"Of mice and men": the relevance of Cometin and Erythropoietin origin for its effects on murine spiral ganglion neuron survival and neurite outgrowth in vitro

Abstract

Neurotrophic factors (NTF) play key roles in the survival of neurons, making them promising candidates for therapy of neurodegenerative diseases. In the case of the inner ear, sensorineural hearing loss (SNHL) is characterized over time by a degeneration of the primary auditory neurons, the spiral ganglion neurons (SGN). It is well known that selected NTF can protect SGN from degeneration, which positively influences the outcome of cochlear implants, the treatment of choice for patients with profound to severe SNHL. However, the outcome of studies investigating protective effects of NTF on auditory neurons are in some cases of high variability. We hypothesize that the factor origin may be one aspect that affects the neuroprotective potential. The aim of this study was to investigate the neuroprotective potential of human and mouse Erythropoietin (EPO) and Cometin on rat SGN. SGN were isolated from neonatal rats (P 2-5) and cultured in serum-free medium. EPO and Cometin of mouse and human origin were added in concentrations of 0.1, 1, and 10 ng/mL and 0.1, 1, and 10 μg/mL, respectively. The SGN survival rate and morphology, and the neurite outgrowth were determined and compared to negative (no additives) and positive (brain-derived neurotrophic factor, BDNF) controls. A neuroprotective effect of 10 μg/mL human Cometin comparable to that obtained with BDNF was observed in the SGN-culture. In contrast, mouse Cometin was ineffective. A similar influence of 10 μg/mL human and mouse and 1 μg/mL human Cometin on the length of regenerated neurites compared to BDNF was also detected. No other Cometin-conditions, and none of the EPO-conditions tested had neuroprotective or neuritogenic effects or influenced the neuronal morphology of the SGN. The neuroprotective effect of 10 μg/mL human Cometin on SGN indicates it is a potentially interesting protein for the supportive treatment of inner ear disorders. The finding that mouse Cometin had no effect on the SGN in the parallel-performed experiments underlines the importance of species origin of molecules being screened for therapeutic purpose.

Keywords: cochlea; drug delivery; inner ear; neurite outgrowth; neuroprotection; pharmacotherapy; species specific.

Figure 1

Example of different morphologies of SGN. 1: monopolar neuron; 2: bipolar neuron; 3: neuron with no neurites, 4: pseudomonopolar neuron; 5: multipolar neuron. Neurons are stained against neurofilament with DAB, contrast of the images is digitally improved. Scale bar: 50 μm.

Figure 2

SGN survival rate after treatment with mouse and human Cometin (A) or EPO (B). No factor-application served as negative control (NC) and 10 ng/mL BDNF served as a positive control (PC). BDNF increased neuronal survival in both test series significantly. 10 μg/mL human Cometin significantly increased the SGN survival rate compared to the NC and all other Cometin conditions and did not differ from PC. In contrast, the addition of 10 ng/mL human EPO significantly decreased SGN survival compared to NC, PC, and the other EPO conditions. No other treatment affected the SGN survival rate significantly compared to the NC. While 10 μg/mL human Cometin significantly improved SGN survival compared to the same concentration of mouse Cometin and the lower human concentrations, the opposite was the case for EPO. Here the 10 ng/mL of human EPO significantly reduced the SGN survival compared to the mouse EPO of the same concentration and the tested lower human concentrations. Asterisks above the error bars indicate the significant differences between the marked experimental conditions and the respective group indicated by the arrowhead. *p < 0.05, **p < 0.01, ***p < 0.001; independent experiments: 3; wells per experiment: 3.

Figure 3

Influence of human and mouse Cometin and EPO on neuronal morphology after 48 h of incubation. SGN were distinguished into monopolar (A,B), bipolar (C,D) and neurons without neurites (E,F) after treatment with Cometin (A,C,E) or EPO (B,D,F). No relevant differences were detected between treatment conditions. Independent experiments: 3, wells per experiment: 3.

Figure 4

Neurite length of Cometin (A) and EPO (B) treated neurons. Neither Cometin nor EPO treatment increased neurite outgrowth compared to the relevant NC. Compared to PC, neurites were shorter for SGN treated with 1 μg/mL mouse Cometin and 0.1 μg/mL mouse and human Cometin. Treatment with 0.1 μg/mL human Cometin mediated the shortest detected neurite length overall. In the case of EPO, neurites were significantly shorter than in PC for all treatments and compared to NC for 10 ng/mL human, and 10 ng/mL, 1 ng/mL, and 0.1 ng/mL mouse EPO. Within the EPO-treated groups, neurites were shortest for 10 ng/mL human EPO. The lower concentrations of human EPO and all concentrations of mouse EPO did not differ from each other in their neurite length. At the same concentrations, 10 ng/mL human EPO resulted in shorter, 1 ng/mL human EPO in longer, and 0.1 ng/mL human EPO in an equivalent neurite length compared to mouse EPO. Asterisks above the error bars indicate the significance of the condition compared to the respective condition indicated by the arrowhead. ***p < 0.001, **p < 0.01, *p < 0.05; independent experiments: 3, wells per experiment: 3.