scholarly journals Manganese-induced Mitochondrial Dysfunction Is Not Detectable at Exposures Below the Acute Cytotoxic Threshold in Neuronal Cell Types

2020 ◽  
Vol 176 (2) ◽  
pp. 446-459
Author(s):  
Emily B Warren ◽  
Miles R Bryan ◽  
Patricia Morcillo ◽  
Keisha N Hardeman ◽  
Michael Aschner ◽  
...  
Keyword(s):  
Cell Death ◽  
Mitochondrial Dysfunction ◽  
Cell Lines ◽  
Mitochondrial Function ◽  
Cell Model ◽  
Neuronal Cell ◽  
Cell Types ◽  
Cell Number ◽  
Striatal Neurons ◽  
Substrate Dependence

Abstract Manganese (Mn) is an essential metal, but excessive exposures have been well-documented to culminate in neurotoxicity. Curiously, the precise mechanisms of Mn neurotoxicity are still unknown. One hypothesis suggests that Mn exerts its toxicity by inhibiting mitochondrial function, which then (if exposure levels are high and long enough) leads to cell death. Here, we used a Huntington’s disease cell model with known differential sensitivities to manganese—STHdhQ7/Q7 and STHdhQ111/Q111 cells—to examine the effects of acute Mn exposure on mitochondrial function. We determined toxicity thresholds for each cell line using both changes in cell number and caspase-3/7 activation. We used a range of acute Mn exposures (0–300 µM), both above and below the cytotoxic threshold, to evaluate mitochondria-associated metabolic balance, mitochondrial respiration, and substrate dependence. In both cell lines, we observed no effect on markers of mitochondrial function at subtoxic Mn exposures (below detectable levels of cell death), yet at supratoxic exposures (above detectable levels of cell death) mitochondrial function significantly declined. We validated these findings in primary striatal neurons. In cell lines, we further observed that subtoxic Mn concentrations do not affect glycolytic function or major intracellular metabolite quantities. These data suggest that in this system, Mn exposure impairs mitochondrial function only at concentrations coincident with or above the initiation of cell death and is not consistent with the hypothesis that mitochondrial dysfunction precedes or induces Mn cytotoxicity.

scholarly journals Increasing the TRPM2 Channel Expression in Human Neuroblastoma SH-SY5Y Cells Augments the Susceptibility to ROS-Induced Cell Death

Cells ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 28 ◽  
Author(s):  
Xinfang An ◽  
Zixing Fu ◽  
Chendi Mai ◽  
Weiming Wang ◽  
Linyu Wei ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Cell Model ◽  
Critical Role ◽  
Neuronal Cell ◽  
Cell Types ◽  
Wild Type ◽  
Human Neuroblastoma ◽  
Channel Activation ◽  
Trpm2 Channel

Human neuroblastoma SH-SY5Y cells are a widely-used human neuronal cell model in the study of neurodegeneration. A recent study shows that, 1-methyl-4-phenylpyridine ion (MPP), which selectively causes dopaminergic neuronal death leading to Parkinson’s disease-like symptoms, can reduce SH-SY5Y cell viability by inducing H2O2 generation and subsequent TRPM2 channel activation. MPP-induced cell death is enhanced by increasing the TRPM2 expression. By contrast, increasing the TRPM2 expression has also been reported to support SH-SY5Y cell survival after exposure to H2O2, leading to the suggestion of a protective role for the TRPM2 channel. To clarify the role of reactive oxygen species (ROS)-induced TRPM2 channel activation in SH-SY5Y cells, we generated a stable SH-SY5Y cell line overexpressing the human TRPM2 channel and examined cell death and cell viability after exposure to H2O2 in the wild-type and TRPM2-overexpressing SH-SY5Y cells. Exposure to H2O2 resulted in concentration-dependent cell death and reduction in cell viability in both cell types. TRPM2 overexpression remarkably augmented H2O2-induced cell death and reduction in cell viability. Furthermore, H2O2-induced cell death in both the wild-type and TRPM2-overexpressing cells was prevented by 2-APB, a TRPM2 inhibitor, and also by PJ34 and DPQ, poly(ADP-ribose) polymerase (PARP) inhibitors. Collectively, our results show that increasing the TRPM2 expression renders SH-SY5Y cells to be more susceptible to ROS-induced cell death and reinforce the notion that the TRPM2 channel plays a critical role in conferring ROS-induced cell death. It is anticipated that SH-SY5Y cells can be useful for better understanding the molecular and signaling mechanisms for ROS-induced TRPM2-mediated neurodegeneration in the pathogenesis of neurodegenerative diseases.

scholarly journals AIF3 splicing switch triggers neurodegeneration

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Shuiqiao Liu ◽  
Mi Zhou ◽  
Zhi Ruan ◽  
Yanan Wang ◽  
Calvin Chang ◽  
...  
Keyword(s):  
Cell Death ◽  
Neurodegenerative Diseases ◽  
Mitochondrial Dysfunction ◽  
Nuclear Translocation ◽  
Chromatin Condensation ◽  
Neuronal Cell ◽  
Adult Brain ◽  
Postmortem Brain ◽  
Mitochondrial Functions

Abstract Background Apoptosis-inducing factor (AIF), as a mitochondrial flavoprotein, plays a fundamental role in mitochondrial bioenergetics that is critical for cell survival and also mediates caspase-independent cell death once it is released from mitochondria and translocated to the nucleus under ischemic stroke or neurodegenerative diseases. Although alternative splicing regulation of AIF has been implicated, it remains unknown which AIF splicing isoform will be induced under pathological conditions and how it impacts mitochondrial functions and neurodegeneration in adult brain. Methods AIF splicing induction in brain was determined by multiple approaches including 5′ RACE, Sanger sequencing, splicing-specific PCR assay and bottom-up proteomic analysis. The role of AIF splicing in mitochondria and neurodegeneration was determined by its biochemical properties, cell death analysis, morphological and functional alterations and animal behavior. Three animal models, including loss-of-function harlequin model, gain-of-function AIF3 knockin model and conditional inducible AIF splicing model established using either Cre-loxp recombination or CRISPR/Cas9 techniques, were applied to explore underlying mechanisms of AIF splicing-induced neurodegeneration. Results We identified a nature splicing AIF isoform lacking exons 2 and 3 named as AIF3. AIF3 was undetectable under physiological conditions but its expression was increased in mouse and human postmortem brain after stroke. AIF3 splicing in mouse brain caused enlarged ventricles and severe neurodegeneration in the forebrain regions. These AIF3 splicing mice died 2–4 months after birth. AIF3 splicing-triggered neurodegeneration involves both mitochondrial dysfunction and AIF3 nuclear translocation. We showed that AIF3 inhibited NADH oxidase activity, ATP production, oxygen consumption, and mitochondrial biogenesis. In addition, expression of AIF3 significantly increased chromatin condensation and nuclear shrinkage leading to neuronal cell death. However, loss-of-AIF alone in harlequin or gain-of-AIF3 alone in AIF3 knockin mice did not cause robust neurodegeneration as that observed in AIF3 splicing mice. Conclusions We identified AIF3 as a disease-inducible isoform and established AIF3 splicing mouse model. The molecular mechanism underlying AIF3 splicing-induced neurodegeneration involves mitochondrial dysfunction and AIF3 nuclear translocation resulting from the synergistic effect of loss-of-AIF and gain-of-AIF3. Our study provides a valuable tool to understand the role of AIF3 splicing in brain and a potential therapeutic target to prevent/delay the progress of neurodegenerative diseases.

Histochemical and Fluorescent Analyses of Mitochondrial Integrity in Chick Auditory Neurons following Deafferentation

2010 ◽  
Vol 21 (03) ◽  
pp. 204-218 ◽  
Author(s):  
Hope Elizabeth Karnes ◽  
Peter Nicholas Scaletty ◽  
Dianne Durham
Keyword(s):  
Cell Death ◽  
Mitochondrial Function ◽  
Apoptotic Cell ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Enzyme Reaction ◽  
Oxidative Enzymes ◽  
Mitochondrial Enzymes ◽  
Auditory Neurons ◽  
Mitotracker Red

Background: Neurons rely exclusively on mitochondrial oxidative phosphorylation to meet cellular energy demands, and disruption of mitochondrial function often precipitates neuronal cell death. Auditory neurons in the chick brain stem (n. magnocellularis [NM]) receive glutamatergic innervation exclusively from ipsilateral eighth nerve afferents. Cochlea removal permanently disrupts afferent support and ultimately triggers apoptotic cell death in 30–50% of ipsilateral, deafferented neurons. Here, we evaluated whether disruption of mitochondrial function occurs during deafferentation-induced neuronal cell death. Purpose: To determine whether mitochondrial dysfunction occurs preferentially within dying NM neurons. Research Design: An experimental study. All birds underwent unilateral cochlea removal. Normally innervated neurons contralateral to surgery served as within-animal controls. Study Sample: Hatchling broiler chickens between 8 and 12 days of age served as subjects. A total of 62 birds were included in the study. Intervention: Cochlea removal was performed to deafferent ipsilateral NM neurons and trigger neuronal cell death. Data Collection and Analysis: Following unilateral cochlea removal, birds were sacrificed 12, 24, 48, or 168 hours later, and brain tissue was harvested. Brainstems were sectioned through NM and evaluated histochemically for oxidative enzyme reaction product accumulation or reacted for Mitotracker Red, an indicator of mitochondrial membrane potential (m) and cytoplasmic TdT-mediated dUTP Nick-End Labeling (TUNEL), an indicator of cell death. Histochemical staining intensities for three mitochondrial enzymes, succinate dehydrogenase (SDH), cytochrome c oxidase (CO), and ATP synthase (ATPase) were measured in individual neurons and compared in ipsilateral and contralateral NM. Comparisons were made using unpaired t-tests (CO) or Kruskal Wallis one way ANOVA followed by Dunn's post hoc pairwise comparisons (ATPase, SDH). Mitotracker Red tissue was examined qualitatively for the presence of and extent of colocalization between Mitotracker Red and TUNEL label in NM. Results: Results showed global upregulation of all three oxidative enzymes within deafferented NM neurons compared to contralateral, unperturbed NM neurons. In addition, differential SDH and ATPase staining intensities were detected across neurons within the ipsilateral nucleus, suggesting functional differences in mitochondrial metabolism across deafferented NM. Quantitative analyses revealed that deafferented neurons with preferentially elevated SDH and ATPase activities represent the subpopulation destined to die following cochlea removal. In addition, Mitotracker Red accumulated intensely within the subset of deafferented NM neurons that also exhibited cytoplasmic TdT-mediated dUTP Nick-End Labeling (TUNEL) and subsequently died. Conclusions: Taken together, our results demonstrate that a subset of deafferented NM neurons, presumably those that die, preferentially upregulates SDH, perhaps via the tricarboxylic acid (TCA) cycle. These same neurons undergo ATPase uncoupling and an eventual loss of Δψm.

scholarly journals Glucose regulates AMP-activated protein kinase activity and gene expression in clonal, hypothalamic neurons expressing proopiomelanocortin: additive effects of leptin or insulin

2007 ◽  
Vol 192 (3) ◽  
pp. 605-614 ◽  
Author(s):  
Fang Cai ◽  
Armen V Gyulkhandanyan ◽  
Michael B Wheeler ◽  
Denise D Belsham
Keyword(s):  
Protein Kinase ◽  
Energy Homeostasis ◽  
Cell Model ◽  
Neuronal Cell ◽  
Cell Types ◽  
Glucose Sensing ◽  
Protein Kinase Activity ◽  
Energy Status ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The mammalian hypothalamus comprises an array of phenotypically distinct cell types that interpret peripheral signals of energy status and, in turn, elicits an appropriate response to maintain energy homeostasis. We used a clonal representative hypothalamic cell model expressing proopiomelanocortin (POMC; N-43/5) to study changes in AMP-activated protein kinase (AMPK) activity and glucose responsiveness. We have demonstrated the presence of cellular machinery responsible for glucose sensing in the cell line, including glucokinase, glucose transporters, and appropriate ion channels. ATP-sensitive potassium channels were functional and responded to glucose. The N-43/5 POMC neurons may therefore be an appropriate cell model to study glucose-sensing mechanisms in the hypothalamus. In N-43/5 POMC neurons, increasing glucose concentrations decreased phospho-AMPK activity. As a relevant downstream effect, we found that POMC transcription increased with 2.8 and 16.7 mM glucose. Upon addition of leptin, with either no glucose or with 5 mM glucose, we found that leptin decreased AMPK activity in N-43/5 POMC neurons, but had no significant effect at 25 mM glucose, whereas insulin decreased AMPK activity at only 5 mM glucose. These results demonstrate that individual hypothalamic neuronal cell types, such as the POMC neuron, can have distinct responses to peripheral signals that relay energy status to the brain, and will therefore be activated uniquely to control neuroendocrine function.

scholarly journals TNFα-induced AMPA-receptor trafficking in CNS neurons; relevance to excitotoxicity?

2004 ◽  
Vol 1 (3) ◽  
pp. 263-273 ◽  
Author(s):  
DMITRI LEONOUDAKIS ◽  
STEVEN P. BRAITHWAITE ◽  
MICHAEL S. BEATTIE ◽  
ERIC C. BEATTIE
Keyword(s):  
Cell Death ◽  
Inflammatory Response ◽  
Hippocampal Neurons ◽  
Neuronal Damage ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Cell Types ◽  
Synaptic Function ◽  
Ampar Trafficking ◽  
Novel Target

Injury and disease in the CNS increases the amount of tumor necrosis factor α (TNFα) that neurons are exposed to. This cytokine is central to the inflammatory response that occurs after injury and during prolonged CNS disease, and contributes to the process of neuronal cell death. Previous studies have addressed how long-term apoptotic-signaling pathways that are initiated by TNFα might influence these processes, but the effects of inflammation on neurons and synaptic function in the timescale of minutes after exposure are largely unexplored. Our published studies examining the effect of TNFα on trafficking of AMPA-type glutamate receptors (AMPARs) in hippocampal neurons demonstrate that glial-derived TNFα causes a rapid (<15 minute) increase in the number of neuronal, surface-localized, synaptic AMPARs leading to an increase in synaptic strength. This indicates that TNFα-signal transduction acts to facilitate increased surface localization of AMPARs from internal postsynaptic stores. Importantly, an excess of surface localized AMPARs might predispose the neuron to glutamate-mediated excitotoxicity and excessive intracellular calcium concentrations, leading to cell death. This suggests a new mechanism for excitotoxic TNFα-induced neuronal death that is initiated minutes after neurons are exposed to the products of the inflammatory response.Here we review the importance of AMPAR trafficking in normal neuronal function and how abnormalities that are mediated by glial-derived cytokines such as TNFα can be central in causing neuronal disorders. We have further investigated the effects of TNFα on different neuronal cell types and present new data from cortical and hippocampal neurons in culture. Finally, we have expanded our investigation of the temporal profile of the action of this cytokine relevant to neuronal damage. We conclude that TNFα-mediated effects on AMPAR trafficking are common in diverse neuronal cell types and very rapid in their onset. The abnormal AMPAR trafficking elicited by TNFα might present a novel target to aid the development of new neuroprotective drugs.

scholarly journals Neuronal apoptosis: signal and cell diversity

1969 ◽  
Vol 40 (1) ◽  
pp. 124-133
Author(s):  
Lina Vanessa Becerra ◽  
Hernán José Pimienta
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Neuronal Response ◽  
Neuronal Cell ◽  
Cell Types ◽  
Point Of View ◽  
Trophic Factors ◽  
Cell Diversity ◽  
Apoptotic Pathways ◽  
The Brain

Programmed cell death occurs as a physiological process during development. In the brain and spinal cord this event determines the number and location of the different cell types. In adulthood, programmed cell death or apoptosis is more restricted but it may play a major role in different acute and chronic pathological entities. However, in contrast to other tissues where apoptosis has been widely documented from a morphological point of view, in the central nervous system complete anatomical evidence of apoptosis is scanty. In spite of this there is consensus about the activation of different signal systems associated to programmed cell death. In the present article we attempt to summarize the main apoptotic pathways so far identified in nervous tissue. Considering that apoptotic pathways are multiple, the neuronal cell types are highly diverse and specialized and that neuronal response to injury and survival depends upon tissue context, (i.e., preservation of connectivity, glial integrity and cell matrix, blood supply and trophic factors availability) what is relevant for the apoptotic process in a sector of the brain may not be important in another.

scholarly journals Drug-Screening Strategies for Inhibition of Virus-Induced Neuronal Cell Death

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2317
Author(s):  
Durbadal Ojha ◽  
Tyson A. Woods ◽  
Karin E. Peterson
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Zika Virus ◽  
Treatment Options ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Virus Production ◽  
Neuronal Stem Cell ◽  
Stem Cell Lines ◽  
Simplex Virus

A number of viruses, including Herpes Simplex Virus (HSV), West Nile Virus (WNV), La Crosse Virus (LACV), Zika virus (ZIKV) and Tick-borne encephalitis virus (TBEV), have the ability to gain access to the central nervous system (CNS) and cause severe neurological disease or death. Although encephalitis cases caused by these viruses are generally rare, there are relatively few treatment options available for patients with viral encephalitis other than palliative care. Many of these viruses directly infect neurons and can cause neuronal death. Thus, there is the need for the identification of useful therapeutic compounds that can inhibit virus replication in neurons or inhibit virus-induced neuronal cell death. In this paper, we describe the methodology to test compounds for their ability to inhibit virus-induced neuronal cell death. These protocols include the isolation and culturing of primary neurons; the culturing of neuroblastoma and neuronal stem cell lines; infection of these cells with viruses; treatment of these cells with selected drugs; measuring virus-induced cell death using MTT or XTT reagents; analysis of virus production from these cells; as well as the basic understanding in mode of action. We further show direct evidence of the effectiveness of these protocols by utilizing them to test the effectiveness of the polyphenol drug, Rottlerin, at inhibiting Zika virus infection and death of neuronal cell lines.

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