Sebastián Pons

Localization of insulin-like growth factor I (IGF-I)-like immunoreactivity in the developing and adult rat brain

The cellular distribution of insulin-like growth factor I (IGF-I) immunoreactivity was examined in the rat brain from embryonic day 15 to maturity. IGF-I immunoreactivity was found in the perikarya of neurons distributed along the entire extension of the neuronal tube in all the embryonic ages studied (E15, E17, E19 and E21). In E21 animals, the majority of immunoreactive neurons was located in the olfactory bulb, cerebral cortex, hippocampus, striatum, diencephalon, mesencephalic colliculi, trigeminal nuclei, trigeminal ganglion and in motoneurons of the brainstem. In 10- and 20-day-old rats, in addition to the above areas, IGF-I immunoreactivity was also observed in capillary walls, ependymal cells, choroid plexus, glial cells and most fiber paths. In postnatal ages, immunoreactivity in neuronal somas was mainly restricted to the cell nuclei. However, IGF-I immunoreactivity in the neuronal cytoplasm was observed in 20-day-old rats treated with colchicine while fiber paths and neuronal cell nuclei were negative in these animals. In the telencephalon of 20-day-old rats injected with colchicine, the most intense immunoreactive neurons were observed in the olfactory bulb, cerebral cortex, tenia tecta, hippocampus, islands of Calleja, septal nuclei, striatum, endopyriform nucleus and amygdala. Most diencephalic nuclei, the substantia nigra, the mesencephalic colliculi, Purkinje cells in the cerebellar cortex and several nuclei in mesencephalon, pons and medulla oblongata were also immunoreactive. In adult rats injected with colchicine, IGF-I immunoreactivity was located in the same areas as in 20-day-old rats. The number of immunoreactive cells and the intensity of the staining was reduced in adult rats as compared to that found in young postnatal animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Bmp2 antagonizes sonic hedgehog-mediated proliferation of cerebellar granule neurones through Smad5 signalling.

During development of the cerebellum, sonic hedgehog (Shh) is directly responsible for the proliferation of granule cell precursors in the external germinal layer. We have looked for signals able to regulate a switch from the Shh-mediated proliferative response to one that directs differentiation of granule neurones. Bone morphogenetic proteins (BMPs) are expressed in distinct neuronal populations within the developing cerebellar cortex. Bmp2 and Bmp4 are expressed in the proliferating precursors and subsequently in differentiated granule neurones of the internal granular layer, whereas Bmp7 is expressed by Purkinje neurones. In primary cultures, Bmp2 and Bmp4, but not Bmp7, are able to prevent Shh-induced proliferation, thereby allowing granule neuron differentiation. Furthermore, Bmp2 treatment downregulates components of the Shh pathway in proliferating granule cell precursors. Smad proteins, the only known BMP receptor substrates capable of transducing the signal, are also differentially expressed in the developing cerebellum: Smad1 in the external germinal layer and Smad5 in newly differentiated granule neurones. Among them, only Smad5 is phosphorylated in vivo and in primary cultures treated with Bmp2, and overexpression of Smad5 is sufficient to induce granule cell differentiation in the presence of Shh. We propose a model in which Bmp2-mediated Smad5 signalling suppresses the proliferative response to Shh by downregulation of the pathway, and allows granule cell precursor to enter their differentiation programme.

β1-Integrins Are Critical for Cerebellar Granule Cell Precursor Proliferation

We have previously shown that mice with a CNS restricted knock-out of the integrin β1 subunit gene (Itgb1-CNSko mice) have defects in the formation of lamina and folia in the cerebral and cerebellar cortices that are caused by disruption of the cortical marginal zones. Cortical structures in postnatal and adult Itgb1-CNSko animals are also reduced in size, but the mechanism that causes the size defect has remained unclear. We now demonstrate that proliferation of granule cell precursors (GCPs) is severely affected in the developing cerebellum of Itgb1-CNSko mice. In the absence of β1 expression, GCPs lose contact with laminin in the meningeal basement membrane, cease proliferating, and differentiate prematurely. In vitro studies provide evidence thatβ1 integrins act at least in part cell autonomously in GCPs to regulate their proliferation. Previous studies have shown that sonic hedgehog (Shh)-induced GCP proliferation is potentiated by the integrin ligand laminin. We show that Shh directly binds to laminin and that laminin–Shh induced cell proliferation is dependent on β1 integrin expression in GCPs. Taken together, these data are consistent with a model in which β1 integrin expression in GCPs is required to recruit a laminin–Shh complex to the surface of GCPs and to subsequently modulate the activity of signaling pathways that regulate proliferation.

Probucol Increases Glutathione Peroxidase-1 Activity and Displays Long-Lasting Protection against Methylmercury Toxicity in Cerebellar Granule Cells

Methylmercury (MeHg) is an environmental neurotoxicant whose molecular mechanisms underlying toxicity remain elusive. Here, we investigated molecular events involved in MeHg-induced neurotoxicity in cultured cerebellar granule cells (CGCs) as well as potential protective strategies for such toxicity. Glutathione peroxidase, isozyme 1 (GPx-1) activity was significantly (p = 0.0017) decreased at 24 h before MeHg-induced neuronal death (day in vitro 4). This event was related to enhanced susceptibilities to hydrogen peroxide or tert-butyl peroxide and increased lipid peroxidation. However, intracellular calcium levels, glutamate uptake, and glutathione levels, as well as glutathione reductase and catalase activities, were not changed by MeHg exposure at this time point. Probucol (PB), a lipid-lowering drug, displayed a long-lasting protective effect against MeHg-induced neurotoxicity. The beneficial effects of PB were correlated with increased GPx-1 activity and decreased lipid peroxidation. The protection afforded by PB was significantly higher when compared to the antioxidants, ascorbic acid and trolox. In vitro studies with the purified GPx-1 proved that MeHg inhibits and PB activates the enzyme activity. Overexpression of GPx-1 prevented MeHg-induced neuronal death. These data indicate that (1) GPx-1 is an important molecular target involved in MeHg-induced neurotoxicity and (2) PB, which increases GPx-1 activity in CGCs, induces enduring protection against such toxicity. The results bring out new insights on the potential therapeutic strategies for poisonings to MeHg and other pathological conditions related to increased production and/or decreased detoxification of peroxides.

Blockade of cannabinoid CB1 receptor function protects against in vivo disseminating brain damage following NMDA-induced excitotoxicity

The ability of cannabinoid CB1 receptors to influence glutamatergic excitatory neurotransmission has fueled interest in how these receptors and their endogenous ligands may interact in conditions of excitotoxic insults. The present study characterized the impact of stimulated and inhibited CB1 receptor function on NMDA‐induced excitotoxicity. Neonatal (6‐day‐old) rat pups received a systemic injection of a mixed CB1/CB2 receptor agonist (WIN55,212–2) or their respective antagonists (SR141716A for CB1 and SR144528 for CB2) prior to an unilateral intrastriatal microinjection of NMDA. The NMDA‐induced excitotoxic damage in the ipsilateral forebrain was not influenced by agonist‐stimulated CB1 receptor function. In contrast, blockade of CB1, but not CB2, receptor activity evoked a robust neuroprotective response by reducing the infarct area and the number of cortical degenerating neurons. These results suggest a critical involvement of CB1 receptor tonus on neuronal survival following NMDA receptor‐induced excitotoxicity in vivo.

In vivo modulation of 5-hydroxytryptamine release in mouse prefrontal cortex by local 5-HT2A receptors: effect of antipsychotic drugs

In the rat, postsynaptic 5‐hydroxytryptamine2A receptors medial prefrontal cortex control the activity of the serotonergic system through changes in the activity of pyramidal neurons projecting to the dorsal raphe nucleus. Here we extend these observations to mouse brain. The prefrontal cortex expresses abundant 5‐ hydroxytryptamine2A receptors, as assessed by immunohistochemistry, Western blots and in situ hybridization procedures. The application of the 5‐hydroxytryptamine2A/2C agonist DOI (100 µm) by reverse dialysis in the medial prefrontal cortex doubled the local release of 5‐hydroxytryptamine. This effect was reversed by coperfusion of tetrodotoxin, and by the selective 5‐hydroxytryptamine2A receptor antagonist M100907, but not by the 5‐hydroxytryptamine2C antagonist SB‐242084. The effect of DOI was also reversed by prazosin (α1‐adrenoceptor antagonist), BAY × 3702 (5‐hydroxytryptamine1A receptor agonist), NBQX (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐4‐propionate/kainic acid antagonist) and 1S,3S‐ACPD (mGluR II/III agonist), but not by dizocilpine (N‐methyl‐d‐aspartate antagonist). α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐4‐propionate mimicked the 5‐hydroxytryptamine elevation produced by DOI, an effect also reversed by BAY × 3702. Likewise, the coperfusion of classical (chlorpromazine, haloperidol) and atypical antipsychotic drugs (clozapine, olanzapine) fully reversed the 5‐hydroxytryptamine elevation induced by DOI. These observations suggest that DOI increases 5‐hydroxytryptamine release in the mouse medial prefrontal cortex through the activation of local 5‐hydroxytryptamine2A receptors by an impulse‐dependent mechanism that involves/requires the activation of local α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐4‐propionate receptors. This effect is reversed by ligands of receptors present in the medial prefrontal cortex, possibly in pyramidal neurons, which are involved in the action of antipsychotic drugs. In particular, the reversal by classical antipsychotics may involve blockade of α1‐adrenoceptors, whereas that of atypical antipsychotics may involve 5‐hydroxytryptamine2A receptors and α1‐adrenoceptors.

Sonic-hedgehog-mediated proliferation requires the localization of PKA to the cilium base.

Cerebellar granular neuronal precursors (CGNPs) proliferate in response to the mitogenic activity of Sonic hedgehog (Shh), and this proliferation is negatively regulated by activation of cAMP-dependent protein kinase (PKA). In the basal state, the PKA catalytic subunits (C-PKA) are inactive because of their association with the regulatory subunits (R-PKA). As the level of cAMP increases, it binds to R-PKA, displacing and thereby activating the C-PKA. Here we report that, in the presence of Shh, inactive C-PKA accumulates at the cilium base of proliferative CGNPs whereas removal of Shh triggers the activation of PKA at this particular location. Furthermore, we demonstrate that the anchoring of the PKA holoenzyme to the cilium base is mediated by the specific binding of the type II PKA regulatory subunit (RII-PKA) to the A-kinase anchoring proteins (AKAPs). Disruption of the interaction between RII-PKA and AKAPs inhibits Shh activity and, therefore, blocks proliferation of CGNP cultures. Collectively, these results demonstrate that the pool of PKA localized to the cilium base of CGNP plays an essential role in the integration of Shh signal transduction.

Bone Morphogenetic Protein 2 Opposes Shh-mediated Proliferation in Cerebellar Granule Cells through a TIEG-1-based Regulation of Nmyc

Nmyc is a potent regulator of cell cycle in cerebellar granular neuron precursors (CGNPs) and has been proposed to be the main effector of Shh (Sonic hedgehog) proliferative activity. Nmyc ectopic expression is sufficient to promote cell autonomous proliferation and can lead to tumorigenesis. Bone morphogenetic protein 2 (BMP2) antagonizes Shh proliferative effect by promoting cell cycle exit and differentiation in CGNPs. Here we report that BMP2 opposes Shh mitogenic activity by blocking Nmyc expression. We have identified TIEG-1 (KLF10) as the intermediary factor that blocks Nmyc expression through the occupancy of the Sp1 sites present in its promoter. We also demonstrate that TIEG-1 ectopic expression in CGNPs induces cell cycle arrest that can lead to apoptosis but fails to promote differentiation. Moreover, TIEG-1 synergizes with BMP2 activity to terminally differentiate CGNPs and independent differentiator signals such as dibutyryl cAMP and prevents apoptosis in TIEG-1 arrested cells. All together, these data strongly suggest that the BMP2 pathway triggers cell cycle exit and differentiation as two separated but coordinated processes, where TIEG-1 acts as a mediator of the cell cycle arrest.

IRS-2 Deficiency Impairs NMDA Receptor-Dependent Long-term Potentiation

The beneficial effects of insulin and insulin-like growth factor I on cognition have been documented in humans and animal models. Conversely, obesity, hyperinsulinemia, and diabetes increase the risk for neurodegenerative disorders including Alzheimers disease (AD). However, the mechanisms by which insulin regulates synaptic plasticity are not well understood. Here, we report that complete disruption of insulin receptor substrate 2 (Irs2) in mice impairs long-term potentiation (LTP) of synaptic transmission in the hippocampus. Basal synaptic transmission and paired-pulse facilitation were similar between the 2 groups of mice. Induction of LTP by high-frequency conditioning tetanus did not activate postsynaptic N-methyl-D-aspartate (NMDA) receptors in hippocampus slices from Irs2−/− mice, although the expression of NR2A, NR2B, and PSD95 was equivalent to wild-type controls. Activation of Fyn, AKT, and MAPK in response to tetanus stimulation was defective in Irs2−/− mice. Interestingly, IRS2 was phosphorylated during induction of LTP in control mice, revealing a potential new component of the signaling machinery which modulates synaptic plasticity. Given that IRS2 expression is diminished in Type 2 diabetics as well as in AD patients, these data may reveal an explanation for the prevalence of cognitive decline in humans with metabolic disorders by providing a mechanistic link between insulin resistance and impaired synaptic transmission.

Climbing fiber deafferentation reduces insulin-like growth factor I (IGF-I) content in cerebellum

The presence of insulin-like growth factor I (IGF-I) and its mRNA in adult rat cerebellum has recently been documented. Previous immunocytochemical studies showed prominent IGF-I-like staining in fibers around Purkinje cell somas. To determine the origin of this IGF-I input to the Purkinje cell we destroyed the inferior olivary complex by either 3-acetylpiridine administration or electrolytical lesions. In both types of lesions we found a similar significant depletion of IGF-I levels in cerebellum (40-50% of controls). No changes were found in cerebellar IGF-I receptors. These results suggest that almost half of the IGF-I content in cerebellum is provided by climbing fiber afferents arising from the inferior olivary complex.