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Premutation in the Fragile X Mental Retardation 1 (FMR1) Gene Affects Maternal Zn-milk and Perinatal Brain Bioenergetics and Scaffolding.

Napoli E, Ross-Inta C, Song G, Wong S, Hagerman R, Gane LW, Smilowitz JT, Tassone F, Giulivi C - Front Neurosci (2016)

Bottom Line: Given that the most significant effects were observed at the end of the lactation period, we hypothesized that KI milk might have a role at compounding the deleterious effects on the FMR1 genetic background.A highly significant milk type × genotype interaction was observed for all three-brain regions, being cortex the most influenced.Finally, lower milk-Zn levels were recorded in milk from lactating women carrying the premutation as well as other Zn-related outcomes (Zn-dependent alkaline phosphatase activity and lactose biosynthesis-whose limiting step is the Zn-dependent β-1,4-galactosyltransferase).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, School of Veterinary Medicine Davis, CA, USA.

ABSTRACT
Fragile X premutation alleles have 55-200 CGG repeats in the 5' UTR of the FMR1 gene. Altered zinc (Zn) homeostasis has been reported in fibroblasts from >60 years old premutation carriers, in which Zn supplementation significantly restored Zn-dependent mitochondrial protein import/processing and function. Given that mitochondria play a critical role in synaptic transmission, brain function, and cognition, we tested FMRP protein expression, brain bioenergetics, and expression of the Zn-dependent synaptic scaffolding protein SH3 and multiple ankyrin repeat domains 3 (Shank3) in a knock-in (KI) premutation mouse model with 180 CGG repeats. Mitochondrial outcomes correlated with FMRP protein expression (but not FMR1 gene expression) in KI mice and human fibroblasts from carriers of the pre- and full-mutation. Significant deficits in brain bioenergetics, Zn levels, and Shank3 protein expression were observed in the Zn-rich regions KI hippocampus and cerebellum at PND21, with some of these effects lasting into adulthood (PND210). A strong genotype × age interaction was observed for most of the outcomes tested in hippocampus and cerebellum, whereas in cortex, age played a major role. Given that the most significant effects were observed at the end of the lactation period, we hypothesized that KI milk might have a role at compounding the deleterious effects on the FMR1 genetic background. A higher gene expression of ZnT4 and ZnT6, Zn transporters abundant in brain and lactating mammary glands, was observed in the latter tissue of KI dams. A cross-fostering experiment allowed improving cortex bioenergetics in KI pups nursing on WT milk. Conversely, WT pups nursing on KI milk showed deficits in hippocampus and cerebellum bioenergetics. A highly significant milk type × genotype interaction was observed for all three-brain regions, being cortex the most influenced. Finally, lower milk-Zn levels were recorded in milk from lactating women carrying the premutation as well as other Zn-related outcomes (Zn-dependent alkaline phosphatase activity and lactose biosynthesis-whose limiting step is the Zn-dependent β-1,4-galactosyltransferase). In premutation carriers, altered Zn homeostasis, brain bioenergetics and Shank3 levels could be compounded by Zn-deficient milk, increasing the risk of developing emotional and neurological/cognitive problems and/or FXTAS later in life.

No MeSH data available.


Related in: MedlinePlus

ZnT4 and ZnT6 gene expression in mammary glands from lactating WT and KI dams. Representative image of the PCR products (obtained by RT-qPCR as described under Methods) that were separated in a 1.3% agarose gel and visualized with ethidium bromide (A). The fragments exhibited the expected sizes of 585 bp (ZnT4) and 537 bp (ZnT6). GAPDH PCR product was separated in a 3% agarose gel electrophoresis with ethidium bromide resulting in a product of the expected of 107 bp (not shown). Intensities of ZnT4 and ZnT6 bands were obtained in a Kodak Imager were normalized by that of GAPDH(B). Bars represent averages ± SEM of 4 individuals/genotype. Statistical analysis was carried out with the Student's t-test between WT and KI. *p < 0.05.
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Figure 9: ZnT4 and ZnT6 gene expression in mammary glands from lactating WT and KI dams. Representative image of the PCR products (obtained by RT-qPCR as described under Methods) that were separated in a 1.3% agarose gel and visualized with ethidium bromide (A). The fragments exhibited the expected sizes of 585 bp (ZnT4) and 537 bp (ZnT6). GAPDH PCR product was separated in a 3% agarose gel electrophoresis with ethidium bromide resulting in a product of the expected of 107 bp (not shown). Intensities of ZnT4 and ZnT6 bands were obtained in a Kodak Imager were normalized by that of GAPDH(B). Bars represent averages ± SEM of 4 individuals/genotype. Statistical analysis was carried out with the Student's t-test between WT and KI. *p < 0.05.

Mentions: In lactating mammary glands from WT dams, both ZnT4 and ZnT6 (normalized to GAPDH) were expressed with a ratio of ZnT6/ZnT4 equal to 15 (Figure 9A). The gene expression of both ZnTs was significantly higher (two-fold) in KI than WT, with no significant changes in the ZnT6/ZnT4 expression ratio compared to WT (Figure 9B). These results suggested a disrupted ZnT expression, which may affect Zn efflux in milk, and as a consequence, the Zn status of the suckling pups.


Premutation in the Fragile X Mental Retardation 1 (FMR1) Gene Affects Maternal Zn-milk and Perinatal Brain Bioenergetics and Scaffolding.

Napoli E, Ross-Inta C, Song G, Wong S, Hagerman R, Gane LW, Smilowitz JT, Tassone F, Giulivi C - Front Neurosci (2016)

ZnT4 and ZnT6 gene expression in mammary glands from lactating WT and KI dams. Representative image of the PCR products (obtained by RT-qPCR as described under Methods) that were separated in a 1.3% agarose gel and visualized with ethidium bromide (A). The fragments exhibited the expected sizes of 585 bp (ZnT4) and 537 bp (ZnT6). GAPDH PCR product was separated in a 3% agarose gel electrophoresis with ethidium bromide resulting in a product of the expected of 107 bp (not shown). Intensities of ZnT4 and ZnT6 bands were obtained in a Kodak Imager were normalized by that of GAPDH(B). Bars represent averages ± SEM of 4 individuals/genotype. Statistical analysis was carried out with the Student's t-test between WT and KI. *p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835505&req=5

Figure 9: ZnT4 and ZnT6 gene expression in mammary glands from lactating WT and KI dams. Representative image of the PCR products (obtained by RT-qPCR as described under Methods) that were separated in a 1.3% agarose gel and visualized with ethidium bromide (A). The fragments exhibited the expected sizes of 585 bp (ZnT4) and 537 bp (ZnT6). GAPDH PCR product was separated in a 3% agarose gel electrophoresis with ethidium bromide resulting in a product of the expected of 107 bp (not shown). Intensities of ZnT4 and ZnT6 bands were obtained in a Kodak Imager were normalized by that of GAPDH(B). Bars represent averages ± SEM of 4 individuals/genotype. Statistical analysis was carried out with the Student's t-test between WT and KI. *p < 0.05.
Mentions: In lactating mammary glands from WT dams, both ZnT4 and ZnT6 (normalized to GAPDH) were expressed with a ratio of ZnT6/ZnT4 equal to 15 (Figure 9A). The gene expression of both ZnTs was significantly higher (two-fold) in KI than WT, with no significant changes in the ZnT6/ZnT4 expression ratio compared to WT (Figure 9B). These results suggested a disrupted ZnT expression, which may affect Zn efflux in milk, and as a consequence, the Zn status of the suckling pups.

Bottom Line: Given that the most significant effects were observed at the end of the lactation period, we hypothesized that KI milk might have a role at compounding the deleterious effects on the FMR1 genetic background.A highly significant milk type × genotype interaction was observed for all three-brain regions, being cortex the most influenced.Finally, lower milk-Zn levels were recorded in milk from lactating women carrying the premutation as well as other Zn-related outcomes (Zn-dependent alkaline phosphatase activity and lactose biosynthesis-whose limiting step is the Zn-dependent β-1,4-galactosyltransferase).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, School of Veterinary Medicine Davis, CA, USA.

ABSTRACT
Fragile X premutation alleles have 55-200 CGG repeats in the 5' UTR of the FMR1 gene. Altered zinc (Zn) homeostasis has been reported in fibroblasts from >60 years old premutation carriers, in which Zn supplementation significantly restored Zn-dependent mitochondrial protein import/processing and function. Given that mitochondria play a critical role in synaptic transmission, brain function, and cognition, we tested FMRP protein expression, brain bioenergetics, and expression of the Zn-dependent synaptic scaffolding protein SH3 and multiple ankyrin repeat domains 3 (Shank3) in a knock-in (KI) premutation mouse model with 180 CGG repeats. Mitochondrial outcomes correlated with FMRP protein expression (but not FMR1 gene expression) in KI mice and human fibroblasts from carriers of the pre- and full-mutation. Significant deficits in brain bioenergetics, Zn levels, and Shank3 protein expression were observed in the Zn-rich regions KI hippocampus and cerebellum at PND21, with some of these effects lasting into adulthood (PND210). A strong genotype × age interaction was observed for most of the outcomes tested in hippocampus and cerebellum, whereas in cortex, age played a major role. Given that the most significant effects were observed at the end of the lactation period, we hypothesized that KI milk might have a role at compounding the deleterious effects on the FMR1 genetic background. A higher gene expression of ZnT4 and ZnT6, Zn transporters abundant in brain and lactating mammary glands, was observed in the latter tissue of KI dams. A cross-fostering experiment allowed improving cortex bioenergetics in KI pups nursing on WT milk. Conversely, WT pups nursing on KI milk showed deficits in hippocampus and cerebellum bioenergetics. A highly significant milk type × genotype interaction was observed for all three-brain regions, being cortex the most influenced. Finally, lower milk-Zn levels were recorded in milk from lactating women carrying the premutation as well as other Zn-related outcomes (Zn-dependent alkaline phosphatase activity and lactose biosynthesis-whose limiting step is the Zn-dependent β-1,4-galactosyltransferase). In premutation carriers, altered Zn homeostasis, brain bioenergetics and Shank3 levels could be compounded by Zn-deficient milk, increasing the risk of developing emotional and neurological/cognitive problems and/or FXTAS later in life.

No MeSH data available.


Related in: MedlinePlus