Research reportCholinergic hypofunction in MeCP2-308 mice: Beneficial neurobehavioural effects of neonatal choline supplementation
Highlights
► Neonatal choline supplementation normalized locomotor activity levels in Mecp2-308 mice. ► Neonatal choline supplementation increased striatal choline acetyl transferase enzymatic activity. ► Neonatal choline increased BDNF and NGF levels in Mecp2-308 and wt mice. ► Signs of hypofunction of central cholinergic populations in Mecp2-308 mice.
Introduction
Rett syndrome (RTT) is a pervasive developmental disorder, primarily affecting girls. RTT causes a wide variety of debilitating symptoms [1]. Mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2) have been found to be responsible for about 90% of classical RTT cases [2], [3]. Although several MeCP2-target genes have been proposed [4], the mechanisms leading to the severe, progressive and specific neuronal dysfunctions when MeCP2 gene is mutated remain to be elucidated. After the discovery of a monogenic origin for RTT, among other null models, a mouse line which expresses the truncated form of Mecp2 gene (Mecp2-308) associated with milder phenotypes in RTT patients, has been generated [5]. This RTT mouse model shows both later onset of symptoms (6 weeks of age) as well as longer life expectation than the null MeCP2 mutants [6], [7]. Symptoms include motor alterations, cognitive disabilities, increased anxiety-like behaviours accompanied by an enhanced physiological response to stress [8], [9], [10].
In RTT mouse models the resonance signal of choline (including glycerophosphocholine, phosphocholine, and to a much lesser extent free choline) has been found either increased [11] or transiently decreased during a restricted postnatal time window, the fifth postnatal week [12]. A detailed developmental profile of choline levels in RTT infants is still missing; so far, one study reported elevated levels of choline (mean age 6 years, but at different stages of the disease) [13] possibly also due to gliosis [14], while in other clinical studies RTT choline levels were comparable to controls [15], [16], [17].
In rodents dietary choline supplementation in the early phases of development has measurable effects in both choline-depleted and choline-normal subjects: increased choline content of the maternal diet yields to increase of some of the choline metabolites (betaine, phosphorylcholine) leaving free choline content surprisingly unaltered [18], [19]; as for CNS effects, developmental choline supplementation enhances transmission at cholinergic synapses, protects against neurodegeneration [20], improves performance on several behavioural tasks [21], [22], and upregulates growth factor expression [23], [24], [25].
Since milk choline content, that depends on dietary choline intake of the mother in mammals [humans: [26]; cows: [27], [28] rodents: [29] and [30] for exhaustive choline radioactive labelling studies], influences offspring circulating choline status [26], the early postnatal choline supplementation in rodents can be easily obtained by supplementing choline to the dams [31].
A dietary choline postnatal supplementation in Mecp2 null male mice improved motor deficits, leaving cognitive deficits unaltered [32]. It also increased NGF expression in both wild-type (wt) and Mecp2 null mice [33], and selectively increased N-acetyl aspartate levels (a marker of neuronal integrity) in Mecp2 null mice [34]. These data suggest that postnatal choline supplementation improves neuronal function and ameliorates some aspects of the Mecp2 null phenotype.
Aim of our study was to investigate the effects of choline supplementation on a different RTT mouse model, the Mecp2 truncated mouse line, which is characterized by a milder phenotype and a more prolonged presymptomatic phase. In the open field test we evaluated the behavioural effects of choline at two different age-points: onset of symptoms (postnatal day, pnd 45), and adulthood (pnd 60). In this latter age-group, also on the basis of cholinergic deficit reported in post-mortem RTT human brains [35], we evaluated the status of the cholinergic function, so far scarcely investigated in RTT mouse models. We then assessed behavioural responses to a challenge with a muscarinic cholinergic antagonist (scopolamine) and activity of choline acetyl transferase (ChAT), the enzyme responsible for acetylcholine synthesis and nerve growth factor (NGF) expression, the trophic factor for basal forebrain cholinergic neurons. In addition, brain derived neurotrophic factor (BDNF) protein and mRNA levels were also evaluated because of functional interactions between Mecp2 and BDNF [36], [37], and evidence pointing to a therapeutic role of BDNF in RTT mouse models [33], [38], [39].
Section snippets
Animals and postnatal choline supplementation
Breeding pairs of MeCP2-308 mice (stock number: 005439 Jackson Laboratories USA) [heterozygous females and hemizygous (hz) males], were housed in polycarbonate transparent cages (33 × 13 × 14 cm) with sawdust bedding. Animals were provided with ad libitum drinking water and a complete pellet diet (Altromin, Germany, including 600 mg/kg choline chloride, which corresponds to approximately 1.9 mg/day standard choline supplementation in non-pregnant mice) and kept on a 12-h dark–light schedule (lights
Dam fluid intake (day 1–25 postpartum)
To control for the effects of choline on fluid intake, we measured the amount of solution drunk throughout treatment. Whereas, as expected, fluid intake rose throughout lactation [F(6, 54) = 11.09; p < .001], this rise was indistinguishable between dams of the two treatment groups [Mean fluid intake (ml) ± SEM: pnd 2: vehicle = 17.07 ± 3.03, n = 5; choline = 16.28 ± 1.63, n = 7; pnd 25: vehicle = 32.84 ± 14.97, n = 5; choline = 32.46 ± 13.82, n = 7]. In choline treated dams the choline supplementation (25 mM in drinking
Discussion
Early choline supplementation has long-term effects upon one aspect of the neurobehavioural phenotype of adult Mecp2-308 mice: a reduction of locomotor activity was evident in 60-day-old Mecp2-308 mice; choline supplementation restored wt-like levels of locomotor activity in these animals.
Despite a large number of behavioural studies in rats [31], and to a lesser extent in mice [22], [50], the exact biological mechanisms triggered by perinatal choline supplementation are not yet fully
Acknowledgements
The authors thank Luigia Cancemi for animal care. Support has been received from the ERARE-EuroRETT network and from ISS-NIH 530F/52 ‘Neurobehavioural phenotyping of genetically modified mouse models of mental retardation’ to LR and Grant from Foundation Jerome Lejeune (France) and Italian Minister of Health (Rare Diseases Call 2008) MECP2 phosphorylation and related kinases in Rett syndrome-ISS Unit to GL.
References (73)
Clinical manifestations and stages of Rett syndrome
Ment Retard Dev Disabil Res Rev
(2002)- et al.
Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2
Nat Genet
(1999) - et al.
The story of Rett syndrome: from clinic to neurobiology
Neuron
(2007) - et al.
MeCP2 in Rett syndrome: transcriptional repressor or chromatin architectural protein?
Curr Opin Genet Dev
(2007) - et al.
Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3
Neuron
(2002) - et al.
Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice
Nat Genet
(2001) - et al.
A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome
Nat Genet
(2001) - et al.
Early postnatal behavioral changes in the Mecp2-308 truncation mouse model of Rett syndrome
Genes Brain Behav
(2010) - et al.
Enhanced anxiety and stress-induced corticosterone release are associated with increased Crh expression in a mouse model of Rett syndrome
Proc Natl Acad Sci U S A
(2006) - et al.
Abnormalities of social interactions and home-cage behavior in a mouse model of Rett syndrome
Hum Mol Genet
(2005)