Autophagy and Apoptosis Are Differentially Induced in Neurons and Astrocytes Treated with an In Vitro Mimic of the Ischemic Penumbra

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.

Cell death and neurodegeneration in the postnatal development of cerebellar vermis in normal and Reeler mice

“…Species-specific responses may be an additional complicating factor in the study of neural death, as well as inter-area anatomical/functional differences. For example, contrasting results were obtained after ischemia or stroke in several mammalian species, as mixed apoptotic/necrotic figures were described in rat (Wei et al, 2006) or mouse (Pamenter et al, 2012), whereas no evidence of apoptosis was described in gerbils (Colbourne et al, 1999), and apoptosis and necrosis occurred in separate neuronal populations in dogs (Martin et al, 2000). Likewise, death in prionic infection was reported to be autophagic by some authors (Liberski et al, 2008) but others denied the occurrence of autophagy and, instead, described a novel, non-apoptotic, non-autophagic form of neuronal death in a transgenic mouse model (Christensen et al, 2010).…”

Sirtuin 3 Mediates Neuroprotection of Ketones against Ischemic Stroke

“…Forkhead box O3a and SOD2 are two main substrates of SIRT3. 21 We examined the total protein levels of SIRT3 and FoxO3a from homogenates in the penumbra. Ketones increased SIRT3 (79.0 ± 7.2% ketones versus 53.7 ± 5.5% NS) and FoxO3a levels (66.0 ± 7.8% ketones versus 36.7 ± 2.6% NS) ( Figures 5A and 5C).…”

Modulation of Mitochondrial Function and Autophagy Mediates Carnosine Neuroprotection Against Ischemic Brain Damage

“…Enhancement of autophagic processes was observed in brain after hypoxic-ischemia, 7 and the occurrence of autophagy measured by conversion of LC3-I to LC3-II during brain ischemia has been confirmed by in vivo imaging. 8 Although controversy exists whether autophagy contributes to cell death or cell survival, 9-11 recent observations using inhibitors or modulators of autophagy revealed that autophagy mediates neuronal cell death during ischemia. 12,13 Wen et al 14 observed autophagy in focal cerebral ischemia, and demonstrated that treatment with inhibitors of autophagy significantly reduced brain damage.…”