Trophic factor and hormonal regulation of neurite outgrowth in sensory neuron-like 50B11 cells

Abstract

Sensory axon integrity and regenerative capacity are important considerations in understanding neuropathological conditions characterized by hyper- or insensitivity. However, our knowledge of mechanisms regulating axon outgrowth are limited by an absence of suitable high-throughput assay systems. The 50B11 cell line generated from rat embryonic dorsal root ganglion neurons offers a promising model for screening assays. Prior characterization shows that these cells express cytoskeletal proteins and genes encoding ion channels and neurotrophin receptors in common with sensory nociceptor neurons. In the present study we further characterized 50B11 cells in regard to their phenotypes and responsiveness to neurotrophic and hormonal factors. 50B11 cells express neuronal cytoplasmic proteins including beta-3 tubulin, peripherin (a marker of unmyelinated neurons), and the pan-neuronal ubiquitin hydrolase, PGP9.5. Only PGP9.5 immunoreactivity was uniformly distributed throughout soma and axons, and therefore presents the best means for visualizing the entire axon arbor. All cells co-express both NGF and GDNF receptors and addition of ligands increased neurite length. 50B11 cells also showed immunoreactivity for the estrogen receptor-α and the angiotensin receptor type II, and both 17-β estradiol and angiotensin II increased outgrowth by differentiated cells. 50B11 cells therefore show features reported previously for primary unmyelinated nociceptor neurons, including responsiveness to classical neurotrophins and hormonal modulators. Coupled with their ease of culture and predictable differentiation, 50B11 cells represent a promising cell line on which to base assays that more clearly reveal mechanisms regulating axon outgrowth and integrity.

Keywords: ANGII; AT2; Axon; DRG; Dorsal root ganglion; E2; ER; GDNF; High throughput screening; Hormones; NGF; Neurotrophic factors; Outgrowth; PGP9.5; angiotensin II; angiotensin II receptor type 2; dorsal root ganglion; estradiol or estrogen; estrogen receptor; glial cell line derived neurotrophic factor; immunoreactivity; ir; nerve growth factor; protein gene product 9.5.

Figure 1

Differentiated 50B11 cells show cytoplasmic proteins that may be useful in analyzing neuronite outgrowth. A. Staining for βIII-tubulin is prominent in the cell body but diminished in distal processes. B. PGP9.5 immunostaining delineates soma and axons in good detail. C. An overlay of A and B shows colocalization of these neuronal markers. D. Peripherin immunohistochemistry shows strong expression in the soma but little in processes. Bar in E=50μm for all panels.

Figure 2

Expression of growth factor receptors by 50B11 cells. Cells were differentiated for 20h and stained for receptor proteins. A. All cells showing neuron-like morphologies showed immunostaining for the NGF receptor, trkA. B. Cells also showed strong immunoreactivity for the GDNF receptor, GFRα1. C. A merged image shows that all differentiated neuron-like cells express both trkA and GFR alpha1. D. Differentiated cells show immunoreactivity for the ANGII receptor, AT2. E. TrkA staining of differentiated cells. F. Immunostaining of the same field as E shows GFRα2. G. Merged image of E and F shows colocalization of trkA and GFRα2. H. Differentiated tubulin βIII positive cells display predominantly nuclear immunoreactivity for estrogen receptor α whereas undifferentiated cells display little or no ERα. Bar in D =50μm for all panels.

Figure 3

Both neurotrophic and hormonal factors increase total neurite length in differentiated 50B11 cells. Total length of all processes emerging from each differentiated neuron 20h after treatment were summed and compared to untreated controls. A. Treatment with 50 ng/ml Nerve Growth Factor (NGF) increased axon length. B. Treatment with 50 ng/ml Glial cell line Derived Neurotrophic Factor (GDNF) also increased total outgrowth from 50B11 cells to an extent similar to NGF. C. Angiotensin II (AngII, 100nM) increased total neurite length. D. Estrogen (E2, 20nM) also increased neurite length. Data are presented as mean +/−s.e.m. *p≤0.001 vs. Control.

Figure 4

Neurotrophins and hormones increase length of the longest neurite in differentiated 50B11 cells. The length of the longest neurite from each cell was measured 20h after treatment. A. NGF (50 ng/ml) increased maximum neurite length. B. GDNF (50ng/ml) also increased axonal length. C. Angiotensin II (AngII, 100nM) increased maximum neurite length. Estrogen (E2, 20nM) induced significantly longer axons in treated neurons as compared to controls. Data are presented as mean +/−s.e.m. *p≤0.001 vs. Control.