Eleonora Calcagno

Reduced AKT/mTOR signaling and protein synthesis dysregulation in a Rett syndrome animal model

Rett syndrome (RTT) is a neurodevelopmental disorder with no efficient treatment that is caused in the majority of cases by mutations in the gene methyl-CpG binding-protein 2 (MECP2). RTT becomes manifest after a period of apparently normal development and causes growth deceleration, severe psychomotor impairment and mental retardation. Effective animal models for RTT are available and show morphofunctional abnormalities of synaptic connectivity. However, the molecular consequences of MeCP2 disruption leading to neuronal and synaptic alterations are not known. Protein synthesis regulation via the mammalian target of the rapamycin (mTOR) pathway is crucial for synaptic organization, and its disruption is involved in a number of neurodevelopmental diseases. We investigated the phosphorylation of the ribosomal protein (rp) S6, whose activation is highly dependent from mTOR activity. Immunohistochemistry showed that rpS6 phosphorylation is severely affected in neurons across the cortical areas of Mecp2 mutants and that this alteration precedes the severe symptomatic phase of the disease. Moreover, we found a severe defect of the initiation of protein synthesis in the brain of presymptomatic Mecp2 mutant that was not restricted to a specific subset of transcripts. Finally, we provide evidence for a general dysfunction of the Akt/mTOR, but not extracellular-regulated kinase, signaling associated with the disease progression in mutant brains. Our results indicate that defects in the AKT/mTOR pathway are responsible for the altered translational control in Mecp2 mutant neurons and disclosed a novel putative biomarker of the pathological process. Importantly, this study provides a novel context of therapeutic interventions that can be designed to successfully restrain or ameliorate the development of RTT.

Genotype-Dependent Activity of Tryptophan Hydroxylase-2 Determines the Response to Citalopram in a Mouse Model of Depression

Polymorphism of tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of brain serotonin (5-HT), is associated with less synthesis of brain 5-HT in DBA/2J and BALB/c than in C57BL/6J and 129/Sv mice. We selected the forced swimming test, a mouse model used to assess the antidepressant potential of drugs, and neurochemical techniques to study strain differences in the response to citalopram, a selective 5-HT reuptake inhibitor. Citalopram reduced immobility time in C57BL/6J and 129/Sv mice but had no such effect in DBA/2J and BALB/c mice. The drug reduced accumulation of 5-hydroxytryptophan (5-HTP), an indicator of 5-HT synthesis, in C57BL/6J and 129/Sv mice but much less in DBA/2J and BALB/c mice. Pretreatment with tryptophan raised 5-HTP accumulation and reinstated the antidepressant-like effect of citalopram in DBA/2J and BALB/c mice, whereas pharmacological inhibition of 5-HT synthesis prevented the effect of citalopram in C57BL/6J and 129/Sv mice. Because there were no strain differences in catecholamine synthesis, locomotor activity, and brain levels of citalopram at the end of the behavioral test, the results suggest that the failure of citalopram to reduce immobility time in DBA/2J and BALB/c mice is attributable to genotype-dependent impairment of 5-HT synthesis. Interstrain comparisons could probably be a useful strategy for understanding the mechanisms underlying the response to selective serotonin reuptake inhibitors.

The 5-HT2A receptor antagonist M100,907 prevents extracellular glutamate rising in response to NMDA receptor blockade in the mPFC

We recently found that intracortical injection of the selective and competitive N‐methyl‐d‐aspartate (NMDA) receptor antagonist 3‐(R)‐2‐carboxypiperazin‐4‐propyl‐1‐phosphonic acid (CPP) impaired attentional performance in rats and blockade of 5‐hydroxytryptamine (5‐HT)2A receptors antagonized this effect. Here, we used the microdialysis technique in conscious rats to study the effect of CPP on extracellular glutamate (GLU) in the medial prefrontal cortex (mPFC) and the regulation of this effect by 5‐HT2A receptors. Intraperitoneal injection of 20 mg/kg CPP increased extracellular GLU in the mPFC (201% of basal levels) but had no effect on 5‐HT. Intracortical infusion of 100 µm CPP increased extracellular GLU (230% of basal values) and 5‐HT (150% of basal values) in the mPFC, whereas 30 µm had no significant effect. The effect of 100 µm CPP on extracellular GLU was abolished by tetrodotoxin, suggesting that neuronal activity is required. Subcutaneous injection of 40 µg/kg M100,907 completely antagonized the effect of 100 µm cpp on extracellular GLU, whereas 10 µg/kg caused only partial attenuation. Likewise, intracortical infusion of 0.1 µm M100,907 completely reversed the increase of extracellular GLU induced by CPP. These findings show that blockade of NMDA receptors in the mPFC is sufficient to increase extracellular GLU locally. The increase of cortical extracellular GLU may contribute to CPP‐induced cognitive deficits and blockade of 5‐HT2A receptors may provide a molecular mechanism for reversing these deficits caused by dysfunctional glutamatergic transmission in the mPFC.

Brain-specific overexpression of trace amine-associated receptor 1 alters monoaminergic neurotransmission and decreases sensitivity to amphetamine.

Trace amines (TAs) such as β-phenylethylamine, p-tyramine, or tryptamine are biogenic amines found in the brain at low concentrations that have been implicated in various neuropsychiatric disorders like schizophrenia, depression, or attention deficit hyperactivity disorder. TAs are ligands for the recently identified trace amine-associated receptor 1 (TAAR1), an important modulator of monoamine neurotransmission. Here, we sought to investigate the consequences of TAAR1 hypersignaling by generating a transgenic mouse line overexpressing Taar1 specifically in neurons. Taar1 transgenic mice did not show overt behavioral abnormalities under baseline conditions, despite augmented extracellular levels of dopamine and noradrenaline in the accumbens nucleus (Acb) and of serotonin in the medial prefrontal cortex. In vitro, this was correlated with an elevated spontaneous firing rate of monoaminergic neurons in the ventral tegmental area, dorsal raphe nucleus, and locus coeruleus as the result of ectopic TAAR1 expression. Furthermore, Taar1 transgenic mice were hyposensitive to the psychostimulant effects of amphetamine, as it produced only a weak locomotor activation and failed to alter catecholamine release in the Acb. Attenuating TAAR1 activity with the selective partial agonist RO5073012 restored the stimulating effects of amphetamine on locomotion. Overall, these data show that Taar1 brain overexpression causes hyposensitivity to amphetamine and alterations of monoaminergic neurotransmission. These observations confirm the modulatory role of TAAR1 on monoamine activity and suggest that in vivo the receptor is either constitutively active and/or tonically activated by ambient levels of endogenous agonist(s).

The 5-HT1A receptor agonist 8-OH-DPAT prevents prefrontocortical glutamate and serotonin release in response to blockade of cortical NMDA receptors

We studied the role of 5‐HT1A receptors in controlling the release of glutamate (GLU) in the medial prefrontal cortex (mPFC) of conscious rats with the in vivo microdialysis technique. The effect of the 5‐HT1A receptor agonist 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin infused in the prefrontal cortex was examined under basal conditions and on the rise of extracellular GLU (+106%) induced by co‐infusion of the competitive N‐methyl‐d‐aspartate receptor antagonist 3‐[(R)‐2‐carboxypiperazin‐4yl]‐propyl‐1‐phosphonic acid (CPP). 8‐OH‐DPAT (0.3 and 3 μm) had no effect on basal extracellular GLU, but the higher concentration completely abolished the rise of extracellular GLU induced by CPP. CPP also increased extracellular serotonin (5‐HT) in the mPFC (+50%) and this effect was antagonized by 3 μm 8‐OH‐DPAT which, by itself, had no effect on basal 5‐HT release. The effects of 8‐OH‐DPAT on extracellular GLU and 5‐HT were reversed by the 5‐HT1A receptor antagonist WAY100 635 (100 μm), indicating a selective involvement of 5‐HT1A receptors. WAY100 635 had no effect by itself. These results show that the stimulation of cortical 5‐HT1A receptors prevents the CPP‐evoked rise of extracellular GLU and 5‐HT and suggest that these effects may contribute to the ability of intracortical 8‐OH‐DPAT to counteract cognitive deficits caused by the blockade of NMDA receptors.

Mapping Pathological Phenotypes in a Mouse Model of CDKL5 Disorder

Mutations in cyclin-dependent kinase-like 5 (CDKL5) cause early-onset epileptic encephalopathy, a neurodevelopmental disorder with similarities to Rett Syndrome. Here we describe the physiological, molecular, and behavioral phenotyping of a Cdkl5 conditional knockout mouse model of CDKL5 disorder. Behavioral analysis of constitutive Cdkl5 knockout mice revealed key features of the human disorder, including limb clasping, hypoactivity, and abnormal eye tracking. Anatomical, physiological, and molecular analysis of the knockout uncovered potential pathological substrates of the disorder, including reduced dendritic arborization of cortical neurons, abnormal electroencephalograph (EEG) responses to convulsant treatment, decreased visual evoked responses (VEPs), and alterations in the Akt/rpS6 signaling pathway. Selective knockout of Cdkl5 in excitatory and inhibitory forebrain neurons allowed us to map the behavioral features of the disorder to separable cell-types. These findings identify physiological and molecular deficits in specific forebrain neuron populations as possible pathological substrates in CDKL5 disorder.

Strain differences in basal and post‐citalopram extracellular 5‐HT in the mouse medial prefrontal cortex and dorsal hippocampus: relation with tryptophan hydroxylase‐2 activity

We used the microdialysis technique to compare basal extracellular serotonin (5‐HT) and the response to citalopram in different strains of mice with functionally different allelic forms of tryptophan hydroxylase‐2 (TPH‐2), the rate‐limiting enzyme in brain 5‐HT synthesis. DBA/2J, DBA/2N and BALB/c mice carrying the 1473G allele of TPH‐2 had less dialysate 5‐HT in the medial prefrontal cortex and dorsal hippocampus (DH) (20–40% reduction) than C57BL/6J and C57BL/6N mice carrying the 1473C allele. Extracellular 5‐HT estimated by the zero‐net flux method confirmed the result of conventional microdialysis. Citalopram, 1.25, 5 and 20 mg/kg, dose‐dependently raised extracellular 5‐HT in the medial prefrontal cortex of C57BL/6J mice, with maximum effect at 5 mg/kg, but had significantly less effect in DBA/2J and BALB/c mice and in the DH of DBA/2J mice. A tryptophan (TRP) load enhanced basal extracellular 5‐HT in the medial prefrontal cortex of DBA/2J mice but did not affect citalopram’s ability to raise cortical and hippocampal extracellular 5‐HT. The impairment of 5‐HT synthesis quite likely accounts for the reduction of basal 5‐HT and the citalopram‐induced rise in mice carrying the mutated enzyme. These findings might explain why DBA/2 and BALB/c mice do not respond to citalopram in the forced swimming test. Although TRP could be a useful strategy to improve the antidepressant effect of citalopram ( Cervo et al. 2005 ), particularly in subjects with low 5‐HT synthesis, the contribution of serotonergic and non‐serotonergic mechanisms to TRP’s effect remains to be elucidated.

GluD1 is a common altered player in neuronal differentiation from both MECP2 -mutated and CDKL5 -mutated iPS cells

Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome.

Endogenous serotonin and serotonin2C receptors are involved in the ability of M100907 to suppress cortical glutamate release induced by NMDA receptor blockade

Blockade of NMDA receptors by intracortical infusion of 3‐(R)‐2‐carboxypiperazin‐4‐propyl‐1‐phosphonic acid (CPP) increases glutamate (GLU) and serotonin (5‐HT) release in the medial prefrontal cortex and impairs attentional performance in the 5‐choice serial reaction time task. These effects are prevented by the 5‐HT2A receptor antagonist, (R)‐(+)‐(2,3‐dimethoxyphenyl)‐1‐[2‐(4‐fluorophenyl)ethyl]‐4‐piperidine methanol (M100907). We explored the roles of endogenous 5‐HT and 5‐HT1A and 5‐HT2C receptors in the mechanisms by which M100907 suppresses CPP‐induced release of cortical GLU and 5‐HT using in vivo microdialysis. CPP raised extracellular GLU and 5‐HT by about 250% and 170% respectively. The 5‐HT synthesis inhibitor, p‐chlorophenylalanine (300 mg/kg), prevented M100907 suppressing CPP‐induced GLU release. The effect of M100907 on these rises of GLU and 5‐HT and attentional performance deficit was mimicked by the 5‐HT2C receptor agonist, (S)‐2‐(6‐chloro‐5‐fluoroindol‐1‐yl)‐1‐methylethylamine fumarate, (Ro60‐0175, 30 μg/kg) while intra‐mPFC (SB242084, 6‐chloro‐5‐methyl‐1‐[[2‐[(2‐methyl‐3‐pyridyl)oxy]‐5‐pyridyl]carbamoyl]‐indoline, 0.1 μM), a 5‐HT2C receptor antagonist, prevented the effect of M100907 on extracellular GLU. The 5‐HT1A receptor antagonist, N‐[2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]ethyl]‐N‐(2‐pyridinyl)cyclohexane carboxenide trihydrochloride (100 μM) abolished the effect of M100907 on the CPP‐induced 5‐HT release. The data show that blockade of 5‐HT2A receptors is not sufficient to suppress the CPP‐induced rise of extracellular GLU and 5‐HT and suggest that M100907 suppresses GLU release induced by CPP by enhancing the action of endogenous 5‐HT on 5‐HT2C receptors.

Glutamate and glutathione interplay in a motor neuronal model of amyotrophic lateral sclerosis reveals altered energy metabolism.

Impairment of mitochondrial function might contribute to oxidative stress associated with neurodegeneration in amyotrophic lateral sclerosis (ALS). Glutamate levels in tissues of ALS patients are sometimes altered. In neurons, mitochondrial metabolism of exogenous glutamine is mainly responsible for the net synthesis of glutamate, which is a neurotransmitter, but it is also necessary for the synthesis of glutathione, the main endogenous antioxidant. We investigated glutathione synthesis and glutamine/glutamate metabolism in a motor neuronal model of familial ALS. In standard culture conditions (with glutamine) or restricting glutamine or cystine, the level of glutathione was always lower in the cell line expressing the mutant (G93A) human Cu, Zn superoxide dismutase (G93ASOD1) than in the line expressing wild-type SOD1. With glutamine the difference in glutathione was associated with a lower glutamate and impairment of the glutamine/glutamate metabolism as evidenced by lower glutaminase and cytosolic malate dehydrogenase activity. d-β-hydroxybutyrate, as an alternative to glutamine as energy substrate in addition to glucose, reversed the decreases of cytosolic malate dehydrogenase activity and glutamate and glutathione. However, in the G93ASOD1 cell line, in all culture conditions the expression of pyruvate dehydrogenase kinase l protein, which down-regulates pyruvate dehydrogenase activity, was induced, together with an increase in lactate release in the medium. These findings suggest that the glutathione decrease associated with mutant SOD1 expression is due to mitochondrial dysfunction caused by the reduction of the flow of glucose-derived pyruvate through the TCA cycle; it implies altered glutamate metabolism and depends on the different mitochondrial energy substrates.