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Rett syndrome (RTT) is a neurodevelopmental disorder that affects mainly females, associated in most cases to mutations in the MECP2 gene. After an apparently normal prenatal and perinatal period, patients display an arrest in growth and in psychomotor development, with autistic behaviour, hand stereotypies and mental retardation. Despite this classical description, researchers always questioned whether RTT patients did have subtle manifestations soon after birth. This issue was recently brought to light by several studies using different approaches that revealed abnormalities in the early development of RTT patients. Our hypothesis was that, in the mouse models of RTT as in patients, early neurodevelopment might be abnormal, but in a subtle manner, given the first descriptions of these models as initially normal. To address this issue, we performed a postnatal neurodevelopmental study in the Mecp2tm1.1Bird mouse. These animals are born healthy, and overt symptoms start to establish a few weeks later, including features of neurological disorder (tremors, hind limb clasping, weight loss). Different maturational parameters and neurological reflexes were analysed in the pre-weaning period in the Mecp2-mutant mice and compared to wild-type littermate controls. We found subtle but significant sex-dependent differences between mutant and wild-type animals, namely a delay in the acquisition of the surface and postural reflexes, and impaired growth maturation. The mutant animals also show altered negative geotaxis and wire suspension behaviours, which may be early manifestations of later neurological symptoms. In the post-weaning period the juvenile mice presented hypoactivity that was probably the result of motor impairments. The early anomalies identified in this model of RTT mimic the early motor abnormalities reported in the RTT patients, making this a good model for the study of the early disease process.
Rett syndrome (RTT) is a major cause of mental retardation in females, affecting 1 per 10 000 to 1 per 22 000 females born (Percy 2002). The ‘classic’ progression of RTT has four stages (Kerr & Engerstrom 2001). Stage I is characterized by an apparently normal development with uneventful prenatal and perinatal periods; in this stage (around 6–18 months) some of the patients learn some words and some are able to walk and feed themselves. In stage II (regression) a deceleration/arrest in the psychomotor development is noticed, with loss of stage I acquired skills, establishment of autistic behaviour and signs of intellectual dysfunction; the hands’ skilful abilities are replaced by stereotypical hand movements, a hallmark of RTT. The pre-school/school years correspond to stage III (pseudo-stationary) and here some improvement can be appreciated, with recovery of previously acquired skills. This is followed by the progressively incapacitating stage IV that can last for years (Hagberg et al. 2002); at this final stage patients develop trunk and gait ataxia, dystonia, autonomic dysfunction (breathing anomalies, sleep and gastrointestinal disturbances) and many of them have a sudden unexplained death in adulthood.
In spite of the classic RTT description, some researchers have questioned whether RTT patients display subtle signs of abnormal development soon after birth (Engerstrom 1992; Kerr 1995; Naidu 1997; Nomura & Segawa 1990). Huppke and colleagues reported on a sample of RTT patients who presented a significantly reduced occipito-frontal circumference, shorter length and lower weight at birth (Huppke et al. 2003). This hypothesis has recently been confirmed by the work of Einspieler and colleagues (Einspieler et al. 2005b), who analysed video records of the first 6 months of life of 22 RTT patients and were able to notice abnormalities in several behaviours. All RTT patients presented an abnormal pattern of spontaneous movements within the first 4 weeks of life, with abnormal ‘fidgety’ movements that were considered a sign of abnormal development (Einspieler et al. 2005a,b). Such abnormal movements were ascribed to problems in the central pattern generators in the brain (Einspieler et al. 2005a; Einspieler & Prechtl 2005). In a different study, midwives and health visitors blinded for the clinical status of the children were able to identify in family videos potential anomalies in the early development of RTT patients, particularly anomalies in physical appearance and hand posture, as well as body movements and postures (Burford 2005). Segawa, in a retrospective study of patients’ clinical files (Segawa 2005), also reported altered presentation of several motor milestones.
Most patients with classic RTT are heterozygous for mutations in the X-linked methyl-CpG binding protein gene (MECP2) (Amir et al. 1999), which encodes the methyl-CpG binding protein, MeCP2; this is known to bind symmetrically methylated CpG dinucleotides, and to recruit the co-repressors Sin3 yeast homologue A and histone deacetylase 1 and histone deacetylase 2 to repress transcription (Jones et al. 1998). When mutated, MeCP2 does not bind or binds ineffectively to its targets and, as a consequence, deregulation of transcription is thought to occur. Animal models of RTT were created in mice, mimicking several motor aspects of RTT and even the more emotional and social aspects of the syndrome (Chen et al. 2001; Guy et al. 2001; Shahbazian et al. 2002). The mutants are born normal and a few weeks later start to present a progressive motor deterioration, despite no gross abnormalities in the brain being noticed. Males carrying the mutation in hemizygosity display an earlier onset and are more severely affected than heterozygous females, probably as the result of X-chromosome inactivation that makes these females mosaics for the expression of the mutation, as is the case for the human condition.
The study presented here was performed using the Mecp2tm1.1Bird (Guy et al. 2001) mouse as a model. These mice were described as presenting no initial phenotype. Male Mecp2tm1.1Bird null animals begin to show symptoms at 3–8 weeks whereas heterozygous female animals manifest the disease at 3 months of age. The phenotype of these animals mimics many of the motor symptoms of RTT: stiff and unco-ordinated gait, reduced spontaneous movement, hind limb clasping, tremor and irregular breathing. Pathologically, no obvious histological abnormalities were detected in peripheral organs or in the brain. However, more recently, Kishi and Macklis reported that in the Mecp2-null mice the neocortical projection layers were thinner and the pyramidal neurons in layer II/ III had smaller somas and less complex dendritic trees in symptomatic animals than in wild-type mice (Kishi & Macklis 2004). Another study in this animal model suggested an essential role of MeCP2 in the mechanisms of synaptic plasticity (LTP and LTD) in the mature hippocampal neurons (Asaka et al. 2005).
The goal of this study was to determine whether the early neurodevelopmental process was altered in the absence of MeCP2 in mice. We assessed the achievement of milestones, considering different maturational and physical growth measures and neurological reflexes, two of the most well-known and most used neurobehavioural testing categories to address neurological disorder (Spear 1990), in the Mecp2tm1.1Bird mouse model of RTT (Mecp2-null males and Mecp2-heterozygous females). We identified an altered developmental progression of the mutant animals since the first postnatal week, in spite of their apparently normal phenotype. The differences seen suggest the presence of mild neurological deficits already at this age; the animals also presented significantly reduced activity, probably as a result of motor impairments early in life. The abnormal achievement of the developmental hallmarks, although transient, could reflect abnormalities that are likely to impact the development of more mature behaviours.