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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

Changes in mitochondrial outcomes in hippocampus, cerebellum and cortex of suckling WT and KI pups nursed on WT or KI dams. At birth, KI pups (n = 24) and WT pups (n = 24) were foster-nursed either on KI dams or WT dams, six pups on each dam. After 21 days, mitochondria were isolated from cortex, cerebellum, and hippocampus and activities of NADH oxidase, succinate oxidase, cytochrome c oxidase, and citrate synthase and RCR were evaluated as described in the Methods section. wt/wt = WT pups nursing on WT milk; ki/ki = KI pups nursing on KI milk. Circles represent WT pups, squares represent KI pups. White symbols represent WT milk and black symbols represent KI milk. Upward or downward arrows represent improvement or worsening, respectively, upon nursing on WT milk or KI milk. Activities (NADH oxidase, succinate oxidase, cytochrome c oxidase) were expressed as nmol × (min × mg protein)−1, normalized by citrate synthase activity and multiplied by 1000. Data are shown as mean ± SEM (from technical replicates of pooled samples). Statistical analysis was performed by Two-way ANOVA, followed by Tukey's post-hoc test for multiple comparisons. The p values are as follows. Hippocampus: p < 0.0001 (a, b, c, g, i, p, q), p = 0.0004 (d, o), p = 0.0002 (e, f), p = 0.0025 (h), p = 0.0008 (j), p = 0.0027 (k), p = 0.0006 (l), p = 0.0262 (m), p = 0.0082 (n); Cerebellum: p = 0.0296 (a), p = 0.0301 (b), p = 0.0039 (c), p < 0.0001 (d, g, h, i, j, k, l, m, n, o, p), p = 0.0023 (e); p = 0.0086 (f); Cortex: p < 0.0001 (a, b, c, d, e, f), p = 0.0047 (g), p = 0.0468 (h). Further statistical details on the genotype, age, and genotype × age effect can be found in Table 4.
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Figure 10: Changes in mitochondrial outcomes in hippocampus, cerebellum and cortex of suckling WT and KI pups nursed on WT or KI dams. At birth, KI pups (n = 24) and WT pups (n = 24) were foster-nursed either on KI dams or WT dams, six pups on each dam. After 21 days, mitochondria were isolated from cortex, cerebellum, and hippocampus and activities of NADH oxidase, succinate oxidase, cytochrome c oxidase, and citrate synthase and RCR were evaluated as described in the Methods section. wt/wt = WT pups nursing on WT milk; ki/ki = KI pups nursing on KI milk. Circles represent WT pups, squares represent KI pups. White symbols represent WT milk and black symbols represent KI milk. Upward or downward arrows represent improvement or worsening, respectively, upon nursing on WT milk or KI milk. Activities (NADH oxidase, succinate oxidase, cytochrome c oxidase) were expressed as nmol × (min × mg protein)−1, normalized by citrate synthase activity and multiplied by 1000. Data are shown as mean ± SEM (from technical replicates of pooled samples). Statistical analysis was performed by Two-way ANOVA, followed by Tukey's post-hoc test for multiple comparisons. The p values are as follows. Hippocampus: p < 0.0001 (a, b, c, g, i, p, q), p = 0.0004 (d, o), p = 0.0002 (e, f), p = 0.0025 (h), p = 0.0008 (j), p = 0.0027 (k), p = 0.0006 (l), p = 0.0262 (m), p = 0.0082 (n); Cerebellum: p = 0.0296 (a), p = 0.0301 (b), p = 0.0039 (c), p < 0.0001 (d, g, h, i, j, k, l, m, n, o, p), p = 0.0023 (e); p = 0.0086 (f); Cortex: p < 0.0001 (a, b, c, d, e, f), p = 0.0047 (g), p = 0.0468 (h). Further statistical details on the genotype, age, and genotype × age effect can be found in Table 4.

Mentions: On the basis of the findings shown thus far we hypothesize that Zn-deficient milk of KI dams would be detrimental on the suckling pups' bioenergetics requirements, whereas milk from WT dams might rescue some of the deficits observed in the premutation mice. To test this hypothesis we designed a cross-fostering experiment in which KI pups nursed on WT dams (WT milk) and WT pups nursed on KI dams (KI milk; Figure 10). We tested mitochondrial outcomes at PND21 from WT and KI in the pups (male hemizygous) nursing on KI milk to elucidate the effect of KI milk on the brain bioenergetics of WT pups, or nursing on WT milk to elucidate the effect of WT milk on the brain bioenergetics of KI pups (Figure 10).


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 mitochondrial outcomes in hippocampus, cerebellum and cortex of suckling WT and KI pups nursed on WT or KI dams. At birth, KI pups (n = 24) and WT pups (n = 24) were foster-nursed either on KI dams or WT dams, six pups on each dam. After 21 days, mitochondria were isolated from cortex, cerebellum, and hippocampus and activities of NADH oxidase, succinate oxidase, cytochrome c oxidase, and citrate synthase and RCR were evaluated as described in the Methods section. wt/wt = WT pups nursing on WT milk; ki/ki = KI pups nursing on KI milk. Circles represent WT pups, squares represent KI pups. White symbols represent WT milk and black symbols represent KI milk. Upward or downward arrows represent improvement or worsening, respectively, upon nursing on WT milk or KI milk. Activities (NADH oxidase, succinate oxidase, cytochrome c oxidase) were expressed as nmol × (min × mg protein)−1, normalized by citrate synthase activity and multiplied by 1000. Data are shown as mean ± SEM (from technical replicates of pooled samples). Statistical analysis was performed by Two-way ANOVA, followed by Tukey's post-hoc test for multiple comparisons. The p values are as follows. Hippocampus: p < 0.0001 (a, b, c, g, i, p, q), p = 0.0004 (d, o), p = 0.0002 (e, f), p = 0.0025 (h), p = 0.0008 (j), p = 0.0027 (k), p = 0.0006 (l), p = 0.0262 (m), p = 0.0082 (n); Cerebellum: p = 0.0296 (a), p = 0.0301 (b), p = 0.0039 (c), p < 0.0001 (d, g, h, i, j, k, l, m, n, o, p), p = 0.0023 (e); p = 0.0086 (f); Cortex: p < 0.0001 (a, b, c, d, e, f), p = 0.0047 (g), p = 0.0468 (h). Further statistical details on the genotype, age, and genotype × age effect can be found in Table 4.
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Related In: Results  -  Collection

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Figure 10: Changes in mitochondrial outcomes in hippocampus, cerebellum and cortex of suckling WT and KI pups nursed on WT or KI dams. At birth, KI pups (n = 24) and WT pups (n = 24) were foster-nursed either on KI dams or WT dams, six pups on each dam. After 21 days, mitochondria were isolated from cortex, cerebellum, and hippocampus and activities of NADH oxidase, succinate oxidase, cytochrome c oxidase, and citrate synthase and RCR were evaluated as described in the Methods section. wt/wt = WT pups nursing on WT milk; ki/ki = KI pups nursing on KI milk. Circles represent WT pups, squares represent KI pups. White symbols represent WT milk and black symbols represent KI milk. Upward or downward arrows represent improvement or worsening, respectively, upon nursing on WT milk or KI milk. Activities (NADH oxidase, succinate oxidase, cytochrome c oxidase) were expressed as nmol × (min × mg protein)−1, normalized by citrate synthase activity and multiplied by 1000. Data are shown as mean ± SEM (from technical replicates of pooled samples). Statistical analysis was performed by Two-way ANOVA, followed by Tukey's post-hoc test for multiple comparisons. The p values are as follows. Hippocampus: p < 0.0001 (a, b, c, g, i, p, q), p = 0.0004 (d, o), p = 0.0002 (e, f), p = 0.0025 (h), p = 0.0008 (j), p = 0.0027 (k), p = 0.0006 (l), p = 0.0262 (m), p = 0.0082 (n); Cerebellum: p = 0.0296 (a), p = 0.0301 (b), p = 0.0039 (c), p < 0.0001 (d, g, h, i, j, k, l, m, n, o, p), p = 0.0023 (e); p = 0.0086 (f); Cortex: p < 0.0001 (a, b, c, d, e, f), p = 0.0047 (g), p = 0.0468 (h). Further statistical details on the genotype, age, and genotype × age effect can be found in Table 4.
Mentions: On the basis of the findings shown thus far we hypothesize that Zn-deficient milk of KI dams would be detrimental on the suckling pups' bioenergetics requirements, whereas milk from WT dams might rescue some of the deficits observed in the premutation mice. To test this hypothesis we designed a cross-fostering experiment in which KI pups nursed on WT dams (WT milk) and WT pups nursed on KI dams (KI milk; Figure 10). We tested mitochondrial outcomes at PND21 from WT and KI in the pups (male hemizygous) nursing on KI milk to elucidate the effect of KI milk on the brain bioenergetics of WT pups, or nursing on WT milk to elucidate the effect of WT milk on the brain bioenergetics of KI pups (Figure 10).

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