induced by HA-Slitrk2. In contrast, AβOs seem to decrease VGLUT1 accumulation induced by HA-NLG1, HA-NLG2, or HA-LRRTM2. AβOs seem not to affect VGAT accumulation induced by HA-NLG1 or HA-NLG2. Scale bar represents 20 μm. (b,c) Quantification of the total intensity of VGLUT1 (b) and VGAT (c) puncta on HEK293 cells expressing the indicated HA-tagged proteins divided by HEK293 cell area. n = 30 cells for each construct from three independent experiments, one-way ANOVA, P < 0.0001. #P < 0.05 and *P < 0.01 for the indicated comparisons between vehicle control and AβO treatment by Bonferroni multiple comparisons tests. n.s., not significant. Data are presented as mean ± SEM.
Mentions: NRXs mediate the presynaptic induction activity of NLGs and LRRTM1/2/3 in hippocampal neurons181920212227282930. We thus tested whether Aβ42 oligomers affect NRX-mediated presynaptic differentiation in coculture-based artificial synapse formation assays (Fig. 4). As reported previously, HEK293 (hereinafter HEK) cells expressing NLG1, NLG2, or LRRTM2 induced the accumulation of the excitatory presynaptic marker VGLUT1. NLG1- or NLG2-expressing HEK cells further induced the accumulation of the inhibitory presynaptic marker VGAT in cocultured hippocampal neurons. Treatment with Aβ42 oligomers significantly decreased VGLUT1 accumulation induced by NLG1, NLG2, or LRRTM2 (Fig. 4a,b). Interestingly, treatment with Aβ42 oligomers had no effect on VGAT accumulation induced by NLG1 or NLG2 (Fig. 4a,c). These data indicate that Aβ treatment diminishes excitatory, but not inhibitory, presynaptic differentiation induced by NRX-interacting synaptic organizers. Notably, treatment with Aβ42 oligomers did not affect TrkC-induced or Slitrk2-induced VGLUT1 accumulation (Fig. 4a,b), which is mediated by RPTPs in cocultured hippocampal neurons22232425, indicating that Aβ treatment diminishes NRX-mediated, but not RPTP-mediated, presynaptic differentiation. Aβ42 oligomers did not affect VGAT accumulation induced by HEK cells expressing Slitrk2 (Fig. 4a,c). The observed phenotypes induced by Aβ42 oligomers are not due to the reduction of surface expression levels of the tested organizers on HEK cells because Aβ42 oligomers did not alter their surface expression (Supplementary Fig. 1). Together these results indicate that Aβ42 oligomers selectively diminish NRX-mediated excitatory presynaptic differentiation and also that RPTP-mediated presynaptic differentiation is insensitive to Aβ42 oligomers.

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.