Optimized Quantities of GDNF Overexpressed by Engineered Astrocytes Are Critical for Protection of Neuroblastoma Cells Against 6-OHDA Toxicity

Abstract

Optimized levels of glial cell line-derived neurotrophic factor (GDNF) are critical for protection of dopaminergic neurons against parkinsonian cell death. Recombinant lentiviruses harboring GDNF coding sequence were constructed and used to infect astrocytoma cell line 1321N1. The infected astrocytes overexpressed GDNF mRNA and secreted an average of 2.2 ng/mL recombinant protein as tested in both 2 and 16 weeks post-infection. Serial dilutions of GDNF-enriched conditioned medium from infected astrocytes added to growing neuroblastoma cell line SK-N-MC resulted in commensurate resistance against 6-OHDA toxicity. SK-N-MC cell survival rate rose from 51% in control group to 84% in the cells grown with astro-CM containing 453 pg secreted GDNF, an increase that was highly significant (P < 0.0001). However, larger volumes of the GDNF-enriched conditioned medium failed to improve cell survival and addition of volumes that contained 1,600 pg or more GDNF further reduced survival rate to below 70%. Changes in cell survival paralleled to changes in the percent of apoptotic cell morphologies. These data demonstrate the feasibility of using astrocytes as minipumps to stably oversecrete neurotrophic factors and further indicate that GDNF can be applied to neuroprotection studies in PD pending the optimization of its concentrations.

Astrocytic modulation of blood brain barrier: perspectives on Parkinson’s disease

“…GDNF, secreted by astrocytes and pericytes, is essential for the survival of dopaminergic neurons, peripheral motor neurons and neurons from the locus coeruleus (Yasuda and Mochizuki, 2010; Allen et al, 2013). In this aspect, GDNF administration by catheter increases dopaminergic neuronal resistance against 6-OHDA toxicity, with preservation of motor functions in rat and rhesus monkey models (Safi et al, 2012). More recently, GDNF was shown to increase the expression of claudin-5 and the transendothelial electrical resistances of brain microvascular ECs, suggesting that it may improve the barrier function of the BBB (Sano et al, 2007).…”

PDGF-BB Protects Mitochondria from Rotenone in T98G Cells

“…The importance of growth factors in neuroprotection has been widely assessed in previous research mainly in neuronal models, as they are important for CNS homeostasis, differentiation, and survival (Falk et al 2009;Lee et al 1997;Mattson et al 1997;Safi et al 2012). Growth factors such as BDNF and bFGF have shown to protect neurons against excitotoxicity through the increased expression of antioxidant enzymes such as Mn-SOD and glutathione reductase (Lee et al 1997;Mattson et al 1997).…”

Complementation of dopaminergic signaling by Pitx3–GDNF synergy induces dopamine secretion by multipotent Ntera2 cells

“…18 2.5 | 6-OHDA treatment and MTT assay The 6-OHDA stock was prepared as described 18 and used to determine the LD 50 of NT2 cells. Primary antibodies against β-tubulin (ab15568; Abcam), Nestin (ab22035; Abcam), and TH (Lnc-1; Millipore) were applied at a 1:1000 working concentration overnight at 4°C.…”