Elastic properties of the cell surface and metabolic profile of an embryonic primary mixed culture of hippocampal neurons under conditions of P2X3 receptor blockade

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P2X3-receptors localized in the hippocampus participate in the transmission of excitation and the formation of synaptic plasticity underlying learning and memory. P2X3-receptors are of great importance in the occurrence of neuropathic pain in epilepsy, acute and inflammatory pain of various genesis and localization as well as in the activation and growth of nerves after traumatic brain injury. The aim of the study was to study the elastic properties of the surface and the metabolic profile of neurons in an embryonic primary mixed hippocampal culture under P2X3-receptor blockade. The study was performed on a primary mixed culture of hippocampal neurons obtained from CD1 mice on the 18th day of gestation (E18). The highly selective blocker 5-(5-iodo-2-isopropyl-4-methoxyphenoxy)pyrimidine-2.4-diamine monochloride salt was selected as a P2X3-receptor blocker. To assess the elastic properties of neurons Young's modulus that characterizes the rigidity of the cell surface was measured. Measurements on an atomic force microscope applying a load in 25 local areas of the cell surface were performed. At each point, the force curves of the cantilever approach and retraction were recorded with subsequent calculation of Young's modulus. The metabolic profile of the neuroglial culture in Energy Phenotype test on a Seahorse HS mini cell metabolism analyzer (USA) was studied. The Young's modulus of the cell surface of neurons in the control was in the range from 6.8 ± 0.1 to 9.7 ± 0.2 kPa, and under the action of the P2X3-receptor blocker in the range from 3.1 ± 0.1 kPa to 8.5 ± 0.1 kPa. Under the conditions of P2X3-receptor blockade on the 5th day of differentiation the Young's modulus of the cell surface was reduced by 62% (p < 0.05), on the 8th day it increased by 22% (p < 0.05) and by the 11th day it decreased by 16.7% (p < 0.05) compared to the control. Aerobic respiration was characteristic of the embryonic hippocampal culture both in the control and with the P2X3-receptor blockade. Consequently, the blockade of the P2X3-receptor did not affect the metabolic profile of the E18 hippocampal culture. The obtained data indicate the direct participation of the P2X3-receptor in the formation of biomechanical properties of the cell surface in the processes of differentiation and signal transduction. It is possible, that the blockade of the P2X3-receptor will be one of the promising molecular targets that can reduce neuronal damage in brain injuries, neuroinflammation, hypoxia, and epilepsy. In addition, the study of the P2X3-receptor blockade can expand the fundamental understanding of the role of the purinergic signaling system in the formation of complex neuronal morphology at early stages of embryonic development under conditions of rapid excitatory signal transmission mediated by the ATP molecule.

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Sobre autores

A. Zelentsova

Belgorod State National Research University

Email: marinaskorkina0077@gmail.com
Rússia, Belgorod

V. Shmigerova

Belgorod State National Research University

Email: marinaskorkina0077@gmail.com
Rússia, Belgorod

Y. Stepenko

Belgorod State National Research University

Email: marinaskorkina0077@gmail.com
Rússia, Belgorod

M. Skorkina

Belgorod State National Research University

Autor responsável pela correspondência
Email: marinaskorkina0077@gmail.com
Rússia, Belgorod

A. Deykin

Belgorod State National Research University

Email: marinaskorkina0077@gmail.com
Rússia, Belgorod

Bibliografia

  1. Papp L, Balázsa T, Köfalvi A, Erdélyi F, Szabó G, Vizi ES, Sperlágh B (2004) P2X receptor activation elicits transporter-mediated noradrenaline release from rat hippocampal slices. J Pharmacol Exp Ther 3: 973–380. https://doi.org/10.1124/jpet.104.066712
  2. Rodrigues RJ, Almeida T, Richardson PJ, Oliveira CR, Cunha RA (2005) Dual presynaptic control by ATP of glutamate release via facilitatory P2X1, P2X2/3, and P2X3 and inhibitory P2Y1, P2Y2, and/or P2Y4 receptors in the rat hippocampus. J Neurosci 25: 6286–6295. https://doi.org/10.1523/JNEUROSCI.0628-05.2005
  3. Pankratov Y, Lalo U, Krishtal OA, Verkhratsky A (2009) P2X receptors and synaptic plasticity. Neuroscience 158: 137–148. https://doi.org/10.1016/j.neuroscience.2008.03.076
  4. Abbracchio MP, Burnstock G (1998) Purinergic signalling: Pathophysiological roles. Jpn J Pharmacol 78: 113–145. https://doi.org/10.1254/jjp.78.113
  5. Burnstock G, Knight GE (2004) Cellular distribution and functions of P2 receptor subtypes in different systems. Int Rev Cytol 240: 31–304. https://doi.org/10.1016/S0074-7696(04)40002-3
  6. Mishra SK, Braun N, Shukla V, Füllgrabe M, Schomerus C, Korf HW, Gachet C, Ikehara Y, Sévigny J, Robson SC, Zimmermann H (2006) Extracellular nucleotide signaling in adult neural stem cells: synergism with growth factor-mediated cellular proliferation. Development 133: 675–684. https://doi.org doi: 10.1242/dev.02233
  7. Brederson JD, Jarvis MF (2008) Homomeric and heteromeric P2X3 receptors in peripheral sensory neurons. Curr Opin Investig Drugs 9: 716–725.
  8. Burnstock G (2015) Physiopathological roles of P2X receptors in the central nervous system. Curr Med Chem 22: 819–844. https://doi.org/10.2174/0929867321666140706130415
  9. Burnstock G (2017) Purinergic signalling: therapeutic developments. Front Pharmacol 8: 661. https://doi.org/10.3389/fphar.2017.00661
  10. Pedata F, Dettori I, Coppi E (2016) Purinergic signalling in brain ischemia. Neuropharmacology 104: 105–130. https://doi.org/10.1016/j.neuropharm.2015.11.007
  11. Heine C, Sygnecka K, Franke H (2016) Purines in neurite growth and astroglia activation. Neuropharmacology 104: 255–271. https://doi.org/10.1016/j.neuropharm.2015.10.022
  12. Heine C, Heimrich B, Vogt J, Wegner A, Illes P, Franke H (2006) P2 receptor-stimulation influences axonal outgrowth in the developing hippocampus in vitro. Neuroscience 138: 303–311. https://doi.org/10.1016/j.neuroscience.2005.11.056
  13. Lommen J, Detken J, Harr K, von Gall C, Ali AAH (2021) Analysis of spatial and temporal distribution of purinergic P2 receptors in the mouse hippocampus. Int J Mol Sci 22: 8078. https://doi.org/10.3390/ijms22158078
  14. Pankratov YV, Lalo UV, Krishtal OA (2002) Role for P2X receptors in long-term potentiation. J Neurosci 22: 8363–8369. https://doi.org/10.1523/JNEUROSCI.22-19-08363.2002
  15. Lüscher C, Malenka RC (2012) NMDA receptor-dependent long-term potentiation and long-term depression (LTP/LTD). Cold Spring Harb Perspect Biol 4: a005710. https://doi.org/10.1101/cshperspect.a005710
  16. Cheung K-K, Burnstock G (2002) Localization of P2X3 receptors and coexpression with P2X2 receptors during rat embryonic neurogenesis. J Comp Neur 443: 368–382. https://doi.org/10.1002/cne.10123
  17. Gong M, Ye S, Li WX, Zhang J, Liu Y, Zhu J, Lv W, Zhang H, Wang J, Lu A, He K (2020) Regulatory function of praja ring finger ubiquitin ligase 2 mediated by the P2rx3/P2rx7 axis in mouse hippocampal neuronal cells. Am J Physiol Cell Physiol 318: C1123–C1135. https://doi.org/10.1152/ajpcell.00070.2019
  18. Devine MJ, Kittler JT (2018) Mitochondria at the neuronal presynapse in health and disease. Nat Rev Neurosci 19: 63–80. https://doi.org/10.1038/nrn.2017.170
  19. Na S, Collin O, Chowdhury F, Tay B, Ouyang M, Wang Y, Wang N (2008) Rapid signal transduction in living cells is a unique feature of mechanotransduction. Proc Natl Acad Sci U S A 105: 6626–6631. https://doi.org/10.1073/pnas.0711704105
  20. Kaech S, Banker G (2006) Culturing hippocampal neurons. Nat Protoc 1: 2406–2415. https://doi.org/10.1038/nprot.2006.356
  21. Carter DS, Alam M, Cai H, Dillon MP, Ford APDW, Gever J (2009) Identification and SAR of novel diaminopyrimidines. Part 1: the discovery of RO-4. a dual P2X3/P2X2/3 antagonist for the treatment of pain. Bioorg Med Chem Lett 19: 1628–1631. https://doi.org/10.1016/j.bmcl.2009.02.003
  22. Spedden E, Staii C (2013) Neuron biomechanics probed by atomic force microscopy. Int J Mol Sci 14: 16124–16140. https://doi.org/10.3390/ijms140816124
  23. Jiang FX, Lin DC, Horkay F, Langrana NA (2011) Probing mechanical adaptation of neurite outgrowth on a hydrogel material using atomic force microscopy. Ann Biomed Eng 39: 706–713. https://doi.org/10.1007/s10439-010-0194-0
  24. Xiong Y, Lee AC, Suter DM, Lee GU (2009) Topography and nanomechanics of live neuronal growth cones analyzed by atomic force microscopy. Biophys J 96: 5060–5072. https://doi.org/10.1016/j.bpj.2009.03.032
  25. Skorkina MY, Fedorova MZ, Sladkova EA, Muravuov AV (2012) The use of nanomechanic sensor for studies of morphofunctional properties of lymphocytes from healthy donors and patients with chronic lymphoblastic leukemia. Bull Exp Biol Med 154: 163–166. https://doi.org/10.1007/s10517-012-1899-x
  26. Lowery LA, Van Vactor D (2009) The trip of the tip: understanding the growth cone machinery. Nat Rev Mol Cell Biol: 332–343. https://doi.org/10.1038/nrm2679
  27. Kronlage C, Schafer-Herte M, Boning D, Oberleithner H, Fels J (2015) Feeling for filaments: quantification of the cortical actin web in live vascular 10endothelium Biophys J 109: 687–698. https://doi.org/10.1016/j.bpj.2015.06.066
  28. Spedden E, White JD, Naumova EN, Kaplan DL, Staii C (2012) Elasticity maps of living neurons measured by combined fluorescence and atomic force microscopy. Biophys J 103: 868–877. https://doi.org/10.1016/j.bpj.2012.08.005
  29. Coles CH, Bradke F (2015) Coordinating neuronal actin-microtubule dynamics. Curr Biol 25: R677–R691. https://doi.org/10.1016/j.cub.2015.06.020
  30. Pollard TD, Cooper J (2009) Actin: a central player in cell shape and movement. Science 326: 1208–1212. https://doi.org/10.1126/science.1175862
  31. Verstegen AM, Tagliatti E, Lignani G, Marte A, Stolero T, Atias M, Corradi A, Valtorta F, Gitler D, Onofri F, Fassio A, Benfenati F (2014) Phosphorylation of synapsin I by cyclin-dependent kinase-5 sets the ratio between the resting and recycling pools of synaptic vesicles at hippocampal synapses. J Neurosci 34: 7266–7280. https://doi.org/10.1523/JNEUROSCI.3973-13.2014
  32. Cesca F, Baldelli P, Valtorta F, Benfenati F (2010) The synapsins: key actors of synapse function and plasticity. Prog Neurobiol 91: 313–348. https://doi.org/10.1016/j.pneurobio.2010.04.006.
  33. Gu Y, Wang C, Li G, Huang LY (2016) EXPRESS: F-actin links Epac-PKC signaling to purinergic P2X3 receptors sensitization in dorsal root ganglia following inflammation. Mol Pain 12: 1744806916660557. https://doi.org/10.1177/1744806916660557
  34. Goswami C, Kuhn J, Heppenstall PA, Hucho T (2010) Importance of non-selective cation channel TRPV4 interaction with cytoskeleton and their reciprocal regulations in cultured cells. PLoS One 5: e11654. https://doi.org/10.1371/journal.pone.0011654
  35. Bele T, Fabbretti E (2016) The scaffold protein calcium/calmodulin-dependent serine protein kinase controls ATP release in sensory ganglia upon P2X3 receptor activation and is part of an ATP keeper complex. J Neurochem 138: 587–597. https://doi.org/10.1111/jnc.13680
  36. Gillespie JM, Hodge JJ (2013) CASK regulates CaMKII autophosphorylation in neuronal growth, calcium signaling, and learning. Front Mol Neurosci 6: 27. https://doi.org/10.3389/fnmol.2013.00027
  37. Gnanasekaran A, Bele T, Hullugundi S, Simonetti M, Ferrari MD, van den Maagdenberg AM, Nistri A, Fabbretti E (2013) Mutated CaV2.1 channels dysregulate CASK/P2X3 signaling in mouse trigeminal sensory neurons of R192Q Cacna1a knock-in mice. Mol Pain 9: 62. https://doi.org/10.1186/1744-8069-9-62
  38. Soares J, Araujo GRS, Santana C, Matias D, Moura-Neto V, Farina M, Frases S, Viana NB, Romão L, Nussenzveig HM, Pontes B (2020) Membrane elastic properties during neural precursor cell differentiation. Cells 9: 1323. https://doi.org/10.3390/cells9061323
  39. Pontes B, Ayala Y, Fonseca AC, Romão LF, Amaral RF, Salgado LT, Lima FR, Farina M, Viana NB, Moura-Neto V, Nussenzveig HM (2013) Membrane elastic properties and cell function. PLoS One 8: e67708. https://doi.org/10.1371/journal.pone.0067708
  40. Abbracchio MP, Ceruti S (2006) Roles of P2 receptors in glial cells: focus on astrocytes. Purinerg Signal 2: 595–604. https://doi.org/10.1007/s11302-006-9016-0
  41. Fumagalli M, Brambilla R, D'Ambrosi N, Volonté C, Matteoli M, Verderio C, Abbracchio MP (2003) Nucleotide-mediated calcium signaling in rat cortical astrocytes: Role of P2X and P2Y receptors. Glia 43: 218–223. https://doi.org/10.1002/glia.10248
  42. Abbracchio MP, Saffrey MJ, Hopker V, Burnstock G (1994) Modulation of astroglial cell proliferation by analogues of adenosine and ATP in primary cultures of rat striatum. Neuroscience 59: 67–76. https://doi.org/10.1016/0306-4522(94)90099-x
  43. Rose J, Brian C, Pappa A, Panayiotidis MI, Franco R (2020) Mitochondrial metabolism in astrocytes regulates brain bioenergetics. Neurotransmission and redox balance. Front Neurosci 14: 536682. https://doi.org/10.3389/fnins.2020.536682
  44. Séguéla P, Haghighi A, Soghomonian JJ, Cooper E (1996) A novel neuronal P2x ATP receptor ion channel with widespread distribution in the brain. J Neurosci 16: 448–455. https://doi.org/10.1523/JNEUROSCI.16-02-00448.1996
  45. Rodrigues RJ, Almeida T, Díaz-Hernández M, Marques JM, Franco R, Solsona C, Miras-Portugal MT, Ciruela F, Cunha RA (2016) Presynaptic P2X1–3 and α3-containing nicotinic receptors assemble into functionally interacting ion channels in the rat hippocampus. Neuropharmacology 105: 241–257. https://doi.org/10.1016/j.neuropharm.2016.01.022
  46. Cunha RA, Vizi ES, Ribeiro JA, Sebastião AM (1996) Preferential release of ATP and its extracellular catabolism as a source of adenosine upon high- but not low-frequency stimulation of rat hippocampal slices. J Neurochem 67: 2180–2187. https://doi.org/10.1046/j.1471-4159.1996.67052180.x
  47. Levy M, Faas GC, Saggau P, Craigen WJ, Sweatt JD (2003) Mitochondrial regulation of synaptic plasticity in the hippocampus. J Biol Chem 278: 17727–17734. https://doi.org/10.1074/jbc.M212878200
  48. Zhou X, Ma LM, Xiong Y, Huang H, Yuan JX, Li RH, Li JN, Chen YM (2016) Upregulated P2X3 receptor expression in patients with intractable temporal lobe epilepsy and in a rat model of epilepsy. Neurochem Res 41: 1263–1273. https://doi.org/10.1007/s11064-015-1820-x
  49. Chen Y, Zhang X, Wang C, Li G, Gu Y, Huang LY (2008) Activation of P2X7 receptors in glial satellite cells reduces pain through downregulation of P2X3 receptors in nociceptive neurons. Proc Natl Acad Sci U S A 105: 16773–16778. https://doi.org/10.1073/pnas.0801793105

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2. Fig. 1. Atomic force spectroscopy procedure of the neuron cell surface on the first day of differentiation in culture: (a) – schematic diagram of sample scanning: cell on a substrate; cantilever that moves along the cell surface; laser beam is tuned to a flexible cantilever console that deflects when moving along the surface and transmits information to a 4-section photodiode, (b) – neuron image obtained by scanning in Nova software with load (1 nN) applied at 25 points on the surface, (c) – approach (blue) and retraction (red) curves at each load application point, based on which the software constructs local elasticity maps, (d) – local elasticity map of the neuron surface (dark areas of the map indicate high values ​​of Young's modulus, and therefore higher surface rigidity).

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3. Fig. 2. Young's modulus of the cell surface of the primary mixed culture of E18 neurons under conditions of P2X3 receptor blockade. * significant differences at p < 0.05 according to Student's criterion.

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4. Fig. 3. Metabolic map of the energy phenotype of the primary mixed neuronal culture: the following respiration regions are indicated: Aerobic – aerobic respiration; Quiescence – respiratory rest; Energy – cellular metabolism based on mixed respiration (oxidative phosphorylation and glycolysis); Glycolytic – anaerobic respiration. The control group is shown in gray, the experimental group (Ro-4 blocker) in black. Filled squares indicate respiration before the blockade of the electron transport chain, unfilled squares indicate respiration after the blockade of the electron transport chain by stressors.

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