Daniela Rossi

Prostaglandins stimulate calcium-dependent glutamate release in astrocytes

Astrocytes in the brain form an intimately associated network with neurons. They respond to neuronal activity and synaptically released glutamate by raising intracellular calcium concentration ([Ca2+]i), which could represent the start of back-signalling to neurons. Here we show that coactivation of the AMPA/kainate and metabotropic glutamate receptors (mGluRs) on astrocytes stimulates these cells to release glutamate through a Ca2+-dependent process mediated by prostaglandins. Pharmacological inhibition of prostaglandin synthesis prevents glutamate release, whereas application of prostaglandins (in particular PGE2) mimics and occludes the releasing action of GluR agonists. PGE2 promotes Ca2+-dependent glutamate release from cultured astrocytes and also from acute brain slices under conditions that suppress neuronal exocytotic release. When applied to the CA1 hippocampal region, PGE2 induces increases in [Ca2+]i both in astrocytes and in neurons. The [Ca2+]i increase in neurons is mediated by glutamate released from astrocytes, because it is abolished by GluR antagonists. Our results reveal a new pathway of regulated transmitter release from astrocytes and outline the existence of an integrated glutamatergic cross-talk between neurons and astrocytes in situ that may play critical roles in synaptic plasticity and in neurotoxicity.

Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain

PrP knockout mice in which only the open reading frame was disrupted (‘Zürich I’) remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, Prnd. A new PrP knockout line, ‘Zürich II’, with a 2.9 kb Prnp deletion, developed this phenotype at ∼10 months (50% morbidity). A single Prnp allele abolished the syndrome. Compound Zürich I/Zürich II heterozygotes had half the Dpl of Zürich II mice and developed symptoms 6 months later. Zürich II mice transgenic for a Prnd‐containing cosmid expressed Dpl at twice the level and became ataxic ∼5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated.

Cell-Penetrating Peptides: From Basic Research to Clinics

The presence of cell and tissue barriers together with the low biomembrane permeability of various therapeutics often hampers systemic drug distribution; thus, most of the available molecules are of limited therapeutic value. Opportunities to increase medicament concentrations in areas that are difficult to access now exist with the advent of cell-penetrating peptides (CPPs), which can transport into the cell a wide variety of biologically active conjugates (cargoes). Numerous preclinical evaluations with CPP-derived therapeutics have provided promising results in various disease models that, in some cases, prompted clinical trials. The outcome of these investigations has thus opened new perspectives for CPP application in the development of unprecedented human therapies that are well tolerated and directed to intracellular targets.

Neuronal and glial glutamate transporters possess an SH-based redox regulatory mechanism

Glutamate uptake into nerve cells and astrocytes via high‐affinity transporters controls the extracellular glutamate concentration in the brain, with major implications for physiological excitatory neurotransmission and the prevention of excitotoxicity. We report here that three recently cloned rat glutamate transporter subtypes, viz. EAAC1 (neuronal), GLT1 and GLAST (glial), possess a redox‐sensing property, undergoing opposite functional changes in response to oxidation or reduction of reactive sulphydryls present in their structure. In particular, thiol oxidation with 5,5′‐dithio‐bis(2–nitrobenzoic) acid (DTNB) and disulphide reduction with dithiothreitol (DTT) result, respectively, in reduced and increased uptake capacity by a preparation of partially purified brain transporters as well as by the three recombinant proteins reconstituted into liposomes. In this model system, EAAC1, GLT1 and GLAST react similarly to DTT/DTNB exposures despite their different contents of cysteines, suggesting that only the conserved residues might be involved in redox modulation. Redox sensitivity is a property of the glutamate transporters also when present in their native cell environment. Thus, by using cultured cortical astrocytes and the whole‐cell patch‐clamp technique we were able to observe dynamic increase and decrease of the glutamate uptake current in response to application of DTT and DTNB in sequence. Moreover, in the same paradigm, DDT‐reversible current inhibition was observed with hydrogen peroxide instead of DTNB, indicating that the SH‐based redox modulatory site is targeted by endogenous oxidants and might constitute an important physiological or pathophysiological regulatory mechanism of glutamate uptake in vivo

Astrocytic dysfunction: Insights on the role in neurodegeneration

For decades, astrocytes have been regarded as passive partners of neurons in central nervous system (CNS) function. Studies of the last 20 years, however, challenged this view by demonstrating that astrocytes possess functional receptors for neurotransmitters and respond to their stimulation via release of gliotransmitters, including glutamate. Notably, astrocytes react to synaptically released neurotransmitters with intracellular calcium ([Ca(2+)]) elevations, which result in the release of glutamate via regulated exocytosis and, possibly, other mechanisms. These findings have led to a new concept of neuron-glia intercommunication where astrocytes play an unsuspected dynamic role by integrating neuronal inputs and modulating synaptic activity. The additional observation that glutamate release from astrocytes is controlled by molecules linked to inflammatory reactions, such as the cytokine tumor necrosis factor alpha (TNFalpha) and prostaglandins (PGs), suggests that glia-to-neuron signalling may be sensitive to changes in the production of these mediators occurring in pathological conditions. Indeed, a local, parenchymal brain inflammatory reaction (neuroinflammation) characterized by astrocytic and microglial activation has been reported in several neurodegenerative disorders, including AIDS dementia complex, Alzheimers disease and amyotrophic lateral sclerosis. This transition may be accompanied by functional de-regulation and even degeneration of the astrocytes with the consequent disruption of the cross-talk normally occurring between these cells and neurons. Incorrect neuron-astrocyte interactions may be involved in neuronal derangement and contribute to disease development. The findings reported in this review suggest that a better comprehension of the glutamatergic interplay between neurons and astrocytes may provide information about normal brain function and also highlight potential molecular targets for therapeutic interventions in pathology.

Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles

Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of ∼800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils. In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.

Focal degeneration of astrocytes in amyotrophic lateral sclerosis

Astrocytes emerge as key players in motor neuron degeneration in Amyotrophic Lateral Sclerosis (ALS). Whether astrocytes cause direct damage by releasing toxic factors or contribute indirectly through the loss of physiological functions is unclear. Here we identify in the hSOD1G93A transgenic mouse model of ALS a degenerative process of the astrocytes, restricted to those directly surrounding spinal motor neurons. This phenomenon manifests with an early onset and becomes significant concomitant with the loss of motor cells and the appearance of clinical symptoms. Contrary to wild-type astrocytes, mutant hSOD1-expressing astrocytes are highly vulnerable to glutamate and undergo cell death mediated by the metabotropic type-5 receptor (mGluR5). Blocking mGluR5 in vivo slows down astrocytic degeneration, delays the onset of the disease and slightly extends survival in hSOD1G93A transgenic mice. We propose that excitotoxicity in ALS affects both motor neurons and astrocytes, favouring their local interactive degeneration. This new mechanistic hypothesis has implications for therapeutic interventions.

Glutamate release from astrocytes in physiological conditions and in neurodegenerative disorders characterized by neuroinflammation

Although glial cells have been traditionally viewed as supportive partners of neurons, studies of the last 20 years demonstrate that astrocytes possess functional receptors for neurotransmitters and other signaling molecules and respond to their stimulation via release of chemical transmitters (called gliotransmitters) such as glutamate, ATP, and d-serine. Notably, astrocytes react to synaptically released neurotransmitters with intracellular calcium ([Ca(2+)](i)) elevations, which result in the release of glutamate via regulated exocytosis and possibly other mechanisms. These findings have led to a new concept of neuron-glia intercommunication where astrocytes play an unsuspected dynamic role by integrating neuronal inputs and modulating synaptic activity. The additional discovery that glutamate release from astrocytes is controlled by molecules linked to inflammatory reactions, such as the cytokine tumor necrosis factor-alpha (TNF-alpha) and prostaglandins, suggests that glia-to-neuron signaling may be sensitive to changes in production of these mediators in pathological conditions. Indeed, a local, parenchymal brain inflammatory reaction (neuroinflammation) characterized by astrocytic and microglial activation has been reported in several neurodegenerative disorders, including Alzheimers disease and AIDS dementia complex. This transition to a reactive state may be accompanied by a disruption of the cross talk normally occurring between astrocytes and neurons and so contribute to disease development. The findings reported in this chapter suggest that a better comprehension of the glutamatergic interplay between neurons and glia may provide information about normal brain function and also highlight possible molecular targets for therapeutic interventions in pathology.

Molecular genetics of ryanodine receptors Ca2+-release channels

The family of ryanodine receptor (RyR) genes encodes three highly related Ca(2+)-release channels: RyR1, RyR2 and RyR3. RyRs are known as the Ca(2+)-release channels that participate to the mechanism of excitation-contraction coupling in striated muscles, but they are also expressed in many other cell types. Actually, in several cells two or three RyR isoforms can be co-expressed and interactive feedbacks among them may be important for generation of intracellular Ca(2+) signals and regulation of specific cellular functions. Important developments have been obtained in understanding the biochemical complexity underlying the process of Ca(2+) release through RyRs. The 3-D structure of these large molecules has been obtained and some regulatory regions have been mapped within these 3-D reconstructions. Recent studies have clarified the role of protein kinases and phosphatases that, by physically interacting with RyRs, appear to play a role in the regulation of these Ca(2+)-release channels. These and other recent advancements in understanding RyR biology will be the object of this review.

HIV-1 gp120 glycoprotein affects the astrocyte control of extracellular glutamate by both inhibiting the uptake and stimulating the release of the amino acid

The mechanisms of HIV‐1 neurotoxicity remain still undefined although the induction of signalling events and a modest inhibition of glutamate uptake induced by the envelope glycoprotein, gp120, have called attention to astrocytes. Here we demonstrate that the levels at which the viral glycoprotein affects glutamate homeostasis of astrocyte cultures are at least two: not only the inhibition of uptake, due to an effect at site(s) away from the transporters of the amino acid but also a slow stimulation of release. The combination of these two events accounts for a considerable steady increase of the extracellular concentration of the excitatory amino acid which could play an important role in the neurotoxicity often observed in AIDS patients.