Limits...
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

Changes in FMRP protein expression in brain from WT and KI mice. (A) Representative Western blots of FMRP and actin protein expression levels in hippocampus and cortex of WT and KI mice. Tubulin was used as loading control. FMRP protein levels of KI mice hippocampus and cerebellum were 37 and 45% of WT at post-natal day (PND) 9, and 41 and 50% of WT at post-natal day 21. In cortex, FMRP protein levels in KI mice were 38% of WT at post-natal day 9. (B) Time-dependent changes in FMRP protein levels in hippocampus, cerebellum, and cortex respectively. Data are reported as mean ± SEM, n = 3–5 per genotype per time point. Statistical analysis was performed by Two-way ANOVA. Post-hoc analysis performed by Tukey's HSD test revealed significant differences between WT and KI, indicated in the figure by asterisk as follows. Hippocampus: ****p < 0.0001; Cerebellum: *p = 0.0110; Cortex: **p = 0.0003, *p = 0.0363, ***p = 0.0001. Statistically significant differences among time points are indicated by letters with the following p values. p = 0.0029 (a), p < 0.0001 (b), p = 0.0009 (c), p = 0.0260 (d), p = 0.0101 (e), p = 0.0033 (f), p = 0.0020 (g). For more statistical details on the genotype, age, and genotype × age effect see Table 3. (C) Correlation between FMRP and actin in the same brain areas. AUD, Arbitrary Units of Densitometry.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835505&req=5

Figure 1: Changes in FMRP protein expression in brain from WT and KI mice. (A) Representative Western blots of FMRP and actin protein expression levels in hippocampus and cortex of WT and KI mice. Tubulin was used as loading control. FMRP protein levels of KI mice hippocampus and cerebellum were 37 and 45% of WT at post-natal day (PND) 9, and 41 and 50% of WT at post-natal day 21. In cortex, FMRP protein levels in KI mice were 38% of WT at post-natal day 9. (B) Time-dependent changes in FMRP protein levels in hippocampus, cerebellum, and cortex respectively. Data are reported as mean ± SEM, n = 3–5 per genotype per time point. Statistical analysis was performed by Two-way ANOVA. Post-hoc analysis performed by Tukey's HSD test revealed significant differences between WT and KI, indicated in the figure by asterisk as follows. Hippocampus: ****p < 0.0001; Cerebellum: *p = 0.0110; Cortex: **p = 0.0003, *p = 0.0363, ***p = 0.0001. Statistically significant differences among time points are indicated by letters with the following p values. p = 0.0029 (a), p < 0.0001 (b), p = 0.0009 (c), p = 0.0260 (d), p = 0.0101 (e), p = 0.0033 (f), p = 0.0020 (g). For more statistical details on the genotype, age, and genotype × age effect see Table 3. (C) Correlation between FMRP and actin in the same brain areas. AUD, Arbitrary Units of Densitometry.

Mentions: Male, hemizygous KI mice were used as a murine model of the FMR1 premutation [CGG repeat expansion = 196 ± 6; (Wenzel et al., 2010)]. This model recapitulates most deficits observed in premutation carriers (Hunsaker et al., 2009, 2011, 2012; Wenzel et al., 2010). Despite the relatively high CGG expansions, the expression of the FMR1 gene product FMRP, was still detectable at PND9, PND21, and PND210 in hippocampus, cerebellum, and cortex and not absent as observed in models of Fragile X syndrome [(Zalfa et al., 2003); Figures 1A,B]. Consistent with other reports (Ludwig et al., 2014), FMRP protein expression decreased with age in all brain areas in WT (Figure 1B); conversely, no age-dependent effect was observed on FMRP expression of KI mice in any of the brain regions tested. At PND9, 40-50% of FMRP control values were observed in hippocampus, cerebellum, and cortex of KI mice, effect that lasted into PND21 for the first two tissues (Figure 1B).


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)

Changes in FMRP protein expression in brain from WT and KI mice. (A) Representative Western blots of FMRP and actin protein expression levels in hippocampus and cortex of WT and KI mice. Tubulin was used as loading control. FMRP protein levels of KI mice hippocampus and cerebellum were 37 and 45% of WT at post-natal day (PND) 9, and 41 and 50% of WT at post-natal day 21. In cortex, FMRP protein levels in KI mice were 38% of WT at post-natal day 9. (B) Time-dependent changes in FMRP protein levels in hippocampus, cerebellum, and cortex respectively. Data are reported as mean ± SEM, n = 3–5 per genotype per time point. Statistical analysis was performed by Two-way ANOVA. Post-hoc analysis performed by Tukey's HSD test revealed significant differences between WT and KI, indicated in the figure by asterisk as follows. Hippocampus: ****p < 0.0001; Cerebellum: *p = 0.0110; Cortex: **p = 0.0003, *p = 0.0363, ***p = 0.0001. Statistically significant differences among time points are indicated by letters with the following p values. p = 0.0029 (a), p < 0.0001 (b), p = 0.0009 (c), p = 0.0260 (d), p = 0.0101 (e), p = 0.0033 (f), p = 0.0020 (g). For more statistical details on the genotype, age, and genotype × age effect see Table 3. (C) Correlation between FMRP and actin in the same brain areas. AUD, Arbitrary Units of Densitometry.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Changes in FMRP protein expression in brain from WT and KI mice. (A) Representative Western blots of FMRP and actin protein expression levels in hippocampus and cortex of WT and KI mice. Tubulin was used as loading control. FMRP protein levels of KI mice hippocampus and cerebellum were 37 and 45% of WT at post-natal day (PND) 9, and 41 and 50% of WT at post-natal day 21. In cortex, FMRP protein levels in KI mice were 38% of WT at post-natal day 9. (B) Time-dependent changes in FMRP protein levels in hippocampus, cerebellum, and cortex respectively. Data are reported as mean ± SEM, n = 3–5 per genotype per time point. Statistical analysis was performed by Two-way ANOVA. Post-hoc analysis performed by Tukey's HSD test revealed significant differences between WT and KI, indicated in the figure by asterisk as follows. Hippocampus: ****p < 0.0001; Cerebellum: *p = 0.0110; Cortex: **p = 0.0003, *p = 0.0363, ***p = 0.0001. Statistically significant differences among time points are indicated by letters with the following p values. p = 0.0029 (a), p < 0.0001 (b), p = 0.0009 (c), p = 0.0260 (d), p = 0.0101 (e), p = 0.0033 (f), p = 0.0020 (g). For more statistical details on the genotype, age, and genotype × age effect see Table 3. (C) Correlation between FMRP and actin in the same brain areas. AUD, Arbitrary Units of Densitometry.
Mentions: Male, hemizygous KI mice were used as a murine model of the FMR1 premutation [CGG repeat expansion = 196 ± 6; (Wenzel et al., 2010)]. This model recapitulates most deficits observed in premutation carriers (Hunsaker et al., 2009, 2011, 2012; Wenzel et al., 2010). Despite the relatively high CGG expansions, the expression of the FMR1 gene product FMRP, was still detectable at PND9, PND21, and PND210 in hippocampus, cerebellum, and cortex and not absent as observed in models of Fragile X syndrome [(Zalfa et al., 2003); Figures 1A,B]. Consistent with other reports (Ludwig et al., 2014), FMRP protein expression decreased with age in all brain areas in WT (Figure 1B); conversely, no age-dependent effect was observed on FMRP expression of KI mice in any of the brain regions tested. At PND9, 40-50% of FMRP control values were observed in hippocampus, cerebellum, and cortex of KI mice, effect that lasted into PND21 for the first two tissues (Figure 1B).

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