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Fig. 1 | Journal of Neuroinflammation

Fig. 1

From: Astrocytic C–X–C motif chemokine ligand-1 mediates β-amyloid-induced synaptotoxicity

Fig. 1

Conditioned medium from Aβ-stimulated astrocytes exaggerates the damaging effects of Aβ at synapses. A Dendritic spine density was measured in neurons transfected at 7DIV with GFP and treated with WTCM astro, TGCM astro and TGCM astro-ID or directly with WTCM and TGCM at 13DIV for 24 h. Representative images are shown. Scale bar is 10 μm. B Quantification of spine density in 10–20 neurons per experimental replicate from eight individual neuron preps (n = 8). TGCM astro caused increased dendritic spine loss relative to treatment with TGCM directly, and this is maintained when Aβ is immunodepleted from the medium. Box indicates data from neurons directly exposed to TGCM or WTCM for clarity. Data was analysed by ordinary one-way ANOVA and Tukey’s post-hoc tests. C Neuronal lysates were immunoblotted using antibodies against PSD-95 (95 kDa), synapsin-1 (77 kDa), cleaved caspase-3 (17–19 kDa) and β-actin (42 kDa). Representative blots are shown. Western blot band intensities were quantified and showed that TGCM astro caused reductions in the abundance of the synaptic markers D PSD-95 and E synapsin-1 and an increase in F cleaved (active) caspase-3, that is maintained when Aβ was immunodepleted (TGCM astro-ID). Data is from eight individual neuron preps (n = 8), each of which have two experimental replicates. Data was analysed by ordinary one-way ANOVA and Tukey’s multiple comparison post-hoc tests. G Neuronal complexity as a measure of neuron health was analysed for all cells in three wells of five biological replicates (n = 5) using Harmony software. This showed H reduced total neurite length, and fewer number of I nodes and J roots following treatment with TGCM astro and TGCM astro-ID, indicating that astrocytic secretions are damaging to neurons. Data was analysed by ordinary one-way ANOVA and Tukey’s multiple comparison post-hoc tests. K Astrocytes were cultured and treated in cell culture inserts, the medium removed by washing and the astrocytes added to neuron cultures. Representative western blots of neuronal lysate from co-culture experiments are shown. Western blot data was quantified by normalising the protein of interest to β-actin levels in the same sample and showed reduced L PSD-95 and M synapsin-1, and N increased levels of cleaved caspase-3 when neurons were co-cultured with astrocytes that had previously been exposed to TGCM (n = 6, data is from six independent experiments, each of which had three technical replicates). Data was analysed using an unpaired t test. Data on graphs is mean ± SEM and is shown as percentage average control (WTCM astro). *p < 0.05, **p < 0.01

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