Non-canonical cyclic AMP SMAD1/5/8 signalling in human granulosa cells.

Development of mammalian ovarian follicles is promoted by the combined action of endocrine cues and paracrine factors. Follicle stimulating hormone (FSH), through the action of cAMP drives follicular growth and development. The oocyte secretes powerful growth factors such as bone morphogenetic protein 15 (BMP15) to regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation through the activation of SMAD1/5/8. This study investigated the role of the cAMP signalling pathway on SMAD1/5/8 action in human granulosa cells. Cyclic AMP enhanced BMP15-induction of a SMAD1/5/8-specific BRE reporter. Moreover, in the absence of BMP ligand, cAMP also activated SMAD1/5/8-induced BRE activity. Cyclic AMP increased canonical downstream targets of BMP signalling such as inhibitor of differentiation (ID) mRNA expression. The observed effects were not mediated by secretion of BMPs as cAMP did not promote BMP ligand mRNA expression and a BMP extracellular antagonist, the BMP type II receptor ectodomain, did not affect cAMP-induced ID mRNA expression. Finally, the ERK1/2 pathway was shown to be required for the maintenance of cAMP-induced SMAD1/5/8 activity. Together our results suggest a novel and non-canonical pathway for cAMP signalling in human granulosa cells. Cyclic AMP appears to promote SMAD1/5/8 pathway activity intracellularly and has the ability to activate canonical SMAD1/5/8 downstream targets. Our results add another layer of complexity to the interactions between endocrine signalling and oocyte-secreted BMP ligands during folliculogenesis. Given the importance of both cAMP and SMAD1/5/8 pathways in follicular development, these interactions are likely required for the fine-tuning of oocyte paracrine signalling by endocrine stimuli.

Astrocytes attenuate oligodendrocyte death in vitro through an alpha(6) integrin-laminin-dependent mechanism.

Oligodendrocyte (OL) death occurs in many disorders of the CNS, including multiple sclerosis and brain trauma. Factors reported to induce OL death include deprivation of growth factors, elevation of cytokines, oxidative stress, and glutamate excitotoxicity. Because astrocytes produce a large amount of growth factors and antioxidants and are a major source of glutamate uptake, we tested the hypothesis that astrocytes may have a protective role for OL survival. We report that when OLs from the adult mouse brain were initiated into tissue culture, DNA fragmentation and chromatin condensation resulted, indicative of apoptosis. OL death was significantly reduced in coculture with astrocytes, but not with fibroblasts, which provided a similar monolayer of cells as astrocytes. The protection of OL demise by astrocytes was not reproduced by its conditioned medium and was not accounted for by several neurotrophic factors. In contrast, interference with the alpha(6) integrin subunit, but not the alpha(1), alpha(2), alpha(3), alpha(4), alpha(5), or alpha(v) integrin chains, negated astrocyte protection of OLs. Furthermore, a function-blocking antibody to alpha(6)beta(1) integrin reduced the ability of astrocytes to promote OL survival. The extracellular matrix ligand for alpha(6)beta(1) is laminin, which is expressed by astrocytes. Significantly, neutralizing antibodies to laminin-2 and laminin-5 inhibited the astrocyte mediation of OL survival. These results implicate astrocytes in promoting OL survival through a mechanism involving the interaction of alpha(6)beta(1) integrin on OLs with laminin on astrocytes. Enhancing this interaction may provide for OL survival in neurological injury.

Kindling of claustrum and insular cortex: comparison to perirhinal cortex in the rat.

The perirhinal cortex has recently been implicated in the kindling of limbic generalized seizures. The following experiments in rats tested the selectivity of the perirhinal cortex's epileptogenic properties by comparing its kindling profile with those of the adjacent insular cortex, posterior (dorsolateral) claustrum and amygdala. The first experiment examined the kindling and EEG profiles, and found that both the claustrum and insular cortex demonstrated rapid epileptogenic properties similar to the perirhinal cortex, including very rapid kindling rates and short latencies to convulsion. Furthermore, electrical stimulation of all three structures led to a two-phase progression through stage-5 seizures which had characteristics of both neocortical and amygdaloid kindling. In a second experiment rats were suspended in a harness to allow for more detailed documentation of both forelimb and hindlimb convulsions. With this procedure we were able to detect subtle yet unique differences in convulsion characteristics from each of the kindled sites and stage-5 seizure phases. Some of these convulsive parameters were correlated with changes in FosB/DeltaFosB protein and BDNF mRNA expression measured two hours after the last convulsion. Overall, it appears that the perirhinal cortex is not unique in its property of rapid epileptogenesis. Moreover, the posterior claustrum exhibited the fastest kindling and most vigorous patterns of clonus, suggesting that it may be even more intimately associated with the motor substrates responsible for limbic seizure generalization than is the perirhinal cortex.

Interleukin-1 is a key regulator of matrix metalloproteinase-9 expression in human neurons in culture and following mouse brain trauma in vivo.

An acute trauma to the CNS rapidly results in the upregulation of inflammatory cytokines that include interleukin-1 (IL-1). We report here that the levels of several matrix metalloproteinases (MMPs) are also elevated following a corticectomy trauma injury to the mouse CNS. The delayed upregulation of MMPs compared to that for IL-1 suggests the possibility that inflammatory cytokines regulate MMP production in CNS trauma. To resolve this, we developed a method to isolate and maintain highly enriched human fetal neurons or astrocytes in culture and examined the regulation by cytokines of the activity of a subgroup of MMPs, the gelatinases (MMP-2 and -9). While both neuronal and astrocytic cultures displayed comparable MMP-2 activity, as evidenced by gelatin zymography, levels of MMP-9 were proportionately higher in neurons compared to astrocytes. Of a variety of cytokines and growth factors tested in vitro, only IL-1beta was effective in increasing the neuronal expression of MMP-9. Finally, an IL-1 receptor antagonist attenuated the increase of neuronal MMP-9 in culture and abolished the injury-induced increase of MMP-9 in the mouse brain. These results implicate IL-1beta as a key regulator of neuronal MMP-9 in culture and of the elevation of MMP-9 that occurs following mouse CNS trauma.