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The mGlu2/3 receptor agonists 4-carboxy-3-hydroxyphenylglycine (4C3HPG) and LY379268 attenuated NMDA toxicity in primary cultures containing both neurons and astrocytes. Neuroprotection was abrogated by PD98059 and LY294002, which inhibit the mitogen activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI-3-K) pathways, respectively. Cultured astrocytes lost the ability to produce transforming growth factor-b1 (TGF-b1) in response to mGlu2/3 receptor agonists when co-incubated with PD98059 or LY294002. As a result, the glial medium was no longer protective against NMDA toxicity. Activation of the MAPK and PI-3-K pathways in cultured astrocytes treated with 4C3HPG or LY379268 was directly demonstrated by an increase in the phosphorylated forms of ERK-1/2 and Akt. Similarly to that observed in the culture, intracerebral or systemic injections of mGlu2/3 receptor agonists enhanced TGF-b1 formation in the rat or mouse caudate nucleus, and this effect was reduced by PD98059. PD98059 also reduced the ability of LY379268 to protect striatal neurons against NMDA toxicity. These results suggest that activation of glial mGlu2/3 receptors induces neuroprotection through the activation of the MAPK and PI-3-K pathways leading to the induction of TGF-b. Keywords: astrocytes, MAP kinase, mGlu receptors, neuroprotection, phosphatidylinositol-3-kinase, TGF-b1.The low success of ionotropic glutamate receptor antagonists in clinical trials (reviewed by Lee et al. 1999) has switched the interest toward metabotropic glutamate (mGlu) receptors as targets for neuroprotective drugs. These receptors form a family of eight subtypes divided into three groups on the basis of sequence homology, pharmacological pro®le and transduction mechanisms. Group-I includes mGlu1 and -5 receptors, which are coupled to polyphosphoinositide hydrolysis and selectively activated by 3,5-dihydroxyphenylglycine (DHPG). Group-II includes mGlu2 and -3 receptors, which are coupled to G i proteins in heterologous expression systems. Compounds are now available that activate these two receptor subtypes with high potency and selectivity, such as (1)-2- Abbreviations used: l-AP4, l-2-amino-4-phosphonobutanoate; 4C3HPG, 4-carboxy-3-hydroxyphenylglycine; DHPG, 3,5-dihydroxyphenylglycine; DIV, days in vitro; GAD, glutamate decarboxylase; GCM, glial conditioned medium; LY354740, (1)-2-aminobicyclo[3,1,0]hexane-2,6-dicarboxylic acid; LY379268, (±)-2-oxa-4-aminobicyclo[3,1,0]hexane-4,6-dicarboxylic acid; LY389795, (±)-2-thia-4-aminobicyclo[3,1,0]hexane-4,6-dicarboxylic acid; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; mGlu, metabotropic glutamate; MAPK, mitogen activated protein kinase; MS, medium stock; PI-3-K, phosphatidylinositol-3-kinase; PPG, 4-phosphonophenylglycine; PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; TGF-b1, transforming growth factor-b1. R e t r a c t e d

Previous studies have identified the mammalian homologue of Bv8 (mBv8), a small protein originally isolated from skin secretions of the frog, Bombina variegata. In situ hybridization showed that mBv8 RNA was widely expressed in the rodent CNS, with high levels being detected in layer II of the cerebral cortex, limbic regions, cerebellar Purkinje cells, and dorsal and ventral horns of the spinal cord. A similar pattern of distribution was found by examining the presence of mBv8 protein by immunocytochemistry. Addition of frog Bv8 to cultured cerebellar granule cells reduced the extent of apoptotic death induced by switching the growing medium from 25 to 5 mM K+. Bv8 could also protect cultured cortical neurons against excitotoxic death. Both effects were prevented by PD98059 and LY294002, which inhibit the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI-3-K) pathways, respectively. In cultured cerebellar granule cells, Bv8 stimulated both the MAPK and the PI-3-K pathways, as revealed by Western blot analysis of phosphorylated p44/p42 MAPKs and phosphorylated Akt, respectively. We conclude that mBv8 acts as an endogenous neurotrophic factor and supports neuronal survival through the activation of the MAPK/PI-3-K pathways.

Kaposis's sarcoma associated herpesvirus (KSHV) has been reported to infect, among others, monocytes and dendritic cells DCs impairing their function. However, the underlying mechanisms remain not completely elucidated yet. Here we show that DC exposure to active or UV-inactivated KSHV resulted in STAT3 phosphorylation. This effect, partially dependent on KSHV-engagement of DC-SIGN, induced a high release of IL-10, IL-6 and IL-23, cytokines that in turn might maintain STAT3 in a phosphorylated state. STAT3 activation also correlated with a block of autophagy in DCs, as indicated by LC3II reduction and p62 accumulation. The IL-10, IL-6 and IL-23 release and the autophagic block could be overcome by inhibiting STAT3 activation, highlighting the role of STAT3 in mediating such effects. In conclusion, here we show that STAT3 activation can be one of the molecular mechanisms leading to KSHV-mediated DC dysfunction, that might allow viral persistence and the onset of KSHV-associated malignancies.

The presence and variant distribution of human herpesvirus 6 (HHV-6) was investigated by a nested polymerase chain reaction (PCR) in 118 biopsies from patients affected by nervous tissue tumor (115 primary tumors and 3 metastasis) and in 31 autopsy samples from the brain of healthy individuals. HHV-6 DNA sequences were detected in normal and neoplastic nervous tissue at a frequency of 32% and 37%, respectively. In both tissues, variant A was three times more frequent than the variant B. Peripheral blood lymphocytes (PBLs) derived from seven tumor affected patients contained the same variant as their respective brain sample, as judged by PCR. The expression of HHV-6 encoded immediate early protein p41 was detected by immunohistochemistry in neoplastic but not in normal brain. This may reflect viral reactivation from latency in immunocompromised patients. The seroepidemiological data indicated a frequency distribution of anti-HHV-6 antibodies in patients with brain tumors similar to that found in healthy donors.

G protein-coupled receptor homologous desensitization is intrinsically related to the function of a class of S/T kinases named G protein-coupled receptor kinases (GRK). The GRK family is composed of six cloned members, named GRK1 to 6. Studies from different laboratories have demonstrated that different calcium sensor proteins (CSP) can selectively regulate the activity of GRK subtypes. In the presence of calcium, rhodopsin kinase (GRK1) is inhibited by the photoreceptor-specific CSP recoverin through direct binding. Several other recoverin homologues (including NCS 1, VILIP 1 and hippocalcin) are also able to inhibit GRK1. The ubiquitous calcium-binding protein calmodulin (CaM) can inhibit GRK5 with a high affinity (IC(50)=40-50 nM). A direct interaction between GRK5 and Ca(2+)/CaM was documented and this binding does not influence the catalytic activity of the kinase, but rather reduced GRK5 binding to the membrane. These studies suggest that CSP act as functional analogues in mediating the regulation of different GRK subtypes by Ca(2+). This mechanism is, however, highly selective with respect to the GRK subtypes: while GRK1, but not GRK2 and GRK5, is regulated by recoverin and other NCS, GRK4, 5 and 6, that belong to the GRK4 subfamily, are potently inhibited by CaM, which had little or no effect on members of other GRK subfamilies.

Several reports have suggested an association of human herpesvirus 6 (HHV-6) with multiple sclerosis. Autoreactive T lymphocytes directed against myelin components seem to contribute to the pathogenesis of the disease. It has been suggested that molecular mimicry between viral and self-antigens might be one of the mechanisms that determine the onset of several autoimmune diseases. Following this hypothesis, the purpose of the present study was to evaluate if HHV-6 could play a role in activating T cells capable of cross-reaction with an important myelin component, the myelin basic protein. T cell lines were established from 22 multiple sclerosis patients and from 16 healthy controls, and their capability to react to both virus and myelin basic protein antigens was compared. The analysis of T cell cross-reactivity in patients and controls did not show significant differences in the HHV-6 ability to activate myelin basic protein-reactive T cells. Similarly, the evaluation of the humoral immune response to HHV-6 in patients and controls did not mirror any abnormality in the HHV-6 status in multiple sclerosis patients. Therefore, although the findings of activity in vitro of T cell lines with dual specificity are consistent with the hypothesis of molecular mimicry, the lack of differences in cross-reactivity between patients and controls do not support molecular mimicry as an important mechanism in the physiopathology of this disease.

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