Partial rescue of MeCP2 deficiency by postnatal activation of MeCP2

Abstract

In humans, mutations in the X-linked MECP2 gene, are the cause of Rett syndrome (RTT), a neurodevelopmental disorder that affects mainly girls. MeCP2 binds to methylated CpGs and is thought to act as a transcriptional repressor. In male mice, deletion or targeted mutation of Mecp2 leads to lethality and causes a neuronal phenotype. Selective mutation of Mecp2 in postnatal neurons results in a similar, although delayed, phenotype, suggesting that the symptoms are caused by MeCP2 deficiency in postmitotic neurons. In agreement with this idea, expression of a Mecp2 transgene in postmitotic neurons of Mecp2-null mutant mice resulted in the phenotypical rescue of the symptoms. To assess whether postnatal activation of MeCP2 in mutant animals could also affect the progression of the disorder, we constructed a conditionally active Mecp2 "rescue transgene" that was activated between P0 and P30. The Mecp2 transgene was under the control of the CAGGS promoter and was activated by using brain specific Cre-mediated recombination. Our results indicate that postnatal, neuron-specific activation of MeCP2 as late as 2-4 weeks of age significantly prolonged the lifespan of mutant animals and delayed the onset of neurologic symptoms.

Fig. 1.

Schematic representation of the rescue construct. (A) Mecp2e2 cDNA (white box), CAGGS promoter (dark gray box), stop cassette (light gray box), LoxP sites (black boxes). The construct was targeted to the Col1a1 locus. Expression of Cre will loop out the stop cassette, allowing the expression of MeCP2e2. (B) Characteristics of the Cre lines used. (C) Western blot analysis of tissue from WT (lanes 1), transgenic (CAGGS LSL Mecp2; Mecp2 −/y, lanes 2), and Mecp2 −/y (KO) mice (lane 3). No MeCP2 signal was detected in brain, lung, and spleen of null animals containing the CAGGS LSL Mecp2 transgene. Anti-GAPDH was used as loading control. (D) Western blot of total brain extracts of 6-week-old animals. Lane 1, endogenous MeCP2 in WT brain. The higher, stronger band corresponds to MeCP2e1, and the lower, fainter band corresponds to the MeCP2e2. Lane 2: Mecp2 −/y (KO); CAGGS LSL Mecp2. Lanes 3 and 4: Nestin and Tau Cre rescued animals, respectively. Lanes 5 and 6: C93 and C159 rescued mice. (E and F) Western blot analysis of CAGGS MeCP2 expression in cortex (Co), hippocampus (Hi), and cerebellum (Ce). Tau and Nestin Cre activate the transgene in all areas (E, lanes 4–9), whereas C93 and C159 activate Mecp2 in cortex and hippocampus (F, lanes 4, 5, 7, and 8) but not in cerebellum (F, lanes 6 and 9). E and F, lanes 1–3 show endogenous MeCP2.

Fig. 2.

Expression of the CAGGS Mecp2 in different areas of the brain. (A) LV cortical neurons from a C93 rescued animal. CAGGS MeCP2 localizes in heterochromatin-rich areas of the nucleus (arrowheads in ii). (i) NeuN (neuronal nuclear-specific marker) in green. (ii) MeCP2 in red. (iii) Merge. (B–D) Percentage of neurons expressing CAGGS MeCP2. For each genotype (mice were at least 3 months old), we analyzed LV of the cortex (M1-S1) (B), hippocampal CA1/CA2 boundary (C), and cerebellum (D). Nestin (N) and Tau Cre (T) rescued animals expressed MeCP2 in 70–90% of neurons in all brain regions surveyed. C93 rescued mice had MeCP2 expression in 50% of the neurons in cortex and hippocampus. C159 rescued mice had maximum expression in the cortex (60% of neurons) but little or no expression elsewhere. No MeCP2-positive cells were found in the cerebellum of C93 and C159 rescued animals. In WT mice, 100% of neurons express MeCP2, whereas no expression was observed in Mecp2 −/y mice (data not shown). At least 100 cells per animal were included in the count. “n” is the number of animals analyzed for each genotype.

Fig. 3.

Rescued animals have higher probability of survival than Mecp2 null littermates. Kaplan–Meier survival curves were generated by plotting the percentage of live mice (y axis) against number of days after birth (x axis). “n” indicates the number of animals analyzed. (A) Nestin (N) and Tau (T) Cre double-transgenic animals lived up to 280 days, whereas control mice died within 106 days after birth. (B) C93 and C159 mice lived up to 140 and 160 days, respectively (C159 vs. null: log rank test P = 0.0431; C93 vs. C159 P = 0.725). (C–E) Spontaneous nocturnal activity was measured by placing the animals in cages equipped with a movement detector (infrared beam). On the y axis, the number of beam interruptions over 10 h, each bar represents a different genotype. Animals were monitored for 5 weeks (C), during the symptomatic stage (5–15 weeks) (D), and later than 15 weeks (E). No significant difference between WT (black bar), mutant (white bar), and rescued mice was found in animals younger than 4 weeks of age. Between 5 and 15 weeks of age, the Mecp2 mutant animals became hypoactive, whereas rescued animals maintained normal levels of activity. At later age (E), the rescued animals showed lower activity that WT controls. Wt, KO, N, T, C93, and C159 are as in A and B.

Fig. 4.

Rescued animals have normal physical development and brain weight. (A) Body weight in grams. We followed the weight of WT (black line) and rescued animals and found no significant difference, whereas null mutant animals (red line) became overweight starting at 5 weeks. By 8 weeks, they were significantly overweight compared with WT and rescued subjects. Nestin Cre*, purple line (n = 4); Tau Cre*, yellow line (n = 3); C93*, green line (n = 5); C159*, blue line (n = 5); WT, black line (n = 4); KO*, red line (n = 4) (*Mecp2 −/y; CAGGS LSL Mecp2). (B) Brain weights in grams. White circles represent WT, and red triangles represent Mecp2 null (*) brains. Green and yellow squares represent brains from C93 (*) rescued animals and Tau Cre* rescued animals, respectively. Paired t test WT vs. transgenic P = 0.000110 (using the first four time points); WT vs. C93 P = 0.0788. (C) Neurons of C93 double-transgenic animals have a normal nuclear size. Two-month-old C93 Cre double-transgenic (gray bar), control WT (black bar), and null transgenic (white bar) littermates were analyzed for the comparison. Images were taken from the primary motor cortex (layer V) at ×40 magnification, and measurements from at least 200 cells were used for each bar. The animals used for the analysis were 8 weeks old. WT, KO, C93 as described above.