Vitamin E prevents lipid peroxidation and iron accumulation in PLA2G6-Associated Neurodegeneration.

BACKGROUND: PLA2G6-Associated Neurodegeneration (PLAN) is a rare neurodegenerative disease with autosomal recessive inheritance, which belongs to the NBIA (Neurodegeneration with Brain Iron Accumulation) group. Although the pathogenesis of the disease remains largely unclear, lipid peroxidation seems to play a central role in the pathogenesis. Currently, there is no cure for the disease. OBJECTIVE: In this work, we examined the presence of lipid peroxidation, iron accumulation and mitochondrial dysfunction in two cellular models of PLAN, patients-derived fibroblasts and induced neurons, and assessed the effects of α-tocopherol (vitamin E) in correcting the pathophysiological alterations in PLAN cell cultures. METHODS: Pathophysiological alterations were examined in fibroblasts and induced neurons generated by direct reprograming. Iron and lipofuscin accumulation were assessed using light and electron microscopy, as well as biochemical analysis techniques. Reactive Oxygen species production, lipid peroxidation and mitochondrial dysfunction were measured using specific fluorescent probes analysed by fluorescence microscopy and flow cytometry. RESULTS: PLAN fibroblasts and induced neurons clearly showed increased lipid peroxidation, iron accumulation and altered mitochondrial membrane potential. All these pathological features were reverted with vitamin E treatment. CONCLUSIONS: PLAN fibroblasts and induced neurons reproduce the main pathological alterations of the disease and provide useful tools for disease modelling. The main pathological alterations were corrected by Vitamin E supplementation in both models, suggesting that blocking lipid peroxidation progression is a critical therapeutic target.

Kainate receptor modulation of glutamatergic synaptic transmission in the CA2 region of the hippocampus.

Kainate (KA) receptors (KARs) are important modulators of synaptic transmission. We studied here the role of KARs on glutamatergic synaptic transmission in the CA2 region of the hippocampus where the actions of these receptors are unknown. We observed that KA depresses glutamatergic synaptic transmission at Schaffer collateral-CA2 synapses; an effect that was antagonized by NBQX (a KA/AMPA receptors antagonist) under condition where AMPA receptors were previously blocked. The study of paired-pulse facilitation ratio, miniature responses, and fluctuation analysis indicated a presynaptic locus of action for KAR. Additionally, we determined the action mechanism for this depression of glutamate release mediated by the activation of KARs. We found that inhibition of protein kinase A suppressed the effect of KAR activation on evoked excitatory post-synaptic current, an effect that was not suppressed by protein kinase C inhibitors. Furthermore, in the presence of Pertussis toxin, the depression of glutamate release mediated by KAR activation was not present, invoking the participation of a Gi/o protein in this modulation. Finally, the KAR-mediated depression of glutamate release was not suppressed by treatments that affect calcium entry trough voltage-dependent calcium channels or calcium release from intracellular stores. We conclude that KARs present at these synapses mediate a depression of glutamate release through a mechanism that involves the activation of G protein and protein kinase A.

Cannabinoid type-1 receptor blockade restores neurological phenotypes in two models for Down syndrome.

Intellectual disability is the most limiting hallmark of Down syndrome, for which there is no gold-standard clinical treatment yet. The endocannabinoid system is a widespread neuromodulatory system involved in multiple functions including learning and memory processes. Alterations of this system contribute to the pathogenesis of several neurological and neurodevelopmental disorders. However, the involvement of the endocannabinoid system in the pathogenesis of Down syndrome has not been explored before. We used the best-characterized preclinical model of Down syndrome, the segmentally trisomic Ts65Dn model. In male Ts65Dn mice, cannabinoid type-1 receptor (CB1R) expression was enhanced and its function increased in hippocampal excitatory terminals. Knockdown of CB1R in the hippocampus of male Ts65Dn mice restored hippocampal-dependent memory. Concomitant with this result, pharmacological inhibition of CB1R restored memory deficits, hippocampal synaptic plasticity and adult neurogenesis in the subgranular zone of the dentate gyrus. Notably, the blockade of CB1R also normalized hippocampal-dependent memory in female Ts65Dn mice. To further investigate the mechanisms involved, we used a second transgenic mouse model overexpressing a single gene candidate for Down syndrome cognitive phenotypes, the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A). CB1R pharmacological blockade similarly improved cognitive performance, synaptic plasticity and neurogenesis in transgenic male Dyrk1A mice. Our results identify CB1R as a novel druggable target potentially relevant for the improvement of cognitive deficits associated with Down syndrome.

Kainate Receptor-Mediated Depression of Glutamate Release Involves Protein Kinase A in the Cerebellum.

Kainate (KA) receptors (KAR) have important modulatory roles of synaptic transmission. In the cerebellum, the action mechanisms of KAR-mediated glutamatergic depression are unknown. We studied these mechanisms by recording evoked excitatory postsynaptic currents (eEPSCs) from cerebellar slices using the whole-cell configuration of the patch-clamp technique. We observed that 3 µM KA decreased the amplitude of eEPSCs and increased the number of failures at the synapses established between parallel fibers (PF) and Purkinje neurons, and the effect was antagonized by NBQX under the condition where AMPA receptors were previously blocked. The inhibition of protein kinase A (PKA) suppressed the effect of KAR activation on eEPSC, and effect was not prevented by protein kinase C inhibitors. Furthermore, in the presence of Pertussis toxin, the depression of glutamate release mediated by KAR activation was prevented, invoking the participation of a Gi/o protein in this modulation. Finally, the KAR-mediated depression of glutamate release was not prevented by blocking calcium-permeable KARs or by treatments that affect calcium release from intracellular stores. We conclude that KARs present at these synapses mediate an inhibition of glutamate release through a mechanism that involves the activation of G-protein and protein kinase A.

Kainate receptors: from synaptic activity to disease.

Kainate receptors (KARs) are glutamate receptors that participate in the postsynaptic transmission of information and in the control of neuronal excitability, as well as presynaptically modulating the release of the neurotransmitters GABA and glutamate. These modulatory effects, general follow a biphasic pattern, with low KA concentrations provoking an increase in GABA and glutamate release, and higher concentrations mediating a decrease in the release of these neurotransmitters. In addition, KARs are involved in different forms of long- and short-term plasticity. Importantly, altered activity of these receptors has been implicated in different central nervous system diseases and disturbances. Here, we describe the pre- and postsynaptic actions of KARs, and the possible role of these receptors in disease, a field that has seen significant progress in recent years.

Metabotropic actions of kainate receptors modulating glutamate release.

Presynaptic kainate (KA) receptors (KARs) modulate GABA and glutamate release in the central nervous system of mammals. While some of the actions of KARs are ionotropic, metabotropic actions for these receptors have also been seen to modulate both GABA and glutamate release. In general, presynaptic KARs modulate glutamate release through their metabotropic actions in a biphasic manner, with low KA concentrations producing an increase in glutamate release and higher concentrations of KA driving weaker release of this neurotransmitter. Different molecular mechanisms are involved in this modulation of glutamate release, with a G-protein independent, Ca2+-calmodulin adenylate cyclase (AC) and protein kinase A (PKA) dependent mechanism facilitating glutamate release, and a G-protein, AC and PKA dependent mechanism mediating the decrease in neurotransmitter release. Here, we describe the events underlying the KAR modulation of glutamatergic transmission in different brain regions, addressing the possible functions of this modulation and proposing future research lines in this field. This article is part of the special Issue on 'Glutamate Receptors - Kainate receptors'.

Astrocyte-mediated switch in spike timing-dependent plasticity during hippocampal development.

Presynaptic spike timing-dependent long-term depression (t-LTD) at hippocampal CA3-CA1 synapses is evident until the 3rd postnatal week in mice, disappearing during the 4th week. At more mature stages, we found that the protocol that induced t-LTD induced t-LTP. We characterized this form of t-LTP and the mechanisms involved in its induction, as well as that driving this switch from t-LTD to t-LTP. We found that this t-LTP is expressed presynaptically at CA3-CA1 synapses, as witnessed by coefficient of variation, number of failures, paired-pulse ratio and miniature responses analysis. Additionally, this form of presynaptic t-LTP does not require NMDARs but the activation of mGluRs and the entry of Ca2+ into the postsynaptic neuron through L-type voltage-dependent Ca2+ channels and the release of Ca2+ from intracellular stores. Nitric oxide is also required as a messenger from the postsynaptic neuron. Crucially, the release of adenosine and glutamate by astrocytes is required for t-LTP induction and for the switch from t-LTD to t-LTP. Thus, we have discovered a developmental switch of synaptic transmission from t-LTD to t-LTP at hippocampal CA3-CA1 synapses in which astrocytes play a central role and revealed a form of presynaptic LTP and the rules for its induction.

Parkin-mediated mitophagy and autophagy flux disruption in cellular models of MERRF syndrome.

Mitochondrial diseases are considered rare genetic disorders characterized by defects in oxidative phosphorylation (OXPHOS). They can be provoked by mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). MERRF (Myoclonic Epilepsy with Ragged-Red Fibers) syndrome is one of the most frequent mitochondrial diseases, principally caused by the m.8344A>G mutation in mtDNA, which affects the translation of all mtDNA-encoded proteins and therefore impairs mitochondrial function. In the present work, we evaluated autophagy and mitophagy flux in transmitochondrial cybrids and fibroblasts derived from a MERRF patient, reporting that Parkin-mediated mitophagy is increased in MERRF cell cultures. Our results suggest that supplementation with coenzyme Q10 (CoQ), a component of the electron transport chain (ETC) and lipid antioxidant, prevents Parkin translocation to the mitochondria. In addition, CoQ acts as an enhancer of autophagy and mitophagy flux, which partially improves cell pathophysiology. The significance of Parkin-mediated mitophagy in cell survival was evaluated by silencing the expression of Parkin in MERRF cybrids. Our results show that mitophagy acts as a cell survival mechanism in mutant cells. To confirm these results in one of the main affected cell types in MERRF syndrome, mutant induced neurons (iNs) were generated by direct reprogramming of patients-derived skin fibroblasts. The treatment of MERRF iNs with Guttaquinon CoQ10 (GuttaQ), a water-soluble derivative of CoQ, revealed a significant improvement in cell bioenergetics. These results indicate that iNs, along with fibroblasts and cybrids, can be utilized as reliable cellular models to shed light on disease pathomechanisms as well as for drug screening.

Characterization of an eutherian gene cluster generated after transposon domestication identifies Bex3 as relevant for advanced neurological functions.

BACKGROUND: One of the most unusual sources of phylogenetically restricted genes is the molecular domestication of transposable elements into a host genome as functional genes. Although these kinds of events are sometimes at the core of key macroevolutionary changes, their origin and organismal function are generally poorly understood. RESULTS: Here, we identify several previously unreported transposable element domestication events in the human and mouse genomes. Among them, we find a remarkable molecular domestication that gave rise to a multigenic family in placental mammals, the Bex/Tceal gene cluster. These genes, which act as hub proteins within diverse signaling pathways, have been associated with neurological features of human patients carrying genomic microdeletions in chromosome X. The Bex/Tceal genes display neural-enriched patterns and are differentially expressed in human neurological disorders, such as autism and schizophrenia. Two different murine alleles of the cluster member Bex3 display morphological and physiopathological brain modifications, such as reduced interneuron number and hippocampal electrophysiological imbalance, alterations that translate into distinct behavioral phenotypes. CONCLUSIONS: We provide an in-depth understanding of the emergence of a gene cluster that originated by transposon domestication and gene duplication at the origin of placental mammals, an evolutionary process that transformed a non-functional transposon sequence into novel components of the eutherian genome. These genes were integrated into existing signaling pathways involved in the development, maintenance, and function of the CNS in eutherians. At least one of its members, Bex3, is relevant for higher brain functions in placental mammals and may be involved in human neurological disorders.

Kainate Receptors: Role in Epilepsy.

Kainate (KA) is a potent neurotoxin that has been widely used experimentally to induce acute brain seizures and, after repetitive treatments, as a chronic model of temporal lobe epilepsy (TLE), with similar features to those observed in human patients with TLE. However, whether KA activates KA receptors (KARs) as an agonist to mediate the induction of acute seizures and/or the chronic phase of epilepsy, or whether epileptogenic effects of the neurotoxin are indirect and/or mediated by other types of receptors, has yet to be satisfactorily elucidated. Positing a direct involvement of KARs in acute seizures induction, as well as a direct pathophysiological role of KARs in the chronic phase of TLE, recent studies have examined the specific subunit compositions of KARs that might underly epileptogenesis. In the present mini-review, we discuss the use of KA as a convulsant in the experimental models of acute seizures of TLE, and consider the involvement of KARs, their subunit composition and the mode of action in KAR-mediated epilepsy. In acute models, evidence points to epileptogenesis being precipitated by an overall depression of interneuron GABAergic transmission mediated by GluK1 containing KARs. On glutamatergic principal cell in the hippocampus, GluK2-containing KARs regulate post-synaptic excitability and susceptibility to KA-mediated epileptogenesis. In chronic models, a role GluK2-containing KARs in the hippocampal CA3 region provokes limbic seizures. Also observed in the hippocampus, is a 'reactive plasticity', where MF sprouting is seen with target granule cells at aberrant synapses recruiting de novo GluR2/GluR5 heteromeric KARs. Finally, in human epilepsy and animal models, astrocytic expression of GluK1, 2, 4, and 5 is reported.

Cerebellar Kainate Receptor-Mediated Facilitation of Glutamate Release Requires Ca2+-Calmodulin and PKA.

We elucidated the mechanisms underlying the kainate receptor (KAR)-mediated facilitatory modulation of synaptic transmission in the cerebellum. In cerebellar slices, KA (3 µM) increased the amplitude of evoked excitatory postsynaptic currents (eEPSCs) at synapses between axon terminals of parallel fibers (PF) and Purkinje neurons. KA-mediated facilitation was antagonized by NBQX under condition where AMPA receptors were previously antagonized. Inhibition of protein kinase A (PKA) suppressed the effect of KA on glutamate release, which was also obviated by the prior stimulation of adenylyl cyclase (AC). KAR-mediated facilitation of synaptic transmission was prevented by blocking Ca2+ permeant KARs using philanthotoxin. Furthermore, depletion of intracellular Ca2+ stores by thapsigargin, or inhibition of Ca2+-induced Ca2+-release by ryanodine, abrogated the synaptic facilitation by KA. Thus, the KA-mediated modulation was conditional on extracellular Ca2+ entry through Ca2+-permeable KARs, as well as and mobilization of Ca2+ from intracellular stores. Finally, KAR-mediated facilitation was sensitive to calmodulin inhibitors, W-7 and calmidazolium, indicating that the increased cytosolic [Ca2+] sustaining KAR-mediated facilitation of synaptic transmission operates through a downstream Ca2+/calmodulin coupling. We conclude that, at cerebellar parallel fiber-Purkinje cell synapses, presynaptic KARs mediate glutamate release facilitation, and thereby enhance synaptic transmission through Ca2+-calmodulin dependent activation of adenylyl cyclase/cAMP/protein kinase A signaling.