Cell interactions in the CNS and their consequences for neuronal apoptosis

Abstract

The central nervous system consists of an intricate network of neurons and glial cells that depend on reciprocal communication to develop and maintain the vital functions of the brain. if the balance is disturbed by injury or disease, two cell types in particular, microglia and astrocytes, respond by producing factors that help combat the infection or delimit the affected area. The microglial response can be beneficial promoting neuronal survival, however it can also cause harm to the surrounding cells. To further investigate this balance, and subsequently for studies of neuronal apoptosis, different cell culture techniques spanning from complex primary co-cultures to one-dimensional cell line studies were applied.

Fetal mouse brain cells were dissociated and cultured under gyratory rotation to form three-dimensional aggregates allowing for cell-cell contact between neurons and glia. The aggregates were characterized in respect to cell contents, viability, expression of neural transmitters, and response to mitogens. The aggregates presented a glia:neuron ratio of 1:20 at day 4 changing to 1:4 until day 16, which correlates to the increased postnatal proliferation of glia in vivo. Both neurons and astrocytes matured during the culture period in regard to increased expression of synaptic proteins and GFAP respectively.

Treatment of the aggregates with EGF induced proliferation of nestin positive cells that were designated as neural precursor cells due to their ability to renew, propagate, and differentiate into neurons and macroglia. Neural precursor cells were maintained in the aggregates for more than two months in culture. This aggregate culture system provides a useful tool for in vivolike in vitro studies combining cellular complexity with a controlled environment.

Studies of neuron-microglia interactions were initiated using a cell line-based model system where SH-SY5Y neuroblastoma cells were co-cultured with THP-1 monocytes. As a means to morphologically detect and quantify potential monocyte-induced neuronal apoptosis, an automated fluorescence imaging system, ImageXpress (IX), was utilized. To validate the system, SH-SY5Y cell apoptosis was induced by rotenone. The treatment resulted in chromatin condensation and nucleus size reduction, parameters that were used in combination with nuclear intensity to distinguish apoptotic cells from normal cells. The IX system proved to be a suitable tool for detecting rotenone-induced neuronal apoptosis.

However, activated THP-1 cells did not induce apoptosis in SH-SY5Y cells, as detected with the IX system. These results were confirmed by an ELISA-based apoptosis assay. Subsequently, we cultured SH-SY5Y cells in the presence of medium that had been conditioned (CM) by activated THP-1 cells for 24h, hence containing higher levels of potentially neuromodulatory factors than could be achieved in the co-culture experiments. We observed that CM from LPS and LPS+IFN-gamma stimulated monocytes induced neuronal apoptosis in a dose dependent manner. The effect of LPS+IFN-gamma stimulation was partially mediated by TNF-a. We concluded that unphysiological ratios of monocytes to neurons are needed to induce neurotoxicity in this system.

Finally, we wanted to investigate whether microglia could rescue damaged neurons from apoptosis, or possibly delay the course. To map a window where apoptosis was initiated, but not committed, SH-SY5Y cells were treated with doses of UV irradiation. We found that activation of JNK and c-Jun was induced early after insult, followed by cytochrome c (cyt c) translocation, cleavage of caspase-3, and ensuing apoptosis. JNK-inhibition induced a 60% reduction in c-Jun phosphorylation (Ser63/Ser73), but did not prevent mitochondrial cyt c release or apoptosis, indicting that JNK is not essential for UV-included apoptosis in SH-SY5Y cells. We conclude that UV irradiation is a complex stress that may induce multiple mechanisms contributing to apoptosis.