Gennaro Schettini

Chemokines and their receptors in the central nervous system.

Chemokines are a family of proteins associated with the trafficking of leukocytes in physiological immune surveillance and inflammatory cell recruitment in host defence. They are classified into four classes based on the positions of key cystiene residues: C, CC, CXC, and CX3C. Chemokines act through both specific and shared receptors that all belong to the superfamily of G-protein-coupled receptors. Besides their well-established role in the immune system, several recent reports have demonstrated that these proteins also play a role in the central nervous system (CNS). In the CNS, chemokines are constitutively expressed by microglial cells, astrocytes, and neurons, and their expression can be increased after induction with inflammatory mediators. Constitutive expression of chemokines and chemokine receptors has been observed in both developing and adult brains, and the role played by these proteins in the normal brain is the object of intense study by many research groups. Chemokines are involved in brain development and in the maintenance of normal brain homeostasis; these proteins play a role in the migration, differentiation, and proliferation of glial and neuronal cells. The chemokine stromal cell-derived factor 1 and its receptor, CXCR4, are essential for life during development, and this ligand-receptor pair has been shown to have a fundamental role in neuron migration during cerebellar formation. Chemokine and chemokine receptor expression can be increased by inflammatory mediators, and this has in turn been associated with several acute and chronic inflammatory conditions. In the CNS, chemokines play an essential role in neuroinflammation as mediators of leukocyte infiltration. Their overexpression has been implicated in different neurological disorders, such as multiple sclerosis, trauma, stroke, Alzheimers disease, tumor progression, and acquired immunodeficiency syndrome-associated dementia. An emerging area of interest for chemokine action is represented by the communication between the neuroendocrine and the immune system. Chemokines have hormone-like actions, specifically regulating the key host physiopathological responses of fever and appetite. It is now evident that chemokines and their receptors represent a plurifunctional family of proteins whose actions on the CNS are not restricted to neuroinflammation. These molecules constitute crucial regulators of cellular communication in physiological and developmental processes.

Characterization of chemokines and their receptors in the central nervous system: physiopathological implications.

Chemokines represent key factors in the outburst of the immune response, by activating and directing the leukocyte traffic, both in lymphopoiesis and in immune surveillance. Neurobiologists took little interest in chemokines for many years, until their link to acquired immune deficiency syndrome‐associated dementia became established, and thus their importance in this field has been neglected. Nevertheless, the body of data on their expression and role in the CNS has grown in the past few years, along with a new vision of brain as an immunologically competent and active organ. A large number of chemokines and chemokine receptors are expressed in neurons, astrocytes, microglia and oligodendrocytes, either constitutively or induced by inflammatory mediators. They are involved in many neuropathological processes in which an inflammatory state persists, as well as in brain tumor progression and metastasis. Moreover, there is evidence for a crucial role of CNS chemokines under physiological conditions, similar to well known functions in the immune system, such as proliferation and developmental patterning, but also peculiar to the CNS, such as regulation of neural transmission, plasticity and survival.

Glial and neuronal cells express functional chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor 1

Abstract : Chemokines are a family of proteins that chemoattract and activate cells by interacting with specific receptors on the surface of their targets. The chemokine stromal cell‐derived factor 1, (SDF1), binds to the seventransmembrane G protein‐coupled CXCR4 receptor and acts to modulate cell migration, differentiation, and proliferation. CXCR4 and SDF1 are reported to be expressed in various tissues including brain. Here we show that SDF1 and CXCR4 are expressed in cultured cortical type I rat astrocytes, cortical neurons, and cerebellar granule cells. In cortical astrocytes, prolonged treatment with lipopolysaccharide induced an increase of SDF1 expression and a down‐regulation of CXCR4, whereas treatment with phorbol esters did not affect SDF1 expression and down‐modulated CXCR4 receptor expression. We also demonstrated the ability of human SDF1α (hSDF1α) to increase the intracellular calcium level in cultured astrocytes and cortical neurons, whereas in the same conditions, cerebellar granule cells did not modify their intracellular calcium concentration. Furthermore, in cortical astrocytes, the simultaneous treatment of hSDF1α with the HIV‐1 capside glycoprotein gp120 inhibits the cyclic AMP formation induced by forskolin treatment.

Stromal cell-derived factor-1α induces astrocyte proliferation through the activation of extracellular signal-regulated kinases 1/2 pathway

Stromal cell‐derived factor‐1 (SDF‐1), the ligand of the CXCR4 receptor, is a chemokine involved in chemotaxis and brain development that also acts as co‐receptor for HIV‐1 infection. We previously demonstrated that CXCR4 and SDF‐1α are expressed in cultured type‐I cortical rat astrocytes, cortical neurones and cerebellar granule cells. Here, we investigated the possible functions of CXCR4 expressed in rat type‐I cortical astrocytes and demonstrated that SDF‐1α stimulated the proliferation of these cells in vitro. The proliferative activity induced by SDF‐1α in astrocytes was reduced by PD98059, indicating the involvement of extracellular signal‐regulated kinases (ERK1/2) in the astrocyte proliferation induced by CXCR4 stimulation. This observation was further confirmed showing that SDF‐1α treatment selectively activated ERK1/2, but not p38 or stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK). Moreover, both astrocyte proliferation and ERK1/2 phosphorylation, induced by SDF‐1α, were inhibited by pertussis toxin (PTX) and wortmannin treatment indicating the involvement of a PTX sensitive G‐protein and of phosphatidyl inositol‐3 kinase in the signalling of SDF‐1α. In addition, Pyk2 activation represent an upstream components for the CXCR4 signalling to ERK1/2 in astrocytes. To our knowledge, this is the first report demonstrating a proliferative effect for SDF‐1α in primary cultures of rat type‐I astrocytes, and showing that the activation of ERK1/2 is responsible for this effect. These data suggest that CXCR4/SDF‐1 should play an important role in physiological and pathological glial proliferation, such as brain development, reactive gliosis and brain tumour formation.

Expression of CXC chemokine receptors 1-5 and their ligands in human glioma tissues : Role of CXCR4 and SDF1 in glioma cell proliferation and migration

Chemokines have been involved in cellular processes associated to malignant transformation such as proliferation, migration and angiogenesis. The expression of five CXC chemokine receptors and their main ligands was analysed by RT-PCR in 31 human astrocytic neoplasms. The mRNAs for all the receptors analysed were identified in a high percentage of tumours, while their ligands showed lower expression. CXCR4 and SDF1 were the most frequently mRNA identified (29/31 and 13/31 of the gliomas studied, respectively). Thus, we further analysed the cell localization of CXCR4 and SDF1 in immunohistochemistry experiments. We show a marked co-localization of CXCR4 and SDF1 in tumour cells, mainly evident in psudolpalisade and microcystic degeneration areas and in the vascular endothelium. In addition, hSDF1alpha induced a significant increase of DNA synthesis in primary human glioblastoma cell cultures and chemotaxis in a glioblastoma cell line. These results provide evidence of the expression of multiple CXC chemokines and their receptors in brain tumours and that in particular CXCR4 and SDF1 sustain proliferation and migration of glioma cells to promote malignant progression.

Signal Transduction through Tyrosine-phosphorylated C-terminal Fragments of Amyloid Precursor Protein via an Enhanced Interaction with Shc/Grb2 Adaptor Proteins in Reactive Astrocytes of Alzheimer's Disease Brain

The proteolytic processing of amyloid precursor protein (APP) through the formation of membrane-bound C-terminal fragments (CTFs) and of soluble β-amyloid peptides likely influences the development of Alzheimers disease (AD). We show that in human brain a subset of CTFs are tyrosine-phosphorylated and form stable complexes with the adaptor protein ShcA. Grb2 is also part of these complexes, which are present in higher amounts in AD than in control brains. ShcA immunoreactivity is also greatly enhanced in patients with AD and occurs at reactive astrocytes surrounding cerebral vessels and amyloid plaques. A higher amount of phospho-ERK1,2, likely as result of the ShcA activation, is present in AD brains. In vitroexperiments show that the ShcA-CTFs interaction is strictly confined to glial cells when treated with thrombin, which is a well known ShcA and ERK1,2 activator and a regulator of APP cleavage. In untreated cells ShcA does not interact with either APP or CTFs, although they are normally generated. Altogether these data suggest that CTFs are implicated in cell signaling via Shc transduction machinery, likely influencing MAPK activity and glial reaction in AD patients.

Amyloid precursor protein and presenilin1 interact with the adaptor GRB2 and modulate ERK1,2 signaling

The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-β peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-β peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.

Chemokines and their receptors in the CNS: expression of CXCL12/SDF-1 and CXCR4 and their role in astrocyte proliferation.

The study of chemokine role in the CNS indubitably represents an important step to understanding many aspects of brain pathology, physiology and development. Here we discuss our recent research on the expression of chemokines and chemokine receptors in brain tissues and in cultured CNS cells, with particular regard to the CXCL12/SDF-1-CXCR4 system. We showed their expression in both glial and neuronal cells in basal conditions and their modulation upon stimulation. We demonstrated that CXCL12/SDF-1 in vitro act as a growth factor for astrocytes by stimulating their proliferation, a phenomenon that could represent the basis of pathological conditions such as gliosis and malignant transformation. We investigated the signal transduction pathways, identifying in the sequential activation of G-protein-PI-3Kinase-ERK1/2 the main signaling cascade linked to the CXCL12/SDF-1-induced proliferation in astrocytes.

Prion protein fragment 106-126 induces apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in the GH3 cell line.

The prion diseases are transmissible neurodegenerative pathologies characterized by the accumulation of altered forms of the prion protein (PrP), termed PrPSc, in the brain. Previous studies have shown that a synthetic peptide homologous to residues 106–126 of PrP (PrP 106–126) maintains many characteristics of PrPSc, i.e., the ability to form amyloid fibrils and to induce apoptosis in neurons. We have investigated the intracellular mechanisms involved in the cellular degeneration induced by PrP 106–126, using the GH3 cells as a model of excitable cells. When assayed in serum‐deprived conditions (48 hr), PrP 106–126 (50 μM) induced cell death time‐dependently, and this process showed the characteristics of the apoptosis. This effect was specific because a peptide with a scrambled sequence of PrP 106–126 was not effective. Then we performed microfluorimetric analysis of single cells to monitor intracellular calcium concentrations and showed that PrP 106–126 caused a complete blockade of the increase in the cytosolic calcium levels induced by K+ (40 mM) depolarization. Conversely, the scrambled peptide was ineffective. The L‐type voltage‐sensitive calcium channel blocker nicardipine (1 μM) also induced apoptosis in GH3 cells, suggesting that the blockade of Ca2+ entry through this class of calcium channels may cause GH3 apoptotic cell death. We thus analyzed, by means of electrophysiological studies, whether Prp 106–126 modulate L‐type calcium channels activity and demonstrated that the apoptotic effect of PrP 106–126 was due to a dose‐dependent inactivation of the L‐type calcium channels. These data demonstrate that the prion protein fragment 106–126 induces a GH3 apoptotic cell death inducing a selective inhibition of the activity of the L‐type voltage‐sensitive calcium channels. J. Neurosci. Res. 54:341–352, 1998.

Expression of the chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1 in human brain tumors and their involvement in glial proliferation in vitro

Abstract: Chemokines are a family of proteins that chemoattract and activate cells by interacting with specific receptors on the surface of their targets. They are grouped into four classes based on the position of key cysteine residues: C, CC, CXC, and CX3C. Stromal cell‐derived factor 1 (SDF1), the ligand of the CXCR4 receptor, is a CXC chemokine involved in chemotaxis and brain development that also acts as coreceptor for HIV‐1 infection. It has been proposed that CXCR4 is overexpressed and required for proliferation in human brain tumor cells. We previously demonstrated that CXCR4 and SDF1 are expressed in culture of cortical type I rat astrocytes, cortical neurons, and cerebellar granule cells. In this study, we analyzed the expression of CXCR4 and SDF1 in four human brain tumor tissues, showing that CXCR4 is expressed in all tumors analyzed, whereas SDF1 is expressed only in two tumor tissues. We also investigated the possible functions of CXCR4 expressed in rat type I cortical astrocytes, demonstrating that SDF1α stimulates the proliferation of these cells in vitro. Moreover, we studied by western blot the intracellular pathway involved in cell proliferation, demonstrating that SDF1α induces the ERK1/2 phosphorylation that is reduced by the PD98059 compound, an MEK inhibitor.