Prevention of the degeneration of human dopaminergic neurons in an astrocyte co‐culture system allowing endogenous drug metabolism

Abstract

BACKGROUND AND PURPOSEFew neuropharmacological model systems use human neurons. Moreover, available test systems rarely reflect functional roles of co-cultured glial cells. There is no human in vitro counterpart of the widely used 1-methyl-4-phenyl-tetrahydropyridine (MPTP) mouse model of Parkinson's disease EXPERIMENTAL APPROACHWe generated such a model by growing an intricate network of human dopaminergic neurons on a dense layer of astrocytes. In these co-cultures, MPTP was metabolized to 1-methyl-4-phenyl-pyridinium (MPP + ) by the glial cells, and the toxic metabolite was taken up through the dopamine transporter into neurons. Cell viability was measured biochemically and by quantitative neurite imaging, siRNA techniques were also used. KEY RESULTSWe initially characterized the activation of PARP. As in mouse models, MPTP exposure induced (poly-ADP-ribose) synthesis and neurodegeneration was blocked by PARP inhibitors. Several different putative neuroprotectants were then compared in mono-cultures and co-cultures. Rho kinase inhibitors worked in both models; CEP1347, ascorbic acid or a caspase inhibitor protected mono-cultures from MPP + toxicity, but did not protect co-cultures, when used alone or in combination. Application of GSSG prevented degeneration in co-cultures, but not in mono-cultures. The surprisingly different pharmacological profiles of the models suggest that the presence of glial cells, and the in situ generation of the toxic metabolite MPP + within the layered cultures played an important role in neuroprotection. CONCLUSIONS AND IMPLICATIONSOur new model system is a closer model of human brain tissue than conventional cultures. Its use for screening of candidate neuroprotectants may increase the predictiveness of a test battery.

Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation

“…Quantification of neuronal area was based on selective immunostaining (see below) of neuronal βIIItubulin (Tuj1 antibody) and high-content imaging: An Array-Scan VTI HCS Reader (Cellomics, PA) was used for image acquisition. Thirty fields per well (330 μm × 330 μm size each) were imaged and analyzed (Efremova et al 2015). The software recognizes the Tuj-positive pixels and calculates the total pixel number per well.…”

“…We used here a recently established co-culture model (Efremova et al 2015) of LUHMES neurons and IMA astrocytes. Three-dimensional imaging by confocal microscopy confirmed that neurons are mostly positioned in a layer above the astrocytes, and that both neurites and somata establish large contact interfaces with astrocytes ( Fig.…”

“…For some applications focusing on the neurodegeneration in co-cultures, immortalized astrocyte cell lines provide a simple, but robust source of astrocytic factors or metabolic capacity (Efremova et al 2015;Lee et al 2013a, b) to be tested on associated neurons. In cases where the toxic factors are non-proteinaceous, cells from two different species can be used, and they provide some analytical advantages.…”

“…These cells are conditionally immortalized, and they assume neuronal morphological, electrophysiological, and neurochemical properties upon the switching off of the v-myc transgene by tetracycline. The cells can be cultured on a layer of astrocytes (Efremova et al 2015;Kuegler et al 2012), and they form highly reproducible co-cultures when combined with the immortalized murine astrocytic cell line IMA (Schildknecht et al 2012). The latter cells can metabolize drugs, such as 1-methyl-4-phenyl-tetrahydropyridine (MPTP), and are well characterized in their inflammatory response.…”

“…The latter cells can metabolize drugs, such as 1-methyl-4-phenyl-tetrahydropyridine (MPTP), and are well characterized in their inflammatory response. The LUHMES-IMA co-culture allowed multiple pharmacological interventions on the level of neurons and astrocytes (Efremova et al 2015).…”