Roberto Gradini

Prenatal restraint stress generates two distinct behavioral and neurochemical profiles in male and female rats

Prenatal Restraint Stress (PRS) in rats is a validated model of early stress resulting in permanent behavioral and neurobiological outcomes. Although sexual dimorphism in the effects of PRS has been hypothesized for more than 30 years, few studies in this long period have directly addressed the issue. Our group has uncovered a pronounced gender difference in the effects of PRS (stress delivered to the mothers 3 times per day during the last 10 days of pregnancy) on anxiety, spatial learning, and a series of neurobiological parameters classically associated with hippocampus-dependent behaviors. Adult male rats subjected to PRS (“PRS rats”) showed increased anxiety-like behavior in the elevated plus maze (EPM), a reduction in the survival of newborn cells in the dentate gyrus, a reduction in the activity of mGlu1/5 metabotropic glutamate receptors in the ventral hippocampus, and an increase in the levels of brain-derived neurotrophic factor (BDNF) and pro-BDNF in the hippocampus. In contrast, female PRS rats displayed reduced anxiety in the EPM, improved learning in the Morris water maze, an increase in the activity of mGlu1/5 receptors in the ventral and dorsal hippocampus, and no changes in hippocampal neurogenesis or BDNF levels. The direction of the changes in neurogenesis, BDNF levels and mGlu receptor function in PRS animals was not consistent with the behavioral changes, suggesting that PRS perturbs the interdependency of these particular parameters and their relation to hippocampus-dependent behavior. Our data suggest that the epigenetic changes in hippocampal neuroplasticity induced by early environmental challenges are critically sex-dependent and that the behavioral outcome may diverge in males and females.

Expression of Activated Notch3 in Transgenic Mice Enhances Generation of T Regulatory Cells and Protects against Experimental Autoimmune Diabetes

Thymic-derived dysregulated tolerance has been suggested to occur in type 1 diabetes via impaired generation of CD4+CD25+ T regulatory cells, leading to autoimmune β cell destruction. In this study, we demonstrate that Notch3 expression is a characteristic feature of CD4+CD25+ cells. Furthermore, streptozotocin-induced autoimmune diabetes fails to develop in transgenic mice carrying the constitutively active intracellular domain of Notch3 in thymocytes and T cells. The failure to develop the disease is associated with an increase of CD4+CD25+ T regulatory cells, accumulating in lymphoid organs, in pancreas infiltrates and paralleled by increased expression of IL-4 and IL-10. Accordingly, CD4+ T cells from Notch3-transgenic mice inhibit the development of hyperglycemia and insulitis when injected into streptozotocin-treated wild-type mice and display in vitro suppressive activity. These observations, therefore, suggest that Notch3-mediated events regulate the expansion and function of T regulatory cells, leading to protection from experimental autoimmune diabetes and identify the Notch pathway as a potential target for therapeutic intervention in type 1 diabetes.

Native group-III metabotropic glutamate receptors are coupled to the mitogen-activated protein kinase/phosphatidylinositol-3-kinase pathways.

We used cultured cerebellar granule cells to examine whether native group‐III metabotropic glutamate (mGlu) receptors are coupled to the mitogen‐activated protein kinase (MAPK) and phosphatidylinositol‐3‐kinase (PI‐3‐K) pathways. Cultured granule cells responded to the group‐III mGlu receptor agonist, L‐2‐amino‐4‐phosphonobutanoate (l‐AP4), with an increased phosphorylation and activity of MAPKs (ERK‐1 and ‐2) and an increased phosphorylation of the PI‐3‐K target, protein kinase B (PKB/AKT). These effects were attenuated by the group‐III antagonists, α‐methyl‐serine‐O‐phosphate (MSOP) and (R,S)‐α‐cyclopropyl‐4‐phosphonophenylglycine (CPPG), or by pretreatment of the cultures with pertussis toxin. l‐AP4 also induced the nuclear translocation of β‐catenin, a downstream effector of the PI‐3‐K pathway. To assess the functional relevance of these mechanisms we examined the ability of l‐AP4 to protect granule cells against apoptosis by trophic deprivation, induced by lowering extracellular K+ from 25 to 10 mm. Neuroprotection by l‐AP4 was attenuated by MSOP and abrogated by the compounds PD98059 and UO126, which inhibit the MAPK pathway, or by the compound LY294002, which inhibits the PI‐3‐K pathway. Taken together, these results show for the first time that native group‐III mGlu receptors are coupled to MAPK and PI‐3‐K, and that activation of both pathways is necessary for neuroprotection mediated by this particular class of receptors.

Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid.

Cultured mouse D3 embryonic stem (ES) cells differentiating into embryoid bodies (EBs) expressed several Wnt isoforms, nearly all isotypes of the Wnt receptor Frizzled and the Wnt/Dickkopf (Dkk) co‐receptor low‐density lipoprotein receptor‐related protein (LRP) type 5. A 4‐day treatment with retinoic acid (RA), which promoted neural differentiation of EBs, substantially increased the expression of the Wnt antagonist Dkk‐1, and induced the synthesis of the Wnt/Dkk‐1 co‐receptor LRP6. Recombinant Dkk‐1 applied to EBs behaved like RA in inducing the expression of the neural markers nestin and distal‐less homeobox gene (Dlx‐2). Recombinant Dkk‐1 was able to inhibit the Wnt pathway, as shown by a reduction in nuclear β‐catenin levels. Remarkably, the antisense‐ or small interfering RNA‐induced knockdown of Dkk‐1 largely reduced the expression of Dlx‐2, and the neuronal marker β‐III tubulin in EBs exposed to RA. These data suggest that induction of Dkk‐1 and the ensuing inhibition of the canonical Wnt pathway is required for neural differentiation of ES cells.

Induction of the wnt inhibitor, dickkopf-1, is associated with neurodegeneration related to temporal lobe epilepsy.

Summary:  Inhibition of the Wnt pathway by the secreted glycoprotein, Dickkopf‐1 (Dkk‐1) has been related to processes of excitotoxic and ischemic neuronal death. We now report that Dkk‐1 is induced in neurons of the rat olfactory cortex and hippocampus degenerating in response to seizures produced by systemic injection of kainate (12 mg/kg, i.p.). There was a tight correlation between Dkk‐1 expression and neuronal death in both regions, as shown by the different expression profiles in animals classified as “high” and “low” responders to kainate. For example, no induction of Dkk‐1 was detected in the hippocampus of low responder rats, in which seizures did not cause neuronal loss. Induction of Dkk‐1 always anticipated neuronal death and was associated with a reduction in nuclear levels of β‐catenin, which reflects an ongoing inhibition of the canonical Wnt pathway. Intracerebroventricular injections of Dkk‐1 antisense oligonucleotides (12 nmol/2 μL) substantially reduced kainate‐induced neuronal damage, as did a pretreatment with lithium ions (1 mEq/kg, i.p.), which rescue the Wnt pathway by acting downstream of the Dkk‐1 blockade. Taken collectively, these data suggest that an early inhibition of the Wnt pathway by Dkk‐1 contributes to neuronal damage associated with temporal lobe epilepsy. We also examined Dkk‐1 expression in the hippocampus of epileptic patients and their controls. A strong Dkk‐1 immunolabeling was found in six bioptic samples and in one autoptic sample from patients with mesial temporal lobe epilepsy associated with hippocampal sclerosis. Dkk‐1 expression was undetectable or very low in autoptic samples from nonepileptic patients or in bioptic samples from patients with complex partial seizures without neuronal loss and/or reactive gliosis in the hippocampus. Our data raise the attractive possibility that drugs able to rescue the canonical Wnt pathway, such as Dkk‐1 antagonists or inhibitors of glycogen synthase kinase‐3β, reduce the development of hippocampal sclerosis in patients with temporal lobe epilepsy.

Subcellular localization of the BRCA1 gene product in mitotic cells

The product of the hereditary breast cancer susceptibility gene BRCA1 is a multifunctional protein involved in the maintenance of genomic integrity, in transcriptional coactivation, and in the control of cell growth. BRCA1‐deficient cells manifest chromosomal instability. During mitosis, BRCA1 is known to interact with γ‐tubulin in the centrosomes, key elements of the mitotic spindle. Using confocal microscopy and immunogold electron microscopy, we investigated the distribution of endogenous BRCA1 relative to mitotic spindle markers in breast cancer cells. By confocal analysis, BRCA1 and β‐tubulin colocalized to microtubules of the mitotic spindle and to the centrosomes. Immunogold electron microscopy confirmed these results and further revealed that BRCA1 and α‐tubulin codistributed to the walls of the centrioles and to pericentriolar fibers at centrosomes. During chromatid segregation, codistribution was also detected along individual spindle microtubules and at sites of insertion of microtubules on chromosomes. At cytokinesis, BRCA1 and α‐tubulin codistributed to the midbody. Coimmunoprecipitation supported the association of full‐length BRCA1 with α‐ and β‐tubulin. These results are consistent with an involvement of BRCA1 in the dynamics of the mitotic spindle and in the segregation of duplicated chromosomes.

GD3 glycosphingolipid contributes to Fas-mediated apoptosis via association with ezrin cytoskeletal protein.

Efficiency of Fas‐mediated apoptosis of lymphoid cells is regulated, among other means, by a mechanism involving its association with ezrin, a cytoskeletal protein belonging to the 4.1 family of proteins. In the present work, we provide evidence for a further molecule that associates to ezrin in Fas‐triggered apoptosis, the disialoganglioside GD3. In fact, as an early event, GD3 redistributed in membrane‐associated domains in uropods and co‐localized with ezrin. Co‐immunoprecipitation analyses confirmed this result, indicating a GD3–ezrin association. Altogether, these results are suggestive for a role of GD3 in Fas/ezrin‐mediated apoptosis, supporting the view that uropods contain a multimolecular signaling complex involved in Fas‐mediated apoptosis.

Surface Expression of the IFN-γR2 Chain Is Regulated by Intracellular Trafficking in Human T Lymphocytes

The surface and cytoplasmic expressions of the transducing chain (IFN-γR2) of the heterodimeric IFN-γ receptor on human T lymphocytes have been investigated. We show that its surface expression is low, whereas high cytoplasmic levels are found in both resting and PHA-activated T lymphocytes. This low expression does not prevent activated T cells from responding to IFN-γ, because it induces IFN-regulatory factor 1 expression. Low surface IFN-γR2 expression appears to be due to recycling between cytoplasmic stores and the cell surface, which does not depend on signals mediated by endogenous IFN-γ, because IFN-γR2 surface expression is low, and its internalization is equally observed in patients with inherited IFN-γR1 gene deficiency and in healthy donors. Moreover, IFN-γR2 internalization in T lymphoblasts from healthy donors was not affected by the presence of anti-IFN-γ-neutralizing or anti-IFN-γR1-blocking mAb. In conclusion, these data illustrate a new mechanism whereby human T cells limit the surface expression of IFN-γR2 in a ligand-independent manner.

Induction of the Wnt antagonist Dickkopf-1 is involved in stress-induced hippocampal damage.

The identification of mechanisms that mediate stress-induced hippocampal damage may shed new light into the pathophysiology of depressive disorders and provide new targets for therapeutic intervention. We focused on the secreted glycoprotein Dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt pathway, involved in neurodegeneration. Mice exposed to mild restraint stress showed increased hippocampal levels of Dkk-1 and reduced expression of β-catenin, an intracellular protein positively regulated by the canonical Wnt signalling pathway. In adrenalectomized mice, Dkk-1 was induced by corticosterone injection, but not by exposure to stress. Corticosterone also induced Dkk-1 in mouse organotypic hippocampal cultures and primary cultures of hippocampal neurons and, at least in the latter model, the action of corticosterone was reversed by the type-2 glucocorticoid receptor antagonist mifepristone. To examine whether induction of Dkk-1 was causally related to stress-induced hippocampal damage, we used doubleridge mice, which are characterized by a defective induction of Dkk-1. As compared to control mice, doubleridge mice showed a paradoxical increase in basal hippocampal Dkk-1 levels, but no Dkk-1 induction in response to stress. In contrast, stress reduced Dkk-1 levels in doubleridge mice. In control mice, chronic stress induced a reduction in hippocampal volume associated with neuronal loss and dendritic atrophy in the CA1 region, and a reduced neurogenesis in the dentate gyrus. Doubleridge mice were resistant to the detrimental effect of chronic stress and, instead, responded to stress with increases in dendritic arborisation and neurogenesis. Thus, the outcome of chronic stress was tightly related to changes in Dkk-1 expression in the hippocampus. These data indicate that induction of Dkk-1 is causally related to stress-induced hippocampal damage and provide the first evidence that Dkk-1 expression is regulated by corticosteroids in the central nervous system. Drugs that rescue the canonical Wnt pathway may attenuate hippocampal damage in major depression and other stress-related disorders.

Evidence for cell surface association between CXCR4 and ganglioside GM3 after gp120 binding in SupT1 lymphoblastoid cells

CXCR4 (fusin) is a chemokine receptor which is involved as a coreceptor in gp120 binding to the cell surface. In this study we provide evidence that binding of gp120 triggers CXCR4 recruitment to glycosphingolipid‐enriched microdomains. Scanning confocal microscopy showed a nearly complete localization of CXCR4 within GM3‐enriched plasma membrane domains of SupT1 cells and coimmunoprecipitation experiments revealed that CXCR4 was immunoprecipitated by IgG anti‐GM3 after gp120 pretreatment. These findings reveal that gp120 binding induces a strict association between CXCR4 and ganglioside GM3, supporting the view that GM3 and CXCR4 are components of a functional multimolecular complex critical for HIV‐1 entry.