Noemi Morello

Heme scavenging and the other facets of hemopexin.

Hemopexin is an acute-phase plasma glycoprotein, produced mainly by the liver and released into plasma, where it binds heme with high affinity. Other sites of hemopexin synthesis are the nervous system, skeletal muscle, retina, and kidney. The only known receptor for the heme-hemopexin complex is the scavenger receptor, LDL receptor-related protein (LRP)1, which is expressed in most cell types, thus indicating multiple sites of heme-hemopexin complex recovery. The better-characterized function of hemopexin is heme scavenging at the systemic level, consisting of the transport of heme to the liver, where it is catabolyzed or used for the synthesis of hemoproteins or exported to bile canaliculi. This is important both in physiologic heme management for heme-iron recycling and in pathologic conditions associated with intravascular hemolysis to prevent the prooxidant and proinflammatory effects of heme. Other than scavenging heme, the heme-hemopexin complex has been shown to be able to activate signaling pathways, thus promoting cell survival, and to modulate gene expression. In this review, the importance of heme scavenging by hemopexin, as well as the other emerging functions of this protein, are discussed.

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.

Haemopexin affects iron distribution and ferritin expression in mouse brain

Haemopexin (Hx) is an acute phase plasma glycoprotein, mainly produced by the liver and released into plasma where it binds heme with high affinity and delivers it to the liver. This system provides protection against free heme‐mediated oxidative stress, limits access by pathogens to heme and contributes to iron homeostasis by recycling heme iron. Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF). Recently, a comparative proteomic analysis has shown an increase of Hx in CSF from patients with Alzheimer’s disease, thus suggesting its involvement in heme detoxification in brain. Here, we report that Hx is synthesised in brain by the ventricular ependymal cells. To verify whether Hx is involved in heme scavenging in brain, and consequently, in the control of iron level, iron deposits and ferritin expression were analysed in cerebral regions known for iron accumulation. We show a twofold increase in the number of iron‐loaded oligodendrocytes in the basal ganglia and thalamus of Hx‐null mice compared to wild‐type controls. Interestingly, there was no increase in H‐ and L‐ferritin expression in these regions. This condition is common to several human neurological disorders such as Alzheimer’s disease and Parkinson’s disease in which iron loading is not associated with an adequate increase in ferritin expression. However, a strong reduction in the number of ferritin‐positive cells was observed in the cerebral cortex of Hx‐null animals. Consistent with increased iron deposits and inadequate ferritin expression, malondialdehyde level and Cu–Zn superoxide dismutase‐1 expression were higher in the brain of Hx‐null mice than in that of wild‐type controls. These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron‐related neurodegenerative diseases.

Pharmacological enhancement of mGlu5 receptors rescues behavioral deficits in SHANK3 knock-out mice.

SHANK3 (also called PROSAP2) genetic haploinsufficiency is thought to be the major cause of neuropsychiatric symptoms in Phelan-McDermid syndrome (PMS). PMS is a rare genetic disorder that causes a severe form of intellectual disability (ID), expressive language delays and other autistic features. Furthermore, a significant number of SHANK3 mutations have been identified in patients with autism spectrum disorders (ASD), and SHANK3 truncating mutations are associated with moderate to profound ID. The Shank3 protein is a scaffold protein that is located in the postsynaptic density (PSD) of excitatory synapses and is crucial for synapse development and plasticity. In this study, we investigated the molecular mechanisms associated with the ASD-like behaviors observed in Shank3Δ11−/− mice, in which exon 11 has been deleted. Our results indicate that Shank3 is essential to mediating metabotropic glutamate receptor 5 (mGlu5)-receptor signaling by recruiting Homer1b/c to the PSD, specifically in the striatum and cortex. Moreover, augmenting mGlu5-receptor activity by administering 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide ameliorated the functional and behavioral defects that were observed in Shank3Δ11−/− mice, suggesting that pharmaceutical treatments that increase mGlu5 activity may represent a new approach for treating patients that are affected by PMS and SHANK3 mutations.

p140Cap Regulates Memory and Synaptic Plasticity through Src-Mediated and Citron-N-Mediated Actin Reorganization

A major challenge in the neuroscience field is the identification of molecules and pathways that control synaptic plasticity and memory. Dendritic spines play a pivotal role in these processes, as the major sites of excitatory synapses in neuronal communication. Previous studies have shown that the scaffold protein p140Cap localizes into dendritic spines and that its knockdown negatively modulates spine shape in culture. However, so far, there is no information on its in vivo relevance. By using a knock-out mouse model, we here demonstrate that p140Cap is a key element for both learning and synaptic plasticity. Indeed, p140Cap−/− mice are impaired in object recognition test, as well as in LTP and in LTD measurements. The in vivo effects of p140Cap loss are presumably attenuated by noncell-autonomous events, since primary neurons obtained from p140Cap−/− mice show a strong reduction in number of mushroom spines and abnormal organization of synapse-associated F-actin. These phenotypes are most likely caused by a local reduction of the inhibitory control of RhoA and of cortactin toward the actin-depolymerizing factor cofilin. These events can be controlled by p140Cap through its capability to directly inhibit the activation of Src kinase and by its binding to the scaffold protein Citron-N. Altogether, our results provide new insight into how protein associated with dynamic microtubules may regulate spine actin organization through interaction with postsynaptic density components.

A Role for Hemopexin in Oligodendrocyte Differentiation and Myelin Formation

Myelin formation and maintenance are crucial for the proper function of the CNS and are orchestrated by a plethora of factors including growth factors, extracellular matrix components, metalloproteases and protease inhibitors. Hemopexin (Hx) is a plasma protein with high heme binding affinity, which is also locally produced in the CNS by ependymal cells, neurons and glial cells. We have recently reported that oligodendrocytes (OLs) are the type of cells in the brain that are most susceptible to lack of Hx, as the number of iron-overloaded OLs increases in Hx-null brain, leading to oxidative tissue damage. In the current study, we found that the expression of the Myelin Basic Protein along with the density of myelinated fibers in the basal ganglia and in the motor and somatosensory cortex of Hx-null mice were strongly reduced starting at 2 months and progressively decreased with age. Myelin abnormalities were confirmed by electron microscopy and, at the functional level, resulted in the inability of Hx-null mice to perform efficiently on the Rotarod. It is likely that the poor myelination in the brain of Hx-null mice was a consequence of defective maturation of OLs as we demonstrated that the number of mature OLs was significantly reduced in mutant mice whereas that of precursor cells was normal. Finally, in vitro experiments showed that Hx promotes OL differentiation. Thus, Hx may be considered a novel OL differentiation factor and the modulation of its expression in CNS may be an important factor in the pathogenesis of human neurodegenerative disorders.

Developmental abnormalities of cortical interneurons precede symptoms onset in a mouse model of Rett syndrome

Rett syndrome (RTT, MIM312750), a neurodevelopmental disorder predominantly occurring in females, is caused in the majority of cases by sporadic mutations in the gene encoding the transcriptional modulator methyl‐CpG‐binding protein 2 (MECP2). In mice, impaired MeCP2 function results in severe motor, cognitive, and emotional defects. The lack of Mecp2 in γ‐aminobutyric acid‐(GABA) releasing forebrain interneurons (INs) recapitulate many RTT features, however, the role of this gene in the development of the cortical inhibitory system is still unknown. Here, we found that MeCP2 expression varies among the three major classes of cortical INs and its nuclear localization differs between neuronal types. The density of calretinin+ and parvalbumin+ INs increases in Mecp2 knockout mice (Mecp2−/y) already at early post‐natal developmental stages. In contrast, the density of somatostatin+ INs is not affected. We also found that the development of multipolar‐calretinin+ INs is selectively affected by the absence of Mecp2. Additionally, we show that in Mecp2 heterozygous female mice, a model closely mimicking human RTT condition, IN abnormalities are similar to those observed in Mecp2−/y mice. Together, our study indicates that loss of function of Mecp2 strongly interferes with the correct establishment of the neocortical inhibitory system producing effects that are specific to different IN subtypes.

Effects of Forced Swimming Stress on ERK and Histone H3 Phosphorylation in Limbic Areas of Roman High- and Low-Avoidance Rats

Stressful events evoke molecular adaptations of neural circuits through chromatin remodeling and regulation of gene expression. However, the identity of the molecular pathways activated by stress in experimental models of depression is not fully understood. We investigated the effect of acute forced swimming (FS) on the phosphorylation of the extracellular signal-regulated kinase (ERK)1/2 (pERK) and histone H3 (pH3) in limbic brain areas of genetic models of vulnerability (RLA, Roman low-avoidance rats) and resistance (RHA, Roman high-avoidance rats) to stress-induced depression-like behavior. We demonstrate that FS markedly increased the density of pERK-positive neurons in the infralimbic (ILCx) and the prelimbic area (PrLCx) of the prefrontal cortex (PFCx), the nucleus accumbens, and the dorsal blade of the hippocampal dentate gyrus to the same extent in RLA and RHA rats. In addition, FS induced a significant increase in the intensity of pERK immunoreactivity (IR) in neurons of the PFCx in both rat lines. However, RHA rats showed stronger pERK-IR than RLA rats in the ILCx both under basal and stressed conditions. Moreover, the density of pH3-positive neurons was equally increased by FS in the PFCx of both rat lines. Interestingly, pH3-IR was higher in RHA than RLA rats in PrLCx and ILCx, either under basal conditions or upon FS. Finally, colocalization analysis showed that in the PFCx of both rat lines, almost all pERK-positive cells express pH3, whereas only 50% of the pH3-positive neurons is also pERK-positive. Moreover, FS increased the percentage of neurons that express exclusively pH3, but reduced the percentage of cells expressing exclusively pERK. These results suggest that (i) the distinctive patterns of FS-induced ERK and H3 phosphorylation in the PFCx of RHA and RLA rats may represent molecular signatures of the behavioural traits that distinguish the two lines and (ii) FS-induced H3 phosphorylation is, at least in part, ERK-independent.

Homer1b/c clustering is impaired in Phelan-McDermid Syndrome iPSCs derived neurons

The panels show representative images of hNP-derived neurons and dendrites from control and PMS patients, which, after infection with a lentivirus expressing Homer-GFP, were differentiated in neuronal differentiation medium for 80 days. The staining (right panel) shows that GFP-Homer1b/c clusters in iPSC-derived neurons colocalize with the presynaptic marker Synaptophysin. Scale bar, 10 μm. The results are shown as bar diagrams. The data are presented as the mean± s.e.m. of three independent experiments and we used n= 2 independent hNP for each individuals. *Po0.05. For more information on this topic, please refer to the article by Vicidomini et al. on pages 689–702.

Loss of Mecp2 Causes Atypical Synaptic and Molecular Plasticity of Parvalbumin-Expressing Interneurons Reflecting Rett Syndrome–Like Sensorimotor Defects

Abstract Rett syndrome (RTT) is caused in most cases by loss-of-function mutations in the X-linked gene encoding methyl CpG-binding protein 2 (MECP2). Understanding the pathological processes impacting sensory-motor control represents a major challenge for clinical management of individuals affected by RTT, but the underlying molecular and neuronal modifications remain unclear. We find that symptomatic male Mecp2 knockout (KO) mice show atypically elevated parvalbumin (PV) expression in both somatosensory (S1) and motor (M1) cortices together with excessive excitatory inputs converging onto PV-expressing interneurons (INs). In accordance, high-speed voltage-sensitive dye imaging shows reduced amplitude and spatial spread of synaptically induced neuronal depolarizations in S1 of Mecp2 KO mice. Moreover, motor learning-dependent changes of PV expression and structural synaptic plasticity typically occurring on PV+ INs in M1 are impaired in symptomatic Mecp2 KO mice. Finally, we find similar abnormalities of PV networks plasticity in symptomatic female Mecp2 heterozygous mice. These results indicate that in Mecp2 mutant mice the configuration of PV+ INs network is shifted toward an atypical plasticity state in relevant cortical areas compatible with the sensory-motor dysfunctions characteristics of RTT.