Neurotransmitters, KCl and antioxidants rescue striatal neurons from apoptotic cell death in culture

Iacovitti, Lorraine; Stull, Natalie D.; Mishizen, Amanda

doi:10.1016/s0006-8993(98)00955-x

Cited by 41 publications

(29 citation statements)

References 46 publications

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“…Possibly, in the latter model, test factors do not serve as neurotrophins but rather act to rescue cells from stress-induced death. Further suggestive of this possibility is the fact that other classes of substances, such as antioxidants, anti-apoptotics and depolarizing agents, will rescue DA neurons in low density mixed cell cultures (Iacovitti et al, 1999;Stull et al, 2002) but did not sustain PDN viability even in high density cultures in our studies. Alternatively, it is possible that the majority of DA neurons (VTA versus SN) present in PDN cultures is critically different from that in VM cultures.…”

Section: Discussionmentioning

confidence: 55%

“…After FACS, cells were pelleted at 1000 RPM (at room temperature), re-suspended in phosphate-buffered saline with added glucose (6 mg/ml) and transplanted into rats (see below) or plated into culture at a density of 1.5 × 10 5 cells/0.5 cm 2 . In vitro, cells were routinely maintained on serum-free defined media (DM) or media supplemented with 10% fetal bovine serum (SM) as described previously (Iacovitti et al, 1999;Stull et al, 2002). In some cases (see Table 1), DM was supplemented with added growth substances in the following concentrations: all growth factors at 10 and/or 100 ng/ml, including GDNF family members (GDNF, neurturin, artemin, persphin) TGFβ-3, the neurotrophin family members (NGF, brain derived neurotrophic factor [BDNF], neurotrophin 3 [NT3]); fibroblast growth factor (FGF) 2 and 8; sonic hedgehog (SHH); epidermal growth factor (EGF), cytokines interleukin (IL) 1 and 2 and leukemia growth factor (LIF); depolarizing agents 40μM KCl, 20μM DA, antiapoptotic agents, including the caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK at 100μM, 250μM trolox (water soluble vitamin E), 100μM melatonin.…”

Section: Cell Dissociation Facs Sorting and Culture Methodsmentioning

confidence: 99%

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Purified mouse dopamine neurons thrive and function after transplantation into brain but require novel glial factors for survival in culture

Donaldson

Marshall

Yang

et al. 2005

Molecular and Cellular Neuroscience

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Cell replacement therapy in Parkinson's disease depends on a reliable source of purified dopamine (DA) neurons (PDN) and the identification of factors relevant to their survival. Our goal was to genetically tag and purify by flow cytometry embryonic midbrain DA neurons from a transgenic mouse line carrying 11 kb of human tyrosine hydroxylase promoter driving expression of the enhanced green fluorescent protein (GFP) for studies in vivo and in vitro. A 99% purification of GFP + cells was achieved. When transplanted into 6-hydroxydopamine-treated rat striatum, PDN survived, became well-integrated and produced recovery from amphetamine-induced motor behaviors. However, when grown in culture, PDN died within days of plating. No known growth factors prevented PDN death as did incubation with novel factors in glia/glial-conditioned media. We conclude that GFP-tagged DA neurons can be purified to homogeneity and can survive and function when grown with glial factors in vitro or after transplantation in vivo.

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Section: Discussionmentioning

confidence: 55%

Section: Cell Dissociation Facs Sorting and Culture Methodsmentioning

confidence: 99%

Purified mouse dopamine neurons thrive and function after transplantation into brain but require novel glial factors for survival in culture

Donaldson

Marshall

Yang

et al. 2005

Molecular and Cellular Neuroscience

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“…Although low KCl media initiate apoptosis in this preparation (Gallo et al 1987;Peng et al 1991) this is prevented by NMDA receptor activation (Balázs et al 1988;Bhave et al 1999) and thus is not a factor in the present experimental design. This is important, as there is ample evidence that apoptotic neuronal cell death is ameliorated by antioxidants (Patel 1998;Iacovitti et al 1999;González-Polo et al 2003).…”

Section: Discussionmentioning

confidence: 99%

Relationships between superoxide levels and delayed calcium deregulation in cultured cerebellar granule cells exposed continuously to glutamate

Vesce

Kirk

Nicholls

2004

Journal of Neurochemistry

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The relationship is investigated between superoxide levels in single cultured rat cerebellar granule neurons exposed continuously to glutamate in low KCl medium and the deregulation of cytoplasmic Ca 2+ . Cells that maintain a regulated cytoplasmic-free Ca 2+ and mitochondrial polarization in the presence of glutamate show no increase in superoxide levels until the onset of cytoplasmic Ca 2+ deregulation. Oxidative stress of mitochondrial origin is readily detectable, as the inhibitors rotenone and antimycin A markedly increase superoxide levels with no effect on cytoplasmic-free Ca 2+ . The potent cell-permeant superoxide dismutase/catalase mimetic manganese tetrakis (N-ethylpyridinium-2yl) porphyrin, MnTE-PyP, abolishes the deregulation-related increase in superoxide but has no effect on deregulation itself. A combination of catalase with the free radical scavenger 4-hydroxy-TEMPO also fails to reduce deregulation. Following the loss of Ca 2+ homeostasis nuclei undergo condensation; this morphological change is not inhibited by MnTE-PyP and cannot account for the increased ethidium fluorescence. Phospholipase A 2 inhibitors decrease the deregulation-related increase in superoxide without protecting against deregulation. In conclusion, our study indicates that deregulation is not caused by NMDA receptor-mediated oxidative stress as NMDA receptor activation does not increase superoxide levels until the onset of deregulation. Furthermore, the majority of superoxide is produced in the cytoplasm rather than in mitochondria.

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“…3). Given that depolarization is known to delay apoptotic cell death (Iacovitti et al, 1999), it was possible that the addition of KCl simply enhanced survival of the precursor cell population. To determine whether KCl was truly activating a latent population, rather than merely keeping the hippocampal precursors alive, we repeated our experiment using Bax knockout mice, which lack programmed cell death in the hippocampus (Sun et al, 2004).…”

Section: Depolarization Activates Hippocampal Stem Cells In Vitromentioning

confidence: 99%

Latent Stem and Progenitor Cells in the Hippocampus Are Activated by Neural Excitation

Walker¹,

White²,

Black³

et al. 2008

J. Neurosci.

107

130

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The regulated production of neurons in the hippocampus throughout life underpins important brain functions such as learning and memory. Surprisingly, however, studies have so far failed to identify a resident hippocampal stem cell capable of providing the renewable source of these neurons. Here, we report that depolarizing levels of KCl produce a threefold increase in the number of neurospheres generated from the adult mouse hippocampus. Most interestingly, however, depolarizing levels of KCl led to the emergence of a small subpopulation of precursors (approximately eight per hippocampus) with the capacity to generate very large neurospheres (Ͼ250 m in diameter). Many of these contained cells that displayed the cardinal properties of stem cells: multipotentiality and self-renewal. In contrast, the same conditions led to the opposite effect in the other main neurogenic region of the brain, the subventricular zone, in which neurosphere numbers decreased by ϳ40% in response to depolarizing levels of KCl. Most importantly, we also show that the latent hippocampal progenitor population can be activated in vivo in response to prolonged neural activity found in status epilepticus. This work provides the first direct evidence of a latent precursor and stem cell population in the adult hippocampus, which is able to be activated by neural activity. Because the latent population is also demonstrated to reside in the aged animal, defining the precise mechanisms that underlie its activation may provide a means to combat the cognitive deficits associated with a decline in neurogenesis.

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Neurotransmitters, KCl and antioxidants rescue striatal neurons from apoptotic cell death in culture

Cited by 41 publications

References 46 publications

Purified mouse dopamine neurons thrive and function after transplantation into brain but require novel glial factors for survival in culture

Purified mouse dopamine neurons thrive and function after transplantation into brain but require novel glial factors for survival in culture

Relationships between superoxide levels and delayed calcium deregulation in cultured cerebellar granule cells exposed continuously to glutamate

Latent Stem and Progenitor Cells in the Hippocampus Are Activated by Neural Excitation

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