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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

Role of milk Zn in brain bioenergetics and synapse scaffolding in premutation carriers. Milk-Zn concentrations followed a pseudo-first order kinetics over the lactation period (A). Milk Zn concentrations from control and premutation donors decreased with the post-partum period, ranging from 30 ± 3 μM at 6–8 weeks to 15 ± 2 μM at 18–20 weeks. The LN values of the Zn concentrations (in μM) were plotted against lactation week. The published values were obtained from Nagra et al. (Nagra, 1989) and shown as white squares. Regression parameters are as follows: for published [Zn], r2 = 0.997, y = −0.09x + 4.3; for this study, r2 = 0.647, y = −0.07x + 4.0. P value for slopes = 0.458; P value for intercepts = 0.986. ALP activity [expressed as nmol × (min × mg protein)−1] in human breast milk from control donors increased linearly from 8 to 18–20 weeks (B). The average ALP activity of controls was 3.3 ± 0.3 nmol × (min × mg protein)−1 or 46 ± 1 μmol × (min × l)−1 and within reported values [40 to 60 μmol × (min × l)−1; (Worth et al., 1981; Coburn et al., 1992)]. Linear regression analyses were performed with control values (reported as mean ± SEM) for both outcomes (r2 = 0.638 and 0.639 for Zn concentration and ALP activity, respectively). The 95% CIs performed with control values are shown with dotted lines. Individual premutation carriers' values for Zn concentrations and ALP activities are shown in Table 5. ALP activities in controls were not affected by vitamin and mineral supplementation [supplemented: 3.7 ± 0.4 vs. not supplemented: 3.2 ± 0.4 nmol × (min × mg protein)−1; p = 0.624]. A model integrating experimental data based on altered Zn homeostasis, mitochondrial dysfunction [current study and (Ross-Inta et al., 2010; Napoli et al., 2011)], and the putative effect of defective synaptic scaffolding in premutation individuals is shown in (C). During lactation, Zn uptake is exerted by ZIP5, 8 and 10, then excreted to milk via ZnT4/ZnT2 with a lower flux through ZnT6 (ZnT10) in the Golgi [Adapted from (Kelleher et al., 2012)]. The presence of the CGG expansion in FMR1, as it is the case with premutation carriers, may ensue in a lower expression of FMRP, which may affect the cytoskeleton (e.g., actin, Shank3), and the expression or function of membrane transporter such as ZnT4/T6. This situation would ensue in the suboptimal delivery of Zn into milk, mitochondrial dysfunction, and synaptic dysregulation. The arrows indicate the direction of Zn transport from maternal blood to milk.
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Figure 11: Role of milk Zn in brain bioenergetics and synapse scaffolding in premutation carriers. Milk-Zn concentrations followed a pseudo-first order kinetics over the lactation period (A). Milk Zn concentrations from control and premutation donors decreased with the post-partum period, ranging from 30 ± 3 μM at 6–8 weeks to 15 ± 2 μM at 18–20 weeks. The LN values of the Zn concentrations (in μM) were plotted against lactation week. The published values were obtained from Nagra et al. (Nagra, 1989) and shown as white squares. Regression parameters are as follows: for published [Zn], r2 = 0.997, y = −0.09x + 4.3; for this study, r2 = 0.647, y = −0.07x + 4.0. P value for slopes = 0.458; P value for intercepts = 0.986. ALP activity [expressed as nmol × (min × mg protein)−1] in human breast milk from control donors increased linearly from 8 to 18–20 weeks (B). The average ALP activity of controls was 3.3 ± 0.3 nmol × (min × mg protein)−1 or 46 ± 1 μmol × (min × l)−1 and within reported values [40 to 60 μmol × (min × l)−1; (Worth et al., 1981; Coburn et al., 1992)]. Linear regression analyses were performed with control values (reported as mean ± SEM) for both outcomes (r2 = 0.638 and 0.639 for Zn concentration and ALP activity, respectively). The 95% CIs performed with control values are shown with dotted lines. Individual premutation carriers' values for Zn concentrations and ALP activities are shown in Table 5. ALP activities in controls were not affected by vitamin and mineral supplementation [supplemented: 3.7 ± 0.4 vs. not supplemented: 3.2 ± 0.4 nmol × (min × mg protein)−1; p = 0.624]. A model integrating experimental data based on altered Zn homeostasis, mitochondrial dysfunction [current study and (Ross-Inta et al., 2010; Napoli et al., 2011)], and the putative effect of defective synaptic scaffolding in premutation individuals is shown in (C). During lactation, Zn uptake is exerted by ZIP5, 8 and 10, then excreted to milk via ZnT4/ZnT2 with a lower flux through ZnT6 (ZnT10) in the Golgi [Adapted from (Kelleher et al., 2012)]. The presence of the CGG expansion in FMR1, as it is the case with premutation carriers, may ensue in a lower expression of FMRP, which may affect the cytoskeleton (e.g., actin, Shank3), and the expression or function of membrane transporter such as ZnT4/T6. This situation would ensue in the suboptimal delivery of Zn into milk, mitochondrial dysfunction, and synaptic dysregulation. The arrows indicate the direction of Zn transport from maternal blood to milk.

Mentions: Milk from healthy donors had an average Zn concentration of 26 ± 2 μM (1.7 ± 0.1 mg/l), within the normal range reported before [1.0–1.7 mg/l at 3–5 months of lactation evaluated by atomic absorption spectrometry (Krebs et al., 1995)], with 86% of the Zn being in the labile form. In agreement with data previously reported (Nagra, 1989; Krebs et al., 1995), milk Zn concentrations from control and premutation women were reciprocally correlated with the postpartum period (Figure 11A), following a pseudo-first order kinetics with a biological half-life of 8 weeks (Figure 11). Although 10 of the 25 control donors were taking vitamins and mineral supplements (including Zn) during lactation, no significant differences in Zn concentrations were observed between these two groups, in agreement with the observed lack of correlation between milk or plasma Zn concentrations and maternal Zn intake (Krebs et al., 1995). In samples from premutation carriers, the average milk Zn concentration at 3–5 months of lactation was 56% of control values (14 ± 3 μM; p = 0.020). Specifically, milk Zn concentrations from two carriers at 8–9 lactation weeks and from one carrier at 19–20 weeks were significantly lower than the 95% CI (Figure 11A, Table 6). Zn levels in the remaining premutation milk samples, obtained after week 15, followed the decrease observed in control milk (Figure 11A, Table 6). Although the sample size is small, the incidence of low Zn concentration was higher in carriers compared to controls (60% vs. 24%; test for one proportion p = 0.0595; 95% CI = 0.2307–0.8824).


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)

Role of milk Zn in brain bioenergetics and synapse scaffolding in premutation carriers. Milk-Zn concentrations followed a pseudo-first order kinetics over the lactation period (A). Milk Zn concentrations from control and premutation donors decreased with the post-partum period, ranging from 30 ± 3 μM at 6–8 weeks to 15 ± 2 μM at 18–20 weeks. The LN values of the Zn concentrations (in μM) were plotted against lactation week. The published values were obtained from Nagra et al. (Nagra, 1989) and shown as white squares. Regression parameters are as follows: for published [Zn], r2 = 0.997, y = −0.09x + 4.3; for this study, r2 = 0.647, y = −0.07x + 4.0. P value for slopes = 0.458; P value for intercepts = 0.986. ALP activity [expressed as nmol × (min × mg protein)−1] in human breast milk from control donors increased linearly from 8 to 18–20 weeks (B). The average ALP activity of controls was 3.3 ± 0.3 nmol × (min × mg protein)−1 or 46 ± 1 μmol × (min × l)−1 and within reported values [40 to 60 μmol × (min × l)−1; (Worth et al., 1981; Coburn et al., 1992)]. Linear regression analyses were performed with control values (reported as mean ± SEM) for both outcomes (r2 = 0.638 and 0.639 for Zn concentration and ALP activity, respectively). The 95% CIs performed with control values are shown with dotted lines. Individual premutation carriers' values for Zn concentrations and ALP activities are shown in Table 5. ALP activities in controls were not affected by vitamin and mineral supplementation [supplemented: 3.7 ± 0.4 vs. not supplemented: 3.2 ± 0.4 nmol × (min × mg protein)−1; p = 0.624]. A model integrating experimental data based on altered Zn homeostasis, mitochondrial dysfunction [current study and (Ross-Inta et al., 2010; Napoli et al., 2011)], and the putative effect of defective synaptic scaffolding in premutation individuals is shown in (C). During lactation, Zn uptake is exerted by ZIP5, 8 and 10, then excreted to milk via ZnT4/ZnT2 with a lower flux through ZnT6 (ZnT10) in the Golgi [Adapted from (Kelleher et al., 2012)]. The presence of the CGG expansion in FMR1, as it is the case with premutation carriers, may ensue in a lower expression of FMRP, which may affect the cytoskeleton (e.g., actin, Shank3), and the expression or function of membrane transporter such as ZnT4/T6. This situation would ensue in the suboptimal delivery of Zn into milk, mitochondrial dysfunction, and synaptic dysregulation. The arrows indicate the direction of Zn transport from maternal blood to milk.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 11: Role of milk Zn in brain bioenergetics and synapse scaffolding in premutation carriers. Milk-Zn concentrations followed a pseudo-first order kinetics over the lactation period (A). Milk Zn concentrations from control and premutation donors decreased with the post-partum period, ranging from 30 ± 3 μM at 6–8 weeks to 15 ± 2 μM at 18–20 weeks. The LN values of the Zn concentrations (in μM) were plotted against lactation week. The published values were obtained from Nagra et al. (Nagra, 1989) and shown as white squares. Regression parameters are as follows: for published [Zn], r2 = 0.997, y = −0.09x + 4.3; for this study, r2 = 0.647, y = −0.07x + 4.0. P value for slopes = 0.458; P value for intercepts = 0.986. ALP activity [expressed as nmol × (min × mg protein)−1] in human breast milk from control donors increased linearly from 8 to 18–20 weeks (B). The average ALP activity of controls was 3.3 ± 0.3 nmol × (min × mg protein)−1 or 46 ± 1 μmol × (min × l)−1 and within reported values [40 to 60 μmol × (min × l)−1; (Worth et al., 1981; Coburn et al., 1992)]. Linear regression analyses were performed with control values (reported as mean ± SEM) for both outcomes (r2 = 0.638 and 0.639 for Zn concentration and ALP activity, respectively). The 95% CIs performed with control values are shown with dotted lines. Individual premutation carriers' values for Zn concentrations and ALP activities are shown in Table 5. ALP activities in controls were not affected by vitamin and mineral supplementation [supplemented: 3.7 ± 0.4 vs. not supplemented: 3.2 ± 0.4 nmol × (min × mg protein)−1; p = 0.624]. A model integrating experimental data based on altered Zn homeostasis, mitochondrial dysfunction [current study and (Ross-Inta et al., 2010; Napoli et al., 2011)], and the putative effect of defective synaptic scaffolding in premutation individuals is shown in (C). During lactation, Zn uptake is exerted by ZIP5, 8 and 10, then excreted to milk via ZnT4/ZnT2 with a lower flux through ZnT6 (ZnT10) in the Golgi [Adapted from (Kelleher et al., 2012)]. The presence of the CGG expansion in FMR1, as it is the case with premutation carriers, may ensue in a lower expression of FMRP, which may affect the cytoskeleton (e.g., actin, Shank3), and the expression or function of membrane transporter such as ZnT4/T6. This situation would ensue in the suboptimal delivery of Zn into milk, mitochondrial dysfunction, and synaptic dysregulation. The arrows indicate the direction of Zn transport from maternal blood to milk.
Mentions: Milk from healthy donors had an average Zn concentration of 26 ± 2 μM (1.7 ± 0.1 mg/l), within the normal range reported before [1.0–1.7 mg/l at 3–5 months of lactation evaluated by atomic absorption spectrometry (Krebs et al., 1995)], with 86% of the Zn being in the labile form. In agreement with data previously reported (Nagra, 1989; Krebs et al., 1995), milk Zn concentrations from control and premutation women were reciprocally correlated with the postpartum period (Figure 11A), following a pseudo-first order kinetics with a biological half-life of 8 weeks (Figure 11). Although 10 of the 25 control donors were taking vitamins and mineral supplements (including Zn) during lactation, no significant differences in Zn concentrations were observed between these two groups, in agreement with the observed lack of correlation between milk or plasma Zn concentrations and maternal Zn intake (Krebs et al., 1995). In samples from premutation carriers, the average milk Zn concentration at 3–5 months of lactation was 56% of control values (14 ± 3 μM; p = 0.020). Specifically, milk Zn concentrations from two carriers at 8–9 lactation weeks and from one carrier at 19–20 weeks were significantly lower than the 95% CI (Figure 11A, Table 6). Zn levels in the remaining premutation milk samples, obtained after week 15, followed the decrease observed in control milk (Figure 11A, Table 6). Although the sample size is small, the incidence of low Zn concentration was higher in carriers compared to controls (60% vs. 24%; test for one proportion p = 0.0595; 95% CI = 0.2307–0.8824).

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