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. 2019 Jan 3;10:53. doi: 10.1038/s41467-018-07971-8

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Effect of APP C-terminus editing on neuronal physiology. a AAV9-sgRNA and AAV9-Cas9 expression vectors. Note that the sgRNA vector co-expresses GFP and the Cas9 is tagged to HA, for identification of transduced neurons. b Cultured hippocampal neurons were transduced with AAV9s carrying mo-APP-sgRNA/Cas9 (or Cas9 only) and immunoblotted with the Y188 and 22C11 antibodies (with GSI). Note attenuation of CTFs by the mo-APP-sgRNA. c Neurons were transfected (at the time of plating) with APP CRISPR. Neuritic/axon outgrowth was analyzed after 5-6 days. Neurons were transfected or infected at DIV7 with APP CRISPR, and synapse structure/function was analyzed after 14–17 days. d Top: Representative images of neurons transfected with the mo-APP-sgRNA/Cas9 (or Cas9 only). Bottom: Quantifications of axon length and number of neurites/branches; note that there was no significant difference (mean ± SEM; axon length: 30 cells for control and 27 cells for mo-APP-sgRNA from two independent experiments, p = 0.2462; neurite number: 35 cells for control and 31 cells for mo-APP-sgRNA from two independent experiments, p = 0.2289; branch number: 27 cells for both conditions from two independent experiments, p = 0.6008 by two-tailed t-test; ns = non-significant). Scale bar 20 μm. e Neurons were infected with mo-APP-sgRNA/Cas9 (or Cas9 only), and fixed/stained with the presynaptic marker VAMP2. Note that the presynaptic density (VAMP2 puncta) was similar in both groups (mean ± SEM of VAMP2 staining along 27 dendrites for control and 25 dendrites for mo-APP-sgRNA from two independent experiments, p = 0.3132 by two-tailed t-test). Scale bar 10 μm. f Neurons were transfected with mo-APP-sgRNA/Cas9 (or Cas9 only as controls). Spine density in both groups was similar (mean ± SEM of 18 dendrites for control and 16 dendrites for mo-APP-sgRNA from two independent experiments, p = 0.7456 by two-tailed t-test). Scale bar 10 μm. g Miniature excitatory postsynaptic currents (mEPSC) were recorded from neurons infected with AAV9-APP-sgRNA/Cas9. Top: Representative mEPSC traces in control and mo-APP-sgRNA transduced neurons. Corresponding alignments of mEPSCs with average (white traces) are shown on right. Bottom: Cumulative histograms of mEPSC amplitude, 20–80% rise-time and inter-event interval in mo-APP-sgRNA/Cas9 and the Cas9-only infected neurons (note no significant differences). Source data are provided as a Source Data file