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Fig. 2 | Molecular Neurodegeneration

Fig. 2

From: Dynamic presenilin 1 and synaptotagmin 1 interaction modulates exocytosis and amyloid β production

Fig. 2

Design of the cell-permeable peptide and its application in primary neurons. a Schematic representation of the PS1-Syt1 complex. The PS1 fragment used for the design of the cell-permeable peptide to inhibit the PS1-Syt1 interaction is indicated in red. The adjacent image presents the fusion of the HIV1 TAT domain with the respective PS1 fragment. For initial experiments the peptide was conjugated with fluorescein (FITC) to visualize intracellular delivery. Scramble peptide was used as a control. b Representative images demonstrate successful delivery of 5 μM TAT-FITC and TAT-PS1-LNT-FITC (green) peptides to the primary neurons after two-hour incubation. The cells were counterstained with phalloidin (red) and DAPI (blue) to visualize the actin cytoskeleton and the nuclei, respectively; scale bar 10 μm; the magnified inserts demonstrate efficient entry of the FITC-labeled peptides into neuronal cell bodies and processes. c Analysis of the cytotoxicity of the scramble and PS1-LNT peptides in neurons after two-hour incubation at the respective concentrations. The % cytotoxicity was determined using lactase dehydrogenase (LDH) activity assay. The data are normalized to the 1% TX-100 condition, considered to cause 100% toxicity. There is negligible toxicity of scramble and PS1-LNT peptides used at 2 μM and 5 μM final concentrations. The data are presented as mean ± SEM, n = 37 for 5 μM, n = 13 for 10 μM and n = 7 for the remaining conditions. Statistical significance was determined using two-tailed unpaired Student’s t-test, ***p < 0.001. d Representative western blot shows efficient endoproteolysis of the full length PS1 (PS1-FL) to yield PS1-NTF and PS1-CTF in control and PS1-LNT pre-treated neurons. The graph presents quantification of the optical density of the respective bands. The ratio between PS1-CTF or PS1-NTF and PS1-FL was used to estimate endoproteolysis efficiency. The data are presented as mean ± SEM, n = 7. Statistical significance was determined using two-tailed unpaired Student’s t-test, ns p > 0.05

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