Friedreich ataxia-induced pluripotent stem cell-derived neurons show a cellular phenotype that is corrected by a benzamide HDAC inhibitor.

We employed induced pluripotent stem cell (iPSC)-derived neurons obtained from Friedreich ataxia (FRDA) patients and healthy subjects, FRDA neurons and CT neurons, respectively, to unveil phenotypic alterations related to frataxin (FXN) deficiency and investigate if they can be reversed by treatments that upregulate FXN. FRDA and control iPSCs were equally capable of differentiating into a neuronal or astrocytic phenotype. FRDA neurons showed lower levels of iron­sulfur (Fe­S) and lipoic acid-containing proteins, higher labile iron pool (LIP), higher expression of mitochondrial superoxide dismutase (SOD2), increased reactive oxygen species (ROS) and lower reduced glutathione (GSH) levels, and enhanced sensitivity to oxidants compared with CT neurons, indicating deficient Fe­S cluster biogenesis, altered iron metabolism, and oxidative stress. Treatment with the benzamide HDAC inhibitor 109 significantly upregulated FXN expression and increased Fe­S and lipoic acid-containing protein levels, downregulated SOD2 levels, normalized LIP and ROS levels, and almost fully protected FRDA neurons from oxidative stress-mediated cell death. Our findings suggest that correction of FXN deficiency may not only stop disease progression, but also lead to clinical improvement by rescuing still surviving, but dysfunctional neurons.

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. An analysis with cyclic voltammetry on the purified protein showed that this structural modification severely reduces the ability of the protein to store iron. In order to analyze the impact of the mutation in vivo, we generated mouse models for the some pathogenic human FTL gene in FVB and C57BL/6J strains. Transgenic mice in the FVB background showed high accumulation of the mutated ferritin in brain where it correlated with increased iron deposition with age, as scored by magnetic resonance imaging. Notably, the accumulation of iron-ferritin bodies was accompanied by signs of oxidative damage. In the C57BL/6 background, both the expression of the mutant ferritin and the iron levels were lower than in the FVB strain. Nevertheless, also these mice showed oxidative alterations in the brain. Furthermore, post-natal hippocampal neurons obtained from these mice experienced a marked increased cell death in response to chronic iron overload and/or acute oxidative stress, in comparison to wild-type neurons. Ultrastructural analyses revealed an accumulation of lipofuscin granules associated with iron deposits, particularly enriched in the cerebellum and striatum of our transgenic mice. Finally, experimental subjects were tested throughout development and aging at 2-, 8- and 18-months for behavioral phenotype. Rotarod test revealed a progressive impaired motor coordination building up with age, FTL mutant old mice showing a shorter latency to fall from the apparatus, according to higher accumulation of iron aggregates in the striatum. Our data show that our 498-499InsTC mouse models recapitulate early pathological and clinical traits of the human neuroferritinopathy, thus providing a valuable model for the study of the disease. Finally, we propose a mechanistic model of lipofuscine formation that can account for the etiopathogenesis of human neuroferritinopathy.

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. Our work investigates the mechanisms of iron entry in cultures of quiescent and activated hippocampal astrocytes. Our data confirm that the main source of iron is the non-transferrin-bound iron (NTBI) and show the involvement of two different routes for its entry: the resident transient receptor potential (TRP) channels in quiescent astrocytes and the de novo expressed divalent metal transporter 1 (DMT1) in activated astrocytes, which accounts for a potentiation of iron entry. Overall, our data suggest that at rest, but even more after activation, astrocytes have the potential to buffer the excess of iron, thereby protecting neurons from iron overload. These findings further extend our understanding of the protective role of astrocytes under the conditions of iron-mediated oxidative stress observed in several neurodegenerative conditions.

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. In this work, we investigated how activation affects the competence of astrocytes to handle iron overload and the ensuing oxidative stress. METHODS: Cultures of pure cortical astrocytes were preincubated with proinflammatory cytokines (interleukin-1ß and tumor necrosis factor α) or conditioned medium from lipopolysaccharide-activated microglia to promote activation and then exposed to a protocol of iron overload. RESULTS: We 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. CONCLUSIONS: We 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.

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 Fe²âº 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.

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. In particular, we studied the ability of CGRP and adrenomedullin to modulate microglia activation, i.e. its competence of producing and releasing pro-inflammatory cytokines/chemokines that are known to play a crucial role in neuroinflammation. In this work we show that the two neuropeptides exert a potent inhibitory effect on lipopolysaccharide-induced microglia activation in vitro, with strong inhibition of the release of pro-inflammatory mediators (such as NO, cytokines and chemokines). Both CGRP and adrenomedullin are known to promote cAMP elevation, this second messenger cannot fully account for the observed inhibitory effects, thereby suggesting that other signaling pathways are involved. Interestingly, the inhibitory effect of CGRP and adrenomedullin appears to be stimulus specific, since direct activation with pro-inflammatory cytokines was not affected. Our findings clarify aspects of microglia activation, and contribute to the comprehension of the switch from reparative to detrimental function that occurs when glia is exposed to different conditions. Moreover, they draw the attention to potential targets for novel pharmacological intervention in pathologies characterized by glia activation and neuroinflammation.

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. 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.

ß-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 ß-secretase that cleaves the amyloid-ß precursor protein, thus allowing the production of amyloid-ß, 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-ß in brain, we have investigated the expression of these two enzymes in primary cultures of astrocytes. We show that astrocytes possess ß-secretase activity and produce amyloid-ß 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 ß-secretase activity, also depending on the differential responsivity of the brain regions.

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. Indeed, chronic microglia activation is commonly recognized as a hallmark of many neurological disorders, especially at an early stage. Many complex pathways, including cytoskeletal remodeling, are involved in the control of the microglial phenotypical and morphological changes that occur during activation. In this work, we focused on the small GTPase Gα13 and its role at the crossroad between RhoA and Rac1 signaling when microglia is exposed to pro-inflammatory stimulation. We propose the direct involvement of Gα13 in the cytoskeletal rearrangements mediated by FAK, LIMK/cofilin, and Rac1 during microglia activation. In fact, we show that Gα13 knockdown significantly inhibited LPS-induced microglial cell activation, in terms of both changes in morphology and migration, through the modulation of FAK and one of its downstream effectors, Rac1. In conclusion, we propose Gα13 as a critical factor in the regulation of morphological and functional properties of microglia during activation, which might become a target of intervention for the control of microglia inflammation.

Casein Kinase 2 dependent phosphorylation of eIF4B regulates BACE1 expression in Alzheimer's disease.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder. Increased Aß 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 Aß 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.

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. The stimulation was not blocked by CGRP8-37, a specific antagonist, suggesting the activation of other related receptors. CGRP did not affect survival of Purkinje cells, granule cells or astrocytes. The selective expression of Receptor Component Protein (RCP) (a component of CGRP receptor family) in astrocytes points to a role of these cells as mediators of CGRP effect. Finally, in pure cerebellar astrocyte cultures CGRP induced a transient morphological differentiation from flat, polygonal to stellate form. It is concluded that CGRP influences Purkinje cell dendrite growth in vitro, most likely through the involvement of astrocytes.

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 Ca2+ 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. In the SPG7 animal model we evaluate the potential improvement of the motor defect, neuroinflammation and neurodegeneration by means of an mPTP inducer, the benzodiazepine Bz-423. FINDINGS: We demonstrate that paraplegin is required for efficient transient opening of the mPTP, that is impaired in both SPG7 patients-derived fibroblasts and primary neurons from Spg7-/- mice. We show that dysregulation of mPTP opening at the pre-synaptic terminal impairs neurotransmitter release leading to ineffective synaptic transmission. Lack of paraplegin impairs mPTP flickering by a mechanism involving increased expression and activity of sirtuin3, which promotes deacetylation of cyclophilin D, thus hampering mPTP opening. Pharmacological treatment with Bz-423, which bypasses the activity of CypD, normalizes synaptic transmission and rescues the motor impairment of the SPG7 mouse model. INTERPRETATION: mPTP targeting opens a new avenue for the potential therapy of this form of spastic paraplegia. FUNDING: Telethon Foundation grant (TGMGCSBX16TT); Dept. of Defense, US Army, grant W81XWH-18-1-0001.

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. An up-regulation of the protein has been described in some AD patients without a change in transcript levels. The features of BACE1 5' untranslated region (5' UTR), such as the length, GC content, evolutionary conservation and presence of upstream AUGs (uAUGs), indicate an important regulatory role of this 5' UTR in translational control. We demonstrate that, in brain and pancreas, almost all of the native BACE1 mRNA contains the full-length 5' UTR. RNA transfection and in vitro translation show that translation is mainly inhibited by the presence of the uAUGs. We provide a mutational analysis that highlight the second uAUG as the main inhibitory element while mutations of all four uAUGs fully de-repress translation. Furthermore, we have evidence that a sequence within the region 222-323 of the BACE1 5' UTR has a stimulatory effect on translation that might depend on the presence of trans-acting factors.

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. Based on this evidence, it is expected that the pharmacology of ASIC1cannot be properly characterized by single-cell voltage-clamp, an experimental condition in which membrane potential is maintained close to resting values. In particular, these measurements do not take into account the influence of the membrane potential depolarization induced by ASIC1a activation during acidosis or neuronal activity. We show here the voltage-dependence of some small molecules antagonists (diminazene, amiloride and a new patented drug from Merck), but not of Psalmotoxin 1, a peptide binding to regions other than the pore. We also demonstrate that the opening of ASIC1a induced by moderate acidosis determines a depolarization sufficient to change the affinity of small molecule antagonists. The characterization of this mechanism was performed on CHO-K1 expressing ASIC1a and further confirmed in hippocampal neurons in culture. Finally, perforated-patch experiments indicate that intracellular modulations do not play a role in the voltage-dependent binding of small molecules. Since ASIC1a activation promotes a membrane depolarization that may influence the binding of small molecules, we propose to adopt experimental methods that do not interfere with the membrane potential for the drug screening of ASIC1a modulators.

Upregulation of Peroxiredoxin 3 Protects Afg3l2-KO Cortical Neurons In Vitro 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 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.

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. CGRP triggered transient increases in intracellular Ca(2+) in astrocytes in culture as well as in acute slices. Responses were observed in the concentration range of 1 nm to 1 mm, in both the cell body and its processes. The calcium transients were dependent on release from intracellular stores as they were blocked by thapsigargin but not by the absence of extracellular calcium. In addition, after CGRP application a further delayed transient increase in calcium activity could be observed. Finally, cerebellar astrocytes from neonatal mice expressed receptor component protein, a component of the CGRP receptor, as revealed by immunofluorescence and confocal microscopy. It is thus proposed that the CGRP-containing afferent fibers in the cerebellum (the climbing fibers) modulate calcium in astrocytes by releasing the neuropeptide during development and hence possibly influence the differentiation of Purkinje cells.

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.

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. Using new tools, which range from high-throughput microscopy to in cell infrared, we prove that overexpression of the frataxin gene affects the cellular metabolism. It also leads to a significant increase of oxidative stress and labile iron pool levels. These cellular alterations are similar to those observed when the gene is partly silenced, as occurs in Friedreich's ataxia patients. Our data suggest that the levels of frataxin must be tightly regulated and fine-tuned, with any imbalance leading to oxidative stress and toxicity.

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. Both DAG production and PKCgamma translocation were localized processes, typically observed within discrete microdomains along the dendritic branches. Markedly, intermediate-strength NMDA receptor (NMDAR) activation or moderate electrical stimulation generated Ca2+ but no DAG signals, whereas mGluR activation generated DAG but no Ca2+ signals. Both receptors were needed for PKCgamma activation. This suggests that a coincidence detection process exists between iGluRs and mGluRs that relies on a molecular coincidence detection process based on the corequirement of Ca2+ and DAG for PKCgamma activation. Nevertheless, the requirement for costimulation with mGluRs could be overcome for maximal NMDAR stimulation through a direct production of DAG via activation of the Ca2+-sensitive PLCdelta (phospholipase Cdelta) isoform. In a second important exception, mGluRs were sufficient for PKCgamma activation in neurons in which Ca2+ stores were loaded by previous electrical activity. Together, the dual activation requirement for PKCgamma provides a plausible molecular interpretation for different synergistic contributions of mGluRs to long-term potentiation and other synaptic plasticity processes.

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. Ser504 phosphorylation increases eIF4B recruitment to the pre-initiation complex and influences eIF4B localization at synapses. Moreover, Ser504 phosphorylation modulates the translation of protein kinase Mζ. Therefore, by sensing synaptic activity, eIF4B could adjust translation to neuronal needs, promoting adaptive changes in synaptic plasticity. We also show that Ser504 phosphorylation is increased in vivo in a rat model of epilepsy during epileptogenesis i.e. when translation drives maladaptive synaptic changes. We propose eIF4B as a mediator between neuronal activity and translation, with relevance in the control of synaptic plasticity.