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5-HT were decreased in specific brain regions of Mecp2-null mice when compared to wt controls; namely in the PFCx and in the MCx. The fact that differences in the serotonergic system were not noticed before at earlier ages in the Mecp2-null mice could be attributed to the lack of sensitivity given the gross dissection of brain regions (whole brain or forebrain) used in the analysis. Additionally, no differences of the biogenic amine levels were found between Mecp2-null and wt neonates (Ide et al. 2005; Viemari et al. 2005) or at postnatal day 14 (Ide et al. 2005); we have not analysed brains at this age given the technical difficulty to appropriately dissect the regions of interest. So, at this time, we can only conclude that onset of the serotonergic imbalance occurs before the age of three weeks.
The diffuse monoaminergic modulatory systems of the brain originate in a core of subcortical nuclei and send extensive projections to several brain areas (figure 4.13) (Herlenius and Lagercrantz 2001). No differences in the levels of biogenic amines were found between Mecp2-null and wt mice in the D/MRN and in the SN-VTA brain regions where 5-HT and DA, respectively, are produced, both at three and at eight weeks of age (figure 4.7 and 4.8). The most obvious differences that we found were a reduction in the levels of NE and 5-HT in the PFCx, MCx and cerebellum of Mecp2-null mice as compared to wt controls (figures 4.3, 4.4 and 4.10), which are known for their involvement in higher and mid- level motor control, in the planning of movement. This aspect of RTT phenotype is well modelled in the Mecp2-null mouse we used for the current study already at an early age (see chapter 3).
Figure 4.13. Brain modulatory monoaminergic systems (adapted from (Herlenius and Lagercrantz 2001).
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The PFCx, together with the CPu, plays a role in motor control, in the integration of the new presented situation with former memories, in order to envisage possible outcomes of an action. On the other hand, the MCx sets a plan in order to achieve the aimed outcome. The cerebellar input is related to the coordination of the movement and, finally, execution of the movement is determined by brainstem nuclei and the spinal cord.
A disturbance in the crosstalk between all these areas, even subtle, may result in a serious motor deficit. The data we obtained in this study in the Mecp2-null mice showed that the above-referred regions presented an impaired bioaminergic modulation. This could explain some of the motor behavioural problems exhibited by this model and also the phenotypic manifestations of the human RTT patients: the non-directed and wide-base walking gait (when acquired), the hand stereotypies and the dyspraxia (figure 4.14).
Figure 4.14. Brain structures involved in the motor control. Represented are the differences found at each brain region of the Mecp2-null mouse model. (Arrows: orange, regions involved in the “high level” control of movement, green, regions involved in the “mid level” control of movement and blue, regions involved in the
“low- level” control of movement.
Brainstem (Vestibular nuclei)
Motor cortex Prefrontal cortex
Cerebellum Caudate-putamen
Spinal cord EXECUTION
COORDINATION PLANNING
ORDER Goal directed
Thalamus
Motor Control
↓HVA, ↓5-HIAA
↓5-HT to
↓NE, ↓5-HT, ↓5-HIAA
↓DOPAC
↑5-HT to
↓NE; ↓5-HT
↑5-HT to
8 weeks of age:
↓NE, ↓DA ↓5-HT;
↓5-HIAA
Dyspraxia Wide-base walking
Upper- and lower-extremities descoordination
↓NE
Abnormal milestones Loss of
purposeful hand use Repetitive movements Acquired microcephaly
↓Epilepsy threshold
3 weeks of age:
↑NE; ↑ 5-HT; ↑ 5-HT
↓DA
↓NET, ↓Adrα2a
↓Htr2a, ↓Htr3a
↓Htr2a, ↓Htr3a
Brainstem (Vestibular nuclei)
Motor cortex Prefrontal cortex
Cerebellum Caudate-putamen
Spinal cord EXECUTION
COORDINATION PLANNING
ORDER Goal directed
Thalamus
Motor Control
↓HVA, ↓5-HIAA
↓5-HT to
↓NE, ↓5-HT, ↓5-HIAA
↓DOPAC
↑5-HT to
↓NE; ↓5-HT
↑5-HT to
8 weeks of age:
↓NE, ↓DA ↓5-HT;
↓5-HIAA
Dyspraxia Wide-base walking
Upper- and lower-extremities descoordination
↓NE
Abnormal milestones Loss of
purposeful hand use Repetitive movements Acquired microcephaly
↓Epilepsy threshold
3 weeks of age:
↑NE; ↑ 5-HT; ↑ 5-HT
↓DA
↓NET, ↓Adrα2a
↓Htr2a, ↓Htr3a
↓Htr2a, ↓Htr3a
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What could be the physiological significance of the observed alterations of neurotransmitters and their metabolites, and what could be their impact on the developing brain?
The noradrenergic, dopaminegic and serotonergic systems achieve their modulatory role partly through an influence on neuronal maturation; in the regulation of physiological processes such as synaptic transmission, synaptic modification (dendritic length and arborisation, spine density and morphology) and neuronal adaptation (influencing LTP) (Hasselmo 1995; Berger-Sweeney and Hohmann 1997). For example, the apical dendritic branches of the infralimbic pyramidal neurons in SERT ko mice, which are characterized by high levels of extracellular 5-HT, were significantly increased in length relative to wt mice (Wellman et al. 2007). Interestingly, alterations in all these processes have already been reported in the brains of human RTT patients (Armstrong et al. 1995; Armstrong 2001; Armstrong 2002; Armstrong 2005) and in its different mouse models (Kishi and Macklis 2004; Asaka et al. 2006; Moretti et al. 2006)
The performance on behavioural tasks is also affected by monoamine dysregulation.
A depletion of NE is related to an impaired performance in attention paradigms, whereas 5-HT is more related to the postural control and locomotor function; its influence, through the descending pathways, in the central pattern generators of locomotion has been described (Pflieger et al. 2002; Vinay et al. 2002). DA is more closely linked to motor response initiation (Hauber 1998), which we have seen to be impaired in this mouse model.
Primarily affected brain regions in RTT
Our results clearly implicate a dysfunction of the noradrenergic and serotonergic pathways in the neuropathology of Mecp2-null mice since the earlier stages. The main affected brain areas are those involved in the higher and mid-level motor control, such as the prefrontal cortex and the motor cortex. The hippocampus and cerebellum seem to play a role only in the later stages of the disease. Altered bioaminergic modulation in these brain regions could be responsible for important components of the phenotype present in human RTT patients, partially modelled in these Mecp2-null mice.
The neurochemical changes detected in the vestibular area also support our previous results on the abnormal development of neurological reflexes of the Mecp2-null
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mice (chapter 3-I), which suggested an impaired neurodevelopment of pathways within the brainstem, particularly vestibular nuclei (Santos et al. 2006b). In the present study, we could confirm that this abnormal early motor development may be in part due to a dysfunction of the noradrenergic system, as Mecp2-null mice presented reduced levels of NE as compared to wt controls, already at 3-weeks of age, in the vestibular nuclei.
In the CPu of Mecp2-null mice we found that the levels of HVA (a metabolite of DA) and the turnover rate of 5-HIAA/5-HT were decreased. Impaired DA transmission within CPu delays motor initiation whereas enhanced serotonergic activity promotes akinesia (for a review see Hauber 1998), which is in agreement with our behavioural data (see chapter 3-II).
In summary, our data on neurochemical measurements suggests that the effect of Mecp2 mutation upon the brain modulatory monoaminergic systems is reflected in several of their projection target regions and not in the regions of their origin. In this way, MeCP2 may affect not the synthesis of monoamines but instead affect their release, their degradation or the pathways that are activated by monoamine receptor stimulation. We can say that the disease has a progressive course at the neurochemical level given that, overall, the mean differences detected between Mecp2-null and wt mice were higher at eight weeks of age than at three weeks of age.
Cerebellar involvement and RTT progression
Little attention has been given to the cerebellum, in respect to RTT pathology.
However, our data showed that neurochemically the cerebellum, although not affected from the beginning, becomes progressively involved, being severely altered at later stages, as has been described for RTT patients (Gotoh et al. 2001). At eight weeks of age the noradrenergic, dopaminergic and serotonergic pathways were significantly impaired (figure 4.10A,B), highlighting the importance of the cerebellum in the later phases and in the progression of the disorder. The cerebellum is the area of the brain responsible for coordinating muscular activity and complex movement. The serotonergic innervation to the cerebellum affects all parts of the cerebellar circuitry (for a review see Schweighofer et al. 2004) and disturbances of the cerebellar input have been related to cerebellar ataxia (Trouillas 1993) and to changes in spontaneous behavioural activity (Mendlin et al. 1996).
The cerebellar noradrenergic modulation is also very important, and noradrenergic terminals make close contacts with granule cell and Purkinje cell dendrites; NE levels
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have also been related to cerebellar learning (for a review see Schweighofer et al. 2004).
The cerebellum of RTT patients exhibited a progressive atrophy with loss of specific neurons, such as Purkinje neurons (Oldfors et al. 1990; Armstrong 2002).
The hippocampus and cognitive defects in RTT
In the hippocampus, no major neurochemical differences were found in the Mecp2-null as compared to wt mice, although at eight weeks of age there was a clear tendency for decreased levels of NE, 5-HT and HVA (figure 4.6A). Additionally, the interaction age x genotype may suggest an involvement of the hippocampus in the later stages of the disease. At three weeks of age, we observed that Mecp2-null mice performed as well as wt controls in the homing test, which assesses spatial learning in young juveniles (data not shown). Our data on neurotransmitter levels was in agreement with this unimpaired learning at three weeks of age. At eight weeks of age, given their severe motor impairment, it is impossible to perform any kind of learning task in this mouse model.
However, it has been reported that when symptomatic (mean eight weeks of age), but not at asymptomatic ages, Mecp2-null mice exhibited an impaired hippocampal LTP (Asaka et al. 2006), which underlies some forms of learning and memory, further supporting our neurochemical data. In another model of RTT, with an hypomorphic MECP2 allele (Mecp2308/Y), the performance of the Mecp2308/Y males in hippocampal-dependent learning and memory tasks was also significantly impaired and synaptic deficits at the hippocampus (LTP and LTD) were reported (Moretti et al. 2006).
Possible causes
The cause for the altered biogenic amine levels in these brain regions of the Mecp2-null mice remains elusive. A defect in the synthesis of monoamines does not appear to be the cause of the deregulated neuromodulation found in the Mecp2-null mice, as no differences in the levels of these amines were found in the regions of production of 5-HT and DA (D/MRN and SN-VTA, respectively).
In order to determine the mechanism by which the Mecp2-null mice exhibit decreased levels of NE and 5-HT we have explored some possible causes of such a difference. For example, a reduction in the levels of 5-HT could result from a reduction in the number of 5-HT fibres that innervate a given region. In the PFCx and MCx of Mecp2-null the levels of 5-HT were reduced as compared to wt mice at three weeks of age;
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however, this was not accompanied by a statistically significant reduction in the number of 5-HT positive fibres that innervate these regions.
As a transcriptional repressor, the absence of MeCP2 could also, directly or indirectly, affect the levels of expression of NE and 5-HT receptors and/or transporters. In fact, differences in the expression levels of other downstream target genes involved in neurotransmission had already been reported in Mecp2-null mice, such as the Dlx5 (Horike et al. 2005) and the human GABRB3 genes (Samaco et al. 2005) (for a review see Santos et al. 2006a).
NE and 5-HT release is modulated by the α2 adrenergic receptors (Baraban and Aghajanian 1980; Baraban and Aghajanian 1981). The action of NE is terminated, in part, by its uptake into presynaptic noradrenergic neurons by the plasma-membrane NET, and serotonergic neurotransmission is regulated by clearance of 5-HT from the extracellular space by SERT. The serotonergic Htr2a is highly expressed in the frontal cortex and Htr3a, also expressed in the cortex, is the only 5-HT receptor that it is not G-protein-coupled but ligand-gated Na/K channel. Both the Htr2a and Htr3a act as heteroreceptors by regulating the synthesis and/or the release of other neurotransmitters, such as GABA and glutamate, which are involved in learning and memory.
Interestingly, we found that in the PFCx of Mecp2-null mice, the mRNA levels of the NE transporter, of the adrenergic receptor Adrα2a and the mRNA levels of the serotonin receptors Htr2a and Htr3a were reduced as compared to wt levels. Additionally, in the MCx of the Mecp2-null mice also the levels of Htr2a and Htr3a receptors were reduced.
We have observed a decreased expression levels of three receptors (Adrα2a, Htr2a and Htr3a) from 3 weeks of age, which were maintained at low levels at eight weeks of age. If the cause of the reduced levels of both 5-HT and NE was in the low levels of the neurotransmitter itself, then through time the receptors would have adapted to the condition, by increasing their expression levels in order to compensate that dysregulation, which does not happen. However, our data appear to indicate that the problem must be at the transcription level. MeCP2 is a repressor and must be regulating the repression of another receptor that in turn modulates the expression of Adrα2a, Htr2a and Htr3a receptors, as their transcription was reduced in the Mecp2-null mice. Since 5-HT receptor levels may be crucial for strengthening of the synapses during development, this reduction
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may have as a consequence the loss of serotonergic synapses and a posterior decrease of serotonin. A further decrease of this neurotransmitter may result from the decrease of the Adrα2a receptor, which regulates the release of 5-HT and NE.
The lower levels of NET in the Mecp2-null mice are more likely to be a consequence of a chronic depletion of NE. It was shown that NET ko mice have increased levels of Adrα2a (Gilsbach et al. 2006). This again suggests that, in a normal situation, the receptor adapts to compensate the levels of its neurotransmitter,.
The PFCx has an important role in both cognitive and executive functions and is one of the brain structures involved in “higher level” control of movement, in the planning of an action (for a review see Berger-Sweeney and Hohmann 1997; Dalley et al. 2004; Arnsten and Li 2005). The action of NE is particularly relevant in the PFCx (reviewed in Dalley et al. 2004; Arnsten and Li 2005) and mediated by the adrenergic receptor α2A (Franowicz et al. 2002). We showed that in the PFCx of Mecp2-null mice the levels of Adrα2A receptor were reduced and this fact may affect the performance of the Mecp2-null mouse in the planning of a motor action. Moreover, the increased levels of extracellular NE of the NET ko mice is related to a decreased vulnerability to seizures (Kaminski et al. 2005). The lower levels of NE in the Mecp2-null mice may thus contribute to the seizures presented by most of the RTT patients.
Both 5-HT and NE were shown to induce an increase in the frequency and amplitude of excitatory postsynaptic potentials (EPSPs) in apical dendrites of neocortex and medial prefrontal cortex layer V pyramidal cells (Aghajanian and Marek 1997) and these effects are mediated by the serotonergic receptor Htr2a but not the adrenergic receptor Adrα2A (Marek and Aghajanian 1999). Interestingly, the Mecp2-null mouse we studied (1) has, as we showed here, decreased levels of 5-HT, NE neurotransmitters as well as of Htr2a receptor in PFCx and MCx, and (2) has reduced amplitude and frequency of mEPSPs in cortical pyramidal cells (Dani et al. 2005; Nelson et al. 2006). In this way, this data seems to suggest a role for NE and 5-HT, through Htr2a, in the neuronal activity levels of Mecp2-null mice.
Beyond the altered expression levels of the transcripts analyzed it would also be useful to analyze their binding activity/functional binding. It would be interesting to evaluate the binding activity of these monoaminergic receptors, through pharmacological
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studies, in the Mecp2-null mice, in order to clarify their involvement in the decreased availability of NE and 5-HT in these brain regions.
In the PFCx and MCx the reduction observed in the levels of 5-HT are accompanied by an increase in the turnover of this neurotransmitter. This evidence may suggest that the regulation of 5-HT turnover is compromised. Assessment of the enzymatic activity of monoamine oxidases may provide clues as to the biochemical basis of this increased turnover rate. Additionally, it would also be important to evaluate the expression levels of vesicular monoamine transporter, which could be further contributing to the decrease of 5-HT and NE in the Mecp2-null mice.
Our future studies will also address whether manipulation of the noradrenergic and serotonergic systems with agonists and antagonists influence the phenotype, in particular the motor performance, of Mecp2-null mice, and should provide further evidence as to the mechanism of neurotransmitter imbalances in this model. This knowledge should be helpful in defining future therapeutic approaches to RTT.
CHAPTER 5
INCREASED NEUROGENESIS IN THE HIPPOCAMPUS OF Mecp2-NULL MICE
The results described in this chapter are included in the following manuscript (in preparation):
Mónica Santos, Andreia Teixeira-Castro, Anabela Silva-Fernandes, Hugo Tavares, Nuno Sousa and Patrícia Maciel. “Increased neurogenesis in the hippocampal subgranular zone of Mecp2-null mice.”
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