Background & Aims

Painful diabetic neuropathy (PDN) is a debilitating and intractable complication of diabetes with patients suffering from a painful, burning sensation in their extremities. Current available treatments have limited effect in masking the pain without remediating the underlying mechanisms of the disease. The cellular hallmarks of PDN are cutaneous nerve-fiber degeneration and the hyperexcitability of the DRG neurons. Keratinocytes are closely juxtaposed to cutaneous nerve terminals, enabling bidirectional communication between keratinocytes and cutaneous nerves. One such ubiquitous mode of communication that is understudied in the skin is extracellular vesicles (EVs), namely exosomes, which are secreted nanoparticles that can produce substantial transcriptional and translational changes. However, the role of keratinocyte-derived exosomes in mediating DRG neuron hyperexcitability and axonal degeneration in PDN is unknown.

Methods

Using primary adult mouse keratinocytes cultures, we’ve begun characterizing keratinocyte-derived exosomes (KDEs) in the high-fat diet (HFD) induced mouse model of PDN and their potential role on DRG excitability and neurite growth both in vitro and in vivo. Using size exclusion chromatography, we have isolated enriched KDEs and are performing an extensive, unbiased molecular characterization via proteomics and RNAsequencing in mice before translating this with the recently received patient samples.

Results

Nanoparticle tracking analysis and negative stain electron microscopy suggest different rates of exosome secretion between RD and HFD mouse keratinocytes while comprehensive proteomics revealed significant differences in their molecular cargo in the HFD keratinocyte-derived exosomes. In addition, using an in vivo EV reporter mouse, we have demonstrated that keratinocyte originating nanoparticles are trafficked into the DRG neuron cell body.

Conclusions

Using the unbiased molecular characterization methods to study keratinocyte-derived exosomes is a novel investigation into the neuron-keratinocyte communication in diabetic skin. We have found a significant difference in their cargo in our PDN mouse model as well as demonstrated the ability for retrograde trafficking of these exosomes to the DRG. Our results could be translated into new topical interventions, which could fulfil the unmet need for new therapies for both small-fiber degeneration and neuropathic pain in diabetes.

References

1. Diagnosis and classification of diabetes mellitus. Diabetes Care, 2011. 34 Suppl 1(Suppl 1): p. S62-9.
2. Menke, A., et al., Prevalence of and Trends in Diabetes Among Adults in the United States, 1988- 2012. Jama, 2015. 314(10): p. 1021-9.
3. Spallone, V., et al., Painful diabetic polyneuropathy: approach to diagnosis and management.
Clin J Pain, 2012. 28(8): p. 726-43.
4. Zimmet, P.Z., et al., Diabetes: a 21st century challenge. Lancet Diabetes Endocrinol, 2014. 2(1):
p. 56-64.
5. Finnerup, N.B., et al., Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis. Lancet Neurol, 2015. 14(2): p. 162-73.
6. Latremoliere, A. and C.J. Woolf, Central sensitization: a generator of pain hypersensitivity by central neural plasticity. J Pain, 2009. 10(9): p. 895-926.
7. Divisova, S., et al., Intraepidermal nerve-fibre density as a biomarker of the course of neuropathy in patients with Type 2 diabetes mellitus. Diabet Med, 2016. 33(5): p. 650-4.
8. Jayaraj, N.D., et al., Reducing CXCR4-mediated nociceptor hyperexcitability reverses painful diabetic neuropathy. J Clin Invest, 2018. 128(6): p. 2205-2225.
9. Menichella, D.M., et al., Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice. Mol Pain, 2016. 12.
10. George, D., et al., Mitochondrial Calcium Uniporter Deletion Prevents Painful Diabetic Neuropathy by Restoring Mitochondrial Morphology and Dynamics. The Journal of Pain, 2021. 22(5): p. 581.
11. Coleman, M.P. and V.H. Perry, Axon pathology in neurological disease: a neglected therapeutic target. Trends Neurosci, 2002. 25(10): p. 532-7.
12. Persson, A.K., et al., Sodium Channels, Mitochondria, and Axonal Degeneration in Peripheral Neuropathy. Trends Mol Med, 2016. 22(5): p. 377-390.
13. Talagas, M., et al., Keratinocytes Communicate with Sensory Neurons via Synaptic-like Contacts.
Ann Neurol, 2020. 88(6): p. 1205-1219.
14. Moehring, F., et al., Keratinocytes mediate innocuous and noxious touch via ATP-P2X4 signaling.
eLife, 2018. 7: p. e31684.
15. Wang, Y. and D.T. Graves, Keratinocyte Function in Normal and Diabetic Wounds and Modulation by FOXO1. Journal of Diabetes Research, 2020. 2020: p. 3714704.
16. Pastar, I., et al., Epithelialization in Wound Healing: A Comprehensive Review. Advances in Wound Care, 2014. 3(7): p. 445-464.
17. Hessvik, N.P. and A. Llorente, Current knowledge on exosome biogenesis and release. Cellular and Molecular Life Sciences, 2018. 75(2): p. 193-208.
18. Wei, H., et al., Regulation of exosome production and cargo sorting. Int J Biol Sci, 2021. 17(1): p. 163-177.
19. Rastogi, S., et al., The Evolving Landscape of Exosomes in Neurodegenerative Diseases: Exosomes Characteristics and a Promising Role in Early Diagnosis. Int J Mol Sci, 2021. 22(1).
20. Elmehrath, A.O., Y.T. Sonbol, and M.Y. Farghal, Exosomes in Neurodegenerative Disorders, in Role of Exosomes in Biological Communication Systems, F.A. Alzahrani and I.M. Saadeldin, Editors. 2021, Springer Singapore: Singapore. p. 183-206.
21. Dai, J., et al., Exosomes: key players in cancer and potential therapeutic strategy. Signal Transduction and Targeted Therapy, 2020. 5(1): p. 145.
22. Fan, Y., Z. Chen, and M. Zhang, Role of exosomes in the pathogenesis, diagnosis, and treatment of central nervous system diseases. Journal of Translational Medicine, 2022. 20(1): p. 291.
23. He, X., et al., Emerging roles of exosomal miRNAs in diabetes mellitus. Clin Transl Med, 2021.
11(6): p. e468.
24. Chang, W. and J. Wang, Exosomes and Their Noncoding RNA Cargo Are Emerging as New Modulators for Diabetes Mellitus. Cells, 2019. 8(8).
25. Sun, Y., et al., Human Mesenchymal Stem Cell Derived Exosomes Alleviate Type 2 Diabetes
Mellitus by Reversing Peripheral Insulin Resistance and Relieving ?-Cell Destruction. ACS Nano, 2018. 12(8): p. 7613-7628.
26. He, Q., et al., Mesenchymal stem cell-derived exosomes exert ameliorative effects in type 2 diabetes by improving hepatic glucose and lipid metabolism via enhancing autophagy. Stem Cell Res Ther, 2020. 11(1): p. 223.
27. Lo Cicero, A., et al., Exosomes released by keratinocytes modulate melanocyte pigmentation. Nat Commun, 2015. 6: p. 7506.
28. Nasiri, G., et al., Shedding light on the role of keratinocyte-derived extracellular vesicles on skin- homing cells. Stem Cell Research & Therapy, 2020. 11(1): p. 421.
29. Zhou, X., et al., Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing. ACS Nano, 2020. 14(10): p. 12732-12748.
30. Jiang, M., et al., Keratinocyte exosomes activate neutrophils and enhance skin inflammation in psoriasis. Faseb j, 2019. 33(12): p. 13241-13253.
31. Kotzerke, K., et al., Immunostimulatory activity of murine keratinocyte-derived exosomes. Exp Dermatol, 2013. 22(10): p. 650-5.
32. Inoue, K., J. Hosoi, and M. Denda, Extracellular ATP Has Stimulatory Effects on the Expression and Release of IL-6 Via Purinergic Receptors in Normal Human Epidermal Keratinocytes. Journal of Investigative Dermatology, 2007. 127(2): p. 362-371.
33. Fan, S.J., et al., Glutamine deprivation alters the origin and function of cancer cell exosomes.
Embo j, 2020. 39(16): p. e103009.
34. Fukuta, T., A. Nishikawa, and K. Kogure, Low level electricity increases the secretion of extracellular vesicles from cultured cells. Biochemistry and Biophysics Reports, 2020. 21: p. 100713.
35. Oh, H.J., et al., Convective exosome-tracing microfluidics for analysis of cell-non-autonomous neurogenesis. Biomaterials, 2017. 112: p. 82-94.
36. Li, B., et al., RhoA triggers a specific signaling pathway that generates transforming microvesicles in cancer cells. Oncogene, 2012. 31(45): p. 4740-4749.
37. Moshourab, R., Y. Schmidt, and H. Machelska, Skin–Nerve Preparation to Assay the Function of Opioid Receptors in Peripheral Endings of Sensory Neurons, in Opioid Receptors: Methods and Protocols, S.M. Spampinato, Editor. 2015, Springer New York: New York, NY. p. 215-228.

Presenting Author

James Coy-Dibley

Poster Authors

James Coy-Dibley

Department of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, IL

Lead Author

E-mail

Topics

  • Specific Pain Conditions/Pain in Specific Populations: Neuropathic Pain - Peripheral