Autophagy and apoptosis are differentially induced in neurons and astrocytes treated with an in vitro mimic of the ischemic penumbra - PubMed (original) (raw)

Autophagy and apoptosis are differentially induced in neurons and astrocytes treated with an in vitro mimic of the ischemic penumbra

Matthew E Pamenter et al. PLoS One. 2012.

Abstract

The development of clinical stroke therapies remains elusive. The neuroprotective efficacies of thousands of molecules and compounds have not yet been determined; however, screening large volumes of potential targets in vivo is severely rate limiting. High throughput screens (HTS) may be used to discover promising candidates, but this approach has been hindered by the lack of a simple in vitro model of the ischemic penumbra, a clinically relevant region of stroke-afflicted brain. Recently, our laboratory developed such a mimic (ischemic solution: IS) suitable for HTS, but the etiology of stress pathways activated by this model are poorly understood. The aim of the present study was to determine if the cell death phenotype induced by IS accurately mimics the in vivo penumbra and thus whether our model system is suitable for use in HTS. We treated cultured neuron and astrocyte cell lines with IS for up to 48 hrs and examined cellular energy state ([ATP]), cell and organelle morphology, and gene and molecular profiles related to stress pathways. We found that IS-treated cells exhibited a phenotype of mixed apoptosis/autophagy characteristic of the in vivo penumbra, including: (1) short-term elevation of [ATP] followed by progressive ATP depletion and Poly ADP Ribose Polymerase cleavage, (2) increased vacuole number in the cytoplasm, (3) mitochondrial rupture, decreased mitochondrial and cristae density, release of cytochrome C and apoptosis inducing factor, (4) chromatin condensation, nuclear lamin A and DNA cleavage, fragmentation of the nuclear envelope, and (5) altered expression of mRNA and proteins consistent with autophagy and apoptosis. We conclude that our in vitro model of the ischemic penumbra induces autophagy and apoptosis in cultured neuron and astrocyte cell lines and that this mimic solution is suitable for use in HTS to elucidate neuroprotective candidates against ischemic penumbral cell death.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. IS induces extensive autophagic vacuolization of neuronal cytoplasm and depletes [ATP] in neurons and astrocytes.

IS-treated neurons and oligomycin-treated neurons and astrocytes exhibit extensive cytoplasmic vacuolization, organelle digestion, and [ATP] changes characteristic of autophagy. (A) Sample TEM images of neurons (upper panels) and astrocytes (lower panels) treated as indicated for 24 hrs. Arrows indicate vacuoles. (B) Summary of vacuole density in the cytoplasm by volume. TEM experiments were repeated 2–3 times and 10–20 cells were examined from each treatment group. (C) Summary of change in [ATP] with time from neurons (open symbols) and astrocytes (closed symbols) treated as indicated through 48 hrs from >10 different experiments. (D) Summary of fold-change in protein expression from western blot analysis of PARP cleavage in samples treated for 6 hrs. Changes were normalized to α–actin expression in the same sample. (E) Sample western blots from (D). Blots are representative of 3 separate experiments. Data are mean ± SEM. Asterisks (*) indicate significant difference from untreated controls (p<0.05). Treatments: control (DMEM/F12), ischemic solution (IS), 2.5 µm staurosporine (STS), and 10 µm oligomycin A (Oligo A).

Figure 2

Figure 2. IS induces apoptotic Annexin V translocation in neurons and astrocytes.

(A) Sample paired DIC (left panels) and Annexin V and DAPI (green and blue fluorescence, respectively; right panels) confocal microscopy images of neurons and astrocytes treated as indicated for 24 hrs. Images are representative of 4 separate experiments. (B) Summary of the ratio of Annexin V-positive stained cells to DAPI-stained nuclei. (C) Summary of Annexin V fluorescence from neurons or astrocytes treated in 96-well microplates as indicated for 24 hrs. Data are mean ± SEM. Asterisks (*) indicated significant difference from untreated controls (p<0.05).

Figure 3

Figure 3. IS causes mitochondrial fission and membrane rupture.

(A) Sample TEM images of mitochondria from neurons (upper) and astrocytes (lower) treated as indicated for 24 hrs. (B) Sample western blots of apoptosis inducing factor (AIF) and cytochrome C (Cyto C) release from samples treated as indicated for 6 hrs. Blots are representative of 3 separate experiments.

Figure 4

Figure 4. IS-treated nuclei exhibit apoptotic chromatin condensation, nuclear envelope fragmentation into apoptotic bodies, and laddered DNA cleavage.

(A) Sample TEM images of nuclei from neurons (upper panels) and astrocytes (lower panels) treated as indicated for 24 hrs. White arrows indicate condensed chromatin beads and fragmenting nuclei. (B) Summary of nuclear volume density from samples in (A). (C) Summary of chromatin volume density from samples in (A). Note: nuclei were too fragmented in oligomycin A-treated astrocytes for quantification. (D) Sample western blots of lamin A cleavage in samples treated as indicated for 6 hrs. Blots are representative of 3 separate experiments. (E) Summary of fold-change in protein expression from (D) normalized to α–actin expression in the same sample. (F&G) Sample conventional agarose gel electrophoresis gel images of DNA fragmentation from neurons (F) and astrocytes (G) treated as indicated for 0, 12, 24, or 48 hrs. Gels are representative of 4 separate experiments. Data are mean ± SEM. Asterisks (*) indicate significant difference from untreated controls (p<0.05).

Figure 5

Figure 5. IS upregulates autophagy and apoptotic pathways in neurons and astrocytes.

(A) Sample Western blots of autophagy- and apoptosis related protein expression from neurons (left panels) and astrocytes (right panels) treated as indicated for 6 hrs. (B) Summary of fold-change in protein expressions from (A) normalized to α–actin expression in the same sample. Data are mean ± SEM from 3 separate experiments for each protein, cell type, and treatment. Asterisks (*) indicate significant difference from untreated controls (p<0.05).

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