174 research outputs found

    Neuroprotective signaling mechanisms in the mammalian brain

    Get PDF
    Summary Alzheimer’s, Parkinson’s disease and other degenerative disorders are characterized by increased neuronal death. Deregulation of several physiological processes, as a consequence of the neuronal death, occurs especially in the regions of the central nervous system involved in learning and memory. Therefore, preserving normal neuronal functions is of great importance. Design and implementation of new therapeutics in clinical trials in order to prevent and stop neuronal death is quite challenging since neurodegenerative processes underlie complex signaling. Therefore, studying and revealing these neuronal processes would lead to more effective therapeutics. One model to study in vitro the molecular mechanisms underlying neurodegeneration is the glutamate-induced excitotoxicity paradigm, where neurons are treated with glutamate to induce neuronal death and several substances are tested for their potency to prevent this neuronal death. Hitherto, a number of studies showed that the cytokine TNF-a is able to promote neuroprotection against glutamate-induced excitotoxicity in cortical neurons. TNF-a is a known cytokine with increased expression in the brain and in the cerebrospinal serum of human patients with neurodegenerative disorders. The main focus of this thesis was to investigate TNF-a-mediated neuroprotective molecular mechanisms in the mammalian brain. Chapters 2 and 4 of this thesis investigate the neuroprotective mechanisms by which TNF-a enhances neuronal resistance against glutamate-induced excitotoxicity. First, the kinetics of the neuroprotective effect of TNF-a in primary cortical neurons was studied. Short-term exposure of neurons for 2-4 h to TNF-a did not rescue neurons from the glutamate-induced cellular death. However, after 6-8 h or longer of TNF-a treatment neuronal survival was significantly increased. It was previously shown that long term (24 h) TNF-a treatment causes an increase of PKB/Akt phosphorylation. PKB/Akt phosphorylation in cortical neurons is associated with increased cellular survival. Chapter 2 describes the PKB/Akt kinetics upon TNF-a treatment. Since PTEN is the major negative regulator of PKB/Akt activation the PTEN expression upon TNF-a treatment was investigated. In previous studies, an increase of PTEN expression in cortical neurons was shown to induce neuronal death. Short term TNF-a treatment (2-4 h) induced an increase of PTEN expression and a decrease of PKB/Akt phosphorylation. However, long term TNF-a treatment (6-8 to 24 h) resulted in a decrease of PTEN expression and augmented PKB/Akt phosphorylation in a time dependent manner. The fact that high levels of PTEN coincided with low levels of PKB/Akt phosphorylation and vice versa is in accordance with the inverse relationship between PTEN and PKB/Akt activation. To assess the contribution of both TNF-receptors, TNF-R1 and/or TNF-R2 to the neuroprotective TNF-a mediated pathway, PKB/Akt and PTEN expression in TNF-R1-/- and TNF-R2-/- neurons was investigated. It appeared that in TNF-R1-/- neurons, PKB/Akt was phosphorylated after long term TNF-a treatment with visible effects after 6-8 h, while in TNF-R2-/- PKB/Akt phosphorylation was down regulated in a TNF-a treatment dependent manner (Chapter 2). Taken together, these results suggested that TNF-a minimizes the consequences of excitotoxic events through PKB/Akt signaling. Since PKB/Akt pathway could be regulated by cyclic adenosine monophosphate (cAMP)-dependent signaling as well, chapter 3 investigated the effect of two mainstream pathways initiated by cAMP, cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) on PKB/Akt phosphorylation in primary cortical neurons. PKA activation led to a decrease of PKB/Akt phosphorylation, whereas activation of Epac increased PKB/Akt phosphorylation. PKA, PKB/Akt and Epacs were all shown to be complexed with the neuronal A-kinase anchoring protein 150 (AKAP150). Particularly, activation of Epac2 increased phosphorylation of PKB/Akt complexed to AKAP150, whereas silencing of cellular Epac2 diminished PKB/Akt phosphorylation. From experiments using PKA binding deficient AKAP150 and PKA disrupting anchoring to AKAP150 peptides, AKAP150 was found to act as a key regulator in the two cAMP pathways that control PKB/Akt phosphorylation. Although the involvement of the TNF-R2, PKB/Akt and NF-kB were suggested to be important in TNF-a induced neuroprotective mechanisms, data on potential downstream targets of these signalling pathways were not available. Therefore, in Chapter 4 we investigated SK channels as possible downstream molecules of TNF-a signaling, since NF-kB regulates the expression of SK2 channels. SK channels modulate synaptic excitability and neuronal activity by contributing to afterhyperpolarization processes. In our studies, we investigated the action of highly specific agonists (NS309, CyPPA) and antagonists (apamin) of SK channels on cortical neurons. The finding that NS309 and CyPPA mediated increased cellular survival against glutamate toxicity, while apamin partially reverted the neuroprotective effect elicited by TNF-a treatment demonstrated the crucial role of SK channel activity in the TNF-a induced neuroprotective mechanisms. Interestingly, a commercially available drug, lovastatin commonly used for coronary artery disease specifically enhanced TNF-R2 expression in HUVEC cells. Lovastatin was proposed as neuroprotective agent for several neurodegenerative disorders. Although both lovastatin and TNF-a were shown to be neuroprotective, so far their signaling pathways had been studied independently. The study described in chapter 5 shows that lovastatin can induce TNF-R2 expression in primary cortical neurons. It was demonstrated that NF-kB is responsible for the lovastatin-dependent increase of TNF-R2 expression (Chapter 5). Since in TNF-R1-/- neurons inhibition of PKB/Akt activity reverted the protective effect induced by lovastatin it was concluded that lovastatin-induced TNF-R2 expression contributes to neuronal survival through PKB/Akt activation. Chapter 6 consolidates the role of PKB/Akt as a downstream molecule in lovastatin-mediated neuroprotective signaling. Using an in vivo model, in which NMDA was infused in the nucleus basalis of Meynert (MNB), located in the basal forebrain cholinergic complex, the effect of lovastatin on cortical cholinergic fibers was investigated. Since the basal forebrain provides the major input in cholinergic projections to the cerebral cortex and hippocampus the cortical cholinergic target area was quantified to determine the magnitude of cholinergic fiber loss produced by NMDA lesions in MNB. Lovastatin treatment significantly protected cholinergic neurons and their cortical projections against NMDA induced cell death in MNB. Lovastatin-mediated neuroprotection was shown to be dependent on protein kinase B (PKB)/Akt signaling since inhibition of PKB/Akt with LY294002 blocked the lovastatin-induced neuroprotective effect (Chapter 6). In addition, lovastatin treatment improved memory deficits in NMDA-lesioned mice. Taken together, our studies suggest potential for therapeutic manipulation of TNF-a signaling in the treatment of neurodegenerative disorders.

    PEG out through the pores with the help of ESCRTIII

    Get PDF
    Ferroptosis is a form of programmed cell death with particular hallmarks, such as oxidative stress, increased calcium fluxes, and altered cellular morphology. In ferroptosis, the disruption of plasma membrane is the step that culminates into cell death. By inducing ferroptosis with Erastin-1 and RSL3 in various human cellular models, Pedrera et al. tracked the behaviour of several hallmarks of ferroptosis and demonstrated that lipid peroxidation precedes cytosolic calcium rise and plasma membrane breakdown, which is dependent on nanopore formation. Ferroptotic cell death is inhibited by osmotically active protectants of proper size that can prevent water flux through nanopores

    Extending the analogy between intracellular motion in mammalian cells and glassy dynamics

    Get PDF
    How molecules, organelles, and foreign objects move within living cells has been studied in organisms ranging from bacteria to human cells. In mammalian cells, in particular, cellular vesicles move across the cell using motor proteins that carry the vesicle down the cytoskeleton to their destination. We have recently noted several similarities between the motion of such vesicles and that in disordered, “glassy”, systems, but the generality of this observation remains unclear. Here we follow the motion of mitochondria, the organelles responsible for cell energy production, in mammalian cells over timescales from 50 ms to 70 s. Qualitative observations show that single mitochondria remain within a spatially limited region for extended periods of time, before moving longer distances relatively quickly. The displacement distribution is roughly Gaussian for shorter distances (≲ 0.05 µm) but exhibits exponentially decaying tails at longer distances (up to 0.40 µm). This behaviour is well-described by a model developed to describe the motion in glassy systems. These observations are extended to in total 3 different objects (mitochondria, lysosomes and nano-sized beads enclosed in vesicles), 3 different mammalian cell types (HEK 293, HeLa, and HT22), from 2 different organisms (human and mouse). Further evidence that supports glass-like characteristics of the motion is a difference between the time it takes to move a longer distance for the first time and subsequent times, as well as a weak ergodicity breaking of the motion. Overall, we demonstrate the ubiquity of glass-like motion in mammalian cells, providing a different perspective on intracellular motion

    Human pluripotent stem cells for the modelling and treatment of respiratory diseases

    Get PDF
    Respiratory diseases are among the leading causes of morbidity and mortality worldwide, representing a major unmet medical need. New chemical entities rarely make it into the clinic to treat respiratory diseases, which is partially due to a lack of adequate predictive disease models and the limited availability of human lung tissues to model respiratory disease. Human pluripotent stem cells (hPSCs) may help fill this gap by serving as a scalable human in vitro model. In addition, human in vitro models of rare genetic mutations can be generated using hPSCs. hPSC-derived epithelial cells and organoids have already shown great potential for the understanding of disease mechanisms, for finding new potential targets by using high-throughput screening platforms, and for personalised treatments. These potentials can also be applied to other hPSC-derived lung cell types in the future. In this review, we will discuss how hPSCs have brought, and may continue to bring, major changes to the field of respiratory diseases by understanding the molecular mechanisms of the pathology and by finding efficient therapeutics

    Calcium-activated potassium channels:Implications for aging and age-related neurodegeneration

    Get PDF
    Population aging, as well as the handling of age-associated diseases, is a worldwide increasing concern. Among them, Alzheimer's disease stands out as the major cause of dementia culminating in full dependence on other people for basic functions. However, despite numerous efforts, in the last decades, there was no new approved therapeutic drug for the treatment of the disease. Calcium-activated potassium channels have emerged as a potential tool for neuronal protection by modulating intracellular calcium signaling. Their subcellular localization is determinant of their functional effects. When located on the plasma membrane of neuronal cells, they can modulate synaptic function, while their activation at the inner mitochondrial membrane has a neuroprotective potential via the attenuation of mitochondrial reactive oxygen species in conditions of oxidative stress. Here we review the dual role of these channels in the aging phenotype and Alzheimer's disease pathology and discuss their potential use as a therapeutic tool

    Mitochondrial dysfunction in neurodegenerative diseases:A focus on iPSC-derived neuronal models

    Get PDF
    Progressive neuronal loss is a hallmark of many neurodegenerative diseases, including Alzheimer's and Parkinson's disease. These pathologies exhibit clear signs of inflammation, mitochondrial dysfunction, calcium deregulation, and accumulation of aggregated or misfolded proteins. Over the last decades, a tremendous research effort has contributed to define some of the pathological mechanisms underlying neurodegenerative processes in these complex brain neurodegenerative disorders. To better understand molecular mechanisms responsible for neurodegenerative processes and find potential interventions and pharmacological treatments, it is important to have robust in vitro and pre-clinical animal models that can recapitulate both the early biological events undermining the maintenance of the nervous system and early pathological events. In this regard, it would be informative to determine how different inherited pathogenic mutations can compromise mitochondrial function, calcium signaling, and neuronal survival. Since post-mortem analyses cannot provide relevant information about the disease progression, it is crucial to develop model systems that enable the investigation of early molecular changes, which may be relevant as targets for novel therapeutic options. Thus, the use of human induced pluripotent stem cells (iPSCs) represents an exceptional complementary tool for the investigation of degenerative processes. In this review, we will focus on two neurodegenerative diseases, Alzheimer's and Parkinson's disease. We will provide examples of iPSC-derived neuronal models and how they have been used to study calcium and mitochondrial alterations during neurodegeneration

    The Potential of Ferroptosis-Targeting Therapies for Alzheimer's Disease:From Mechanism to Transcriptomic Analysis

    Get PDF
    Alzheimer’s disease (AD), the most common form of dementia, currently affects 40–50 million people worldwide. Despite the extensive research into amyloid β (Aβ) deposition and tau protein hyperphosphorylation (p-tau), an effective treatment to stop or slow down the progression of neurodegeneration is missing. Emerging evidence suggests that ferroptosis, an iron-dependent and lipid peroxidation-driven type of programmed cell death, contributes to neurodegeneration in AD. Therefore, how to intervene against ferroptosis in the context of AD has become one of the questions addressed by studies aiming to develop novel therapeutic strategies. However, the underlying molecular mechanism of ferroptosis in AD, when ferroptosis occurs in the disease course, and which ferroptosis-related genes are differentially expressed in AD remains to be established. In this review, we summarize the current knowledge on cell mechanisms involved in ferroptosis, we discuss how these processes relate to AD, and we analyze which ferroptosis-related genes are differentially expressed in AD brain dependant on cell type, disease progression and gender. In addition, we point out the existing targets for therapeutic options to prevent ferroptosis in AD. Future studies should focus on developing new tools able to demonstrate where and when cells undergo ferroptosis in AD brain and build more translatable AD models for identifying anti-ferroptotic agents able to slow down neurodegeneration
    • …
    corecore