Acute NMDA toxicity in cultured rat cerebellar granule neurons is accompanied by autophagy induction and late onset autophagic cell death phenotype
© Sadasivan et al; licensee BioMed Central Ltd. 2010
Received: 2 December 2008
Accepted: 18 February 2010
Published: 18 February 2010
Autophagy, an intracellular response to stress, is characterized by double membrane cytosolic vesicles called autophagosomes. Prolonged autophagy is known to result in autophagic (Type II) cell death. This study examined the potential role of an autophagic response in cultured cerebellar granule neurons challenged with excitotoxin N-methyl-D-aspartate (NMDA).
NMDA exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in both cell bodies and neurites as early as 3 hours post-treatment. Elevated levels of Beclin-1 and the autophagosome-targeting LC3-II were also observed following NMDA exposure. Prolonged exposure of the cultures to NMDA (8-24 h) generated MDC-, LC3-positive autophagosomal bodies, concomitant with the neurodegenerative phase of NMDA challenge. Lysosomal inhibition studies also suggest that NMDA-treatment diverted the autophagosome-associated LC3-II from the normal lysosomal degradation pathway. Autophagy inhibitor 3-methyladenine significantly reduced NMDA-induced LC3-II/LC3-I ratio increase, accumulation of autophagosomes, and suppressed NMDA-mediated neuronal death. ATG7 siRNA studies also showed neuroprotective effects following NMDA treatment.
Collectively, this study shows that autophagy machinery is robustly induced in cultured neurons subjected to prolonged exposure to excitotoxin, while autophagosome clearance by lysosomal pathway might be impaired. Our data further show that prolonged autophagy contributes to cell death in NMDA-mediated excitotoxicity.
Autophagy is an intracellular pathway that is activated in response to cell stress. It is a phenomenon where the cytoplasmic organelles in the cell are engulfed by double membrane vesicles called the autophagosomes and delivered to the lysosomes where the organelles are broken down by lysosomal proteases and the amino acids recycled back into the cell machinery to aid cell survival [1, 2]. Some of the key autophagy protein (Atg) identified to be involved in this process are Atg4, Atg6, Atg8, Atg12 and Atg5 . Autophagy has been reported to be vital in the development of the central nervous system [4, 5]. It has also been documented to be constitutively active in the healthy neurons and aid survival . Researchers have used a number of tools to study and interpret autophagy induction . For example, an elevated level of Bcl-2-binding protein Beclin-1 (Atg6) has been documented to be indicative of autophagy induction. Another protein marker for autophagy induction extensively studied, is the lipidated form of microtubule associated protein light chain-3 (MAP-LC3) found on the outer and to a lesser extent the inner membrane of the double membrane of the autophagosome.
Programmed cell death among neurons in the central nervous system is a regulated process. Neurons undergo either apoptotic (type I) or autophagic (type II) cell death or oncotic/necrotic (type III) depending on the nature of insult [8, 9]. Acute excitotoxic insults resulting from the use of glutamate in primary culture has been shown to induce both oncotic and apoptotic cell death in neurons [10, 11]. Increased excitation of the glutamate receptors by its ligand has been shown to cause an imbalance in the ionic gradient in neurons, resulting in an increase in the calcium and sodium levels intracellularly leading to oncosis. At the same time, this excessive activation in neurons has also been demonstrated to activate the endonucleases, causing internucleosomal DNA fragmentation, thus resulting in apoptosis. Though extensive studies have been conducted on apoptotic cell death mechanisms, the biochemical mechanisms and the exact definition of "autophagic cell death" is poorly understood [12–16].
Autophagic vacuoles have been shown to accumulate in affected neurons of several neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Wang et al., (2006) recently demonstrated that the induction of autophagy was associated with axonal degeneration in Purkinje cells in Lurcher mice. More recent experimental evidence has also shown the upregulation of autophagy protein Beclin-1 (Atg-6) and/or to LC3-II/LC3-I ratio increase in different rodent models of traumatic brain injury (TBI) [17–20].
Excitotoxicity via overactivation of ionotropic glutamate receptor subtype N-methyl-D-aspartate (NMDA)-receptor, is one of the documented hallmark events that occur following acute brain injury [21, 22]. Hence we sought to examine if autophagy is a general response during excitotoxic NMDA challenge by using rat cerebellar granule neuronal (CGN) cultures in vitro. In addition, we hypothesize that autophagy and possible autophagic cell death might also participate in NMDA excitotoxicity.
Acute NMDA exposure induces autophagy in cerebellar granule neurons in culture
Autophagy protein markers beclin-1 and LC3-II are up-regulated following early phase of NMDA exposure
Autophagy inhibitor 3-Methyladenine (3-MA) effectively suppresses NMDA-induced autophagy
Interaction between NMDA challenge and LC3-II flux and turnover
Cell death in NMDA-treated neurons was alleviated by 3-MA
NMDA-induced caspase-3 activation is suppressed by 3-MA
ATG7 disruption results in neuroprotection following NMDA exposure
Discussion and Conclusions
Autophagy induction occurs in the central nervous system under conditions of stress/starvation or protein aggregating neurodegenerative diseases [5, 28–30]. This study has shown that acute excitotoxicity by NMDA exposure can act as a stressor to induce autophagy in cerebellar neurons. Glutamate excitotoxicity has previously been documented as one of the pathways of cell death following experimental traumatic brain injury [10, 31, 32]. Erlich and colleagues  demonstrated an increase in beclin-1 expression in mice following traumatic brain injury suggesting that autophagy is upregulated around the regions of injury to support the cells under duress and help dispose of damaged components. Recently, there has also been suggestive evidence for the involvement of autophagy in chronic neurodegenerative diseases such as Parkinson's disease and Huntington disease [34–36].
In our experiments we observed an increase in the autophagy protein LC3 immunostaining and the monodansylcadaverine (MDC) positive autophagosomes following NMDA treatment as compared to control samples (Figs 1, 2 and 3). The NMDA treatment also increased the levels of LC3-I when compared to the controls at earlier time periods (3 and 6 h). This transient enhancement of the LC3-I protein levels in comparison to control indicates an enhanced capability of the cells to launch an autophagic response (data not shown). There was also an increase in LC3-II levels or LC3-II/LC3-I ratio (from about 0.60 to 1.30) (Fig 5) following NMDA treatment, as measured by quantitative immunoblots. This suggests that there may be a pool of LC3-II being generated following NMDA exposure that is translocated to the outer membrane of the autophagosomes [37, 38].
Evidence from other studies demonstrated the induction of autophagy and subsequent neuronal death in spinal cord motor neurons and organotypic hippocampal cultures, following glutamate receptor-mediated injury [39, 40]. According to one study, a buildup of autophagosomes could be observed in the axonal terminals of neurons in Lurcher mice . We extended their findings by demonstrating that NMDA in cultured neurons resulted in robust autophagosome formation throughout the cell bodies and neurites. The presence of unusually large stained autophagosomal bodies 24 hours following NMDA exposure, suggests a breakdown in the turnover machinery of the autophagosomes. Also, the presence of autophagosome accumulation in the neurons at a time when neuronal death was observed, points towards the fact that enhanced autophagy may be pushing the cells towards autophagic cell death.
The NMDA-induced L3-II accumulation could be a result of either LC3-I to LC-3-II conversion or it could signify defects in LC3-II turnover. We found that both lysosomal protease inhibition and proteasome pathway inhibition significantly elevated LC3-II protein levels compared to controls, suggesting that there are at least two pathways of LC3-II turnover. NMDA treatment conditions seem to render the cells refractory to further elevation of LC3-II protein levels with co-treatment with E64d and surprisingly with lactacystin. The significance of proteasome involvement in LC3-II clearance will need further investigation. Our data suggest the proteasome system is a novel, alternative pathway for clearance of LC3-II [either membrane-associated pool or non-membrane-associated associated (cytosolic) pool] under conditions of stress.
Since prolonged autophagy has been shown to result in autophagic cell death (type II), we hypothesized that this form of cell death may be a crucial component to NMDA excitotoxicity. To test this hypothesis we employed the autophagy inhibitor 3-MA and examined whether autophagy inhibition could alleviate NMDA-mediated neuronal death. The results showed effective inhibition of the NMDA-induced increase of the lipidated LC3-II protein, the latter being important in the stabilization of the autophagosomal membrane. Also, a strong decrease in the LC3 immunostaining and the MDC-positive autophagosome staining with 3-MA co-treatment following NMDA exposure were observed as well. 3-MA co-treatment in neurons [45–47] also resulted in significant protection against NMDA-induced cell death (Fig 8). To avoid complete reliance on interpretation of 3-MA treatment effects, we used the siRNA approach to investigate the role of autophagy in NMDA mediated neurotoxicity . Employing ATG7 siRNA significantly decreased LDH release (a marker for compromised cell membrane integrity) from neurons into the culture medium, suggesting its neuroprotective effects (Fig 10).
Apoptotic and autophagy pathways are intricately intertwined in the cell [42–44]. In our experiments we observed that NMDA-induced caspase-3 enzyme activation and breakdown of spectrin were 3-MA sensitive. Also, intervention with the pan caspase inhibitor IDN-6556 (20 μM) resulted in partial but significant protection against NMDA-mediated excitotoxicity, suggesting the involvement of other cell death pathways. These observations suggest that autophagy and caspase-3 activation may be interlinked.
In summary, based on several lines of evidence, this study shows that autophagosomes accumulated in neurons when subjected to direct excitotoxic NMDA exposure in a simple culture paradigm. Secondly, based on (i) the presence of MDC- and LC3-densely stained autophagosomes following NMDA treatment (but not amino acid starvation paradigm) and (ii) the refractory response of LC3-II to E64d treatment in the presence of NMDA, it appears that NMDA exposure impairs or diverts the LC3-II/autophagosomes from normal clearance. Autophagy inhibition via 3-MA and ATG7 siRNA provides neuroprotection against NMDA toxicity.
Taken together, we propose that NMDA neurotoxic stress triggers the neurons into an autophagic response. Impairment in autophagosomes clearance causes the neurons to be 'stuck" in the early autophagosome accumulation mode, which appears to be detrimental to cell survival. In other words, autophagy under NMDA toxicity paradigm in fact contributes to the neuronal cell injury/cell death process. Obviously, more research into this direction will be needed to confirm this hypothesis. It is also equally important to examine how widespread this autophagic response phenomenon is in other neurotoxic conditions either in culture or in vivo.
Chemicals and Antibodies
N-methyl D aspartate (NMDA), 3-methyladenine (MA), monodansylcadaverine (MDC), lysosomal protease inhibitor E64D and proteasome blocker lactacystin were purchased from Sigma Laboratories (St. Louis, MO). Prolong Antifade was purchased from Molecular Probes (Eugene, OR). Fetal bovine serum and Dulbecco's modified eagle's medium (DMEM) was from Gibco laboratories (Grand Island, NY). Antibodies mouse monoclonal anti-αII-spectrin and rabbit polyclonal anti-caspase-3 specific spectrin breakdown product of 120 kDa (SBDP120) [26, 27] were made in-house as was pan caspase inhibitor IDN-6556 . Anti-β-actin antibody was purchased from Sigma laboratories (St.Louis, MO), anti-NeuN antibody was obtained from Chemicon Laboratories (Temecula, CA) and anti-LC3 antibody from Novus Biologicals (Littleton, CO).
Female pregnant Sprague Dawley rats (Harlan Laboratories) were received and housed in individual cages. The animals were maintained on a 12 h light/dark cycle with food and water ad libitum. All the experimental procedures in the animals were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and the protocols were approved by the UF IACUC.
Cell cultures and treatments
Cerebellar cultures were obtained from dissociated cerebella of 6-8 day old Sprague Dawley rat pups (Harlan Laboratories) and plated in Dulbecco's modified eagle's medium (DMEM) supplemented with 25 mM glucose, 25 mM KCl, 10% fetal bovine serum on culture dishes (Nunc plates, Fisher). 1β-arabinofuranosylcytosine (10 μM) was added to the culture medium 22 hours after plating to prevent the proliferation on non-neuronal cells for 48 hours. On the 8th day following harvesting, the neurons were exposed to different treatment conditions and subsequent experimental end points. The neurons were treated with or without NMDA (200 μM) for different time periods and the cells were eventually lysed with triton based lysis buffer for protein immunoblots. The other treatment condition involved a co-treatment of NMDA with 3-methyladenine (3-MA, 10 mM). For fluorescent microscopy, the neurons were cultured on glass coverslips coated with poly-l-lysine and treated in a similar manner as the cultures on plates. Positive control to induce autophagy involved subjecting CGNs to amino acid starvation by incubating them in Hank's buffer with 25 mM glucose (with calcium, magnesium) and vitamins.
Cerebellar cells plated on coverslips were fixed using freshly prepared 4% paraformaldehyde solution for 10 min at 4°C, washed in pure methanol and then permeabilized with 1× Tris buffered saline -Tween-20 (TBST, Sigma Laboratories, St. Louis, MO). Following TBST washing, the cells were incubated in 5% normal goat serum (NGS) at 37°C for 30 minutes before incubating with the primary antibody microtubule associated light chain-3 (LC-3; Atg8; 1:1000) in 5% NGS overnight at 4°C. On the following day, the coverslips were washed 3 times with 1× TBST and incubated with the Alexa Fluor (Molecular Probes, Carlsbad, CA) red or green-conjugated secondary antibodies (1:3000) for 1 hour at 37°C. The coverslips were then washed with 1× TBST and then mounted with the mounting medium Vectashield (Burlingame, CA) and observed under the microscope.
Autophagosome labeling with MDC
The neurons on the coverslips were incubated with a fluorescent dye monodansylcadaverine (MDC; 0.05 mM) in phosphate buffered saline (PBS) after 6, 12, 16 and 24 hour NMDA challenges at 37°C for 10 minutes, to observe for autophagosomes . The cells were washed 2 times with PBS, mounted using Antifade solution (Prolong Antifade) and immediately observed by Zeiss fluorescence microscope. Following prolonged NMDA, we observed unusually large autophagosomal bodies, which are defined as having a size at least 5-time the size of normal sized autophagosomes. For their quantification of both average sized autophagosomes and unusually large autophagosomal bodies, for each experimental condition/well, 4 randomly selected fields per well (400× magnification) are used and more than 20 cells in each field are analyzed with counted.
Western Blot Analysis
The cerebellar neuronal lysates were collected at different time points after treatment with the appropriate media using lysis buffer containing 1% (v/v) Triton X-100, 5 mM EGTA, 5 mM EDTA, 150 mM NaCl and 20 mM Tris HCl (pH 7.4). The protein content was determined using DC Protein Assay (Bio-Rad, Hercules, CA) and the protein concentration was standardized to 1 μg/μL. Twenty micrograms of protein were subjected to SDS-PAGE gel electrophoresis on 4-20% or 6% Tris-glycine gels (Invitrogen, Carlsbad, CA) and then transferred onto PVDF membrane on a semi-dry electro transferring unit (Bio-Rad). Following the transfer, the membranes were blocked in 5% nonfat dry milk in 1× Tris buffered saline with Tween-20 (TBST) and probed overnight with primary antibody at 4°C. The following day, the membranes were washed with TBST and probed with either secondary peroxidase conjugated anti-rabbit or the biotinylated anti-mouse antibody. Immunoreactivity was detected by either using streptavidin alkaline phosphatase conjugate tertiary antibody or enhanced chemiluminescence (ECL) reaction (Amersham, Piscataway, NJ). Densitometric quantification of the bands was performed using ImageJ software (version 1.29×; NIH, Bethesda, MD).
Lactate Dehydrogenase (LDH) Release Assay for Cell Death
Lactate dehydrogenase release assay was performed to assess cell death by measuring the release of lactate dehydrogenase into the medium from damaged cells due to necrosis and secondary necrosis following apoptosis  or autophagic cell death. Culture medium, 25 μL, was collected after 0, 3, 6, 12, 16, 20 hours and 1 day in 96-well flat bottom plates. An equivalent volume (25 μL) of detection reagent (CytoTox One Reagent, Promega, Madison, WI), was added to each well containing the culture medium and incubated for 30 minutes in the dark at room temperature. Absorbance was measured (wavelength: 490 nm) using a colorimetric microplate reader (SpectraMax Gemini, Molecular Devices). Six replicates for each time point per experiment were assayed and three such experiments were performed. The arbitrary density unit values were plotted against time.
Caspase-3 enzymatic activity assay
To assay for caspase-3 activity, control, NMDA-treated and NMDA/3-MA co-treated granule neurons from three different wells (12 and 24 h) were scraped in a buffer containing 20 mM Tris-HC1 (pH 7.4 at 4°C), 150 mM NaC1, 1 mM dithiothreitol, 5 mM EDTA, 5 mM EGTA, and 1% (vol/vol) Triton X-100 for 1 h. The cleared lysates were mixed with 50% (vol/vol) glycerol. Cell lysates were assayed with 100 μM acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-MCA; Bachem Bioscience), 100 mM HEPES, 10% glycerol, 1 mM EDTA, 10 mM dithiothreitol. Fluorescence (excitation, 380 ± 15 nm; emission, 460 ± 15 nm) was measured at 60 minutes using a fluorescent microplate reader (SpectraMax Gemini EM, Molecular Devices) as described previously .
ATG7 short interfering RNA (siRNA) preparation and transfection
Small interfering RNA (siRNA) targeting rat ATG7 (5'-GCAUCAUCUUUGAAGUGAA-3'; Sigma), or pooled non-targeting control siRNA (5'-AUGAACGUGAAUUGCUCAA-3', 5'-UAAGGCUAUGAAGAGUAC-3', 5'-AUGUAUUGGCCUGUAUUAG-3', and 5'-UAGCGACUAAACACAUCAA-3'; Dharmacon)  were purchased commercially and used. Primary neurons were transfected on day 4 of culture preparation, with 20 nmol of ATG7 or control siRNA using Lipofectamine 2000 (Invitrogen), 72 hours before subjecting to NMDA challenge. The efficacy of ATG7 knockdown was assessed by Western blot using an antibody against Atg7 (Sigma).
One-way ANOVA with Tukey post hoc test was used to draw comparisons between groups in the LDH assay. Data was plotted as means ± S.E.M. (standard error of the mean). A value of p ≤ 0.05 was considered to be significant. Student's t-test was performed to draw statistical comparisons between two treatment groups and a p < 0.05 was considered to be statistically significant.
- 3 MA:
microtubule associated protein-light chain 3.
The authors wish to thank Drs. William Dunn, Jr. and Lucia Notterpek (University of Florida) for their insights and opinions in this work. This work was supported by the Department of Defense grant DAMMED-03-1-0066 and NIH grant R01 NS049175-01-A1. KKW and RLH own stocks and receive royalties from and are executive officers of Banyan Biomarkers Inc. and as such may benefit financially as a result of the outcome of this research or work reported in this publication.
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