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Guanosine and GMP increase the number of granular cerebellar neurons in culture: dependence on adenosine A2A and ionotropic glutamate receptors

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Abstract

The guanine-based purines (GBPs) have essential extracellular functions such as modulation of glutamatergic transmission and trophic effects on neurons and astrocytes. We previously showed that GBPs, such as guanosine-5′-monophosphate (GMP) or guanosine (GUO), promote the reorganization of extracellular matrix proteins in astrocytes, and increase the number of neurons in a neuron-astrocyte co-culture protocol. To delineate the molecular basis underlying these effects, we isolated cerebellar neurons in culture and treated them with a conditioned medium derived from astrocytes previously exposed to GUO or GMP (GBPs-ACM) or, directly, with GUO or GMP. Agreeing with the previous studies, there was an increase in the number of β-tubulin III-positive neurons in both conditions, compared with controls. Interestingly, the increase in the number of neurons in the neuronal cultures treated directly with GUO or GMP was more prominent, suggesting a direct interaction of GBPs on cerebellar neurons. To investigate this issue, we assessed the role of adenosine and glutamate receptors and related intracellular signaling pathways after GUO or GMP treatment. We found an involvement of A2A adenosine receptors, ionotropic glutamate N-methyl-D-aspartate (NMDA), and non-NMDA receptors in the increased number of cerebellar neurons. The signaling pathways extracellular-regulated kinase (ERK), calcium-calmodulin-dependent kinase-II (CaMKII), protein kinase C (PKC), phosphatidilinositol-3′-kinase (PI3-K), and protein kinase A (PKA) are also potentially involved with GMP and GUO effect. Such results suggest that GMP and GUO, and molecules released in GBPs-ACM promote the survival or maturation of primary cerebellar neurons or both via interaction with adenosine and glutamate receptors.

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Abbreviations

ACM:

Astrocytic-conditioned medium

A1R:

Adenosine A1 receptors

A2AR:

Adenosine A2A receptors

AMPA:

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

CaMKII:

Calcium-calmodulin dependent kinase-II

Chel:

Chelerythrine

DIP:

Dipyridamole

DPCPX:

1,3-Dipropyl-8-cyclopentylxanthine

ECM:

Extracellular matrix proteins

ERK:

Extracellular-regulated kinase

GAMS:

c-d-Glutamylaminomethylsulphonic acid

GBPs-ACM:

Guanine-based purines-astrocytic-conditioned medium

GMP:

Guanosine-5′-monophosphate

GUO:

Guanosine

iGluRs:

Ionotropic glutamate receptors

MK-801:

(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-iminemaleate

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NMDA:

N-methyl-D-aspartate

8-PT:

8-Phenyltheophylline

PI3-K:

Phosphatidilinositol-3′-kinase

PKA:

Protein kinase A

PKC:

Protein kinase C

Wort:

Wortmannin

ZM24138:

4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol

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Funding

Research supported by grants from the Brazilian funding agencies: CAPES (Coordenação do Pessoal de Ensino Superior) – Project Procad-CAPES; and CAPES-PVE 052/2012; CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) – Projects IBN-Net # 01.06.0842-00 and INCT for Excitotoxicity and Neuroprotection; FAPESC (Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina) – Project NENASC. C.I.T. is recipient of CNPq productivity fellowship. All authors have materially participated in the research and/or article preparation.

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Author notes

  1. Helena Decker

    Present address: Max Planck Florida Institute for Neuroscience, Jupiter, FL, 33458, USA

  2. Helena Decker and Tetsade C. B. Piermartiri contributed equally to this work.

Authors and Affiliations

  1. Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Trindade, 88040-900, Florianópolis, SC, Brasil

    Helena Decker, Tetsade C. B. Piermartiri, Sheila S. Francisco, Tharine Dal-Cim & Carla I. Tasca

  2. Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Trindade, 88040-900, Florianópolis, SC, Brasil

    Cláudia B. Nedel

  3. Departamento de Anatomia, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21941-590, Rio de Janeiro, RJ, Brasil

    Luciana F. Romão & Vivaldo Moura-Neto

  4. Programa de Pós-graduação em Nanociências, Universidade Franciscana, Santa Maria, RS, Brasil

    Carina R. Boeck

  5. Instituto Estadual do Cérebro Paulo Niemeyer da Secretaria de Estado de Saúde do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brasil

    Vivaldo Moura-Neto

Authors
  1. Helena Decker
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  2. Tetsade C. B. Piermartiri
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  4. Luciana F. Romão
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  6. Tharine Dal-Cim
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  9. Carla I. Tasca
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Corresponding author

Correspondence to Carla I. Tasca.

Ethics declarations

The procedures used in this study complied with the guidelines on animal care of the UFSC Ethics Committee on the Use of Animals (CEUA), which follow the Principles of laboratory animal care from NIH (2011).

Conflicts of interest

Helena Decker declares that she has no conflict of interest.

Tetsade C. B. Piermartiri declares that she has no conflict of interest.

Cláudia B. Nedel declares that she has no conflict of interest.

Luciana F. Romão declares that she has no conflict of interest.

Sheila S. Francisco declares that she has no conflict of interest.

Tharine Dal-Cim declares that she has no conflict of interest.

Carina R. Boeck declares that she has no conflict of interest.

Vivaldo Moura-Neto declares that he has no conflict of interest.

Carla I. Tasca declares that she has no conflict of interest.

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Decker, H., Piermartiri, T.C.B., Nedel, C.B. et al. Guanosine and GMP increase the number of granular cerebellar neurons in culture: dependence on adenosine A2A and ionotropic glutamate receptors. Purinergic Signalling 15, 439–450 (2019). https://doi.org/10.1007/s11302-019-09677-y

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