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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

Protein expression levels of mitochondrial ATPase β-subunit in brain from KI mice. ATPase β-subunit protein expression (normalized to GAPDH) was evaluated at PND9, 21 and 210 in hippocampus, cerebellum, and cortex lysates. Asterisk in the immunoblot image of hippocampus denotes a 90 CGG KI sample. Densitometry data for this sample have not been taken into account for averages calculation of KI. Cortex samples were ran in two different gels (PND9 and PND21-210) and are shown separately. Data are reported as mean ± SEM, n = 3–5 per genotype per time point, ran in triplicates. Statistical analysis was performed by Two-way ANOVA. Post-hoc analysis was performed by Tukey's HSD test for multiple comparisons. Significant difference between WT and KI are indicated by asterisks as follows. ****p < 0.0001; *p < 0.0492. Statistically significant differences among time points are indicated by letters as follows: p < 0.0001 (a, c), p = 0.0087 (b), p = 0.0222 (d). Further, statistical details on the genotype, age, and genotype × age effect can be found in Table 3. AUD, Arbitrary Units of Densitometry.
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Figure 3: Protein expression levels of mitochondrial ATPase β-subunit in brain from KI mice. ATPase β-subunit protein expression (normalized to GAPDH) was evaluated at PND9, 21 and 210 in hippocampus, cerebellum, and cortex lysates. Asterisk in the immunoblot image of hippocampus denotes a 90 CGG KI sample. Densitometry data for this sample have not been taken into account for averages calculation of KI. Cortex samples were ran in two different gels (PND9 and PND21-210) and are shown separately. Data are reported as mean ± SEM, n = 3–5 per genotype per time point, ran in triplicates. Statistical analysis was performed by Two-way ANOVA. Post-hoc analysis was performed by Tukey's HSD test for multiple comparisons. Significant difference between WT and KI are indicated by asterisks as follows. ****p < 0.0001; *p < 0.0492. Statistically significant differences among time points are indicated by letters as follows: p < 0.0001 (a, c), p = 0.0087 (b), p = 0.0222 (d). Further, statistical details on the genotype, age, and genotype × age effect can be found in Table 3. AUD, Arbitrary Units of Densitometry.

Mentions: Mitochondrial protein levels (relative to total protein) were evaluated by measuring the expression of the abundant protein ATPase β-subunit of Complex V [ATPB; (Elfering et al., 2004; Haynes et al., 2010), Figure 3]. Lower levels of ATPB were noted at PND21 and lasting into adulthood (PND210) in hippocampus of KI mice, while in cerebellum significant ATPB deficits were observed only at PND21 (Figure 3). No statistically significant differences were observed in cortex at any time point (Figure 3).


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)

Protein expression levels of mitochondrial ATPase β-subunit in brain from KI mice. ATPase β-subunit protein expression (normalized to GAPDH) was evaluated at PND9, 21 and 210 in hippocampus, cerebellum, and cortex lysates. Asterisk in the immunoblot image of hippocampus denotes a 90 CGG KI sample. Densitometry data for this sample have not been taken into account for averages calculation of KI. Cortex samples were ran in two different gels (PND9 and PND21-210) and are shown separately. Data are reported as mean ± SEM, n = 3–5 per genotype per time point, ran in triplicates. Statistical analysis was performed by Two-way ANOVA. Post-hoc analysis was performed by Tukey's HSD test for multiple comparisons. Significant difference between WT and KI are indicated by asterisks as follows. ****p < 0.0001; *p < 0.0492. Statistically significant differences among time points are indicated by letters as follows: p < 0.0001 (a, c), p = 0.0087 (b), p = 0.0222 (d). Further, statistical details on the genotype, age, and genotype × age effect can be found in Table 3. AUD, Arbitrary Units of Densitometry.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Protein expression levels of mitochondrial ATPase β-subunit in brain from KI mice. ATPase β-subunit protein expression (normalized to GAPDH) was evaluated at PND9, 21 and 210 in hippocampus, cerebellum, and cortex lysates. Asterisk in the immunoblot image of hippocampus denotes a 90 CGG KI sample. Densitometry data for this sample have not been taken into account for averages calculation of KI. Cortex samples were ran in two different gels (PND9 and PND21-210) and are shown separately. Data are reported as mean ± SEM, n = 3–5 per genotype per time point, ran in triplicates. Statistical analysis was performed by Two-way ANOVA. Post-hoc analysis was performed by Tukey's HSD test for multiple comparisons. Significant difference between WT and KI are indicated by asterisks as follows. ****p < 0.0001; *p < 0.0492. Statistically significant differences among time points are indicated by letters as follows: p < 0.0001 (a, c), p = 0.0087 (b), p = 0.0222 (d). Further, statistical details on the genotype, age, and genotype × age effect can be found in Table 3. AUD, Arbitrary Units of Densitometry.
Mentions: Mitochondrial protein levels (relative to total protein) were evaluated by measuring the expression of the abundant protein ATPase β-subunit of Complex V [ATPB; (Elfering et al., 2004; Haynes et al., 2010), Figure 3]. Lower levels of ATPB were noted at PND21 and lasting into adulthood (PND210) in hippocampus of KI mice, while in cerebellum significant ATPB deficits were observed only at PND21 (Figure 3). No statistically significant differences were observed in cortex at any time point (Figure 3).

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