Advanced Glycation End-Products Sensitize Human Sensory-Like Neuron Cells to Capsaicin-Induced Calcium Influx

Summary

Increased collagen-derived advanced glycation end products (AGEs) are consistently linked to painful diseases. Here, we investigated whether glycation sensitizes sensory neurons to capsaicin excitation.

Abstract

Increased collagen-derived advanced glycation end products (AGEs) are consistently linked to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. Human sensory-like neurons differentiated from the SH-SY5Y cell line gain pro-nociceptive functions when exposed to AGEs by releasing substance P and upregulating the transient receptor potential vanilloid 1 (TRPV1) expression. Here, we investigated whether this receptor was functionally active and whether the glycation process sensitizes sensory neurons to capsaicin excitation. Sensory-like neuron cells were obtained from the differentiation of SH-SY5Y cells with all-trans-retinoic acid and brain-derived neurotrophic factor. Incubation with glycated collagen extracellular matrix (ECM-GC) simulated a pro-nociceptive stimulus. Control cells were incubated with a non-glycated extracellular collagen matrix (ECM-NC). Fluo-8 Calcium Flux Assay Kit was used to assess calcium influx, which was stimulated by capsaicin. The results show that glycation increases calcium influx compared with cells treated with normal collagen, suggesting that sensory-like neurons express functional TRPV1 channels and that glycation increases capsaicin excitation. These data indicate AGEs hypersensitive sensory-like neuron cells, triggering pro-nociceptive signaling. Together, our results suggest that we established a functional model responsive to capsaicin that can be useful for screening candidates for managing painful conditions.

Introduction

Glycation is a non-enzymatic, irreversible, and spontaneous process in which proteins, such as collagen, bind to reducing sugar molecules, resulting in advanced glycation end products (AGEs). AGEs may activate cellular membrane receptors, triggering intracellular pathways activation, such as extracellular signal-regulated protein kinase (ERK) 1/2, p38 mitogen-activated protein kinase (MAPK), and c-jun n-terminal kinases (JNKs), rho-GTPases, phosphoinositol-3-kinase (PI3K), Janus kinase/signal transducer and activator of transcription (JAK/STAT), and protein kinase C (PKC), increasing proinflammatory molecules release and oxidative stress¹. Glyc....

Protocol

1. SH-SY5Y culture and differentiation into sensory-like neuron cells

NOTE 1: All steps present in this section need to be done under a laminar flow hood, and all solutions and supplies need to be sterile.

1. First, prepare a culture flask (25 cm²) by adding 5 mL of culture medium.
   NOTE: For thawing, expansion, and maintenance of this cell type, use a mixture of culture medium: Dulbecco Modified Eagle's and Ham's Medium F12 (DMEM/F12) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin.
2. Then, remove the cryotube containing the SH-SY5Y cells from the liq....

Results

SH-SY5Y cells differentiation into sensory-like neuron
High-content screening images demonstrate that the protocol of neuronal differentiation changes SH-SY5Y cell morphology. The sensory-like neuron cells (differentiated cells) display a rounded cell body that projects an extensive network of neurofilaments. They form branches of more elongated neurite projections connecting surrounding neurons, which is consistent with mature neuron features (Figure 3)

Discussion

Nociceptors are specialized subsets of sensory neurons that mediate pain. These cells express voltage-gated and ligand ion channels, such as TRPV1, whose activation leads to calcium influx and the release of neuropeptides and neurotransmitters that regulate nociceptive transmission. Here, we describe a protocol for differentiating SH-SY5Y into sensory-like neuron cells to evaluate capsaicin-induced calcium influx^8,9. Importantly, we showed that mimicking a pro-no.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
All-trans retinoic acid	Tocris	695
BDNF	Tocris	TOCR-2837
BDNF	Sigma-Aldrich	B3795
Butterfly type 23GA sterile	Beckton Dickinson Asepto	38833814	Scalp vein set
Capsaicin	Sigma-Aldrich	M2028
D-glucose	Sigma-Aldrich	G5767
DMEM/F12	Gibco	12500062	Basal medium
D-ribose	Sigma-Aldrich	R7500
D-threose	Sigma-Aldrich	T7392
Fluo-8 Calcium Flux Assay Kit	Abcam	ab112129	No wash
Heat-inactivated fetal bovine serum	Gibco	A5670801
High Content Screening	Molecular Devices
LASX software	Leica Microsystems		Microscopy software
Leica TCS SP8	Leica Microsystems	Leica TCS SP8	Confocal microscope
Penicillin-streptomycin	Gibco	15140130
Petri dish (35/10 mm)	Greiner bio-one	627965
Rat tail type I collagen	Corning	354236
SH-SY5Y	Merck	94030304-1VL	Neuroblastoma cell line