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"The divalent metal transporter 1 (DMT1) is the best characterized Fe²⁺ transporter involved in cellular iron uptake in mammals. Four possible isoforms have been identified as a result of alternative promoter (DMT1-1A and DMT1-1B) and alternative splicing involving the C-terminus and producing transcripts with or without an iron responsive element [DMT1-IRE⁺ and DMT1-IRE⁻, respectively]. Despite the general importance of DMT1 in controlling iron homeostasis, the distribution and the role of the transporter in the CNS is still controversial. In this study, we characterize the expression of DMT1 in hippocampal neurons and astrocytes. We found that the main isoform endogenously expressed is DMT1-1B\/IRE⁺, which shows cytoplasmic distribution, colocalization with late endosome\/lysosome markers and iron regulation, as expected from the presence of an iron responsive element. Our results also show that DMT1-1B\/IRE⁺ isoform does not sustain iron entry, even after its neuronal over-expression. Overall, our results argue against a physiological role of the endogenous DMT1 in neuronal iron uptake but do not exclude that, under pathological conditions, the expression of other DMT1 isoforms might contribute to iron overload."

"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 Fe(2+) (the main component of NTBI pool) is sufficient to promote Fe(2+) 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 Ca(2+) concentration ([Ca(2+) ](i) ). The enhancement of Fe(2+) 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 Fe(2+) and Ca(2+) 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."

"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."

Niemann-Pick type A is a disease characterized by the absence of a functional SMPD1 (acidic sphingomyelinase) gene and the abnormal accumulation of sphingomyelin. Under these conditions, also sphingosylphosphocholine (SPC, a sphingomyelin metabolite) accumulates in various tissues, including the brain, where it might act as a toxic stimulus, contributing to the appearance of the neurological symptoms. We studied the effects of SPC on astrocytic and neuronal cultures from rat. In particular, we investigated the possibility that SPC acts on astrocytes and that this represents the first step leading to neurodegeneration. Our results show that acute administration of SPC to astrocytes in culture promotes Ca2+ responses and a release of glutamate that, in turn, leads to cytosolic [Ca2+] elevation in neurons. We also show that chronic stimulation by SPC leads astrocytes to proliferate, but can also change their phenotype towards an activated state that might contribute to the inflammatory responses. Interestingly, upon acute SPC stimulation, activated astrocytes release more glutamate. In conclusion, we show that both chronic and acute exposure to SPC can constitute harmful signals that may have a role in the sequence of events leading to neurodegeneration.

Diabetic retinopathy (DR) is a common complication of diabetes mellitus and is the major cause of vision loss in the working-age population. Although DR is traditionally considered a microvascular disease, an increasing body of evidence suggests that neurodegeneration is an early event that occurs even before the manifestation of vasculopathy. Accordingly, attention should be devoted to the complex neurodegenerative process occurring in the diabetic retina, also considering possible functional alterations in non-neuronal cells, such as glial cells. In this work, we investigate functional changes in Müller cells, the most abundant glial population present within the retina, under experimental conditions that mimic those observed in DR patients. More specifically, we investigated on the Müller cell line rMC-1 the effect of high glucose, alone or associated with activation processes and oxidative stress. By fluorescence microscopy and cellular assays approaches, we studied the alteration of functional properties, such as reactive oxygen species production, antioxidant response, calcium homeostasis, and mitochondrial membrane potential. Our results demonstrate that hyperglycaemic-like condition per se is well-tolerated by rMC-1 cells but makes them more susceptible to a pro-inflammatory environment, exacerbating the effects of this stressful condition. More specifically, rMC-1 cells exposed to high glucose decrease their ability to counteract oxidative stress, with consequent toxic effects. In conclusion, our study offers new insights into Müller cell pathophysiology in DR and proposes a novel in vitro model which may prove useful to further investigate potential antioxidant and anti-inflammatory molecules for the prevention and/or treatment of DR.

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. Increased Aβ production plays a fundamental role in the pathogenesis of the disease and BACE1, the protease that triggers the amyloidogenic processing of APP, is a key protein and a pharmacological target in AD. Changes in neuronal activity have been linked to BACE1 expression and Aβ generation, but the underlying mechanisms are still unclear. We provide clear evidence for the role of Casein Kinase 2 in the control of activity-driven BACE1 expression in cultured primary neurons, organotypic brain slices, and murine AD models. More specifically, we demonstrate that neuronal activity promotes Casein Kinase 2 dependent phosphorylation of the translation initiation factor eIF4B and this, in turn, controls BACE1 expression and APP processing. Finally, we show that eIF4B expression and phosphorylation are increased in the brain of APPPS1 and APP-KI mice, as well as in AD patients. Overall, we provide a definition of a mechanism linking brain activity with amyloid production and deposition, opening new perspectives from the therapeutic standpoint.

Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more affected than others, possibly because of the high expression of an altered gene or the presence of particular features that make them more susceptible to insults. On the other hand, resilient neurons may display the ability to develop antioxidant defenses, particularly in diseases of mitochondrial origin, where oxidative stress might contribute to the neurodegenerative process. In this work, we investigated the oxidative stress response of embryonic fibroblasts and cortical neurons obtained from Afg3l2-KO mice. AFG3L2 encodes a subunit of a protease complex that is expressed in mitochondria and acts as both quality control and regulatory enzyme affecting respiration and mitochondrial dynamics. When cells were subjected to an acute oxidative stress protocol, the survival of AFG3L2-KO MEFs was not significantly influenced and was comparable to that of WT; however, the basal level of the antioxidant molecule glutathione was higher. Indeed, glutathione depletion strongly affected the viability of KO, but not of WT MEF, thereby indicating that oxidative stress is more elevated in KO MEF even though well controlled by glutathione. On the other hand, when cortical KO neurons were put in culture, they immediately appeared more vulnerable than WT to the acute oxidative stress condition, but after few days in vitro, the situation was reversed with KO neurons being more resistant than WT to acute stress. This compensatory, protective competence was not due to the upregulation of glutathione, rather of two mitochondrial antioxidant proteins: superoxide dismutase 2 and, at an even higher level, peroxiredoxin 3. This body of evidence sheds light on the capability of neurons to activate neuroprotective pathways and points the attention to peroxiredoxin 3, an antioxidant enzyme that might be critical for neuronal survival also in other disorders affecting mitochondria.