Premutation in the Fragile X Mental Retardation 1 (FMR1) Gene Affects Maternal Zn-milk and Perinatal Brain Bioenergetics and Scaffolding

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.

Keywords: FMR1; Shank3; bioenergetics; brain; milk; mitochondria; premutation; zinc.

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.

Figure 2

Brain bioenergetics of KI mice during neurodevelopment and adulthood. Mitochondria were isolated from cortex, cerebellum, and hippocampus of WT and KI pups as described in the Methods section. Activities of NADH oxidase, succinate oxidase, and cytochrome c oxidase (CCO), and respiratory control ratio (RCR) were evaluated at PND0 (cerebellum and cortex only, due to the scarcity of hippocampal tissue), PND7, PND21, and PND210. Data are reported as mean ± SEM, n = 3–7 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 by asterisks as follows NADH oxidase: ^*p = 0.0468; Succinate oxidase: ^*p = 0.0466 at PND21, ^*p = 0.0423 at PND210; Cytochrome c oxidase: ^*p = 0.0484; Coupling: ^**p = 0.001, ^***p = 0.0004, ^*p = 0.0496. Letters indicate statistically significant differences amongst time points as follows. NADH oxidase: p = 0.0291 (a), p = 0.0033 (b), p = 0.0166 (c), p = 0.0320 (d), p = 0.0036 (e), p = 0.0001 (f), p = 0.0113 (g), p = 0.0032 (h), p = 0.0004 (i); Succinate oxidase: p < 0.0001 (a), p = 0.0002 (b), p = 0.0240 (c), p = 0.0434 (d), p = 0.0283 (e), p = 0.0009 (f), p = 0.0250 (g), p = 0.0013 (h); Cytochrome c oxidase: p = 0.0053 (a), p = 0.0035 (b). Coupling: p = 0.0003 (a), p = 0.0010 (b). Further statistical details on the genotype, age, and genotype × age effect can be found in the legend of Table 3.

Figure 3

Protein expression levels of mitochondrial ATPase β-subunit in brain from KI mice. ATPase β-subunit protein expression (normalized to GAPDH) was evaluated at PND9, 21 and 210 in hippocampus, cerebellum, and cortex lysates. Asterisk in the immunoblot image of hippocampus denotes a 90 CGG KI sample. Densitometry data for this sample have not been taken into account for averages calculation of KI. Cortex samples were ran in two different gels (PND9 and PND21-210) and are shown separately. 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. Post-hoc analysis was performed by Tukey's HSD test for multiple comparisons. Significant difference between WT and KI are indicated by asterisks as follows. ^****p < 0.0001; ^*p < 0.0492. Statistically significant differences among time points are indicated by letters as follows: p < 0.0001 (a, c), p = 0.0087 (b), p = 0.0222 (d). Further, statistical details on the genotype, age, and genotype × age effect can be found in Table 3. AUD, Arbitrary Units of Densitometry.

Figure 4

Correlation between mitochondrial outcomes and FMRP or FMR1 expression levels. The correlation between FMRP and FMR1 expression with a mitochondrial outcome (i.e., coupling between ATP synthesis and electron transfer or RCRu) was carried out using human primary dermal from controls, premutation carriers (105–180 CGG), and full mutation carriers (>200 CGG repeats). RCRu, FMR1, and FMRP levels were expressed as percentage of control values. Data are shown as mean ± SEM for controls, permutation, and full mutation carriers.

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.

Figure 6

Brain Zn concentrations in WT and KI mice. Total Zn levels were measured in whole hippocampus, cerebellum and cortex homogenates from WT and KI mice. Data are shown as mean ± SEM, n = 3–6 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 at individual time-points are indicated by asterisks as follows: Hippocampus: ^*p = 0.0425; ^**p = 0.0070; Cerebellum: ^*p = 0.0158; ^***p = 0.0003. Statistically significant differences among time points are indicated by letters as follows: p = 0.0190 (a), p = 0.0144 (b), p = 0.0003 (c). Further statistical details on the genotype, age, and genotype × age effect can be found in Table 3.

Figure 7

Correlations among Zn levels, selected mitochondrial outcomes, and protein expression of FMRP and Shank3, in brain of WT and KI mice. A significant direct correlation was noted between average Zn levels and activities of CCO and CS and RCR for all the time points analyzed (A). CCO and CS activities were expressed as nmol × (min × mg protein)⁻¹. CCO activity has been normalized to citrate synthase and multiplied by 100. All the other mitochondrial outcomes did not show a correlation with Zn. A direct correlation was also observed between Zn levels and both FMRP and Shank3 protein expression (individual values) in brains of WT and KI when all time points were combined (B,C). Dotted lines illustrate the 95% CI obtained with WT and KI values.

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.

Figure 9

ZnT4 and ZnT6 gene expression in mammary glands from lactating WT and KI dams. Representative image of the PCR products (obtained by RT-qPCR as described under Methods) that were separated in a 1.3% agarose gel and visualized with ethidium bromide (A). The fragments exhibited the expected sizes of 585 bp (ZnT4) and 537 bp (ZnT6). GAPDH PCR product was separated in a 3% agarose gel electrophoresis with ethidium bromide resulting in a product of the expected of 107 bp (not shown). Intensities of ZnT4 and ZnT6 bands were obtained in a Kodak Imager were normalized by that of GAPDH (B). Bars represent averages ± SEM of 4 individuals/genotype. Statistical analysis was carried out with the Student's t-test between WT and KI. ^*p < 0.05.

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.

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., ; 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., ; 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.