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1.
Figure 2

Figure 2. Accumulation of neurexin-CTFs in PS/β-secretase-deficient hippocampal neurons.. From: Presenilin/?-Secretase Regulates Neurexin Processing at Synapses.

A) Cultured hippocampal neurons (3–5 DIV) were non-infected (control) or infected with recombinant lentivirus (LV) expressing: GFP, PS1 or PS1 D385A. The levels of neurexins (Nrx), PS1, N-cadherin (N-cadh) and CASK were analyzed by Western blotting 9–10 days after infection. Non-infected cultures and cultures infected with GFP were treated with vehicle or DAPT for the last 14 hours. FLα: full-length α-neurexins; FLβ: full-length β-neurexins. B) Hippocampal neurons were non-infected or infected with LV-GFP, LV-HA-βNrx1 or LV-HA-βNrx1 ΔC and treated with vehicle or DAPT, as above. The expression of HA-tagged β-neurexin proteins and neurexin-CTFs was analyzed by Western blotting with an anti-HA and cyto-neurexin antibodies, respectively. α-Actin levels were used as loading control.

Carlos A. Saura, et al. PLoS One. 2011;6(4):e19430.
2.
Figure 4

Figure 4. Accumulation of neurexin-CTFs in synaptic fractions of PS cDKO mice.. From: Presenilin/?-Secretase Regulates Neurexin Processing at Synapses.

A) Brain lysates from PS1+/+, PS1+/− and PS1−/− mouse embryos (E 17.5) or 2 month-old control wild-type (WT) and PS cDKO mice were analyzed by Western blotting with the cyto-neurexin (Nrx), PS1 and N-cadherin (N-cadh) antibodies, as indicated. Neurexin-CTFs and N-cadherin-CTF levels increased coinciding with decreased PS1 expression in animals lacking PS. The results were replicated in at least two independent mice per condition, as shown. B) Accumulation of neurexin-CTFs in purified synaptosomal and synaptic fractions from PS cDKO mice. Cortical brain lysates and pre- and post-synaptic fractions obtained from synaptosomal preparations from control wild-type (WT) and PS cDKO mice were analyzed by Western blotting with the cyto-neurexin, PS1, synaptophysin (SYP) and PSD95 antibodies. Neurexin-CTFs levels are markedly increased in synaptosomes and presynaptic fractions of PS cDKO mice. Synaptophysin and PSD-95 are specifically enriched in the presynaptic and postsynaptic fractions, respectively.

Carlos A. Saura, et al. PLoS One. 2011;6(4):e19430.
3.
Figure 1

Figure 1. Processing of neurexins by metalloprotease- and γ-secretase-dependent cleavages.. From: Presenilin/?-Secretase Regulates Neurexin Processing at Synapses.

A) HEK293 cells were transfected with vector or HA-βNrx1 and treated with DMSO (control), GM6001 or DAPT, as indicated. Left panels: Western blot experiments of cell lysates analyzed with the cyto-neurexin antibody. Right panel: Western blot with an anti-HA antibody of HA-immunoprecipitates of conditioned media of transfected cells. A cell lysate of HEK293 cells transfected with HA-βNrx1 was included in the same blot to show βNrx-FL. Arrow points at IgG heavy chains in immunoprecipitates. B) HEK293 cells transfected with vector, HA-βNrx1, HA-βNrx2 or HA-αNrx1 were incubated with vehicle or DAPT, as indicated. The presence of neurexin-CTFs was analyzed by Western blotting with the cyto-neurexin antibody. C) COS cells transfected with HA-βNrx1 (upper panels) or HA-βNrx2 (lower panels) were incubated with DMSO (control) or with DAPT alone or in combination with TAPI-1, GL189, TAPI-1 and GL189, or GM6001 inhibitors, as indicated. Neurexin-CTFs levels were studied by Western blotting with the cyto-neurexin antibody. α-Actin levels were used as loading control.

Carlos A. Saura, et al. PLoS One. 2011;6(4):e19430.
4.
Figure 6

Figure 6. Recruitment of neurexins to HA-Nlg1-mediated synapses in PS1-deficient neurons.. From: Presenilin/?-Secretase Regulates Neurexin Processing at Synapses.

A) Confocal images showing the recruitment of neurexins to HA-Nlg1 synapses in hippocampal cultures overexpressing PS1 or PS1 D385. Hippocampal neurons were infected at 3 DIV with LV-GFP, LV-PS1 or LV-PS1 D385, as indicated. The cultures were transfected 9 days after infection (12 DIV) with HA-Nlg1 and the localization of HA-Nlg1 (middle panels, red in the colocalization), and neurexins (lower panels, blue in the colocalization) was analyzed by immunofluorescence 2–3 days after transfection. The fluorescence signal obtained with the cyto-neurexin antibody is increased in dendrites expressing PS1 D385. B) Recruitment of neurexins to HEK293 cells expressing HA-Nlg1 in neuronal cultures overexpressing PS1 proteins. Hippocampal neurons infected with LV-GFP, LV-PS1 or LV-PS1 D385 at 3 DIV were co-cultured at 12 DIV with HEK293 cells expressing HA-Nlg1 for 24 hours. The cultures were co-stained with HA and neurexin antibodies, as above. The expression of exogenous PS1 proteins in these hemisynapses is restricted to axons contacting HA-Nlg1 expressing cells. C, D) Quantification of neurexin recruitment to dendrites (C) or HEK293 cells (D) expressing HA-Nlg1. Pooled data collected from three independent experiments are shown (n>24 cells per condition). Data are expressed as means ± SEMs. ***P<0.001. Scale bars: 5 µm.

Carlos A. Saura, et al. PLoS One. 2011;6(4):e19430.
5.
Figure 5

Figure 5. Recruitment of PS1 to neurexin/neuroligin mediated synapses.. From: Presenilin/?-Secretase Regulates Neurexin Processing at Synapses.

A) Confocal images of cultured hippocampal neurons transfected with GFP or HA-Nlg1 at 10–13 DIV and stained with PS1 (PS1 N-terminal antibody, red in the colocalization) and cyto-neurexin antibodies (blue in the colocalization) two days after transfection. PS1 is recruited along neurexins to HA-Nlg1 transfected dendrites. B) Hippocampal neurons (14–16 DIV) were co-cultured with HEK293 cells expressing GFP or HA-Nlg1 for 24 hours. The recruitment of PS1 and neurexins to the transfected HEK293 cells was analyzed by immunofluorescence with PS1 (Ab-2 antibody, red in the colocalization) and cyto-neurexin antibodies (blue in the colocalization). The Ab-2 antibody does not recognize human PS1 expressed in HEK293 cells. In A) and B) HA-Nlg1 expression was analyzed with a HA antibody and GFP expression was detected by direct fluorescence. C, D) Quantitative analysis of the recruitment of PS1 and neurexins to dendrites (C) or HEK293 cells (D) transfected with GFP or HA-Nlg1, as indicated. Recruitment was quantified as the relative area occupied by each specific marker in the transfected cell. Data collected from three (C, n = 25 cells per condition) or two (D, n = 20 cells per condition) independent experiments are shown. Asterisks indicate significant statistical differences (*P<0.05; ***P<0.001). Data are expressed as means ± SEMs. Scale bars: 5 µm.

Carlos A. Saura, et al. PLoS One. 2011;6(4):e19430.
6.
Figure 3

Figure 3. The processing of β-neurexin-1 is differentially affected by FAD-linked PS1 mutants.. From: Presenilin/?-Secretase Regulates Neurexin Processing at Synapses.

A) PS+/+ or PS−/− MEF cells were transfected with vector or co-transfected with HA-βNrx1 and the following plasmids: empty vector, PS1 WT or the FAD-linked mutants PS1 M146L, PS1 H163R, PS1 C410Y or PS1 ΔE9. Cell lysates were subjected to Western blotting analysis with the cyto-neurexin (Nrx), N-cadherin (N-cadh), PS1 and Nicastrin (NCT) antibodies. To study PS1 expression, two different PS1 antibodies recognizing both mouse (m) and human PS1 (PS1 loop antibody) or mouse PS1 (Ab-2 antibody) were used. The endoproteolysis of PS1 (∼45 kDa) generates 18–20 kDa CTFs. The expression of PS1 WT and FAD-associated PS1 mutants increased the mature form of Nicastrin recognized as a slower migrating band in Western blot experiments. The FAD-linked PS1 mutants C410Y and ΔE9 show deficient PS1 endoproteolysis and fail to rescue neurexin- and N-Cadherin-CTFs processing. β-Actin levels were used as loading control. B) Quantitative analysis of HA-βNrx1 CTFs relative levels. Data represents mean ± SD of three independent experiments. Statistical significant differences are shown (***P<0.001).

Carlos A. Saura, et al. PLoS One. 2011;6(4):e19430.

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