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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

Brain Shank3 protein levels in WT and KI mice. (A) Western blots for Shank3 protein expression levels in hippocampus, cerebellum, and cortex were performed at PND 9, 21, and 210 from WT (white) and KI (black) mice as described in details in the Materials and Methods Section. Further, images taken with acetone-precipitated samples and normalized by either tubulin or GADPH are found under Supplementary Figure 1. (B) Densitometry data is shown as mean ± SEM, n = 4–5 individuals per genotype, per time point, ran individually. Statistical analysis was performed by two-way ANOVA, followed by Tukey's HSD post-hoc test for multiple comparisons. Statistically significant differences between WT and KI are indicated by asterisks as follows: Hippocampus: **p = 0.0054; Cerebellum: ****p < 0.0001. Statistically significant differences among time points are indicated by letters as follows: p = 0.0074 (a), p = 0.0267 (b), p < 0.0001 (c, e, f), p = 0.0217(d), p = 0.0041 (g). Further, statistical details on the genotype, age, and genotype × age effect can be found in Table 3. (C) Correlation between FMRP levels and Shank3 protein expression in brain of WT and KI mice. Both proteins have been normalized by tubulin, used as loading control. Shown are individual data points collected in cerebellum, hippocampus, and cortex of WT and KI mice at PND9, PND21, and PND210. PND210 is shown in a separate plot due to the difference in protein expression of both FMRP and Shank3 at this time point, relative to the previous ones. Dotted lines are 95% CI constructed with WT and KI values. AUD, Arbitrary Units of Densitometry.
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Figure 8: Brain Shank3 protein levels in WT and KI mice. (A) Western blots for Shank3 protein expression levels in hippocampus, cerebellum, and cortex were performed at PND 9, 21, and 210 from WT (white) and KI (black) mice as described in details in the Materials and Methods Section. Further, images taken with acetone-precipitated samples and normalized by either tubulin or GADPH are found under Supplementary Figure 1. (B) Densitometry data is shown as mean ± SEM, n = 4–5 individuals per genotype, per time point, ran individually. Statistical analysis was performed by two-way ANOVA, followed by Tukey's HSD post-hoc test for multiple comparisons. Statistically significant differences between WT and KI are indicated by asterisks as follows: Hippocampus: **p = 0.0054; Cerebellum: ****p < 0.0001. Statistically significant differences among time points are indicated by letters as follows: p = 0.0074 (a), p = 0.0267 (b), p < 0.0001 (c, e, f), p = 0.0217(d), p = 0.0041 (g). Further, statistical details on the genotype, age, and genotype × age effect can be found in Table 3. (C) Correlation between FMRP levels and Shank3 protein expression in brain of WT and KI mice. Both proteins have been normalized by tubulin, used as loading control. Shown are individual data points collected in cerebellum, hippocampus, and cortex of WT and KI mice at PND9, PND21, and PND210. PND210 is shown in a separate plot due to the difference in protein expression of both FMRP and Shank3 at this time point, relative to the previous ones. Dotted lines are 95% CI constructed with WT and KI values. AUD, Arbitrary Units of Densitometry.

Mentions: Deficits in Shank3 protein expression—evaluated by Western blots—were noticeable at PND9, PND21, and PND210 in hippocampus and cerebellum from KI mice (Figure 8 and Supplementary Figure 1). These results are consistent with other studies reporting Shank3 protein expression being brain-region/cell-type specific and developmentally regulated (Wang et al., 2014). Of note, although Shank3 is a postsynaptic density protein, several studies have shown that Shank3 expression pattern in whole brain areas mirrors that observed at the synaptic regions (Han et al., 2013; Kouser et al., 2013) providing support for the Shank3 expression in total homogenates from brain regions. Furthermore, the amount of tissue collected from each pup, especially at early time points—i.e., PND9 and PND21—would not have been sufficient as starting material for postsynaptic density preparations.


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)

Brain Shank3 protein levels in WT and KI mice. (A) Western blots for Shank3 protein expression levels in hippocampus, cerebellum, and cortex were performed at PND 9, 21, and 210 from WT (white) and KI (black) mice as described in details in the Materials and Methods Section. Further, images taken with acetone-precipitated samples and normalized by either tubulin or GADPH are found under Supplementary Figure 1. (B) Densitometry data is shown as mean ± SEM, n = 4–5 individuals per genotype, per time point, ran individually. Statistical analysis was performed by two-way ANOVA, followed by Tukey's HSD post-hoc test for multiple comparisons. Statistically significant differences between WT and KI are indicated by asterisks as follows: Hippocampus: **p = 0.0054; Cerebellum: ****p < 0.0001. Statistically significant differences among time points are indicated by letters as follows: p = 0.0074 (a), p = 0.0267 (b), p < 0.0001 (c, e, f), p = 0.0217(d), p = 0.0041 (g). Further, statistical details on the genotype, age, and genotype × age effect can be found in Table 3. (C) Correlation between FMRP levels and Shank3 protein expression in brain of WT and KI mice. Both proteins have been normalized by tubulin, used as loading control. Shown are individual data points collected in cerebellum, hippocampus, and cortex of WT and KI mice at PND9, PND21, and PND210. PND210 is shown in a separate plot due to the difference in protein expression of both FMRP and Shank3 at this time point, relative to the previous ones. Dotted lines are 95% CI constructed with WT and KI values. AUD, Arbitrary Units of Densitometry.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Brain Shank3 protein levels in WT and KI mice. (A) Western blots for Shank3 protein expression levels in hippocampus, cerebellum, and cortex were performed at PND 9, 21, and 210 from WT (white) and KI (black) mice as described in details in the Materials and Methods Section. Further, images taken with acetone-precipitated samples and normalized by either tubulin or GADPH are found under Supplementary Figure 1. (B) Densitometry data is shown as mean ± SEM, n = 4–5 individuals per genotype, per time point, ran individually. Statistical analysis was performed by two-way ANOVA, followed by Tukey's HSD post-hoc test for multiple comparisons. Statistically significant differences between WT and KI are indicated by asterisks as follows: Hippocampus: **p = 0.0054; Cerebellum: ****p < 0.0001. Statistically significant differences among time points are indicated by letters as follows: p = 0.0074 (a), p = 0.0267 (b), p < 0.0001 (c, e, f), p = 0.0217(d), p = 0.0041 (g). Further, statistical details on the genotype, age, and genotype × age effect can be found in Table 3. (C) Correlation between FMRP levels and Shank3 protein expression in brain of WT and KI mice. Both proteins have been normalized by tubulin, used as loading control. Shown are individual data points collected in cerebellum, hippocampus, and cortex of WT and KI mice at PND9, PND21, and PND210. PND210 is shown in a separate plot due to the difference in protein expression of both FMRP and Shank3 at this time point, relative to the previous ones. Dotted lines are 95% CI constructed with WT and KI values. AUD, Arbitrary Units of Densitometry.
Mentions: Deficits in Shank3 protein expression—evaluated by Western blots—were noticeable at PND9, PND21, and PND210 in hippocampus and cerebellum from KI mice (Figure 8 and Supplementary Figure 1). These results are consistent with other studies reporting Shank3 protein expression being brain-region/cell-type specific and developmentally regulated (Wang et al., 2014). Of note, although Shank3 is a postsynaptic density protein, several studies have shown that Shank3 expression pattern in whole brain areas mirrors that observed at the synaptic regions (Han et al., 2013; Kouser et al., 2013) providing support for the Shank3 expression in total homogenates from brain regions. Furthermore, the amount of tissue collected from each pup, especially at early time points—i.e., PND9 and PND21—would not have been sufficient as starting material for postsynaptic density preparations.

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