Elena Boggio

Early Environmental Enrichment Moderates the Behavioral and Synaptic Phenotype of MeCP2 Null Mice

BACKGROUND Rett syndrome (RTT) is an X-linked progressive neurodevelopmental disorder characterized by a variety of symptoms including motor abnormalities, mental retardation, anxiety, and autism. Most of RTT cases are caused by mutations of MeCP2. In mice, impaired MeCP2 function results in synaptic deficits associated with motor, cognitive, and emotional alterations. Environmental enrichment (EE) is a rearing condition that enhances synapse formation and plasticity. Previous studies analyzing the effects of postweaning EE found limited effects on motor performance of male MeCP2 mutants. However, EE during early postnatal development produces powerful effects on neural development and plasticity. Thus, we tested whether early EE could ameliorate several phenotypes of male homozygous and female heterozygous MeCP2 mutants. METHODS We investigated the effects of early EE on motor coordination, structural and functional synaptic plasticity, and brain-derived neurotrophic factor expression in male MeCP2 null mice. Anxiety-related behavior and spatial learning was analyzed in heterozygous MeCP2 female mice. RESULTS In male mutants, EE modified excitatory and to a lesser extent inhibitory synaptic density in cerebellum and cortex, reversed the cortical long-term potentiation deficit and augmented cortical brain-derived neurotrophic factor levels. Environmental enrichment also ameliorated motor coordination and motor learning. In female heterozygous mice, a model closely mimicking some aspects of RTT symptoms, EE rescued memory deficits in the Morris water maze and decreased anxiety-related behavior. CONCLUSIONS Early EE dramatically improves several phenotypes of MeCP2 mutants. Thus, environmental factors should be taken into account when analyzing phenotypes of MeCP2 knockout mice, an accepted model of RTT. Early EE might be beneficial in RTT patients.

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.

The short-time structural plasticity of dendritic spines is altered in a model of Rett syndrome

The maturation of excitatory transmission comes about through a developmental period in which dendritic spines are highly motile and their number, form and size are rapidly changing. Surprisingly, although these processes are crucial for the formation of cortical circuitry, little is known about possible alterations of these processes in brain disease. By means of acute in vivo 2-photon imaging we show that the dynamic properties of dendritic spines of layer V cortical neurons are deeply affected in a mouse model of Rett syndrome (RTT) at a time around P25 when the neuronal phenotype of the disease is still mild. Then, we show that 24h after a subcutaneous injection of IGF-1 spine dynamics is restored. Our study demonstrates that spine dynamics in RTT mice is severely impaired early during development and suggest that treatments for RTT should be started very early in order to reestablish a normal period of spine plasticity.

Variations of the perforin gene in patients with multiple sclerosis

Perforin is involved in cell-mediated cytotoxicity and mutations of its gene (PRF1) cause familial hemophagocytic lymphohistiocytosis (FLH2). PRF1 sequencing in 190 patients with multiple sclerosis and 268 controls detected two FLH2-associated variations (A91V, N252S) in both groups and six novel mutations (C999T, G1065A, G1428A, A1620G, G719A, C1069T) in patients. All together, carriers of these variations were more frequent in patients than in controls (phenotype frequency: 17 vs 9%, P=0.0166; odds ratio (OR)=2.06, 95% confidence interval (CI): 1.13–3.77). Although A91V was the most frequent variation and displayed a trend of association with multiple sclerosis (MS) in the first population of patients and controls (frequency of the 91V allele: 0.076 vs 0.043, P=0.044), we used it as a marker to confirm PRF1 involvement in MS and assessed its frequency in a second population of 966 patients and 1520 controls. Frequency of the 91V allele was significantly higher in patients than in controls also in the second population (0.075 vs 0.058%, P=0.019). In the combined cohorts of 1156 patients and 1788 controls, presence of the 91V allele in single or double dose conferred an OR=1.38 (95% CI=1.10–1.74). These data suggest that A91V and possibly other perforin variations indicate susceptibility to MS.

Visual stimulation activates ERK in synaptic and somatic compartments of rat cortical neurons with parallel kinetics.

Background Extracellular signal-regulated kinase (ERK) signalling pathway plays a crucial role in regulating diverse neuronal processes, such as cell proliferation and differentiation, and long-term synaptic plasticity. However, a detailed understanding of the action of ERK in neurons is made difficult by the lack of knowledge about its subcellular localization in response to physiological stimuli. To address this issue, we have studied the effect of visual stimulation in vivo of dark-reared rats on the spatial-temporal dynamics of ERK activation in pyramidal neurons of the visual cortex. Methodology/Principal Findings Using immunogold electron microscopy, we show that phosphorylated ERK (pERK) is present in dendritic spines, both at synaptic and non-synaptic plasma membrane domains. Moreover, pERK is also detected in presynaptic axonal boutons forming connections with dendritic spines. Visual stimulation after dark rearing during the critical period causes a rapid increase in the number of pERK-labelled synapses in cortical layers I–II/III. This visually-induced activation of ERK at synaptic sites occurs in pre- and post-synaptic compartments and its temporal profile is identical to that of ERK activation in neuronal cell bodies. Conclusions/Significance Visual stimulation in vivo increases pERK expression at pre- and post-synaptic sites of axo-spinous junctions, suggesting that ERK plays an important role in the local modulation of synaptic function. The data presented here support a model in which pERK can have early and late actions both centrally in the cell nucleus and peripherally at synaptic contacts.

Serum levels of osteopontin are increased in SIRS and sepsis

ObjectiveIn sepsis, dysregulation of the immune response leads to rapid multiorgan failure and death. Accurate and timely diagnosis is lifesaving and should discriminate sepsis from the systemic inflammatory response syndrome (SIRS) caused by non-infectious agents. Osteopontin acts as an extracellular matrix component or a soluble cytokine in inflamed tissues. Its exact role in immune response and sepsis remains to be elucidated. Therefore, we investigated the role of osteopontin in SIRS and sepsis.DesignProspective, observational study.SettingIntensive care unit of a university hospital.Patients and participantsFifty-six patients with SIRS or sepsis and 56 healthy subjects were enrolled.InterventionsWe analyzed the serum levels of osteopontin and TH1–TH2 cytokines and investigated the role of osteopontin on interleukin 6 secretion by monocytes.Measurements and main resultsSerum osteopontin levels were strikingly higher in patients than in controls and in sepsis than in SIRS, and decreased during the resolution of both the disorders. Receiver operating characteristic curves showed that osteopontin levels have discriminative power between SIRS and sepsis with an area under the curve of 0.796. Osteopontin levels directly correlated with those of interleukin 6 and in vitro, recombinant osteopontin increased interleukin 6 secretion by monocytes in both the absence and presence of high doses of lipopolysaccharide.ConclusionThese data suggest that osteopontin might be a mediator involved in the pathogenesis of SIRS and sepsis, possibly by supporting interleukin 6 secretion.Descriptor45. SIRS/Sepsis: clinical studies.

Synaptic Determinants of Rett Syndrome

There is mounting evidence showing that the structural and molecular organization of synaptic connections is affected both in human patients and in animal models of neurological and psychiatric diseases. As a consequence of these experimental observations, it has been introduced the concept of synapsopathies, a notion describing brain disorders of synaptic function and plasticity. A close correlation between neurological diseases and synaptic abnormalities is especially relevant for those syndromes including also mental retardation in their symptomatology, such as Rett syndrome (RS). RS (MIM312750) is an X-linked dominant neurological disorder that is caused in the majority of cases by mutations in methyl-CpG-binding protein 2 (MeCP2). This review will focus on the current knowledge of the synaptic alterations produced by mutations of the gene MeCP2 in mouse models of RS and will highlight prospects experimental therapies currently in use. Different experimental approaches have revealed that RS could be the consequence of an impairment in the homeostasis of synaptic transmission in specific brain regions. Indeed, several forms of experience-induced neuronal plasticity are impaired in the absence of MeCP2. Based on the results presented in this review, it is reasonable to propose that understanding how the brain is affected by diseases such as RS is at reach. This effort will bring us closer to identify the neurobiological bases of human cognition.

Hippocampal CA1 Pyramidal Neurons of Mecp2 Mutant Mice Show a Dendritic Spine Phenotype Only in the Presymptomatic Stage

Alterations in dendritic spines have been documented in numerous neurodevelopmental disorders, including Rett Syndrome (RTT). RTT, an X chromosome-linked disorder associated with mutations in MECP2, is the leading cause of intellectual disabilities in women. Neurons in Mecp2-deficient mice show lower dendritic spine density in several brain regions. To better understand the role of MeCP2 on excitatory spine synapses, we analyzed dendritic spines of CA1 pyramidal neurons in the hippocampus of Mecp2tm1.1Jae male mutant mice by either confocal microscopy or electron microscopy (EM). At postnatal-day 7 (P7), well before the onset of RTT-like symptoms, CA1 pyramidal neurons from mutant mice showed lower dendritic spine density than those from wildtype littermates. On the other hand, at P15 or later showing characteristic RTT-like symptoms, dendritic spine density did not differ between mutant and wildtype neurons. Consistently, stereological analyses at the EM level revealed similar densities of asymmetric spine synapses in CA1 stratum radiatum of symptomatic mutant and wildtype littermates. These results raise caution regarding the use of dendritic spine density in hippocampal neurons as a phenotypic endpoint for the evaluation of therapeutic interventions in symptomatic Mecp2-deficient mice. However, they underscore the potential role of MeCP2 in the maintenance of excitatory spine synapses.

Thrombin Cleavage of Osteopontin Modulates Its Activities in Human Cells In Vitro and Mouse Experimental Autoimmune Encephalomyelitis In Vivo

Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α 4 β 1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases.

Variations of the UNC13D Gene in Patients with Autoimmune Lymphoproliferative Syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is caused by genetic defects decreasing Fas function and is characterized by lymphadenopathy/splenomegaly and expansion of CD4/CD8 double-negative T cells. This latter expansion is absent in the ALPS variant named Dianzani Autoimmune/lymphoproliferative Disease (DALD). In addition to the causative mutations, the genetic background influences ALPS and DALD development. We previously suggested a disease-modifying role for the perforin gene involved in familial hemophagocytic lymphohistiocytosis (FHL). The UNC13D gene codes for Munc13-4, which is involved in perforin secretion and FHL development, and thus, another candidate for a disease-modifying role in ALPS and DALD. In this work, we sequenced UNC13D in 21 ALPS and 20 DALD patients and compared these results with sequences obtained from 61 healthy subjects and 38 multiple sclerosis (MS) patients. We detected four rare missense variations in three heterozygous ALPS patients carrying p.Cys112Ser, p.Val781Ile, and a haplotype comprising both p.Ile848Leu and p.Ala995Pro. Transfection of the mutant cDNAs into HMC-1 cells showed that they decreased granule exocytosis, compared to the wild-type construct. An additional rare missense variation, p.Pro271Ser, was detected in a healthy subject, but this variation did not decrease Munc13-4 function. These data suggest that rare loss-of-function variations of UND13D are risk factors for ALPS development.