Effects of Trace Metal Profiles Characteristic for Autism on Synapses in Cultured Neurons

Figure 3

An ASD biometal profile affects cell health and synapse numbers in vitro. (a) Hippocampal neurons were grown from DIV 10 to DIV 14 in cell culture media containing different sets of trace metals: control cells (Ctrl) were grown in Neurobasal medium. ASD1 cells were grown in Neurobasal medium with addition of putative toxic metals (0.5 μM Cd, Cu, Hg, and 2 μM Pb). ASD2 cells were grown in trace metal depleted Neurobasal medium that was reconstituted only for Mg and Ca, with addition of the putative toxic metals (0.5 μM Cd, Cu, Hg, and 2 μM Pb). The amount of cell death was calculated assessing the number of neuronal apoptotic nuclei (identified by MAP2 and DAPI staining) per optic field (from 5 fields of view) normalized against the total number of neurons per optic field. A significant reduction in cell health can be observed in cells deficient in Fe and Zn and subjected to toxic metals (ASD2). (b) The number of primary, secondary, and tertiary dendrites was investigated from 10 cells per condition. Cells were stained with MAP2 antibody. As signs of cell death, neurons show fragmentation (pinching off) dendrites, starting with branches more distal from the soma. Dendrites showing signs of fragmentation were not counted. Corresponding to the increase in cell death, neurons growing under ASD2 conditions showed significantly increased signs of dendritic fragmentation. (c) Synapses were labeled using Bassoon and Homer1b/c fluorescence and the number of immunoreactive puncta was measured per 10 μm dendrite length on primary dendrites (3 dendrites per cell, 10 cells in total per group). Merged images show additional staining of nuclei using DAPI (cyan) and MAP2 (blue). A significant reduction can be seen in cells growing in medium resembling the biometal profile found in ASD patients (ASD2). (d) Expression levels of NMDA receptor subunits (GluN1, GluN2a, and GluN2b) and SHANK genes (Shank1, Shank2, and Shank3) were measured by qRT-PCR. Virtual mRNA concentrations are shown averaging from three replicates and normalized against HMBS. A significant decrease of GluN1 and GluN2a mRNA expression levels can be seen in cells grown under ASD2 conditions. Under ASD1 conditions, GluN2b levels significantly increase. A significant reduction in gene expression levels can also be observed in Shank family members under ASD2 conditions, which is significant for Shank1, Shank2, and Shank3. (e) Immunocytochemistry of hippocampal neurons DIV 14 grown under control, ASD1, and ASD2 conditions. The fluorescence intensity of Shank positive puncta was measured using antibodies specific for Shank1, Shank2, and Shank3. Exemplary images (upper panel) and quantification of average puncta signal intensity of 10 cells per condition (lower panel). Merged images show additional DAPI staining of the nucleus. Neurons grown under ASD1 conditions show a significant decrease of synaptic Shank proteins, while neurons under ASD2 conditions did not show a reduction. (f) Analysis of protein expression levels in synaptic (P2) fractions of NMDAR subunits, proteins of the Shank family, and ZnT-1 from three independent experiments normalized against β-III-tubulin or actin. Neurons grown in ASD2 medium show a significant reduction of GluN2a receptor subunits and a trend towards a decrease of GluN2b. In contrast, synaptic ZnT-1 shows a strong upregulation. The expression of Shank family members is reduced under ASD2 conditions (lower left panel). The reduction is significant for Shank1 and Shank2 and seen as a clear trend for Shank3. Exemplary bands are shown in the lower right panel.

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