Fabio Grohovaz

Synergistic Control of Protein Kinase Cγ Activity by Ionotropic and Metabotropic Glutamate Receptor Inputs in Hippocampal Neurons

Conventional protein kinase C (PKC) isoforms are abundant neuronal signaling proteins with important roles in regulating synaptic plasticity and other neuronal processes. Here, we investigate the role of ionotropic and metabotropic glutamate receptor (iGluR and mGluR, respectively) activation on the generation of Ca2+ and diacylglycerol (DAG) signals and the subsequent activation of the neuron-specific PKCγ isoform in hippocampal neurons. By combining Ca2+ imaging with total internal reflection microscopy analysis of specific biosensors, we show that elevation of both Ca2+ and DAG is necessary for sustained translocation and activation of EGFP (enhanced green fluorescent protein)-PKCγ. Both DAG production and PKCγ translocation were localized processes, typically observed within discrete microdomains along the dendritic branches. Markedly, intermediate-strength NMDA receptor (NMDAR) activation or moderate electrical stimulation generated Ca2+ but no DAG signals, whereas mGluR activation generated DAG but no Ca2+ signals. Both receptors were needed for PKCγ activation. This suggests that a coincidence detection process exists between iGluRs and mGluRs that relies on a molecular coincidence detection process based on the corequirement of Ca2+ and DAG for PKCγ activation. Nevertheless, the requirement for costimulation with mGluRs could be overcome for maximal NMDAR stimulation through a direct production of DAG via activation of the Ca2+-sensitive PLCδ (phospholipase Cδ) isoform. In a second important exception, mGluRs were sufficient for PKCγ activation in neurons in which Ca2+ stores were loaded by previous electrical activity. Together, the dual activation requirement for PKCγ provides a plausible molecular interpretation for different synergistic contributions of mGluRs to long-term potentiation and other synaptic plasticity processes.

Iron handling in hippocampal neurons: activity-dependent iron entry and mitochondria-mediated neurotoxicity

The characterization of iron handling in neurons is still lacking, with contradictory and incomplete results. In particular, the relevance of non‐transferrin‐bound iron (NTBI), under physiologic conditions, during aging and in neurodegenerative disorders, is undetermined. This study investigates the mechanisms underlying NTBI entry into primary hippocampal neurons and evaluates the consequence of iron elevation on neuronal viability. Fluorescence‐based single cell analysis revealed that an increase in extracellular free Fe2+ (the main component of NTBI pool) is sufficient to promote Fe2+ entry and that activation of either N‐methyl‐d‐aspartate receptors (NMDARs) or voltage operated calcium channels (VOCCs) significantly potentiates this pathway, independently of changes in intracellular Ca2+ concentration ([Ca2+]i). The enhancement of Fe2+ influx was accompanied by a corresponding elevation of reactive oxygen species (ROS) production and higher susceptibility of neurons to death. Interestingly, iron vulnerability increased in aged cultures. Scavenging of mitochondrial ROS was the most powerful protective treatment against iron overload, being able to preserve the mitochondrial membrane potential and to safeguard the morphologic integrity of these organelles. Overall, we demonstrate for the first time that Fe2+ and Ca2+ compete for common routes (i.e. NMDARs and different types of VOCCs) to enter primary neurons. These iron entry pathways are not controlled by the intracellular iron level and can be harmful for neurons during aging and in conditions of elevated NTBI levels. Finally, our data draw the attention to mitochondria as a potential target for the treatment of the neurodegenerative processes induced by iron dysmetabolism.

Succesful transplantation of human islets in recipients bearing a kidney graft

Abstract.Aims/hypothesis: Islet transplantation is a minimally invasive approach to curing Type I (insulin-dependent) diabetes mellitus. Success has recently been reported in patients receiving solitary islet transplants but the outcome in patients receiving islets together with, or after, kidney transplants has been limited and unpredictable. Methods: Here we report successful islet transplantation in a cohort of 15 patients with Type I diabetes who were followed for at least 1 year after islet transplantation, after having already received kidney allografts because of end-stage nephropathy. Results: C-peptide after transplantation was higher than 0.17 nmol/l in all 15 recipients, reflecting the absence of primary non-function. Insulin requirement was reduced by over 50 % in all but one patient, and insulin independence was achieved in 10 (66 %) recipients, five of whom now have stable, prolonged insulin independence, well controlled fasting glycaemia, a substantial first-phase and normal second-phase response to glucose, normal insulin sensitivity (HOMA analyses) and HbA1 c of under 6.2 % (33, 26, 18, 13 and 12 months after transplantation respectively). Of importance for patient management, an assessment of fasting blood glucose and proinsulin values following overnight withdrawal of insulin administration one month after transplantation was a potent predictor of insulin independence, and could be used to decide patients who should have further islet preparations. Conclusion/interpretation: These findings support the use of islet transplantation as a cure for Type I diabetes in patients with severe complications. [Diabetologia (2002) 45: 77–84]

Complex translational regulation of BACE1 involves upstream AUGs and stimulatory elements within the 5′ untranslated region

BACE1 is the protease responsible for the production of amyloid-β peptides that accumulate in the brain of Alzheimers disease (AD) patients. BACE1 expression is regulated at the transcriptional, as well as post-transcriptional level. Very high BACE1 mRNA levels have been observed in pancreas, but the protein and activity were found mainly in brain. An up-regulation of the protein has been described in some AD patients without a change in transcript levels. The features of BACE1 5′ untranslated region (5′ UTR), such as the length, GC content, evolutionary conservation and presence of upstream AUGs (uAUGs), indicate an important regulatory role of this 5′ UTR in translational control. We demonstrate that, in brain and pancreas, almost all of the native BACE1 mRNA contains the full-length 5′ UTR. RNA transfection and in vitro translation show that translation is mainly inhibited by the presence of the uAUGs. We provide a mutational analysis that highlight the second uAUG as the main inhibitory element while mutations of all four uAUGs fully de-repress translation. Furthermore, we have evidence that a sequence within the region 222-323 of the BACE1 5′ UTR has a stimulatory effect on translation that might depend on the presence of trans-acting factors.

Biological activities of Bv8 analogues

The small protein Bv8, secreted by the skin of the frog Bombina variegata, belongs to a novel family of secreted proteins whose orthologues have been identified in snakes (MIT) and in mammals (prokineticins (PKs)). A characteristic feature of this protein family is the same N‐terminal sequence, AVITGA, and the presence of 10 cysteines with identical spacing in the C‐terminal domain. Two closely related G protein‐coupled receptors that mediate signal transduction of Bv8/PKs have been cloned (PK‐R1 and PK‐R2). In mammals, the Bv8/PK protein family is involved in a number of biological activities such as ingestive behaviours, circadian rhythms, angiogenesis and pain sensitization. In an attempt to identify the structural determinants required for the pronociceptive activity of Bv8, we prepared Bv8 derivatives lacking one (des‐Ala‐Bv8) or two (des‐Ala‐Val‐Bv8) residues from the N‐terminus. des‐Ala‐Bv8 displayed a receptor affinity five times lower than that of Bv8, it was five times less potent in inducing [Ca2+]i transients and in causing p42/p44 MAPK phosphorylation in CHO‐cells expressing PK‐R1 and PK‐R2. Moreover, dA‐Bv8 was about 20 times less potent than Bv8 in inducing hyperalgesia in rats. The deletion of the first two amino acids of Bv8 abolished any biological activity both ‘in vitro’ and ‘in vivo’; however, des‐AlaVal‐Bv8 is able to antagonize the Bv8‐induced hyperalgesia, binding the PK‐Rs on peripheral and central projections of the primary sensitive neurons.

HIV‐1 gp120 Glycoprotein Induces [Ca2+]i Responses not only in Type‐2 but also Type‐1 Astrocytes and Oligodendrocytes of the Rat Cerebellum

Cultures of cerebellar cortex cells were exposed to the HIV‐1 envelope glycoprotein, gp120, and investigated for cytosolic Ca2+ ion concentration ([Ca2+]i) changes by the fura‐2 ratio videoimaging technique while bathed in complete, Na+‐free or Mg2+‐free Krebs‐Ringer media. At the end of the [Ca2+]i experiments the cells were fixed and immunoidentified through the revelation of markers specific for neurons (microtubule associated protein‐2), type‐2 (A2B5) or all (glial fibrillary acidic protein) astrocytes, oligodendrocytes (galactocerebroside) or microglia (F4/80 antibody). In complete medium, rapid biphasic (spike‐plateau) responses induced by gp120 (0.1–1 nM) were observed in a subpopulation of type‐2 astrocytes. In addition, slow but progressive responses were observed in other type‐2 cells and oligodendrocytes, whereas type‐1 astrocytes showed small responses, if any, and granule neurons did not respond at all. Use of Na+‐free medium (a condition that blocked another gp120‐induced response, cytosolic alkalinization) resulted in an increase in [Ca2+]i response that was appreciable not only in type‐2 but also in most type‐1 astrocytes, possibly because of the inhibition of the Na+/Ca2+ exchanger and the ensuing decrease in Ca2+ extrusion. Granule neurons, including those in direct contact with responsive astrocytes, remained unresponsive, even when the experiments were carried out in Mg2+‐free medium supplemented with glycine, a condition that favours activation of the glutamatergic N‐methyl‐D‐aspartate (NMDA) receptor. The results obtained demonstrate that sensitivity to gp120 is a property of not only a few type‐2 astrocytes but of the majority of cerebellar glial cells, which, however, do not respond to the protein with glutamate release, as indicated by the negative results obtained with NMDA‐receptor‐expressing granule neurons. Single glial cell [Ca2+]i increase, the faster and most sensitive effect of gp120 revealed in the brain so far, could be ultimately employed to reveal CD4‐independent transmembrane signalling machanisms of the viral protein that, at the moment, remain almost entirely unknown.

Iron uptake in quiescent and inflammation-activated astrocytes: A potentially neuroprotective control of iron burden

Astrocytes play a crucial role in proper iron handling within the central nervous system. This competence can be fundamental, particularly during neuroinflammation, and neurodegenerative processes, where an increase in iron content can favor oxidative stress, thereby worsening disease progression. Under these pathological conditions, astrocytes undergo a process of activation that confers them either a beneficial or a detrimental role on neuronal survival. Our work investigates the mechanisms of iron entry in cultures of quiescent and activated hippocampal astrocytes. Our data confirm that the main source of iron is the non-transferrin-bound iron (NTBI) and show the involvement of two different routes for its entry: the resident transient receptor potential (TRP) channels in quiescent astrocytes and the de novo expressed divalent metal transporter 1 (DMT1) in activated astrocytes, which accounts for a potentiation of iron entry. Overall, our data suggest that at rest, but even more after activation, astrocytes have the potential to buffer the excess of iron, thereby protecting neurons from iron overload. These findings further extend our understanding of the protective role of astrocytes under the conditions of iron-mediated oxidative stress observed in several neurodegenerative conditions.

Expression of divalent metal transporter 1 in primary hippocampal neurons: reconsidering its role in non‐transferrin‐bound iron influx

J. Neurochem. (2012) 120, 269–278.

Inhibition of lipopolysaccharide-induced microglia activation by calcitonin gene related peptide and adrenomedullin

Calcitonin gene related peptide (CGRP) and adrenomedullin are potent biologically active peptides that have been proposed to play an important role in vascular and inflammatory diseases. Their function in the central nervous system is still unclear since they have been proposed as either pro-inflammatory or neuroprotective factors. We investigated the effects of the two peptides on astrocytes and microglia, cells of the central nervous system that exert a strong modulatory activity in the neuroinflammatory processes. In particular, we studied the ability of CGRP and adrenomedullin to modulate microglia activation, i.e. its competence of producing and releasing pro-inflammatory cytokines/chemokines that are known to play a crucial role in neuroinflammation. In this work we show that the two neuropeptides exert a potent inhibitory effect on lipopolysaccharide-induced microglia activation in vitro, with strong inhibition of the release of pro-inflammatory mediators (such as NO, cytokines and chemokines). Both CGRP and adrenomedullin are known to promote cAMP elevation, this second messenger cannot fully account for the observed inhibitory effects, thereby suggesting that other signaling pathways are involved. Interestingly, the inhibitory effect of CGRP and adrenomedullin appears to be stimulus specific, since direct activation with pro-inflammatory cytokines was not affected. Our findings clarify aspects of microglia activation, and contribute to the comprehension of the switch from reparative to detrimental function that occurs when glia is exposed to different conditions. Moreover, they draw the attention to potential targets for novel pharmacological intervention in pathologies characterized by glia activation and neuroinflammation.

Calcitonin gene-related peptide (CGRP) triggers Ca2+ responses in cultured astrocytes and in Bergmann glial cells from cerebellar slices

The neuropeptide calcitonin gene‐related peptide (CGRP) is transiently expressed in cerebellar climbing fibers during development while its receptor is mainly expressed in astrocytes, in particular Bergmann glial cells. Here, we analyzed the effects of CGRP on astrocytic calcium signaling. Mouse cultured astrocytes from cerebellar or cerebral cortex as well as Bergmann glial cells from acutely isolated cerebellar slices were loaded with the Ca2+ sensor Fura‐2. CGRP triggered transient increases in intracellular Ca2+ in astrocytes in culture as well as in acute slices. Responses were observed in the concentration range of 1 nm to 1 mm, in both the cell body and its processes. The calcium transients were dependent on release from intracellular stores as they were blocked by thapsigargin but not by the absence of extracellular calcium. In addition, after CGRP application a further delayed transient increase in calcium activity could be observed. Finally, cerebellar astrocytes from neonatal mice expressed receptor component protein, a component of the CGRP receptor, as revealed by immunofluorescence and confocal microscopy. It is thus proposed that the CGRP‐containing afferent fibers in the cerebellum (the climbing fibers) modulate calcium in astrocytes by releasing the neuropeptide during development and hence possibly influence the differentiation of Purkinje cells.