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Effects of trace metal profiles characteristic for autism on synapses in cultured neurons.

Hagmeyer S, Mangus K, Boeckers TM, Grabrucker AM - Neural Plast. (2015)

Bottom Line: Additionally, synaptic protein levels of GluN2a and Shanks are reduced.Although Zn supplementation is able to rescue the aforementioned alterations, Zn deficiency is not solely responsible as causative factor.Thus, we conclude that balancing Zn levels in ASD might be a prime target to normalize synaptic alterations caused by biometal dyshomeostasis.

View Article: PubMed Central - PubMed

Affiliation: WG Molecular Analysis of Synaptopathies, Neurology Department, Neurocenter of Ulm University, 89081 Ulm, Germany.

ABSTRACT
Various recent studies revealed that biometal dyshomeostasis plays a crucial role in the pathogenesis of neurological disorders such as autism spectrum disorders (ASD). Substantial evidence indicates that disrupted neuronal homeostasis of different metal ions such as Fe, Cu, Pb, Hg, Se, and Zn may mediate synaptic dysfunction and impair synapse formation and maturation. Here, we performed in vitro studies investigating the consequences of an imbalance of transition metals on glutamatergic synapses of hippocampal neurons. We analyzed whether an imbalance of any one metal ion alters cell health and synapse numbers. Moreover, we evaluated whether a biometal profile characteristic for ASD patients influences synapse formation, maturation, and composition regarding NMDA receptor subunits and Shank proteins. Our results show that an ASD like biometal profile leads to a reduction of NMDAR (NR/Grin/GluN) subunit 1 and 2a, as well as Shank gene expression along with a reduction of synapse density. Additionally, synaptic protein levels of GluN2a and Shanks are reduced. Although Zn supplementation is able to rescue the aforementioned alterations, Zn deficiency is not solely responsible as causative factor. Thus, we conclude that balancing Zn levels in ASD might be a prime target to normalize synaptic alterations caused by biometal dyshomeostasis.

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Related in: MedlinePlus

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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fig3: 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.

Mentions: Cells exposed to ASD2 medium indeed displayed a significant reduction in cell health visible by an increased number of apoptotic nuclei and increased dendritic fragmentation (Figures 3(a) and 3(b)). The number of synapses per 10 μm dendrite length assessed by quantification of Bassoon and Homer1b/c positive puncta was significantly reduced in cells growing in medium resembling the biometal profile found in ASD patients (ASD2) (Figure 3(c)). Along with a reduction in the number of synapses, gene expression levels of synaptic receptors such as NMDAR (GluN1, GluN2a, and GluN2b) as well as the Zn dependent Shank scaffold proteins (Shank1, Shank2, and Shank3) show significant alterations (Figure 3(d)). While the presence of putative toxic metals increased in ASD patients (ASD1) is sufficient to significantly increase GluN2b expression levels, GluN1 and GluN2a and Shank1, Shank2, and Shank3 mRNA levels are only significantly altered in case of an additional Zn and Fe deficiency (ASD2). Fluorescent readouts for Shank proteins show a reduction on protein level under ASD1 conditions, while the remaining synapses under ASD2 condition display normal Shank levels (Figure 3(e)). However, analysis using protein biochemistry shows that, overall, Shank protein concentrations are reduced in the P2 fraction of cells growing under ASD2 condition (Figure 3(f)). Additionally, similar to the decrease of mRNA levels of NMDAR subunits, we detected significantly less GluN2a protein and a trend towards a decrease of GluN2b protein levels in cells grown in ASD2 medium. Furthermore, ZnT-1 expression levels, a Zn exporter recently described as being enriched at postsynapses [16], reacts very sensitively to changes in trace metal concentrations as those induced by application of ASD2 medium (Figure 3(f)).


Effects of trace metal profiles characteristic for autism on synapses in cultured neurons.

Hagmeyer S, Mangus K, Boeckers TM, Grabrucker AM - Neural Plast. (2015)

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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fig3: 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.
Mentions: Cells exposed to ASD2 medium indeed displayed a significant reduction in cell health visible by an increased number of apoptotic nuclei and increased dendritic fragmentation (Figures 3(a) and 3(b)). The number of synapses per 10 μm dendrite length assessed by quantification of Bassoon and Homer1b/c positive puncta was significantly reduced in cells growing in medium resembling the biometal profile found in ASD patients (ASD2) (Figure 3(c)). Along with a reduction in the number of synapses, gene expression levels of synaptic receptors such as NMDAR (GluN1, GluN2a, and GluN2b) as well as the Zn dependent Shank scaffold proteins (Shank1, Shank2, and Shank3) show significant alterations (Figure 3(d)). While the presence of putative toxic metals increased in ASD patients (ASD1) is sufficient to significantly increase GluN2b expression levels, GluN1 and GluN2a and Shank1, Shank2, and Shank3 mRNA levels are only significantly altered in case of an additional Zn and Fe deficiency (ASD2). Fluorescent readouts for Shank proteins show a reduction on protein level under ASD1 conditions, while the remaining synapses under ASD2 condition display normal Shank levels (Figure 3(e)). However, analysis using protein biochemistry shows that, overall, Shank protein concentrations are reduced in the P2 fraction of cells growing under ASD2 condition (Figure 3(f)). Additionally, similar to the decrease of mRNA levels of NMDAR subunits, we detected significantly less GluN2a protein and a trend towards a decrease of GluN2b protein levels in cells grown in ASD2 medium. Furthermore, ZnT-1 expression levels, a Zn exporter recently described as being enriched at postsynapses [16], reacts very sensitively to changes in trace metal concentrations as those induced by application of ASD2 medium (Figure 3(f)).

Bottom Line: Additionally, synaptic protein levels of GluN2a and Shanks are reduced.Although Zn supplementation is able to rescue the aforementioned alterations, Zn deficiency is not solely responsible as causative factor.Thus, we conclude that balancing Zn levels in ASD might be a prime target to normalize synaptic alterations caused by biometal dyshomeostasis.

View Article: PubMed Central - PubMed

Affiliation: WG Molecular Analysis of Synaptopathies, Neurology Department, Neurocenter of Ulm University, 89081 Ulm, Germany.

ABSTRACT
Various recent studies revealed that biometal dyshomeostasis plays a crucial role in the pathogenesis of neurological disorders such as autism spectrum disorders (ASD). Substantial evidence indicates that disrupted neuronal homeostasis of different metal ions such as Fe, Cu, Pb, Hg, Se, and Zn may mediate synaptic dysfunction and impair synapse formation and maturation. Here, we performed in vitro studies investigating the consequences of an imbalance of transition metals on glutamatergic synapses of hippocampal neurons. We analyzed whether an imbalance of any one metal ion alters cell health and synapse numbers. Moreover, we evaluated whether a biometal profile characteristic for ASD patients influences synapse formation, maturation, and composition regarding NMDA receptor subunits and Shank proteins. Our results show that an ASD like biometal profile leads to a reduction of NMDAR (NR/Grin/GluN) subunit 1 and 2a, as well as Shank gene expression along with a reduction of synapse density. Additionally, synaptic protein levels of GluN2a and Shanks are reduced. Although Zn supplementation is able to rescue the aforementioned alterations, Zn deficiency is not solely responsible as causative factor. Thus, we conclude that balancing Zn levels in ASD might be a prime target to normalize synaptic alterations caused by biometal dyshomeostasis.

Show MeSH
Related in: MedlinePlus