Greta Forlani

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.

Functional consequences of mutations in CDKL5, an X-linked gene involved in infantile spasms and mental retardation.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome, West syndrome, and X-linked infantile spasms sharing the common features of generally intractable early seizures and mental retardation. Disease-causing mutations are distributed in both the catalytic domain and in the large COOH terminus. In this report, we examine the functional consequences of some Rett mutations of CDKL5 together with some synthetically designed derivatives useful to underline the functional domains of the protein. The mutated CDKL5 derivatives have been subjected to in vitro kinase assays and analyzed for phosphorylation of the TEY (Thr-Glu-Tyr) motif within the activation loop, their subcellular localization, and the capacity of CDKL5 to interact with itself. Whereas wild-type CDKL5 autophosphorylates and mediates the phosphorylation of the methyl-CpG-binding protein 2 (MeCP2) in vitro, Rett-mutated proteins show both impaired and increased catalytic activity suggesting that a tight regulation of CDKL5 is required for correct brain functions. Furthermore, we show that CDKL5 can self-associate and mediate the phosphorylation of its own TEY (Thr-Glu-Tyr) motif. Eventually, we show that the COOH terminus regulates CDKL5 properties; in particular, it negatively influences the catalytic activity and is required for its proper sub-nuclear localization. We propose a model in which CDKL5 phosphorylation is required for its entrance into the nucleus whereas a portion of the COOH-terminal domain is responsible for a stable residency in this cellular compartment probably through protein-protein interactions.

Modulation of extracellular signal-regulated kinases cascade by chronic Δ9-tetrahydrocannabinol treatment

Acute Delta(9)-tetrahydrocannabinol (THC) injection increased ERK pathway (ERK, pCREB, and c-fos) mostly in the caudate putamen and cerebellum. This effect underwent to homeostatic adaptation after chronic treatment. Moreover, chronic THC exposure induced increases in the ERK cascade (ERK, pCREB, and Fos B) in the prefrontal cortex and hippocampus, suggesting that different neuronal circuits seem to be involved in the early phase and late phase of exposure. The involvement of ERK pathway in cannabinoid chronic exposure was also confirmed in Ras-GRF1 knock out mice, a useful model where cannabinoid-induced ERK activation is lost. In fact, Ras-GRF1 ko mice did not develop tolerance to THC analgesic and hypolocomotor effect. Our data suggest that ERK cascade could play a pivotal role in the induction of synaptic plasticity due to cannabinoid chronic exposure.

Ras/ERK signalling in cannabinoid tolerance: from behaviour to cellular aspects

We investigated the role of the Ras/extracellular‐regulated kinase (ERK) pathway in the development of tolerance to Δ9‐tetrahydrocannabinol (THC)‐induced reduction in spontaneous locomotor activity by a genetic (Ras‐specific guanine nucleotide exchange factor (Ras‐GRF1) knock‐out mice) and pharmacological approach. Pre‐treatment of wild‐type mice with SL327 (50 mg/kg i.p.), a specific inhibitor of mitogen‐activated protein kinase kinase (MEK), the upstream kinase of ERK, fully prevented the development of tolerance to THC‐induced hypolocomotion. We investigated the impact of the inhibition of ERK activation on the biological processes involved in cannabinoid tolerance (receptor down‐regulation and desensitization), by autoradiographic cannabinoid CB1 receptor and cannabinoid‐stimulated [35S]GTPγS binding studies in subchronically treated mice (THC, 10 mg/kg s.c., twice a day for 5 days). In the caudate putamen and cerebellum of Ras‐GRF1 knock‐out mice and SL327 pre‐treated wild‐type mice, CB1 receptor down‐regulation and desensitization did not occur, suggesting that ERK activation might account for CB1 receptor plasticity involved in the development of tolerance to THC hypolocomotor effect. In contrast, the hippocampus and prefrontal cortex showed CB1 receptor adaptations regardless of the genetic or pharmacological inhibition of the ERK pathway, suggesting regional variability in the cellular events underlying the altered CB1 receptor function. These findings suggest that at least in the caudate putamen and cerebellum, the Ras/ERK pathway is essential for triggering the alteration in CB1 receptor function responsible for tolerance to THC‐induced hypomotility.

Temperature effects on the presteady-state and transport-associated currents of GABA cotransporter rGAT1.

The effects of temperature on the γ‐aminobutyric acid (GABA) uptake and on the presteady‐state and transport‐associated currents of the GABA cotransporter, rat γ‐aminobutyric acid transporter 1 (rGAT1), have been studied using heterologous oocyte expression and voltage‐clamp. Increasing temperature from 15 to 30°C increased GABA uptake, diminished the maximal value of the relaxation time constant of the presteady‐state currents and increased the amplitude of the current associated with the transport of GABA. The curve of the presteady‐state charge versus voltage was shifted toward negative potentials by increasing the temperature, while the maximal amount of charge (Q max) remained constant; the τ versus V curve was also negatively shifted by increasing temperatures. Analysis of the outward (α) and inward (β) rate constants as functions of temperature showed that they are affected differently, with a Q 10=3.4 for α and Q 10=1.5 for β. The different temperature coefficients of the rate constants account for the observed shifts. These observations are consistent with a charge moving mechanism based on a conformational change of the protein; the weaker temperature sensitivity of the inward rate constant suggests a rate‐limiting diffusional component on this process.

Mutation K448E in the external loop 5 of rat GABA transporter rGAT1 induces pH sensitivity and alters substrate interactions

1 The effect of the mutation K448E in the rat GABA transporter rGAT1 was studied using heterologous expression in Xenopus oocytes and voltage clamp. 2 At neutral pH, the transport‐associated current vs. voltage (I–V) relationship of the mutated transporter was different from wild‐type, and the pre‐steady‐state currents were shifted towards more positive potentials. The mutated transporter showed an increased apparent affinity for Na+ (e.g. 62 vs. 152 mm at −60 mV), while the opposite was true for GABA (e.g. 20 vs. 13 μm at −60 mV). 3 In both isoforms changes in [Na+]o shifted the voltage dependence of the pre‐steady‐state and of the transport‐associated currents by similar amounts. 4 In the K448E form, the moved charge and the relaxation time constant were shifted by increasing pH towards positive potentials. The transport‐associated current of the mutated transporter was strongly reduced by alkalinization, while acidification slightly decreased and distorted the shape of the I–V curve. Accordingly, uptake of [3H]GABA was strongly reduced in K448E at pH 9.0. The GABA apparent affinity of the mutated transporter was reduced by alkalinization, while acidification had the opposite result. 5 These observations suggest that protonation of negatively charged residues may regulate the Na+ concentration in the proximity of the transporter. Calculation of the unidirectional rate constants for charge movement shows that, in the K448E form, the inward rate constant is increased at alkaline pH, while the outward rate constant does not change, in agreement with an effect due to mass action law. 6 A possible explanation for the complex effect of pH on the transport‐associated current may be found by combining changes in local [Na+]o with a direct action of pH on GABA concentration or affinity. Our results support the idea that the extracellular loop 5 may participate to form a vestibule to which sodium ions must have access before proceeding to the steps involving charge movement.

The MeCP2/YY1 interaction regulates ANT1 expression at 4q35: novel hints for Rett syndrome pathogenesis

Rett syndrome is a severe neurodevelopmental disorder mainly caused by mutations in the transcriptional regulator MeCP2. Although there is no effective therapy for Rett syndrome, the recently discovered disease reversibility in mice suggests that there are therapeutic possibilities. Identification of MeCP2 targets or modifiers of the phenotype can facilitate the design of curative strategies. To identify possible novel MeCP2 interactors, we exploited a bioinformatic approach and selected Ying Yang 1 (YY1) as an interesting candidate. We demonstrate that MeCP2 interacts in vitro and in vivo with YY1, a ubiquitous zinc-finger epigenetic factor regulating the expression of several genes. We show that MeCP2 cooperates with YY1 in repressing the ANT1 gene encoding a mitochondrial adenine nucleotide translocase. Importantly, ANT1 mRNA levels are increased in human and mouse cell lines devoid of MeCP2, in Rett patient fibroblasts and in the brain of Mecp2-null mice. We further demonstrate that ANT1 protein levels are upregulated in Mecp2-null mice. Finally, the identified MeCP2-YY1 interaction, together with the well-known involvement of YY1 in the regulation of D4Z4-associated genes at 4q35, led us to discover the anomalous depression of FRG2, a subtelomeric gene of unknown function, in Rett fibroblasts. Collectively, our data indicate that mutations in MeCP2 might cause the aberrant overexpression of genes located at a specific locus, thus providing new candidates for the pathogenesis of Rett syndrome. As both ANT1 mutations and overexpression have been associated with human diseases, we consider it highly relevant to address the consequences of ANT1 deregulation in Rett syndrome.

Modulation of the Inward Rectifier Potassium Channel IRK1 by the Ras Signaling Pathway

In this study, we investigated the role of Ras and the mitogen-activated protein kinase (MAPK) pathway in the modulation of the inward rectifier potassium channel IRK1. We show that although expression of IRK1 in HEK 293 cells leads to the appearance of a potassium current with strong inward rectifying properties, coexpression of the constitutively active form of Ras (Ras-L61) results in a significant reduction of the mean current density without altering the biophysical properties of the channel. The inhibitory effect of Ras-L61 is not due to a decreased expression of IRK1 since Northern analysis indicates that IRK1 mRNA level is not affected by Ras-L61 co-expression. Moreover, the inhibition can be relieved by treatment with the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD98059. Confocal microscopy analysis of cells transfected with the fusion construct green fluorescent protein-IRK1 shows that the channel is mainly localized at the plasma membrane. Coexpression of Ras-L61 delocalizes fluorescence to the cytoplasm, whereas treatment with PD98059 partially restores the membrane localization. In conclusion, our data indicate that the Ras-MAPK pathway modulates IRK1 current by affecting the subcellular localization of the channel. This suggests a role for Ras signaling in regulating the intracellular trafficking of this channel.

Role of anion-cation interactions on the pre-steady-state currents of the rat Na(+)-Cl(-)-dependent GABA cotransporter rGAT1.

The effects of sodium and chloride on the properties of the sodium‐dependent component of the ‘pre‐steady‐state’ currents of rGAT1, a GABA cotransporter of the Na+‐Cl−‐dependent family, were studied using heterologous oocyte expression and voltage clamp. Reductions in either extracellular sodium or chloride shifted the charge‐voltage (Q‐V) and time constant‐voltage (τ‐V) characteristics of the process towards more negative potentials. The shift induced by sodium (TMA+, tetramethylammonium substitution) was stronger than that induced by chloride (acetate substitution), and the shift of τ was accompanied by a decrease in its maximum value. Increasing extracellular Ca2+ did not produce significant shifts in Q‐V and τ‐V curves. The negative shift of the Q‐V curve upon chloride reduction and the decrease in the value of the relaxation time constant, τ, when either sodium or chloride were lowered, contrasted with the prediction of the Hill‐Boltzmann interpretation of the process. Analysis of the unidirectional rate constants under different conditions revealed that both sodium and chloride shifted the outward rate more than the inward rate; furthermore, the shifts induced by sodium were larger than those induced by chloride. These observations are qualitatively compatible with the existence of a selective vestibule at the mouth of the transporters, acting similarly to a Donnan system.

Major Histocompatibility Complex Class II Transactivator CIITA Is a Viral Restriction Factor That Targets Human T-Cell Lymphotropic Virus Type 1 Tax-1 Function and Inhibits Viral Replication

ABSTRACT Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of an aggressive malignancy of CD4+ T lymphocytes. Since the viral transactivator Tax-1 is a major player in T-cell transformation, targeting Tax-1 protein is regarded as a possible strategy to arrest viral replication and to counteract neoplastic transformation. We demonstrate that CIITA, the master regulator of major histocompatibility complex class II gene transcription, inhibits HTLV-1 replication by blocking the transactivating function of Tax-1 both when exogenously transfected in 293T cells and when endogenously expressed by a subset of U937 promonocytic cells. Tax-1 and CIITA physically interact in vivo via the first 108 amino acids of Tax-1 and two CIITA adjacent regions (amino acids 1 to 252 and 253 to 410). Interestingly, only CIITA 1-252 mediated Tax-1 inhibition, in agreement with the fact that CIITA residues from positions 64 to 124 were required to block Tax-1 transactivation. CIITA inhibitory action on Tax-1 correlated with the nuclear localization of CIITA and was independent of the transcription factor NF-YB, previously involved in CIITA-mediated inhibition of Tax-2 of HTLV-2. Instead, CIITA severely impaired the physical and functional interaction of Tax-1 with the cellular coactivators p300/CBP-associated factor (PCAF), cyclic AMP-responsive element binding protein (CREB), and activating transcription factor 1 (ATF1), which are required for the optimal activation of HTLV-1 promoter. Accordingly, the overexpression of PCAF, CREB, and ATF1 restored Tax-1-dependent transactivation of the viral long-terminal-repeat promoter inhibited by CIITA. These findings strongly support our original observation that CIITA, beside increasing the antigen-presenting function for pathogen antigens, acts as an endogenous restriction factor against human retroviruses by blocking virus replication and spreading.