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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 of precursor and mature CCOIV in brains from KI mice. Representative Western blots of CCOIV (precursor and mature proteins) in hippocampus (A), cerebellum (B) and cortex (C) of WT and KI mice. The densitometry for all the samples is also shown. Data were expressed as Arbitrary Densitometry Units and reported as mean ± SEM. Mature form of CCO4 was normalized by GAPDH. 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, followed by Tukey post-hoc test for multiple comparisons. Statistically significant differences between WT and KI are indicated by asterisks as follows. Hippocampus: *p = 0.0416; ***p = 0.0002; ****p < 0.0001. Cerebellum: *p = 0.0212 for mature CCO at PND21; *p = 0.0197 for P:M at PND21. Statistically significant differences among time points are indicated by letters as follows. Hippocampus: p = 0.0002 (a), p = 0.0172 (b), p = 0.0013 (c), p = 0.0047 (d), p = 0.0086 (e), p < 0.0001 (f), p = 0.0003 (g). Cerebellum: p = 0.0459 (a). Cortex: p = 0.0391 (a), p = 0.0113 (b). Further statistical details on the genotype, age, and genotype × age effect can be found in Table 3. (D) Uncropped version of the Western blot image showing the intensity and mobility of the precursor band relative to the mature protein. The observed molecular weights for the CCOIV precursor and mature forms were, respectively, 20.0 and 17.5 kDa, as extrapolated by the Molecular Weight markers with the use of the Carestream software. This 2.5 kDa difference was close to the theoretical calculated molecular weight (2.4 kDa) of the 22 residues of amino acids (MLATRVFSLVGKRAISTSVCVR) present in the precursor form, which is cleaved by mitochondrial matrix peptidases (Isaya et al., 1991) to produce the mature mitochondrial form of the protein (UniProtKB P13073). Asterisk in the immunoblot image of hippocampus (panels A and D) denotes a 90 CGG KI sample. Densitometry data for this sample have not been included in the averages for KI. For cortex, samples were run in two separate gels (PND9 and PND21-210). AUD, Arbitrary Units of Densitometry.
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Figure 5: Protein expression of precursor and mature CCOIV in brains from KI mice. Representative Western blots of CCOIV (precursor and mature proteins) in hippocampus (A), cerebellum (B) and cortex (C) of WT and KI mice. The densitometry for all the samples is also shown. Data were expressed as Arbitrary Densitometry Units and reported as mean ± SEM. Mature form of CCO4 was normalized by GAPDH. 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, followed by Tukey post-hoc test for multiple comparisons. Statistically significant differences between WT and KI are indicated by asterisks as follows. Hippocampus: *p = 0.0416; ***p = 0.0002; ****p < 0.0001. Cerebellum: *p = 0.0212 for mature CCO at PND21; *p = 0.0197 for P:M at PND21. Statistically significant differences among time points are indicated by letters as follows. Hippocampus: p = 0.0002 (a), p = 0.0172 (b), p = 0.0013 (c), p = 0.0047 (d), p = 0.0086 (e), p < 0.0001 (f), p = 0.0003 (g). Cerebellum: p = 0.0459 (a). Cortex: p = 0.0391 (a), p = 0.0113 (b). Further statistical details on the genotype, age, and genotype × age effect can be found in Table 3. (D) Uncropped version of the Western blot image showing the intensity and mobility of the precursor band relative to the mature protein. The observed molecular weights for the CCOIV precursor and mature forms were, respectively, 20.0 and 17.5 kDa, as extrapolated by the Molecular Weight markers with the use of the Carestream software. This 2.5 kDa difference was close to the theoretical calculated molecular weight (2.4 kDa) of the 22 residues of amino acids (MLATRVFSLVGKRAISTSVCVR) present in the precursor form, which is cleaved by mitochondrial matrix peptidases (Isaya et al., 1991) to produce the mature mitochondrial form of the protein (UniProtKB P13073). Asterisk in the immunoblot image of hippocampus (panels A and D) denotes a 90 CGG KI sample. Densitometry data for this sample have not been included in the averages for KI. For cortex, samples were run in two separate gels (PND9 and PND21-210). AUD, Arbitrary Units of Densitometry.

Mentions: To test whether the import/processing of nDNA-encoded mitochondrial proteins was also affected in the KI mouse model, providing an explanation for the observed defects in OXPHOS, we evaluated the protein expression levels of mature and precursor cytochrome c oxidase subunit IV (CCOIV, Figure 5) in brains from WT and KI mice. The CCOIV Precursor-to-Mature ratios (P:M) were significantly increased in hippocampus (PND21 and PND210) and cerebellum (PND21) of KI mice (Figure 5), consistent with our previous reports.


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 of precursor and mature CCOIV in brains from KI mice. Representative Western blots of CCOIV (precursor and mature proteins) in hippocampus (A), cerebellum (B) and cortex (C) of WT and KI mice. The densitometry for all the samples is also shown. Data were expressed as Arbitrary Densitometry Units and reported as mean ± SEM. Mature form of CCO4 was normalized by GAPDH. 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, followed by Tukey post-hoc test for multiple comparisons. Statistically significant differences between WT and KI are indicated by asterisks as follows. Hippocampus: *p = 0.0416; ***p = 0.0002; ****p < 0.0001. Cerebellum: *p = 0.0212 for mature CCO at PND21; *p = 0.0197 for P:M at PND21. Statistically significant differences among time points are indicated by letters as follows. Hippocampus: p = 0.0002 (a), p = 0.0172 (b), p = 0.0013 (c), p = 0.0047 (d), p = 0.0086 (e), p < 0.0001 (f), p = 0.0003 (g). Cerebellum: p = 0.0459 (a). Cortex: p = 0.0391 (a), p = 0.0113 (b). Further statistical details on the genotype, age, and genotype × age effect can be found in Table 3. (D) Uncropped version of the Western blot image showing the intensity and mobility of the precursor band relative to the mature protein. The observed molecular weights for the CCOIV precursor and mature forms were, respectively, 20.0 and 17.5 kDa, as extrapolated by the Molecular Weight markers with the use of the Carestream software. This 2.5 kDa difference was close to the theoretical calculated molecular weight (2.4 kDa) of the 22 residues of amino acids (MLATRVFSLVGKRAISTSVCVR) present in the precursor form, which is cleaved by mitochondrial matrix peptidases (Isaya et al., 1991) to produce the mature mitochondrial form of the protein (UniProtKB P13073). Asterisk in the immunoblot image of hippocampus (panels A and D) denotes a 90 CGG KI sample. Densitometry data for this sample have not been included in the averages for KI. For cortex, samples were run in two separate gels (PND9 and PND21-210). AUD, Arbitrary Units of Densitometry.
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Figure 5: Protein expression of precursor and mature CCOIV in brains from KI mice. Representative Western blots of CCOIV (precursor and mature proteins) in hippocampus (A), cerebellum (B) and cortex (C) of WT and KI mice. The densitometry for all the samples is also shown. Data were expressed as Arbitrary Densitometry Units and reported as mean ± SEM. Mature form of CCO4 was normalized by GAPDH. 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, followed by Tukey post-hoc test for multiple comparisons. Statistically significant differences between WT and KI are indicated by asterisks as follows. Hippocampus: *p = 0.0416; ***p = 0.0002; ****p < 0.0001. Cerebellum: *p = 0.0212 for mature CCO at PND21; *p = 0.0197 for P:M at PND21. Statistically significant differences among time points are indicated by letters as follows. Hippocampus: p = 0.0002 (a), p = 0.0172 (b), p = 0.0013 (c), p = 0.0047 (d), p = 0.0086 (e), p < 0.0001 (f), p = 0.0003 (g). Cerebellum: p = 0.0459 (a). Cortex: p = 0.0391 (a), p = 0.0113 (b). Further statistical details on the genotype, age, and genotype × age effect can be found in Table 3. (D) Uncropped version of the Western blot image showing the intensity and mobility of the precursor band relative to the mature protein. The observed molecular weights for the CCOIV precursor and mature forms were, respectively, 20.0 and 17.5 kDa, as extrapolated by the Molecular Weight markers with the use of the Carestream software. This 2.5 kDa difference was close to the theoretical calculated molecular weight (2.4 kDa) of the 22 residues of amino acids (MLATRVFSLVGKRAISTSVCVR) present in the precursor form, which is cleaved by mitochondrial matrix peptidases (Isaya et al., 1991) to produce the mature mitochondrial form of the protein (UniProtKB P13073). Asterisk in the immunoblot image of hippocampus (panels A and D) denotes a 90 CGG KI sample. Densitometry data for this sample have not been included in the averages for KI. For cortex, samples were run in two separate gels (PND9 and PND21-210). AUD, Arbitrary Units of Densitometry.
Mentions: To test whether the import/processing of nDNA-encoded mitochondrial proteins was also affected in the KI mouse model, providing an explanation for the observed defects in OXPHOS, we evaluated the protein expression levels of mature and precursor cytochrome c oxidase subunit IV (CCOIV, Figure 5) in brains from WT and KI mice. The CCOIV Precursor-to-Mature ratios (P:M) were significantly increased in hippocampus (PND21 and PND210) and cerebellum (PND21) of KI mice (Figure 5), consistent with our previous reports.

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