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Neuroferritinopathy is a rare autosomal dominant inherited movement disorder caused by alteration of the L-ferritin gene that results in the production of a ferritin molecule that is unable to properly manage iron, leading to the presence of free redox-active iron in the cytosol. This form of iron has detrimental effects on cells, particularly severe for neuronal cells, which are highly sensitive to oxidative stress. Although very rare, the disorder is notable for two reasons. First, neuroferritinopathy displays features also found in a larger group of disorders named Neurodegeneration with Brain Iron Accumulation (NBIA), such as iron deposition in the basal ganglia and extrapyramidal symptoms; thus, the elucidation of its pathogenic mechanism may contribute to clarifying the incompletely understood aspects of NBIA. Second, neuroferritinopathy shows the characteristic signs of an accelerated process of aging; thus, it can be considered an interesting model to study the progress of aging. Here, we will review the clinical and neurological features of neuroferritinopathy and summarize biochemical studies and data from cellular and animal models to propose a pathogenic mechanism of the disorder.

Summary Neuroferritinopathy (NF) is a movement disorder caused by alterations in the L-ferritin gene that generate cytosolic free iron. NF is a unique pathophysiological model for determining the direct consequences of cell iron dysregulation. We established lines of induced pluripotent stem cells from fibroblasts from two NF patients and one isogenic control obtained by CRISPR/Cas9 technology. NF fibroblasts, neural progenitors, and neurons exhibited the presence of increased cytosolic iron, which was also detectable as: ferritin aggregates, alterations in the iron parameters, oxidative damage, and the onset of a senescence phenotype, particularly severe in the neurons. In this spontaneous senescence model, NF cells had impaired survival and died by ferroptosis. Thus, non-ferritin-bound iron is sufficient per se to cause both cell senescence and ferroptotic cell death in human fibroblasts and neurons. These results provide strong evidence supporting the primary role of iron in neuronal aging and degeneration., Graphical Abstract, Highlights • NF is a unique pathophysiological model for analyzing cellular iron dysregulation • NF fibroblast/iPSC-derived NPCs and neurons show a phenotype of iron mobilization • Free iron is sufficient per se to cause both cell senescence and ferroptosis • Iron has a primary role in neuronal aging and degeneration, In this article Sonia Levi and colleagues show that non-ferritin-bound iron is able to cause cell senescence and ferroptotic cell death in human fibroblasts and neurons, underlining the primary role of iron in accelerating the processes of aging and neurodegeneration. These findings provide implications for studies investigating the role of iron in a more general context of neurodegenerative diseases.

In 2001, a new type of human ferritin was identified by searching for homologous sequences to H-ferritin in the human genome. After the demonstration that this ferritin is located specifically in the mitochondrion, it was called mitochondrial ferritin. Studies on the properties of this new type of ferritin have been limited by its very high homology with the cytosolic H-ferritin, which is expressed at higher levels in cells. This great similarity made it difficult to obtain specific antibodies against the mitochondrial ferritin devoid of cross-reactivity with cytosolic ferritin. Thus, the knowledge of the physiological role of mitochondrial ferritin is still incomplete despite 20 years of research. In this review, we summarize the literature on mitochondrial ferritin expression regulation and its physical and biochemical properties, with particular attention paid to the differences with cytosolic ferritin and its role in physiological condition. Until now, there has been no evidence that the alteration of the mitochondrial ferritin gene is causative of any disorder; however, the identified association of the mitochondrial ferritin with some disorders is discussed.

Objectives GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear. Methods We carried out proteomic analysis and monitored [18F]-fluoro-2-deoxy-d-glucose uptake into brain slices of Gdi1 knockout and wild type control mice. d-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP, d-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible Gdi1 knockout mouse carrying the Gdi1 deletion only in adult astrocytes and conducted behavioural tests. Results Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [18F]-fluoro-2-deoxy-d-glucose revealed an increased d-glucose uptake in Gdi1 knockout tissue versus wild type control mice, consistent with the facilitated d-glucose uptake determined by FRET measurements. In mice with Gdi1 deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-d-glucose injection. Conclusions These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice., Highlights • Mutations in human Gdi1, encoding αGDI, a protein controlling vesicle traffic, are responsible for Intellectual Disability. • Gdi1 knockout revealed significant changes in astrocyte-resident glycolytic enzymes and facilitated D-glucose utilization. • Astrocyte-selective Gdi1 deletion impairs working memory, which can be rescued by administration of 2-deoxy-D-glucose. • Astrocyte-based glycolysis is a new target to treat Intellectual Disability.

The adult brain retains endogenous neural stem/precursors cells (eNPCs) in two major neurogenic niches, the subgranular zone (SGZ) in the hippocampus and the subventricular zone (SVZ). While in humans and rodents the SGZ contributes to memory functions by generating new neurons that integrate into hippocampal circuitry, the role of eNPCs of the SVZ is less clear. SVZ-eNPCs contribute to the generation of new neurons fated to the olfactory bulb (OB) in rodents but not in humans where they are thought to contribute to striatal neurogenesis as a result of tissue damage.Here, we show in mice and humans a novel non-neurogenic physiological role of adult SVZ-eNPCs in supporting cognitive functions by regulating striatal neuronal activity. We first provide evidence that GABAergic transmission between parvalbumin-expressing fast-spiking interneurons (FSIs) and medium spiny neurons (MSNs) is tuned by SVZ-eNPCs via secretion of Insulin-Like Growth Factor Binding Protein Like 1 (IGFBPL-1) that, in turn, regulates the Insulin-Like Growth Factor (IGF-1) signalling cascade. Consistently, selective ablation of SVZ-eNPCs and in vivo disruption of the IGF-signalling determine the impairment of intrastriatal coherence. A finding associated with a higher failure rate of GABAergic transmission mediated by FSIs and with striatum-related behavioural dysfunctions impairing decision making. Human validation studies revealed IGFBPL-1 expression in the SVZ and in foetal and induced-pluripotent stem cell-derived NPCs as well as a strong correlation in neurological patients between SVZ pathological damage, reduction of striatum volume and impairment of information processing speed.All in all, our results highlight a novel non-neurogenic homeostatic role exerted by SVZ-eNPCs on striatal GABAergic neurons that might contribute to cognitive processes involving decisions-making tasks.

SummaryThe functional contribution of somatostatin-expressing interneurons (SST-INs) to the synaptic organization of the striatum is poorly understood. Using electrophysiological recordings, optogenetic stimulation, and single-cell PCR analysis, we investigated functional patterns of synaptic connectivity in striatal SST-INs expressing channelrhodopsin-2. Photostimulation of these cells induced both glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitory postsynaptic currents (IPSCs) in striatal spiny projection neurons (SPNs) and fast-spiking interneurons (FSIs). The two synaptic components showed equally fast onset latencies, suggesting a mechanism of co-transmission. Accordingly, single-cell PCR analysis revealed that individual striatal SST-INs expressed mRNAs for both glutamate and GABA vesicular transporters (VGLUT1 and VGAT, respectively). During relatively prolonged optical stimuli (0.5-1s), IPSC arrays consistently outlasted EPSCs. As a result, photostimulation of SST-INs caused a transient burst of action potentials followed by a prolonged inhibition in postsynaptic cells.These data suggest that striatal SST-INs are specialized to locally project synapses exerting a composite excitatory and inhibitory effect through GABA/glutamate co-transmission onto different postsynaptic targets.

KEY POINTS The mechanisms of action of anaesthetics on the living brain are still poorly understood. In this respect, the analysis of the differential effects of anaesthetics on spontaneous and sensory-evoked cortical activity might provide important and novel cues. Here we show that the anaesthetic sevoflurane strongly silences the brain but potentiates in a dose- and frequency-dependent manner the cortical visual response. Such enhancement arises from a linear scaling by sevoflurane of the power-law relation between light intensity and the cortical response. The fingerprint of sevoflurane action suggests that circuit silencing can boost linearly synaptic responsiveness presumably by scaling the number of responding units and/or their correlation following a sensory stimulation. ABSTRACT General anaesthetics, which are expected to silence brain activity, often spare sensory responses. To evaluate differential effects of anaesthetics on spontaneous and sensory-evoked cortical activity, we characterized their modulation by sevoflurane and propofol. Power spectra and the bust-suppression ratio from EEG data were used to evaluate anaesthesia depth. ON and OFF cortical responses were elicited by light pulses of variable intensity, duration and frequency, during light and deep states of anaesthesia. Both anaesthetics reduced spontaneous cortical activity but sevoflurane greatly enhanced while propofol diminished the ON visual response. Interestingly, the large potentiation of the ON visual response by sevoflurane was found to represent a linear scaling of the encoding mechanism for light intensity. To the contrary, the OFF cortical visual response was depressed by both anaesthetics. The selective depression of the OFF component by sevoflurane could be converted into a robust potentiation by the pharmacological blockade of the ON pathway, suggesting that the temporal order of ON and OFF responses leads to a depression of the latter. This hypothesis agrees with the finding that the enhancement of the ON response was converted into a depression by increasing the frequency of light-pulse stimulation from 0.1 to 1 Hz. Overall, our results support the view that inactivity-dependent modulation of cortical circuits produces an increase in their responsiveness. Among the implications of our findings, the silencing of cortical circuits can boost linearly the cortical responsiveness but with negative impact on their frequency transfer and with a loss of the information content of the sensory signal.

Pantothenate Kinase-associated Neurodegeneration (PKAN) belongs to a wide spectrum of diseases characterized by brain iron accumulation and extrapyramidal motor signs. PKAN is caused by mutations in PANK2, encoding the mitochondrial pantothenate kinase 2, which is the first enzyme of the biosynthesis of Coenzyme A. We established and characterized glutamatergic neurons starting from previously developed PKAN Induced Pluripotent Stem Cells (iPSCs). Results obtained by inductively coupled plasma mass spectrometry indicated a higher amount of total cellular iron in PKAN glutamatergic neurons with respect to controls. PKAN glutamatergic neurons, analyzed by electron microscopy, exhibited electron dense aggregates in mitochondria that were identified as granules containing calcium phosphate. Calcium homeostasis resulted compromised in neurons, as verified by monitoring the activity of calcium-dependent enzyme calpain1, calcium imaging and voltage dependent calcium currents. Notably, the presence of calcification in the internal globus pallidus was confirmed in seven out of 15 genetically defined PKAN patients for whom brain CT scan was available. Moreover, we observed a higher prevalence of brain calcification in females. Our data prove that high amount of iron coexists with an impairment of cytosolic calcium in PKAN glutamatergic neurons, indicating both, iron and calcium dys-homeostasis, as actors in pathogenesis of the disease.

Ideally, elucidating the role of specific brain circuits in animal behavior would require the ability to measure activity at all involved synapses, possibly with unrestricted field of view, thus even at those boutons deeply located into the brain. Here, we introduce and validate an efficient scheme reporting synaptic vesicle cycling in vivo. This is based on SynaptoZip, a genetically encoded molecule deploying in the vesicular lumen a bait moiety designed to capture upon exocytosis a labeled alien peptide, Synbond. The resulting signal is cumulative and stores the number of cycling events occurring at individual synapses. Since this functional signal is enduring and measurable both online and ex post, SynaptoZip provides a unique method for the analysis of the history of synaptic activity in regions several millimeters below the brain surface. We show its broad applicability by reporting stimulus-evoked and spontaneous circuit activity in wide cortical fields, in anesthetized and freely moving animals., Visualization of synaptic activity in the living brain is challenging. This study devises a simple and efficient scheme that reports synaptic vesicle recycling in vivo using SynaptoZip, a genetically encoded sensor of past synaptic activities.

The mechanisms of action of anaesthetics on the living brain are still poorly understood. In this respect, the analysis of the differential effects of anaesthetics on spontaneous and sensory-evoked cortical activity might provide important and novel cues. Here we show that the anaesthetic sevoflurane strongly silences the brain but potentiates in a dose- and frequency-dependent manner the cortical visual response. Such enhancement arises from a linear scaling by sevoflurane of the power-law relation between light intensity and the cortical response. The fingerprint of sevoflurane action suggests that circuit silencing can boost linearly synaptic responsiveness presumably by scaling the number of responding units and/or their correlation following a sensory stimulation.General anaesthetics, which are expected to silence brain activity, often spare sensory responses. To evaluate differential effects of anaesthetics on spontaneous and sensory-evoked cortical activity, we characterized their modulation by sevoflurane and propofol. Power spectra and the bust-suppression ratio from EEG data were used to evaluate anaesthesia depth. ON and OFF cortical responses were elicited by light pulses of variable intensity, duration and frequency, during light and deep states of anaesthesia. Both anaesthetics reduced spontaneous cortical activity but sevoflurane greatly enhanced while propofol diminished the ON visual response. Interestingly, the large potentiation of the ON visual response by sevoflurane was found to represent a linear scaling of the encoding mechanism for light intensity. To the contrary, the OFF cortical visual response was depressed by both anaesthetics. The selective depression of the OFF component by sevoflurane could be converted into a robust potentiation by the pharmacological blockade of the ON pathway, suggesting that the temporal order of ON and OFF responses leads to a depression of the latter. This hypothesis agrees with the finding that the enhancement of the ON response was converted into a depression by increasing the frequency of light-pulse stimulation from 0.1 to 1 Hz. Overall, our results support the view that inactivity-dependent modulation of cortical circuits produces an increase in their responsiveness. Among the implications of our findings, the silencing of cortical circuits can boost linearly the cortical responsiveness but with negative impact on their frequency transfer and with a loss of the information content of the sensory signal.

An inductively coupled plasma machine has been modified to be able to apply working powers in the order of 1 kW, thus switching to the real inductive H-mode. The plasma is generated by applying a 13.56 MHz radio-frequency to a λ/4 antenna outside the plasma chamber in low pressure conditions. The working gas is argon at pressure in the range from 10 to 100 Pa. With this high power source we have been able to perform plasma etching on a poly(vinyl-chloride) (PVC) film. In particular the effect of the plasma is the selective removal of hydrogen and chlorine from the sample surface. The action of the high power plasma on the sample has been proved to be much more effective than that of the low power one. Results similar to those obtained with the low power machine at about 300 W for 120 min, have been obtained with the high power source at about 600 W for 30 min. The superficial generation of a conductive layer of double C=C bonds was obtained. The samples have been investigated by means of ATR spectroscopy, FIB/SEM microscopy and micro-electrical measurements, which revealed the change in charge conductivity.

The endoplasmic reticulum (ER) is involved in many functions, including protein folding, redox homeostasis,and Ca2+ storage and signaling. To perform these multiple tasks, the ER is composed of distinct,specialized subregions, amongst which mitochondrial-associated ER membranes (MAM) emerge as key signalinghubs. How these multiple functions are integrated with one another in living cells remains unclear.Results: Here we show that Ero1a, a key controller of oxidative folding and ER redox homeostasis, is enriched inMAM and regulates Ca2+ fluxes. Downregulation of Ero1a by RNA interference inhibits mitochondrial Ca2+fluxes and modifies the activity of mitochondrial Ca2+ uniporters. The overexpression of redox active Ero1aincreases passive Ca2+ efflux from the ER, lowering [Ca2+]ER and mitochondrial Ca2+ fluxes in response to IP3agonists. Innovation: The unexpected observation that Ca2+ fluxes are affected by either increasing or decreasingthe levels of Ero1a reveals a pivotal role for this oxidase in the early secretory compartment and implies a strictcontrol of its amounts. Conclusions: Taken together, our results indicate that the levels, subcellular localization,and activity of Ero1a coordinately regulate Ca2+ and redox homeostasis and signaling in the early secretorycompartment. Antioxid. Redox Signal. 16, 1077–1087.

Calcium (Ca(2+)) waves and Ca(2+) oscillations within cells initiate a wide range of physiological processes including control of cell signaling, gene expression, secretion, and cell migration. A thorough analysis of Ca(2+) waves in glial cells provides information not only about the subcellular location of signaling processing events but also about nonneuronal or intercellular signaling pathways, their timing, routes, spatial domains, and coordination. In this study, three novel image processing methods have been applied to the study of Ca(2+) dynamics in cells. These bring additional information to the methods already available in the literature, providing insight into the analysis of calcium dynamics in fluorescence recordings and defining bidimensional maps that give a complete and detailed description of calcium intracellular behavior. The application of these processing methods to glial cells highlighted the complex 2-D Ca(2+) dynamics phenomena, the location of calcium uptake and release microdomains on the endoplasmic reticulum, and the correlation between different calcium signals inside the cell. A perinuclear zone acting as a filter and regulator of intracellular calcium waves was detected: it acts as a controller of calcium fluxes between the cytoplasm and the nucleus.

In the last decade new ideas were born about the temporal and spatial dynamics of intercellular calcium waves in astrocytes. In this paper we introduce a new approach to analyze the ways in which astrocytes communicate in cultures. We present a method to describe the spatial propagation of Ca(2+) waves in vitro and a technique to compare the activity of different cells in vivo and in vitro under different stimulation conditions. The proposed method resulted to be an interesting way to distinguish different astrocyte clusters, which can be related to the communication characteristics in the network.

One of the main problems that affect fluoroscopic imaging is the difficulty in coupling the recorded activity with the morphological information. The comprehension of fluorescence events in relationship with the internal structure of the cell can be very difficult. At this purpose, we developed a new method able to maximize the fluoroscopic movie quality. The method (Maximum Intensity Enhancement, MIE) works as follow: considering all the frames that compose the fluoroscopic movie, the algorithm extracts, for each pixel of the matrix, the maximal brightness value assumed along all the frames. Such values are collected in a maximum intensity matrix. Then, the method provides the projection of the target molecule oscillations which are present in the DeltaF/F(0) movie onto the maximum intensity matrix. This is done by creating a RGB movie and by assigning to the normalized (DeltaF/F(0)) activity a single channel and by reproducing the maximum intensity matrix on all the frames by using the remaining color channels. The application of such a method to fluoroscopic calcium imaging of astrocyte cultures demonstrated a meaningful enhancement in the possibility to discern the internal and external structure of cells.

Although patients with chronic renal failure are increasing worldwide, many aspects of kidney biology remain to be elucidated. Recent research has uncovered several molecular properties of the glomerular filtration barrier, in which podocytes, highly differentiated, ramified cells that enwrap the glomerular basement membrane, have been reported to be mainly responsible for filter's selectivity. We previously described that podocytes express Rab3A, a GTPase restricted to cell types that are capable of highly regulated exocytosis, such as neuronal cells. Here, we first demonstrate by a proteomic study that Rab3A in podocytes coimmmunoprecipitates with molecules once thought to be synapse specific. We then show that podocytes possess structures resembling synaptic vesicles, which contain glutamate, coexpress Rab3A and synaptotagmin 1, and undergo spontaneous and stimulated exocytosis and recycling, with glutamate release. Finally, from the results of a cDNA microarray study, we describe the presence of a series of neuron- and synapse-specific molecules in normal human glomeruli and confirm the glomerular protein expression of both metabotropic and ionotropic glutamate receptors. These data point toward a synaptic-like mechanism of communication among glomerular cells, which perfectly fits with the molecular composition of the glomerular filter and puts in perspective several previous observations, proposing a different working hypothesis for understanding glomerular signaling dynamics.