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

Figure 4

Synergistic and antagonistic relationships between trace metals and Zn. 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 trace metal depleted Neurobasal medium that was reconstituted for all depleted trace metals (Mg, Ca, Fe, and Zn) according to the manufacturer’s indicated metal concentrations. 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). ASD2 + Zn cells were grown in ASD2 medium except that 50 μM Zn was added to the medium. 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). Zn addition was unable to rescue the observed cell death (ASD2 + Zn). (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. The significant reduction seen under ASD2 conditions could be rescued significantly by addition of Zn. (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). A significant reduction can be seen in cells growing in medium resembling the biometal profile found in ASD patients (ASD2). Addition of Zn rescues the effects seen on synapse density. (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. Supplementation of Zn was able to significantly increase the reduction in mRNA levels seen in cells growing under ASD2 conditions. However, in all cases, mRNA levels still remained significantly decreased compared to controls for NMDAR subunits GluN1 and GluN2a. The expression levels of Shank family members were rescued completely (Shank1 and Shank3). (e) Zn supplementation on DIV 10–14 did not significantly alter fluorescence intensities of synaptic puncta. (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, which is rescued by supplementation of Zn. Similarly, synaptic ZnT-1 levels decrease almost to control levels in ASD2 medium with Zn compared to ASD2 Zn deficient medium. The expression of Shank proteins is reduced under ASD2 conditions and the synaptic concentration of Zn-dependent Shank2 and Shank3 family members is restored by Zn supplementation such that Shank2 levels are no longer significantly decreased under ASD2 + Zn condition (lower left panel). Exemplary bands are shown in the lower right panel. (g) Using ICP-MS, the extracellular metal concentrations of hippocampal cell cultures ( per metal, Mann-Whitney test) was measured with and without the presence of Zn. A reduction in the uptake of Cu, Hg, and Se was seen as trend upon coapplication of Zn, while an increase in uptake of Pb was visible.

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