Background & Aims

Elevated levels of advanced glycation end products (AGEs) derived from glycated collagen (GC) are associated with painful conditions, including diabetic neuropathy and neurodegenerative disorders. We previously demonstrated that human sensory neuron-like cells, derived from SH-SY5Y cell lines, gain pro-nociceptive functions, such as substance P release and increasing in TRPV1 and AGE receptor (RAGE) expressions. In this study, we aimed to investigate whether the glycation process sensitizes sensory neurons to capsaicin and activates signaling pathways associated with nociception, as well as explore the role of RAGE in these processes.

Methods

Sensory neuron-like cells were obtained from the differentiation of SH-SY5Y cells with all-trans-retinoic acid (10 µM) and brain-derived neuron factor (50 ng/µL). In order to mimic a pro-nociceptive environment, cells were incubated with extracellular collagen matrix (ECM- 100 µg/mL). Cell viability was assessed by MTT reduction assay. Capsaicin (1 µM)-induced calcium influx was evaluated by the fluo-8 calcium assay kit capsaicin. The levels of proteins involved in pro-nociceptive signaling pathways, such as ATF-3, MAPK-p38, Akt/PI3K, were quantified by western blot. Mitochondrial fragmentation was evaluated by MitoTracker Green kit and the parameters such as mitochondrial count, mean area, and medium branch junction were evaluated. Mitochondrial oxidation was analyzed using the Mitosox kit. Next, neurite growth assay was quantified by using ?-III tubulin expression. The experiments were conducted in presence or absence of RAGE antagonist (10-100 µM).

Results

The glycation process does not interfere with cell viability. The analysis of calcium influx shows that glycation process increases calcium influx (52.13%) when compared with cells treated with normal collagen and RAGE antagonist blocks this effect. The glycation increases the protein levels of ATF-3 and MAPK-p38 (41.6 % and 78.95 %, respectively) and decreased pAkt 12.3 %. Mitochondrial fragmentation analysis revealed that glycation induces an increase in mitochondrial count (112.5 %) and a decrease in the area and medium branch junctions (16.5 % and 39.57 %, respectively), followed by increased superoxide production (29 %), indicating mitochondrial fragmentation and function impairment. The neurite growth assay revealed that glycation reduces the number of extensions and branches per soma (43.3 %), which is prevented by AGE receptor antagonist.

Conclusions

These data indicate that glycation hypersensitize neuron-like cells to capsaicin and triggers pro-nociceptive and pro-neurodegenerative intracellular signaling, which is mediated by RAGE. Together, our results suggest that we established a functional model that can be useful for screening candidates for the management of painful conditions.

FINANCIAL SUPPORT: Butantan Foundation and Fundação de Amparo à Pesquisa de São Paulo (FAPESP 2016/19391-8, 2021/14831-8 AND 2022/08417-7).

References

Bufalo MC, Almeida ME, Franca IA, Zambelli VO, Martins Sant’anna MB, Kimura LF, et al. Advanced glycation endproducts produced by in vitro glycation of type I collagen modulate the functional and secretory behavior of dorsal root ganglion cells cultivated in two-dimensional system. Exp Cell Res. 2019 Sep 15;382(2).
Bufalo MC, de Almeida MES, Jensen JR, Deocesano-Pereira C, Lichtenstein F, Picolo G, et al. Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds. Cells. 2022 Jan 1;11(2).
Vistoli G, De Maddis D, Cipak A, Zarkovic N, Carini M, Aldini G. Advanced glycoxidation and lipoxidation end products (AGEs and ALEs): an overview of their mechanisms of formation. Free Radic Res [Internet]. 2013 Aug 17;47(sup1):3–27.
Fang Yan S, Marie Schmidt A, Ramasamy R. Receptor for Advanced Glycation Endproducts (RAGE): A Formi-dable Force in the Pathogenesis of the Cardiovascular Complica-tions of Diabetes & Aging. Vol. 7, Current Molecular Medicine. 2007.
Srikanth V, Maczurek A, Phan T, Steele M, Westcott B, Juskiw D, et al. Advanced glycation endproducts and their receptor RAGE in Alzheimer’s disease. Vol. 32, Neurobiology of Aging. 2011. p. 763–77.

Presenting Author

Gessica Sabrina de Assis Silva

Poster Authors

GESSICA SILVA

MSc

BUTANTAN INSTITUTE

Lead Author

E-mail

Mariana Caprio Schiess

Degree

Butantan Institute

Lead Author

E-mail

Michelle Cristiane Bufalo

PhD

Butantan Institute

Lead Author

E-mail

Ana Marisa Chudzinski-Tavassi PhD

Center of Excellence in New Target Discovery (CENTD); Laboratory of Innovation, Butantan Institute.

Lead Author

E-mail

Marcelo Medina de Souza

PhD

Butantan Institute

Lead Author

E-mail

Gisele Picolo

Butantan Institute

Lead Author

E-mail

Talita Glaser

PhD

University of São Paulo

Lead Author

E-mail

Vanessa Zambelli PhD

Butantan Institute

Lead Author

E-mail

Topics

  • Mechanisms: Biological-Molecular and Cell Biology