Rui O. Costa

Endoplasmic reticulum stress occurs downstream of GluN2B subunit of N-methyl-D-aspartate receptor in mature hippocampal cultures treated with amyloid-β 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 (AβO), the species implicated in synaptic loss during the initial disease stages, induce endoplasmic reticulum (ER) stress in cultured neurons. Here, we investigated whether AβO 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 AβO 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 AβO 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 AβO interaction with GluN2B subunits. In addition, AβO‐induced ER stress and hippocampal dysfunction were prevented by ifenprodil, an antagonist of GluN2B subunits, while the GluN2A antagonist NVP‐AAM077 only slightly attenuated AβO‐induced neurotoxicity. Taken together, our results highlight the role of GluN2B subunit of NMDARs on ER stress‐mediated hippocampal dysfunction caused by AβO suggesting that it might be a potential therapeutic target during the early stages of AD.

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 Alzheimers 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.

ER Stress-Mediated Apoptotic Pathway Induced by Aβ Peptide Requires the Presence of Functional Mitochondria

Amyloid-beta (Abeta) peptide plays a significant role in the pathogenesis of Alzheimers 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.

Amyloid β-induced ER stress is enhanced under mitochondrial dysfunction conditions

Previously we reported that endoplasmic reticulum (ER)-mitochondria crosstalk is involved in amyloid-β (Aβ)-induced apoptosis. Now we show that mitochondrial dysfunction affects the ER stress response triggered by Aβ using cybrids that recreate the defect in mitochondrial cytochrome c oxidase (COX) activity detected in platelets from Alzheimers disease (AD) patients. AD and control cybrids were treated with Aβ 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 Aβ-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 Aβ-induced ER stress. This study further supports the close communication between ER and mitochondria during apoptosis in AD.

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

Previously we reported that amyloid-β (Aβ) leads to endoplasmic reticulum (ER) stress in cultured cortical neurons and that ER-mitochondria Ca(2+) transfer is involved in Aβ-induced apoptotic neuronal cell death. In cybrid cells which recreate the defect in mitochondrial cytochrome c oxidase (COX) activity observed in platelets from Alzheimers disease (AD) patients, we have shown that mitochondrial dysfunction affects the ER stress response triggered by Aβ. Here, we further investigated the impact of COX inhibition on Aβ-induced ER dysfunction using a neuronal model. Primary cultures of cortical neurons were challenged with toxic concentrations of Aβ 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 Aβ-induced depletion of ER Ca(2+) content. KCN pre-treatment was also shown to enhance the rise of cytosolic Ca(2+) levels triggered by Aβ 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 Aβ 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 Aβ- 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 Aβ, 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 Aβ-induced ER dysfunction and, to a less extent, neuronal cell death. Furthermore, data supports that the effect of impaired COX on Aβ-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 Aβ-induced ER stress, and strongly suggest that the close communication between ER and mitochondria can be a valuable future therapeutic target in AD.

Leptin and ghrelin prevent hippocampal dysfunction induced by Aβ oligomers.

It was recently established that the stomach-derived ghrelin and the adipokine leptin promote learning and memory through actions within the hippocampus. Changes in the peripheral or brain levels of these peptides were described in Alzheimers disease (AD) patients and were shown to correlate with the severity of cognitive decline. Furthermore, in vivo and in vitro studies demonstrated that leptin or ghrelin can ameliorate amyloid and tau pathologies as well as cognitive deficits. However, the exact role of these peptides in AD is far from being elucidated. To fill this gap, our working hypothesis was that leptin and ghrelin can exert a neuroprotective role in AD suppressing hippocampal dysfunction triggered by synapto- and neurotoxic amyloid-β oligomers (AβO). Using primary cultured hippocampal neurons, we demonstrated that both peptides reduce AβO-induced production of superoxide and mitochondrial membrane depolarization, improving cell survival, and inhibit cell death through a receptor-dependent mechanism. Furthermore, it was shown that in AβO-treated neurons both leptin and ghrelin prevent glycogen synthase kinase 3β activation. Therefore, the evidence gathered in this study revealed that leptin and ghrelin can act as neuroprotective agents able to rescue hippocampal neurons from AβO toxicity, thus highlighting their potential therapeutic role in AD.

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.

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

Abstract 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 IIβ (CaMKIIβ) 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.