Mitochondrial iron deficiency triggers cytosolic iron overload in PKAN hiPS-derived astrocytes.

Disease models of neurodegeneration with brain iron accumulation (NBIA) offer the possibility to explore the relationship between iron dyshomeostasis and neurodegeneration. We analyzed hiPS-derived astrocytes from PANK2-associated neurodegeneration (PKAN), an NBIA disease characterized by progressive neurodegeneration and high iron accumulation in the globus pallidus. Previous data indicated that PKAN astrocytes exhibit alterations in iron metabolism, general impairment of constitutive endosomal trafficking, mitochondrial dysfunction and acquired neurotoxic features. Here, we performed a more in-depth analysis of the interactions between endocytic vesicles and mitochondria via superresolution microscopy experiments. A significantly lower number of transferrin-enriched vesicles were in contact with mitochondria in PKAN cells than in control cells, confirming the impaired intracellular fate of cargo endosomes. The investigation of cytosolic and mitochondrial iron parameters indicated that mitochondrial iron availability was substantially lower in PKAN cells compared to that in the controls. In addition, PKAN astrocytes exhibited defects in tubulin acetylation/phosphorylation, which might be responsible for unregulated vesicular dynamics and inappropriate iron delivery to mitochondria. Thus, the impairment of iron incorporation into these organelles seems to be the cause of cell iron delocalization, resulting in cytosolic iron overload and mitochondrial iron deficiency, triggering mitochondrial dysfunction. Overall, the data elucidate the mechanism of iron accumulation in CoA deficiency, highlighting the importance of mitochondrial iron deficiency in the pathogenesis of disease.

Iron imbalance in neurodegeneration.

Iron is an essential element for the development and functionality of the brain, and anomalies in its distribution and concentration in brain tissue have been found to be associated with the most frequent neurodegenerative diseases. When magnetic resonance techniques allowed iron quantification in vivo, it was confirmed that the alteration of brain iron homeostasis is a common feature of many neurodegenerative diseases. However, whether iron is the main actor in the neurodegenerative process, or its alteration is a consequence of the degenerative process is still an open question. Because the different iron-related pathogenic mechanisms are specific for distinctive diseases, identifying the molecular mechanisms common to the various pathologies could represent a way to clarify this complex topic. Indeed, both iron overload and iron deficiency have profound consequences on cellular functioning, and both contribute to neuronal death processes in different manners, such as promoting oxidative damage, a loss of membrane integrity, a loss of proteostasis, and mitochondrial dysfunction. In this review, with the attempt to elucidate the consequences of iron dyshomeostasis for brain health, we summarize the main pathological molecular mechanisms that couple iron and neuronal death.

Neural precursor cells tune striatal connectivity through the release of IGFBPL1.

The adult brain retains over life endogenous neural stem/precursor cells (eNPCs) within the subventricular zone (SVZ). Whether or not these cells exert physiological functions is still unclear. In the present work, we provide evidence that SVZ-eNPCs tune structural, electrophysiological, and behavioural aspects of striatal function via secretion of insulin-like growth factor binding protein-like 1 (IGFBPL1). In mice, selective ablation of SVZ-eNPCs or selective abrogation of IGFBPL1 determined an impairment of striatal medium spiny neuron morphology, a higher failure rate in GABAergic transmission mediated by fast-spiking interneurons, and striatum-related behavioural dysfunctions. We also found IGFBPL1 expression in the human SVZ, foetal and induced-pluripotent stem cell-derived NPCs. Finally, we found a significant correlation between SVZ damage, reduction of striatum volume, and impairment of information processing speed in neurological patients. Our results highlight the physiological role of adult SVZ-eNPCs in supporting cognitive functions by regulating striatal neuronal activity.

PKAN hiPS-Derived Astrocytes Show Impairment of Endosomal Trafficking: A Potential Mechanism Underlying Iron Accumulation.

PKAN disease is caused by mutations in the PANK2 gene, encoding the mitochondrial enzyme pantothenate kinase 2, catalyzing the first and key reaction in Coenzyme A (CoA) biosynthetic process. This disorder is characterized by progressive neurodegeneration and excessive iron deposition in the brain. The pathogenic mechanisms of PKAN are still unclear, and the available therapies are only symptomatic. Although iron accumulation is a hallmark of PKAN, its relationship with CoA dysfunction is not clear. We have previously developed hiPS-derived astrocytes from PKAN patients showing iron overload, thus recapitulating the human phenotype. In this work, we demonstrated that PKAN astrocytes presented an increase in transferrin uptake, a key route for cellular iron intake via transferrin receptor-mediated endocytosis of transferrin-bound iron. Investigation of constitutive exo-endocytosis and vesicular dynamics, exploiting the activity-enriching biosensor SynaptoZip, led to the finding of a general impairment in the constitutive endosomal trafficking in PKAN astrocytes. CoA and 4-phenylbutyric acid treatments were found to be effective in partially rescuing the aberrant vesicular behavior and iron intake. Our results demonstrate that the impairment of CoA biosynthesis could interfere with pivotal intracellular mechanisms involved in membrane fusions and vesicular trafficking, leading to an aberrant transferrin receptor-mediated iron uptake.

Massive iron accumulation in PKAN-derived neurons and astrocytes: light on the human pathological phenotype.

Neurodegeneration associated with defective pantothenate kinase-2 (PKAN) is an early-onset monogenic autosomal-recessive disorder. The hallmark of the disease is the massive accumulation of iron in the globus pallidus brain region of patients. PKAN is caused by mutations in the PANK2 gene encoding the mitochondrial enzyme pantothenate kinase-2, whose function is to catalyze the first reaction of the CoA biosynthetic pathway. To date, the way in which this alteration leads to brain iron accumulation has not been elucidated. Starting from previously obtained hiPS clones, we set up a differentiation protocol able to generate inhibitory neurons. We obtained striatal-like medium spiny neurons composed of approximately 70-80% GABAergic neurons and 10-20% glial cells. Within this mixed population, we detected iron deposition in both PKAN cell types, however, the viability of PKAN GABAergic neurons was strongly affected. CoA treatment was able to reduce cell death and, notably, iron overload. Further differentiation of hiPS clones in a pure population of astrocytes showed particularly evident iron accumulation, with approximately 50% of cells positive for Perls staining. The analysis of these PKAN astrocytes indicated alterations in iron metabolism, mitochondrial morphology, respiratory activity, and oxidative status. Moreover, PKAN astrocytes showed signs of ferroptosis and were prone to developing a stellate phenotype, thus gaining neurotoxic features. This characteristic was confirmed in iPS-derived astrocyte and glutamatergic neuron cocultures, in which PKAN glutamatergic neurons were less viable in the presence of PKAN astrocytes. This newly generated astrocyte model is the first in vitro disease model recapitulating the human phenotype and can be exploited to deeply clarify the pathogenetic mechanisms underlying the disease.

Mitochondrial Ferritin: Its Role in Physiological and Pathological Conditions.

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.

Pathogenic mechanism and modeling of neuroferritinopathy.

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.

Inhibiting glycolysis rescues memory impairment in an intellectual disability Gdi1-null mouse.

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.

Neuronal Ablation of CoA Synthase Causes Motor Deficits, Iron Dyshomeostasis, and Mitochondrial Dysfunctions in a CoPAN Mouse Model.

COASY protein-associated neurodegeneration (CoPAN) is a rare but devastating genetic autosomal recessive disorder of inborn error of CoA metabolism, which shares with pantothenate kinase-associated neurodegeneration (PKAN) similar features, such as dystonia, parkinsonian traits, cognitive impairment, axonal neuropathy, and brain iron accumulation. These two disorders are part of the big group of neurodegenerations with brain iron accumulation (NBIA) for which no effective treatment is available at the moment. To date, the lack of a mammalian model, fully recapitulating the human disorder, has prevented the elucidation of pathogenesis and the development of therapeutic approaches. To gain new insights into the mechanisms linking CoA metabolism, iron dyshomeostasis, and neurodegeneration, we generated and characterized the first CoPAN disease mammalian model. Since CoA is a crucial metabolite, constitutive ablation of the Coasy gene is incompatible with life. On the contrary, a conditional neuronal-specific Coasy knock-out mouse model consistently developed a severe early onset neurological phenotype characterized by sensorimotor defects and dystonia-like movements, leading to premature death. For the first time, we highlighted defective brain iron homeostasis, elevation of iron, calcium, and magnesium, together with mitochondrial dysfunction. Surprisingly, total brain CoA levels were unchanged, and no signs of neurodegeneration were present.

Harmful Iron-Calcium Relationship in Pantothenate kinase Associated Neurodegeneration.

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.

Stem Cell Modeling of Neuroferritinopathy Reveals Iron as a Determinant of Senescence and Ferroptosis during Neuronal Aging.

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.

Functional mapping of brain synapses by the enriching activity-marker SynaptoZip.

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.

Linear transformation of the encoding mechanism for light intensity underlies the paradoxical enhancement of cortical visual responses by sevoflurane.

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.

Scale Invariant Disordered Nanotopography Promotes Hippocampal Neuron Development and Maturation with Involvement of Mechanotransductive Pathways.

The identification of biomaterials which promote neuronal maturation up to the generation of integrated neural circuits is fundamental for modern neuroscience. The development of neural circuits arises from complex maturative processes regulated by poorly understood signaling events, often guided by the extracellular matrix (ECM). Here we report that nanostructured zirconia surfaces, produced by supersonic cluster beam deposition of zirconia nanoparticles and characterized by ECM-like nanotopographical features, can direct the maturation of neural networks. Hippocampal neurons cultured on such cluster-assembled surfaces displayed enhanced differentiation paralleled by functional changes. The latter was demonstrated by single-cell electrophysiology showing earlier action potential generation and increased spontaneous postsynaptic currents compared to the neurons grown on the featureless unnaturally flat standard control surfaces. Label-free shotgun proteomics broadly confirmed the functional changes and suggests furthermore a vast impact of the neuron/nanotopography interaction on mechanotransductive machinery components, known to control physiological in vivo ECM-regulated axon guidance and synaptic plasticity. Our results indicate a potential of cluster-assembled zirconia nanotopography exploitable for the creation of efficient neural tissue interfaces and cell culture devices promoting neurogenic events, but also for unveiling mechanotransductive aspects of neuronal development and maturation.

Effects of the Concomitant Activation of ON and OFF Retinal Ganglion Cells on the Visual Thalamus: Evidence for an Enhanced Recruitment of GABAergic Cells.

A fundamental question in vision neuroscience is how parallel processing of Retinal Ganglion Cell (RGC) signals is integrated at the level of the visual thalamus. It is well-known that parallel ON-OFF pathways generate output signals from the retina that are conveyed to the dorsal lateral geniculate nucleus (dLGN). However, it is unclear how these signals distribute onto thalamic cells and how these two pathways interact. Here, by electrophysiological recordings and c-Fos expression analysis, we characterized the effects of pharmacological manipulations of the retinal circuit aimed at inducing either a selective activation of a single pathway, OFF RGCs [intravitreal L-(+)-2-Amino-4-phosphonobutyric, L-AP4] or an unregulated activity of all classes of RGCs (intravitreal 4-Aminopyridine, 4-AP). In in vivo experiments, the analysis of c-Fos expression in the dLGN showed that these two manipulations recruited active cells from the same area, the lateral edge of the dLGN. Despite this similarity, the unregulated co-activation of both ON and OFF pathways by 4-AP yielded a much stronger recruitment of GABAergic interneurons in the dLGN when compared to L-AP4 pure OFF activation. The increased activation of an inhibitory thalamic network by a high level of unregulated discharge of ON and OFF RGCs might suggest that cross-inhibitory pathways between opposing visual channels are presumably replicated at multiple levels in the visual pathway, thus increasing the filtering ability for non-informative or noisy visual signals.

A conditional transgenic reporter of presynaptic terminals reveals novel features of the mouse corticospinal tract.

In many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), synaptic alterations precede the demise of the neuronal cell, making synapses a useful vantage point from which to monitor the onset and progression of clinical signs and pathological changes. While murine models of ALS display many features in common with the clinical picture observed in patients, corticospinal tract (CST) involvement is usually less severe in mice than the picture observed in humans. In this paper we describe the characterization of a new conditional transgenic line obtained by targeted integration of a GFP-VAMP2 fusion gene into the Rosa26 locus, and devised to permit the detection of genetically defined presynaptic terminals in wild type mice and murine models of neural disorders. This reporter molecule is selectively enriched in presynaptic boutons, significantly reducing the background signal produced by fibers of passage. The specific features of this reporter line allow us to strongly support the view that murine CST terminals give rise to very few direct contacts with spinal motor neurons. Moreover, the evidence described here reveals the existence of previously uncharacterized, putative direct connections between CST presynaptic boutons and Renshaw neurons in the spinal cord. These results constitute a proof of concept for the potential application of this indicator line to morphological analyses of wild type and diseased synapses.

Ero1α regulates Ca(2+) fluxes at the endoplasmic reticulum-mitochondria interface (MAM).

AIMS: The endoplasmic reticulum (ER) is involved in many functions, including protein folding, redox homeostasis, and Ca(2+) 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 signaling hubs. How these multiple functions are integrated with one another in living cells remains unclear. RESULTS: Here we show that Ero1α, a key controller of oxidative folding and ER redox homeostasis, is enriched in MAM and regulates Ca(2+) fluxes. Downregulation of Ero1α by RNA interference inhibits mitochondrial Ca(2+) fluxes and modifies the activity of mitochondrial Ca(2+) uniporters. The overexpression of redox active Ero1α increases passive Ca(2+) efflux from the ER, lowering [Ca(2+)](ER) and mitochondrial Ca(2+) fluxes in response to IP3 agonists. INNOVATION: The unexpected observation that Ca(2+) fluxes are affected by either increasing or decreasing the levels of Ero1α reveals a pivotal role for this oxidase in the early secretory compartment and implies a strict control of its amounts. CONCLUSIONS: Taken together, our results indicate that the levels, subcellular localization, and activity of Ero1α coordinately regulate Ca(2+) and redox homeostasis and signaling in the early secretory compartment.

Novel image processing methods for the analysis of calcium dynamics in glial cells.

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.

Exploiting the multiplicative nature of fluoroscopic image stochastic noise to enhance calcium imaging recording quality.

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.

Glomerular podocytes contain neuron-like functional synaptic vesicles.

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.