Oxidative stress involving changes in Nrf2 and ER stress in early stages of Alzheimer's disease.

Oxidative stress and endoplasmic reticulum (ER) stress have been associated with Alzheimer's disease (AD) progression. In this study we analyzed whether oxidative stress involving changes in Nrf2 and ER stress may constitute early events in AD pathogenesis by using human peripheral blood cells and an AD transgenic mouse model at different disease stages. Increased oxidative stress and increased phosphorylated Nrf2 (p(Ser40)Nrf2) were observed in human peripheral blood mononuclear cells (PBMCs) isolated from individuals with mild cognitive impairment (MCI). Moreover, we observed impaired ER Ca2+ homeostasis and increased ER stress markers in PBMCs from MCI individuals and mild AD patients. Evidence of early oxidative stress defense mechanisms in AD was substantiated by increased p(Ser40)Nrf2 in 3month-old 3xTg-AD male mice PBMCs, and also with increased nuclear Nrf2 levels in brain cortex. However, SOD1 protein levels were decreased in human MCI PBMCs and in 3xTg-AD mice brain cortex; the latter further correlated with reduced SOD1 mRNA levels. Increased ER stress was also detected in the brain cortex of young female and old male 3xTg-AD mice. We demonstrate oxidative stress and early Nrf2 activation in AD human and mouse models, which fails to regulate some of its targets, leading to repressed expression of antioxidant defenses (e.g., SOD-1), and extending to ER stress. Results suggest markers of prodromal AD linked to oxidative stress associated with Nrf2 activation and ER stress that may be followed in human peripheral blood mononuclear cells.

Inhibition of mitochondrial cytochrome c oxidase potentiates Aß-induced ER stress and cell death in cortical neurons.

Previously we reported that amyloid-ß (Aß) leads to endoplasmic reticulum (ER) stress in cultured cortical neurons and that ER-mitochondria Ca(2+) transfer is involved in Aß-induced apoptotic neuronal cell death. In cybrid cells which recreate the defect in mitochondrial cytochrome c oxidase (COX) activity observed in platelets from Alzheimer's disease (AD) patients, we have shown that mitochondrial dysfunction affects the ER stress response triggered by Aß. Here, we further investigated the impact of COX inhibition on Aß-induced ER dysfunction using a neuronal model. Primary cultures of cortical neurons were challenged with toxic concentrations of Aß upon chemical inhibition of COX with potassium cyanide (KCN). ER Ca(2+) homeostasis was evaluated under these conditions, together with the levels of ER stress markers, namely the chaperone GRP78 and XBP-1, a mediator of the ER unfolded protein response (UPR). We demonstrated that COX inhibition potentiates the Aß-induced depletion of ER Ca(2+) content. KCN pre-treatment was also shown to enhance the rise of cytosolic Ca(2+) levels triggered by Aß and thapsigargin, a widely used ER stressor. This effect was reverted in the presence of dantrolene, an inhibitor of ER Ca(2+) release through ryanodine receptors. Similarly, the increase in GRP78 and XBP-1 protein levels was shown to be higher in neurons treated with Aß or thapsigargin in the presence of KCN in comparison with levels determined in neurons treated with the neurotoxins alone. Although the decrease in cell survival, the activation of caspase-9- and -3-mediated apoptotic cell death observed in Aß- and thapsigargin-treated neurons were also potentiated by KCN, this effect is less pronounced than that observed in Ca(2+) signalling and UPR. Furthermore, in neurons treated with Aß, the potentiating effect of the COX inhibitor in cell survival and death was not prevented by dantrolene. These results show that inhibition of mitochondrial COX activity potentiates Aß-induced ER dysfunction and, to a less extent, neuronal cell death. Furthermore, data supports that the effect of impaired COX on Aß-induced cell death occurs independently of Ca(2+) release through ER ryanodine receptors. Together, our data demonstrate that mitochondria dysfunction in AD enhances the neuronal susceptibility to toxic insults, namely to Aß-induced ER stress, and strongly suggest that the close communication between ER and mitochondria can be a valuable future therapeutic target in AD.

Brain-derived neurotrophic factor-induced regulation of RNA metabolism in neuronal development and synaptic plasticity.

The neurotrophin brain-derived neurotrophic factor (BDNF) plays multiple roles in the nervous system, including in neuronal development, in long-term synaptic potentiation in different brain regions, and in neuronal survival. Alterations in these regulatory mechanisms account for several diseases of the nervous system. The synaptic effects of BDNF mediated by activation of tropomyosin receptor kinase B (TrkB) receptors are partly mediated by stimulation of local protein synthesis which is now considered a ubiquitous feature in both presynaptic and postsynaptic compartments of the neuron. The capacity to locally synthesize proteins is of great relevance at several neuronal developmental stages, including during neurite development, synapse formation, and stabilization. The available evidence shows that the effects of BDNF-TrkB signaling on local protein synthesis regulate the structure and function of the developing and mature synapses. While a large number of studies have illustrated a wide range of effects of BDNF on the postsynaptic proteome, a growing number of studies also point to presynaptic effects of the neurotrophin in the local regulation of the protein composition at the presynaptic level. Here, we will review the latest evidence on the role of BDNF in local protein synthesis, comparing the effects on the presynaptic and postsynaptic compartments. Additionally, we overview the relevance of BDNF-associated local protein synthesis in neuronal development and synaptic plasticity, at the presynaptic and postsynaptic compartments, and their relevance in terms of disease. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Export and Localization > RNA Localization.

Isolation and Culture of Chick Ciliary Ganglion Neurons.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Alterations in GABAA-Receptor Trafficking and Synaptic Dysfunction in Brain Disorders.

GABAA receptors (GABAAR) are the major players in fast inhibitory neurotransmission in the central nervous system (CNS). Regulation of GABAAR trafficking and the control of their surface expression play important roles in the modulation of the strength of synaptic inhibition. Different pieces of evidence show that alterations in the surface distribution of GABAAR and dysregulation of their turnover impair the activity of inhibitory synapses. A diminished efficacy of inhibitory neurotransmission affects the excitatory/inhibitory balance and is a common feature of various disorders of the CNS characterized by an increased excitability of neuronal networks. The synaptic pool of GABAAR is mainly controlled through regulation of internalization, recycling and lateral diffusion of the receptors. Under physiological condition these mechanisms are finely coordinated to define the strength of GABAergic synapses. In this review article, we focus on the alteration in GABAAR trafficking with an impact on the function of inhibitory synapses in various disorders of the CNS. In particular we discuss how similar molecular mechanisms affecting the synaptic distribution of GABAAR and consequently the excitatory/inhibitory balance may be associated with a wide diversity of pathologies of the CNS, from psychiatric disorders to acute alterations leading to neuronal death. A better understanding of the cellular and molecular mechanisms that contribute to the impairment of GABAergic neurotransmission in these disorders, in particular the alterations in GABAAR trafficking and surface distribution, may lead to the identification of new pharmacological targets and to the development of novel therapeutic strategies.

Synaptogenesis Stimulates a Proteasome-Mediated Ribosome Reduction in Axons.

Ribosomes and a subset of cellular mRNAs are trafficked into axons of developing neurons. The axonal localization of translational machinery allows new proteins to be rapidly and locally synthesized during axonal growth and pathfinding. However, in mature neurons, axonal ribosomes are significantly reduced or even absent. The mechanism that elicits this removal is currently unknown. Here, we demonstrate that synapse formation is the trigger for ribosome reduction in mature axons. In vivo analysis shows that axonal ribosome levels decrease in rat brain at a developmental stage coincident with synapse formation. Next, we observe in vitro that different synaptogenic inducers trigger an overall decrease of ribosomal proteins and rRNA in the axons of spinal motor neurons. We further observe that this process is dependent on the ubiquitin-proteasome system but not on autophagy. Together, these data identify synaptogenesis as the long missing biological trigger that leads to ribosome disappearance during axonal maturation.

PROneurotrophins and CONSequences.

Proneurotrophins were initially thought to be simple inactive precursors, only responsible for promoting the folding of the mature domain and for the regulation of the neurotrophin secretory pathway. However, recent evidence shows that proneurotrophins can be secreted to the extracellular space, bind with high affinity to specific receptor complexes and induce activation of the apoptotic machinery with subsequent cell death of different neuronal populations. These pathways can be activated due to injury and to several neurodegenerative disorders, which promote proneurotrophin secretion to the extracellular space. In addition to neuropathology, extracellular proneurotrophins also play a pivotal role in many other cellular mechanisms in the nervous system. Proneurotrophins were shown to mediate synaptic plasticity, namely long-term depression in hippocampal neurons. They are also important in axonal development, and an increase of pro- to mature neurotrophin ratio has been described as a trigger of cell death. The conversion of proneurotrophins into the respective mature form is controlled by the action of several enzymes and regulators. The failure in this regulation is now considered one of the possible mechanisms responsible for pathological cell death associated to proneurotrophins. Here, we discuss the processes behind proneurotrophin action, with particular focus on proBDNF and proNGF and their regulatory pathways. Additionally, we review the most recent studies concerning proneurotrophin involvement in neuronal death, in several disease-associated states in the CNS and PNS, and discuss future avenues of investigation in the proneurotrophin field.

Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons.

This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of cells, while offering minimal cell death that often occurs during dissociation of differentiated neurons. Cells plated under the appropriate conditions, either in Petri-dishes or in multi-well plates, will develop and establish synaptic contacts over time since the neuronal culture medium provides the nutrients and trophic factors required for differentiation. In this protocol we describe the methodology for the preparation of both cortical and hippocampal neuronal cultures.

Mesenchymal stem cells secretome-induced axonal outgrowth is mediated by BDNF.

Mesenchymal stem cells (MSCs) have been used for cell-based therapies in regenerative medicine, with increasing importance in central and peripheral nervous system repair. However, MSCs grafting present disadvantages, such as, a high number of cells required for transplantation and low survival rate when transplanted into the central nervous system (CNS). In line with this, MSCs secretome which present on its composition a wide range of molecules (neurotrophins, cytokines) and microvesicles, can be a solution to surpass these problems. However, the effect of MSCs secretome in axonal elongation is poorly understood. In this study, we demonstrate that application of MSCs secretome to both rat cortical and hippocampal neurons induces an increase in axonal length. In addition, we show that this growth effect is axonal intrinsic with no contribution from the cell body. To further understand which are the molecules required for secretome-induced axonal outgrowth effect, we depleted brain-derived neurotrophic factor (BDNF) from the secretome. Our results show that in the absence of BDNF, secretome-induced axonal elongation effect is lost and that axons present a reduced axonal growth rate. Altogether, our results demonstrate that MSCs secretome is able to promote axonal outgrowth in CNS neurons and this effect is mediated by BDNF.

The RNA-Binding Protein hnRNP K Mediates the Effect of BDNF on Dendritic mRNA Metabolism and Regulates Synaptic NMDA Receptors in Hippocampal Neurons.

Brain-derived neurotrophic factor (BDNF) is an important mediator of long-term synaptic potentiation (LTP) in the hippocampus. The local effects of BDNF depend on the activation of translation activity, which requires the delivery of transcripts to the synapse. In this work, we found that neuronal activity regulates the dendritic localization of the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K) in cultured rat hippocampal neurons by stimulating BDNF-Trk signaling. Microarray experiments identified a large number of transcripts that are coimmunoprecipitated with hnRNP K, and about 60% of these transcripts are dissociated from the protein upon stimulation of rat hippocampal neurons with BDNF. In vivo studies also showed a role for TrkB signaling in the dissociation of transcripts from hnRNP K upon high-frequency stimulation (HFS) of medial perforant path-granule cell synapses of male rat dentate gyrus (DG). Furthermore, treatment of rat hippocampal synaptoneurosomes with BDNF decreased the coimmunoprecipitation of hnRNP K with mRNAs coding for glutamate receptor subunits, Ca2+- and calmodulin-dependent protein kinase IIß (CaMKIIß) and BDNF. Downregulation of hnRNP K impaired the BDNF-induced enhancement of NMDA receptor (NMDAR)-mediated mEPSC, and similar results were obtained upon inhibition of protein synthesis with cycloheximide. The results demonstrate that BDNF regulates specific populations of hnRNP-associated mRNAs in neuronal dendrites and suggests an important role of hnRNP K in BDNF-dependent forms of synaptic plasticity.

Visualizing K48 Ubiquitination during Presynaptic Formation By Ubiquitination-Induced Fluorescence Complementation (UiFC).

In recent years, signaling through ubiquitin has been shown to be of great importance for normal brain development. Indeed, fluctuations in ubiquitin levels and spontaneous mutations in (de)ubiquitination enzymes greatly perturb synapse formation and neuronal transmission. In the brain, expression of lysine (K) 48-linked ubiquitin chains is higher at a developmental stage coincident with synaptogenesis. Nevertheless, no studies have so far delved into the involvement of this type of polyubiquitin chains in synapse formation. We have recently proposed a role for polyubiquitinated conjugates as triggering signals for presynaptic assembly. Herein, we aimed at characterizing the axonal distribution of K48 polyubiquitin and its dynamics throughout the course of presynaptic formation. To accomplish so, we used an ubiquitination-induced fluorescence complementation (UiFC) strategy for the visualization of K48 polyubiquitin in live hippocampal neurons. We first validated its use in neurons by analyzing changing levels of polyubiquitin. UiFC signal is diffusely distributed with distinct aggregates in somas, dendrites and axons, which perfectly colocalize with staining for a K48-specific antibody. Axonal UiFC aggregates are relatively stable and new aggregates are formed as an axon grows. Approximately 65% of UiFC aggregates colocalize with synaptic vesicle clusters and they preferentially appear in the axonal domains of axo-somatodendritic synapses when compared to isolated axons. We then evaluated axonal accumulation of K48 ubiquitinated signals in bead-induced synapses. We observed rapid accumulation of UiFC signal and endogenous K48 ubiquitin at the sites of newly formed presynapses. Lastly, we show by means of a microfluidic platform, for the isolation of axons, that presynaptic clustering on beads is dependent on E1-mediated ubiquitination at the axonal level. Altogether, these results indicate that enrichment of K48 polyubiquitin at the site of nascent presynaptic terminals is an important axon-intrinsic event for presynaptic differentiation.

Amyloid ß-induced ER stress is enhanced under mitochondrial dysfunction conditions.

Previously we reported that endoplasmic reticulum (ER)-mitochondria crosstalk is involved in amyloid-ß (Aß)-induced apoptosis. Now we show that mitochondrial dysfunction affects the ER stress response triggered by Aß using cybrids that recreate the defect in mitochondrial cytochrome c oxidase (COX) activity detected in platelets from Alzheimer's disease (AD) patients. AD and control cybrids were treated with Aß or classical ER stressors and the ER stress-mediated apoptotic cell death pathway was accessed. Upon treatment, we found increased glucose-regulated protein 78 (GRP78) levels and caspase-4 activation (ER stress markers) which were more pronounced in AD cybrids. Treated AD cybrids also exhibited decreased cell survival as well as increased caspase-3-like activity, poli-ADP-ribose-polymerase (PARP) levels and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells. Finally, we showed that Aß-induced caspase-3 activation in both cybrid cell lines was prevented by dantrolene, thus implicating ER Ca(2+) release in ER stress-mediated apoptosis. Our results demonstrate that mitochondrial dysfunction occurring in AD patients due to COX inhibition potentiates cell susceptibility to Aß-induced ER stress. This study further supports the close communication between ER and mitochondria during apoptosis in AD.

Endoplasmic reticulum stress occurs downstream of GluN2B subunit of N-methyl-d-aspartate receptor in mature hippocampal cultures treated with amyloid-ß oligomers.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder affecting both the hippocampus and the cerebral cortex. Reduced synaptic density that occurs early in the disease process seems to be partially due to the overactivation of N-methyl-d-aspartate receptors (NMDARs) leading to excitotoxicity. Recently, we demonstrated that amyloid-beta oligomers (AßO), the species implicated in synaptic loss during the initial disease stages, induce endoplasmic reticulum (ER) stress in cultured neurons. Here, we investigated whether AßO trigger ER stress by an NMDAR-dependent mechanism leading to neuronal dysfunction and analyzed the contribution of GluN2A and GluN2B subunits of this glutamate receptor. Our data revealed that AßO induce ER stress in mature hippocampal cultures, activating ER stress-associated sensors and increasing the levels of the ER chaperone GRP78. We also showed that AßO induce NADPH oxidase (NOX)-mediated superoxide production downstream of GluN2B and impairs ER and cytosolic Ca2+ homeostasis. These events precede changes in cell viability and activation of the ER stress-mediated apoptotic pathway, which was associated with translocation of the transcription factor GADD153 / CHOP to the nucleus and occurred by a caspase-12-independent mechanism. Significantly, ER stress took place after AßO interaction with GluN2B subunits. In addition, AßO-induced ER stress and hippocampal dysfunction were prevented by ifenprodil, an antagonist of GluN2B subunits, while the GluN2A antagonist NVP-AAM077 only slightly attenuated AßO-induced neurotoxicity. Taken together, our results highlight the role of GluN2B subunit of NMDARs on ER stress-mediated hippocampal dysfunction caused by AßO suggesting that it might be a potential therapeutic target during the early stages of AD.

ER stress-mediated apoptotic pathway induced by Abeta peptide requires the presence of functional mitochondria.

Amyloid-beta (Abeta) peptide plays a significant role in the pathogenesis of Alzheimer's disease (AD). Previously we found that Abeta induces both mitochondrial and endoplasmic reticulum (ER) dysfunction leading to apoptosis, and now we address the relevance of ER-mitochondria crosstalk in apoptotic cell death triggered by Abeta peptide. Using mitochondrial DNA-depleted rho0 cells derived from the human NT2 teratocarcinoma cell line, characterized by the absence of functional mitochondria, and the parental rho+ cells, we report here that treatment with the synthetic Abeta1-40 peptide, or the classical ER stressors thapsigargin or brefeldin A, increases GRP78 expression levels and caspase activity, two ER stress markers, and also depletes ER calcium stores. Significantly, we show that the presence of functional mitochondria is required for ER stress-mediated apoptotic cell death triggered by toxic insults such as Abeta. We found that the increase in the levels of the pro-apoptotic transcription factor GADD153/CHOP, which mediates ER stress-induced cell death, as well as caspase-9 and -3 activation and increased number of TUNEL-positive cells, occurs in treated parental rho+ cells but is abolished in rho0 cells. Our results strongly support the close communication between ER and mitochondria during apoptotic cell death induced by the Abeta peptide and provide insights into the molecular cascade of cell death in AD.