Andrea Bergamaschi

Inflammation Triggers Synaptic Alteration and Degeneration in Experimental Autoimmune Encephalomyelitis

Neurodegeneration is the irremediable pathological event occurring during chronic inflammatory diseases of the CNS. Here we show that, in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, inflammation is capable in enhancing glutamate transmission in the striatum and in promoting synaptic degeneration and dendritic spine loss. These alterations occur early in the disease course, are independent of demyelination, and are strongly associated with massive release of tumor necrosis factor-α from activated microglia. CNS invasion by myelin-specific blood-borne immune cells is the triggering event, and the downregulation of the early gene Arc/Arg3.1, leading to the abnormal expression and phosphorylation of AMPA receptors, represents a culminating step in this cascade of neurodegenerative events. Accordingly, EAE-induced synaptopathy subsided during pharmacological blockade of AMPA receptors. Our data establish a link between neuroinflammation and synaptic degeneration and calls for early neuroprotective therapies in chronic inflammatory diseases of the CNS.

Persistent inflammation alters the function of the endogenous brain stem cell compartment.

Endogenous neural stem/precursor cells (NPCs) are considered a functional reservoir for promoting tissue homeostasis and repair after injury, therefore regenerative strategies that mobilize these cells have recently been proposed. Despite evidence of increased neurogenesis upon acute inflammatory insults (e.g. ischaemic stroke), the plasticity of the endogenous brain stem cell compartment in chronic CNS inflammatory disorders remains poorly characterized. Here we show that persistent brain inflammation, induced by immune cells targeting myelin, extensively alters the proliferative and migratory properties of subventricular zone (SVZ)-resident NPCs in vivo leading to significant accumulation of non-migratory neuroblasts within the SVZ germinal niche. In parallel, we demonstrate a quantitative reduction of the putative brain stem cells proliferation in the SVZ during persistent brain inflammation, which is completely reversed after in vitro culture of the isolated NPCs. Together, these data indicate that the inflamed brain microenvironment sustains a non cell-autonomous dysfunction of the endogenous CNS stem cell compartment and challenge the potential efficacy of proposed therapies aimed at mobilizing endogenous precursors in chronic inflammatory brain disorders.

Neural progenitor cells orchestrate microglia migration and positioning into the developing cortex

Microglia are observed in the early developing forebrain and contribute to the regulation of neurogenesis through still unravelled mechanisms. In the developing cerebral cortex, microglia cluster in the ventricular/subventricular zone (VZ/SVZ), a region containing Cxcl12-expressing basal progenitors (BPs). Here we show that the ablation of BP as well as genetic loss of Cxcl12 affect microglia recruitment into the SVZ. Ectopic Cxcl12 expression or pharmacological blockage of CxcR4 further supports that Cxcl12/CxcR4 signalling is involved in microglial recruitment during cortical development. Furthermore, we found that cell death in the developing forebrain triggers microglial proliferation and that this is mediated by the release of macrophage migration inhibitory factor (MIF). Finally, we show that the depletion of microglia in mice lacking receptor for colony-stimulating factor-1 (Csf-1R) reduces BPs into the cerebral cortex.

Laquinimod prevents inflammation-induced synaptic alterations occurring in experimental autoimmune encephalomyelitis

Background There are two generally accepted strategies for treating multiple sclerosis (MS), preventing central nervous system (CNS) damage indirectly through immunomodulatory interventions and/or repairing CNS damage by promoting remyelination. Both approaches also provide neuroprotection since they can prevent, indirectly or directly, axonal damage. Objective Recent experimental and clinical evidence indicates that the novel immunomodulatory drug laquinimod can exert a neuroprotective role in MS. Whether laquinimod-mediated neuroprotection is exerted directly on neuronal cells or indirectly via peripheral immunomodulation is still unclear. Methods C57Bl/6 experimental autoimmune encephalomyelitis (EAE) mice, immunised with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, were treated for 26 days with subcutaneous daily injections of laquinimod (from 1 to 25 mg/kg). Patch clamp electrophysiology was performed on acute brain striatal slices from EAE mice treated with daily (25 mg/kg) laquinimod and on acute brain striatal slices from control mice bathed with laquinimod (1–30 µM). Results Both preventive and therapeutic laquinimod treatment fully prevented the alterations of GABAergic synapses induced by EAE, the first limiting also glutamatergic synaptic alterations. This dual effect might, in turn, have limited glutamatergic excitotoxicity, a phenomenon previously observed early during EAE and possibly correlated with later axonal damage. Furthermore, laquinimod treatment also preserved cannabinoid CB1 receptor sensitivity, normally lost during EAE. Finally, laquinimod per se was able to regulate synaptic transmission by increasing inhibitory post-synaptic currents and, at the same time, reducing excitatory post-synaptic currents. Conclusions Our data suggest a novel neuroprotective mechanism by which laquinimod might in vivo protect from neuronal damage occurring as a consequence of inflammatory immune-mediated demyelination.

MiR-30e and miR-181d control Radial Glia cell proliferation via HtrA1 modulation

The precise mechanisms by which microRNAs (miRNAs) contribute to the dynamic regulation of gene expression during the forebrain development are still partly elusive. Here we show that the depletion of miRNAs in the cerebral cortex and hippocampus, via genetic inactivation of Dicer after the onset of forebrain neurogenesis, profoundly impairs the morphological and proliferative characteristics of neural stem and progenitor cells. The cytoarchitecture and self-renewal potential of radial glial (RG) cells located within the cerebral cortex and the hippocampus were profoundly altered, thus causing a significant derangement of both the post natal dorsal sub-ventricular zone and the dentate gyrus. This effect was attributed to the High-temperature requirement A serine peptidase 1 (HtrA1) gene product whose overexpression in the developing forebrain recapitulated some of the aspects of the Dicer−/− phenotype. MiR-30e and miR-181d were identified as posttranscriptional negative regulators of HtrA1 by binding to its 3′ untranslated region. In vivo overexpression of miR-30e and miR-181d in Dicer−/− forebrain rescued RG proliferation defects.

Cxcl10 enhances blood cells migration in the sub-ventricular zone of mice affected by experimental autoimmune encephalomyelitis.

The peri-ventricular area of the forebrain constitutes a preferential site of inflammation in multiple sclerosis, and the sub-ventricular zone (SvZ) is functionally altered in its animal model experimental autoimmune encephalomyelitis (EAE). The reasons for this preferential localization are still poorly understood. We show here that, in EAE mice, blood-derived macrophages, T and B cells and microglia (Mg) from the surrounding parenchyma preferentially accumulate within the SvZ, deranging its cytoarchitecture. We found that the chemokine Cxcl10 is constitutively expressed by a subset of cells within the SvZ, constituting a primary chemo-attractant signal for activated T cells. During EAE, T cells and macrophages infiltrating the SvZ in turn secrete pro-inflammatory cytokines such as TNFalpha and IFNgamma capable to induce Mg cells accumulation and SvZ derangement. Accordingly, lentiviral-mediated over-expression of IFNgamma or TNFalpha in the healthy SvZ mimics Mg/microglia recruitment occurring during EAE, while Cxcl10 over-expression in the SvZ is able to increase the frequency of peri-ventricular inflammatory lesions only in EAE mice. Finally, we show, by RT-PCR and in situ hybridization, that Cxcl10 is expressed also in the healthy human SvZ, suggesting a possible molecular parallelism between multiple sclerosis and EAE.

Wnt Signaling Has Opposing Roles in the Developing and the Adult Brain That Are Modulated by Hipk1

The canonical Wnt/Wingless pathway is implicated in regulating cell proliferation and cell differentiation of neural stem/progenitor cells. Depending on the context, β-Catenin, a key mediator of the Wnt signaling pathway, may regulate either cell proliferation or differentiation. Here, we show that β-Catenin signaling regulates the differentiation of neural stem/progenitor cells in the presence of the β-Catenin interactor Homeodomain interacting protein kinase-1 gene (Hipk1). On one hand, Hipk1 is expressed at low levels during the entire embryonic forebrain development, allowing β-Catenin to foster proliferation and to inhibit differentiation of neural stem/progenitor cells. On the other hand, Hipk1 expression dramatically increases in neural stem/progenitor cells, residing within the subventricular zone (SVZ), at the time when the canonical Wnt signaling induces cell differentiation. Analysis of mouse brains electroporated with Hipk1, and the active form of β-Catenin reveals that coexpression of both genes induces proliferating neural stem/progenitor cells to escape the cell cycle. Moreover, in SVZ derive neurospheres cultures, the overexpression of both genes increases the expression of the cell-cycle inhibitor P16Ink4. Therefore, our data confirm that the β-Catenin signaling plays a dual role in controlling cell proliferation/differentiation in the brain and indicate that Hipk1 is the crucial interactor able to revert the outcome of β-Catenin signaling in neural stem/progenitor cells of adult germinal niches.

Neural stem cell transplantation induces stroke recovery by upregulating glutamate transporter GLT-1 in astrocytes

Ischemic stroke is the leading cause of disability, but effective therapies are currently widely lacking. Recovery from stroke is very much dependent on the possibility to develop treatments able to both halt the neurodegenerative process as well as to foster adaptive tissue plasticity. Here we show that ischemic mice treated with neural precursor cell (NPC) transplantation had on neurophysiological analysis, early after treatment, reduced presynaptic release of glutamate within the ipsilesional corticospinal tract (CST), and an enhanced NMDA-mediated excitatory transmission in the contralesional CST. Concurrently, NPC-treated mice displayed a reduced CST degeneration, increased axonal rewiring, and augmented dendritic arborization, resulting in long-term functional amelioration persisting up to 60 d after ischemia. The enhanced functional and structural plasticity relied on the capacity of transplanted NPCs to localize in the peri-ischemic and ischemic area, to promote the upregulation of the glial glutamate transporter 1 (GLT-1) on astrocytes and to reduce peri-ischemic extracellular glutamate. The upregulation of GLT-1 induced by transplanted NPCs was found to rely on the secretion of VEGF by NPCs. Blocking VEGF during the first week after stroke reduced GLT-1 upregulation as well as long-term behavioral recovery in NPC-treated mice. Our results show that NPC transplantation, by modulating the excitatory–inhibitory balance and stroke microenvironment, is a promising therapy to ameliorate disability, to promote tissue recovery and plasticity processes after stroke. SIGNIFICANCE STATEMENT Tissue damage and loss of function occurring after stroke can be constrained by fostering plasticity processes of the brain. Over the past years, stem cell transplantation for repair of the CNS has received increasing interest, although underlying mechanism remain elusive. We here show that neural stem/precursor cell transplantation after ischemic stroke is able to foster axonal rewiring and dendritic plasticity and to induce long-term functional recovery. The observed therapeutic effect of neural precursor cells seems to underlie their capacity to upregulate the glial glutamate transporter on astrocytes through the vascular endothelial growth factor inducing favorable changes in the electrical and molecular stroke microenvironment. Cell-based approaches able to influence plasticity seem particularly suited to favor poststroke recovery.

Protein fingerprints of cultured CA3-CA1 hippocampal neurons: comparative analysis of the distribution of synaptosomal and cytosolic proteins

BackgroundAll studies aimed at understanding complex molecular changes occurring at synapses face the problem of how a complete view of the synaptic proteome and of its changes can be efficiently met. This is highly desirable when synaptic plasticity processes are analyzed since the structure and the biochemistry of neurons and synapses get completely reshaped. Because most molecular studies of synapses are nowadays mainly or at least in part based on protein extracts from neuronal cultures, this is not a feasible option: these simplified versions of the brain tissue on one hand provide an homogeneous pure population of neurons but on the other yield only tiny amounts of proteins, many orders of magnitude smaller than conventional brain tissue. As a way to overcome this limitation and to find a simple way to screen for protein changes at cultured synapses, we have produced and characterized two dimensional electrophoresis (2DE) maps of the synaptic proteome of CA3-CA1 hippocampal neurons in culture.ResultsTo obtain 2D maps, hippocampal cultures were mass produced and after synaptic maturation, proteins were extracted following subfractionation procedures and separated by 2D gel electrophoresis. Similar maps were obtained for the crude cytosol of cultured neurons and for synaptosomes purified from CA3-CA1 hippocampal tissue. To efficiently compare these different maps some clearly identifiable reference points were molecularly identified by mass spectrometry and immunolabeling methods. This information was used to run a differential analysis and establish homologies and dissimilarities in these 2D protein profiles.ConclusionBecause reproducible fingerprints of cultured synapses were clearly obtained, we believe that our mapping effort could represent a simple tool to screen for protein expression and/or protein localization changes in CA3-CA1 hippocampal neurons following plasticity.

Down-sizing of neuronal network activity and density of presynaptic terminals by pathological acidosis are efficiently prevented by Diminazene Aceturate.

Local acidosis is associated with neuro-inflammation and can have significant effects in several neurological disorders, including multiple sclerosis, brain ischemia, spinal cord injury and epilepsy. Despite local acidosis has been implicated in numerous pathological functions, very little is known about the modulatory effects of pathological acidosis on the activity of neuronal networks and on synaptic structural properties. Using non-invasive MRI spectroscopy we revealed protracted extracellular acidosis in the CNS of Experimental Autoimmune Encephalomyelitis (EAE) affected mice. By multi-unit recording in cortical neurons, we established that acidosis affects network activity, down-sizing firing and bursting behaviors as well as amplitudes. Furthermore, a protracted acidosis reduced the number of presynaptic terminals, while it did not affect the postsynaptic compartment. Application of the diarylamidine Diminazene Aceturate (DA) during acidosis significantly reverted both the loss of neuronal firing and bursting and the reduction of presynaptic terminals. Finally, in vivo DA delivery ameliorated the clinical disease course of EAE mice, reducing demyelination and axonal damage. DA is known to block acid-sensing ion channels (ASICs), which are proton-gated, voltage-insensitive, Na(+) permeable channels principally expressed by peripheral and central nervous system neurons. Our data suggest that ASICs activation during acidosis modulates network electrical activity and exacerbates neuro-degeneration in EAE mice. Therefore pharmacological modulation of ASICs in neuroinflammatory diseases could represent a new promising strategy for future therapies aimed at neuro-protection.