This review is directed at understanding how neuronal death occurs in two distinct insults, global ischemia and focal ischemia. These are the two principal rodent models for human disease. Cell dea...

Ischemic Cell Death in Brain Neurons

A rapid and simple procedure is presented to obtain nearly pure populations of human neuron-like cells from the SH-SY5Y neuroblastoma cell line. Sequential exposure of SH-SY5Y cells to retinoic acid and brain-derived neurotrophic factor in serum-free medium yields homogeneous populations of cells with neuronal morphology, avoiding the presence of other neural crest derivatives that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5-bromo-2′-deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain-derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron-specific enolase, and growth-associated protein-43 as well as neuronal polarity markers such as tau and microtubule-associated protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the negative staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large-scale biochemical and molecular studies due to its susceptibility to genetic manipulation and the availability of an unlimited amount of cells.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1471-4159.2000.0750991.x

Sequential Treatment of SH‐SY5Y Cells with Retinoic Acid and Brain‐Derived Neurotrophic Factor Gives Rise to Fully Differentiated, Neurotrophic Factor‐Dependent, Human Neuron‐Like Cells

Pharmacological inhibitors of cyclin-dependent kinases

Recovery recapitulates ontogeny

There is increasing evidence that some neuronal death after brain ischaemia is mediated by the action of cysteine-requiring aspartate-directed proteases (caspases), the proteases responsible for apoptosis in mammals, although this form of neuronal death is not always accompanied by the morphological changes that are typical of apoptosis in other tissues. Caspase-mediated neuronal death is more extensive after transient than permanent focal brain ischaemia and may contribute to delayed loss of neurons from the penumbral region of infarcts. The activation of caspases after brain ischaemia is largely consequent on the translocation of Bax, Bak, and other BH3-only members of the Bcl-2 family to the mitochondrial outer membrane and the release of cytochrome c, procaspase-9, and apoptosis activating factor-1 (Apaf-1) from the mitochondrial intermembrane space. How exactly ischaemia induces this translocation is still poorly understood. NF-kappaB, the c-jun N-terminal kinase-c-Jun pathway, p53, E2F1, and other transcription factors are probably all involved in regulating the expression of BH3-only proteins after brain ischaemia, and mitochondrial translocation of Bad from sequestering cytosolic proteins is promoted by inactivation of the serine-threonine kinase, Akt. Other processes that are probably involved in the activation of caspases after brain ischaemia include the mitochondrial release of the second mitochondrial activator of caspases (Smac) or direct inhibitor-of-apoptosis-binding protein with low pI (DIABLO), the accumulation of products of lipid peroxidation, a marked reduction in protein synthesis, and the aberrant reentry of neurons into the cell cycle. Non-caspase-mediated neuronal apoptosis may also occur, but there is little evidence to date that this makes a significant contribution to brain damage after ischaemia. The intracellular processes that contribute to caspase-mediated neuronal death after ischaemia are all potential targets for therapy. However, anti-apoptotic interventions in stroke patients will require detailed evaluation using a range of outcome measures, as some such interventions seem simply to delay neuronal death and others to preserve neurons but not neuronal function.

Apoptosis and brain ischaemia.

Several observations suggest that delayed neuronal death in ischaemia, epilepsy and other brain disorders includes an apoptotic component, involving programmed cell death (PCD). PCD is hypothesized to result, in part, from aberrant control of the cell cycle. Because they are instrumental in mitosis, cyclins D are key markers to evaluate whether neurons indeed progress into the cell cycle in situations of pathology. Therefore, we investigated in rat brains, the expression of cyclins D in the delayed neuronal death that occurs following transient global ischaemia and kainate-induced seizures. Following a four-vessel occlusion insult, quantitative in situ hybridization revealed a highly significant and persistent 100% increase of cyclin D1 mRNA in the vulnerable pyramidal neurons of the CA1 hippocampal region. Ischaemia also induced a smaller and transient cyclin D1 mRNA increase in the resistant CA3 area and dentate gyrus. In contrast, the cyclin D2 and D3 mRNAs, expressed constitutively in the adult rat hippocampus, were not upregulated. Following kainate-induced seizures, cyclin D1 mRNA was induced in the vulnerable CA3 region, and to a lesser extent, in non-vulnerable regions. Cyclin D1 immunohistochemistry revealed increased protein levels in the cytoplasm and nucleus of neurons commited to die after ischaemia. Double labelling experiments indicate that cyclin D1 is also expressed in reactive astrocytes but not in microglial cells. Finally, we report that in neurons, cyclin D1 expression peaks before nuclear condensation and the appearance of DNA fragmentation. We propose that cyclin D1, when expressed at high levels in lesioned neurons, may act as a modulator of apoptosis.

Frédérique Guischard

Papers

Increased cyclin D1 in vulnerable neurons in the hippocampus after ischaemia and epilepsy: a modulator of in vivo programmed cell death?