Figure 6. Distortion of the dendritic tree, microtubule breakdown and mitochondria loss occur in concomitance with decline of presynaptic proteins density in living hippocampal neurons chronically exposed to NH₂htau.

a.-b.-c. Confocal microscopy analysis of double immunofluorescence carried out on mature hippocampal primary neurons (DIV15) exposed for 48h to NH₂htau and its reverse control sequence (1µM). Merge images show the overlay of the three fluorescence channels, Differential Interference Contrast (DIC; gray channel) enables the visualization of the neuritic network, DIC Merge is the composition of the three fluorescence and of the DIC channels. (a): presynaptic synaptophysin (green channel) and dendritic MAP-2 (red channel). Nuclei (blue) were stained with Hoechst 33258 (0.5 mg/ml). Arrowheads and arrows point to MAP2- positive neurites of larger and smaller caliber, respectively. (b): presynaptic α-synuclein (green channel) and neuron-specific cytoskeletal beta III tubulin (red channel). Arrowheads and arrows point to beta III tubulin-positive neurites of larger and smaller caliber, respectively. (c): presynaptic synapsin I (green channel) and mitochondrial marker COX I (red channel). Arrowheads point to synapsin I-labeled presynaptic spots and arrows point to COX I -positive mitochondrial structures. In the merge, DIC and DIC-Merge channels, arrowheads and arrows appear in opposition to give evidence to mitochondria resident at juxstaposed presynaptic sites. Asterisks mark typical punctuate structures immunoreactive for both synapsin I and COX I (yellow dots) representing mitochondria which are localized to presynaptic sites (i.e.synaptic mitochondria). Note the loss of double-stained synapsin I/COX I puncta and the decrease of juxstaposed presynaptic sites/mitochondria in the NH₂htau-treated cultures. Images are representative of at least three independent experiments. Scale bar: A=20 µm ;B-C=10 µm.