Redox and Calcium Alterations of a Müller Cell Line Exposed to Diabetic Retinopathy-Like Environment.

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.

Gα13 Contributes to LPS-Induced Morphological Alterations and Affects Migration of Microglia.

Microglia are the resident immune cells of the CNS that are activated in response to a variety of stimuli. This phenotypical change is aimed to maintain the local homeostasis, also by containing the insults and repair the damages. All these processes are tightly regulated and coordinated and a failure in restoring homeostasis by microglia can result in the development of neuroinflammation that can facilitate the progression of pathological conditions.

Impaired flickering of the permeability transition pore causes SPG7 spastic paraplegia.

Background: Mutations of the mitochondrial protein paraplegin cause hereditary spastic paraplegia type 7 (SPG7), a so-far untreatable degenerative disease of the upper motoneuron with still undefined pathomechanism. The intermittent mitochondrial permeability transition pore (mPTP) opening, called flickering, is an essential process that operates to maintain mitochondrial homeostasis by reducing intra-matrix Ca and reactive oxygen species (ROS) concentration, and is critical for efficient synaptic function.

Methods: We use a fluorescence-based approach to measure mPTP flickering in living cells and biochemical and molecular biology techniques to dissect the pathogenic mechanism of SPG7.

Upregulation of Peroxiredoxin 3 Protects 2-KO Cortical Neurons from Oxidative Stress: A Paradigm for Neuronal Cell Survival under Neurodegenerative Conditions.

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 -KO mice.

Membrane potential changes occurring upon acidification influence the binding of small-molecule inhibitors to ASIC1a.

Acid-sensing ion channels (ASICs) are proton-activated, sodium-permeable channels, highly expressed in both central and peripheral nervous systems. ASIC1a is the most abundant isoform in the central nervous system and is credited to be involved in several neurological disorders including stroke, multiple sclerosis, and epilepsy. Interestingly, the affinity of ASIC1a for two antagonists, diminazene and amiloride, has recently been proposed to be voltage sensitive.

Adding a temporal dimension to the study of Friedreich's ataxia: the effect of frataxin overexpression in a human cell model.

The neurodegenerative disease Friedreich's ataxia is caused by lower than normal levels of frataxin, an important protein involved in iron-sulfur (Fe-S) cluster biogenesis. An important step in designing strategies to treat this disease is to understand whether increasing the frataxin levels by gene therapy would simply be beneficial or detrimental, because previous studies, mostly based on animal models, have reported conflicting results. Here, we have exploited an inducible model, which we developed using the CRISPR/Cas9 methodology, to study the effects of frataxin overexpression in human cells and monitor how the system recovers after overexpression.

eIF4B phosphorylation at Ser504 links synaptic activity with protein translation in physiology and pathology.

Neuronal physiology requires activity-driven protein translation, a process in which translation initiation factors are key players. We focus on eukaryotic initiation factor 4B (eIF4B), a regulator of protein translation, whose function in neurons is undetermined. We show that neuronal activity affects eIF4B phosphorylation and identify Ser504 as a phosphorylation site regulated by casein kinases and sensitive to the activation of metabotropic glutamate receptors.

Iron entry in neurons and astrocytes: a link with synaptic activity.

Iron plays a fundamental role in the development of the central nervous system (CNS) as well as in several neuronal functions including synaptic plasticity. Accordingly, neuronal iron supply is tightly controlled: it depends not only on transferrin-bound iron but also on non-transferrin-bound iron (NTBI), which represents a relevant quote of the iron physiologically present in the cerebrospinal fluid (CSF). Different calcium permeable channels as well as the divalent metal transporter 1 (DMT1) have been proposed to sustain NTBI entry in neurons and astrocytes even though it remains an open issue.

A novel neuroferritinopathy mouse model (FTL 498InsTC) shows progressive brain iron dysregulation, morphological signs of early neurodegeneration and motor coordination deficits.

Neuroferritinopathy is a rare genetic disease with a dominant autosomal transmission caused by mutations of the ferritin light chain gene (FTL). It belongs to Neurodegeneration with Brain Iron Accumulation, a group of disorders where iron dysregulation is tightly associated with neurodegeneration. We studied the 498-499InsTC mutation which causes the substitution of the last 9 amino acids and an elongation of extra 16 amino acids at the C-terminus of L-ferritin peptide.

Involvement of calcitonin gene-related peptide and receptor component protein in experimental autoimmune encephalomyelitis.

Calcitonin Gene-Related Peptide (CGRP) inhibits microglia inflammatory activation in vitro. We here analyzed the involvement of CGRP and Receptor Component Protein (RCP) in experimental autoimmune encephalomyelitis (EAE). Alpha-CGRP deficiency increased EAE scores which followed the scale alpha-CGRP null>heterozygote>wild type.

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

Background: Astrocytes 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.

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.

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

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.

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.

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.

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.

Calcitonin gene-related peptide (CGRP) stimulates purkinje cell dendrite growth in culture.

Previous reports described the transient expression during development of Calcitonin Gene-Related Peptide (CGRP) in rodent cerebellar climbing fibers and CGRP receptor in astrocytes. Here, mixed cerebellar cultures were used to analyze the effects of CGRP on Purkinje cells growth. Our results show that CGRP stimulated Purkinje cell dendrite growth under cell culture conditions mimicking Purkinje cell development in vivo.

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 Ca(2+) sensor Fura-2.

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.

Sphingosylphosphocholine effects on cultured astrocytes reveal mechanisms potentially involved in neurotoxicity in Niemann-Pick type A disease.

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.

BACE1 expression and activity: relevance in Alzheimer's disease.

A turning point of research in Alzheimer's disease was undoubtedly the discovery of BACE1, the amyloid-beta precursor protein-cleaving enzyme that initiates the generation of amyloid-beta, the peptide strongly suspected to be responsible for neuronal malfunction and death. Several research groups started a race to identify the best inhibitor of BACE1 activity. On the other hand, basic researchers are evaluating the changes in BACE1 expression and activity with the aim to better understand the pathogenetic process of the disease.

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-beta peptides that accumulate in the brain of Alzheimer's 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.

Synergistic control of protein kinase Cgamma 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 PKCgamma 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)-PKCgamma.