Romina Macco

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.

β-Secretase activity in rat astrocytes: Translational block of BACE1 and modulation of BACE2 expression

BACE1 and BACE2 are two closely related membrane‐bound aspartic proteases. BACE1 is widely recognized as the neuronal β‐secretase that cleaves the amyloid‐β precursor protein, thus allowing the production of amyloid‐β, i.e. the peptide that has been proposed to trigger the neurodegenerative process in Alzheimer’s disease. BACE2 has ubiquitous expression and its physiological and pathological role is still unclear. In light of a possible role of glial cells in the accumulation of amyloid‐β in brain, we have investigated the expression of these two enzymes in primary cultures of astrocytes. We show that astrocytes possess β‐secretase activity and produce amyloid‐β because of the activity of BACE2, but not BACE1, the expression of which is blocked at the translational level. Finally, our data demonstrate that changes in the astrocytic phenotype during neuroinflammation can produce both a negative as well as a positive modulation of β‐secretase activity, also depending on the differential responsivity of the brain regions.

Astrocytes acquire resistance to iron-dependent oxidative stress upon proinflammatory activation

BackgroundAstrocytes respond to local insults within the brain and the spinal cord with important changes in their phenotype. This process, overall known as “activation”, is observed upon proinflammatory stimulation and leads astrocytes to acquire either a detrimental phenotype, thereby contributing to the neurodegenerative process, or a protective phenotype, thus supporting neuronal survival. Within the mechanisms responsible for inflammatory neurodegeneration, oxidative stress plays a major role and has recently been recognized to be heavily influenced by changes in cytosolic iron levels. In this work, we investigated how activation affects the competence of astrocytes to handle iron overload and the ensuing oxidative stress.MethodsCultures of pure cortical astrocytes were preincubated with proinflammatory cytokines (interleukin-1β and tumor necrosis factor α) or conditioned medium from lipopolysaccharide-activated microglia to promote activation and then exposed to a protocol of iron overload.ResultsWe demonstrate that activated astrocytes display an efficient protection against iron-mediated oxidative stress and cell death. Based on this evidence, we performed a comprehensive biochemical and molecular analysis, including a transcriptomic approach, to identify the molecular basis of this resistance.ConclusionsWe propose the protective phenotype acquired after activation not to involve the most common astrocytic antioxidant pathway, based on the Nrf2 transcription factor, but to result from a complex change in the expression and activity of several genes involved in the control of cellular redox state.

Iron and calcium in the central nervous system: a close relationship in health and sickness.

Iron and calcium are required for general cellular functions, as well as for specific neuronal-related activities. However, a pathological increase in their levels favours oxidative stress and mitochondrial damage, leading to neuronal death. Neurodegeneration can thus be determined by alterations in ionic homoeostasis and/or pro-oxidative-antioxidative equilibrium, two conditions that vary significantly in different kinds of brain cell and also with aging. In the present review, we re-evaluate recent data on NTBI (non-transferrin bound iron) uptake that suggest a strict interplay with the mechanisms of calcium control. In particular, we focus on the use of common entry pathways and on the way cytosolic calcium can modulate iron entry and determine its intracellular accumulation.

Metallothioneins as dynamic markers for brain disease in lysosomal disorders

To facilitate development of novel disease‐modifying therapies for lysosomal storage disorder (LSDs) characterized by nervous system involvement such as metachromatic leukodystrophy (MLD), molecular markers for monitoring disease progression and therapeutic response are needed. To this end, we sought to identify blood transcripts associated with the progression of MLD.

P3-352: Beta-secretase modulation and amyloid-beta production in rat brain primary cultures

Background: The processing of the amyloid-beta precursor protein by a beta-secretase is the rate-limiting step in the production of amyloid-beta within the central nervous system. Two beta-secretases have been identified, BACE1, mainly expressed in neurons, and BACE2, with a more ubiquitous expression pattern. Their expression is tightly regulated at both the transcriptional and translational level. In primary cultures from rat brain, we investigated: i) the regulation of BACE1 expression in neurons; ii) the regulation of beta-secretase activity, in resting and activated astrocytes, and its contribution to amyloid-beta production. Methods: Primary cultures of neurons and astrocytes from rat brain were employed. The translational control of BACE1 expression was evaluated by transfection of reporter genes preceded by the BACE1 transcript leader. For the evaluation of endogenous beta-secretases, we tested their activity by an in vitro assay, and BACE1/2 expression by RNA analysis and Western blotting. Results: In a previous work we demonstrated that the translation driven by the BACE1 transcript leader (as evaluated by reporter genes) is higher in neurons than in astrocytes or other commonly used cell lines. In the present study, we investigated the molecular determinants of this control by analyzing the features of the BACE1 mRNA and monitoring changes in endogenous BACE1 upon various treatments. This analysis revealed the presence of both inhibitory and stimulatory sequences in the transcript leader. Furthermore, we demonstrated the presence of a translational block that is specifically bypassed in neurons. In parallel, we studied how betasecretase activity is regulated in astrocytes. We did not detect BACE1 protein, even under conditions of astrocyte activation induced by various stimuli (including pro-inflammatory cytokines). Our data show that betasecretase activity in astrocytes was due to the expression of BACE2. On the other hand, activated astrocytes displayed a reduced BACE2 expression as well as a decrease in beta-secretase activity. We are currently investigating the role of astrocyte activation on the production of amyloid-beta. Conclusions: BACE1 expression is regulated at the translational level by a neuronal-specific mechanism. Astrocytes do not express BACE1 and, upon activation with pro-inflammatory cytokines, they down-regulate BACE2 expression, thus decreasing beta-secretase activity and, likely, amyloidbeta production.