Neuronal Nitric Oxide Synthase in Cultured Cerebellar Bergmann Glia

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Letter

Neuronal Nitric Oxide Synthase in cultured cerebellar Bergmann Glia: Glutamate-dependent regulation Reynaldo Tiburcio-Félix, Bulmaro Cisneros, Luisa C.R Hernández-Kelly, María A Hernández-Contreras, Julieta Luna-Herrera, Ismael Rea-Hernández, Rosalinda Jiménez-Aguilar, Tatiana N Olivares-Bañuelos, and Arturo Ortega ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00656 • Publication Date (Web): 15 May 2019 Downloaded from http://pubs.acs.org on May 21, 2019

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ACS Chemical Neuroscience

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Neuronal Nitric Oxide Synthase in cultured cerebellar Bergmann Glia:

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Glutamate-dependent regulation

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Reynaldo Tiburcio-Félix1, Bulmaro Cisneros1, Luisa C.R. Hernández-Kelly 2, María A.

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Hernández-Contreras 3, Julieta Luna-Herrera 3, Ismael Rea-Hernández 1, Rosalinda

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Jiménez-Aguilar 4, Tatiana N. Olivares-Bañuelos TN5, Arturo Ortega2

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Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados

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del IPN, Apartado Postal 14-740, Ciudad de México 07360, Mexico.

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2Departamento

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Postal 14-740, Ciudad de México 07360, Mexico.

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3Departamento

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Nacional, Ciudad de México 11340, México

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4Unidad

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Unidad de Alta Especialidad Médica (UMAE). Instituto Mexicano del Seguro Social, Ciudad de

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México 02990, México

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5Instituto

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Baja California, 22860, México.

de Toxicología, Centro de Investigación y de Estudios Avanzados del IPN, Apartado de Inmunología, Escuela Nacional de Ciencias Biológicas del Instituto Politécnico

de Cuidados Intensivos Pediátricos. Hospital General La Raza Gaudencio González Garza.

de Investigaciones Oceanológicas, Universidad Autónoma de Baja California, Ensenada,

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Correspondance

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Arturo Ortega, PhD Departamento de Toxicología Cinvestav-IPN Apartado Postal 14-740 Ciudad de México, 07360 México [email protected] ORCID ID https://orcid.org/0000-0002-9594-8114

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ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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Glutamate exerts its actions through the activation of membrane receptors expressed in

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neurons and glia cells. The signaling properties of glutamate transporters have been

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characterized recently, suggesting a complex array of signaling transactions triggered by

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presynaptic released glutamate. In the cerebellar molecular layer, glutamatergic synapses are

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surrounded by Bergmann glia cells, compulsory participants of glutamate turnover and

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supply to neurons. Since a glutamate-dependent increase in cGMP levels has been described

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in these cells and the nitric oxide-cGMP signaling cascade increases their glutamate uptake

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activity, we describe here the Bergmann glia expression of neuronal nitric oxide synthetase.

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An augmentation of neuronal nitric oxide synthase was found upon glutamate exposure. This

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effect is mediated by glutamate transporters and is related to an increase in the stability of

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the enzyme. These results strengthen the notion of a complex regulation of glial glutamate

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uptake that supports neuronal glutamate signaling.

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KEYWORDS

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Bergmann glia, neuronal Nitric Oxide synthetase, glutamate transporters, cGMP, protein

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stability

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ABBREVIATIONS

AMPA

-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid

Asp

D-Aspartate

BGC

Bergmann Glia Cells

cGMP

Cyclic Guanosine Monophosphate

CHIP

Hsc70-interacting Protein

CHX

Cycloheximide

CNS

Central Nervous System

CNQX

6-cyano-7-Nitroquinoxaline-2,3-dione

EAAT

Excitatory Amino Acid Transporter 1

EAAT1

Excitatory Amino Acid Transporter

EAATs

Excitatory Amino Acid Transporters

GLAST

Glutamate/Aspartate Transporter

Gln

Glutamine

GLT-1

Glutamate Transporter 1

Glu

Glutamate

GluRs

Glutamate Receptors

GRIA

Glutamate Ionotropic Receptor AMPA type

GRIK

Glutamate Ionotropic Receptor Kainate type

GRIN

Glutamate Ionotropic Receptor NMDA type

GRM1

Metabotropic Glutamate Receptor subtype 1

GRM2


GRM3


KA

Kainate

mGluRs

Metabotropic Glutamate Receptors

NMDA

N-methyl-D aspartate

nNOS

Neuronal Nitric Oxide Synthase

NO

Nitric Oxide

PKG

Protein Kinase G or cGMP-dependent Protein Kinase

SNAT2

Sodium dependent Neutral Amino acid Transporter 2



SNAT3

Sodium dependent Neutral Amino acid Transporter 3

SNP

Sodium Nitroprussiate

TBOA

DL-threo-β-Benzyloxyaspartic Acid

YY1

Yin Yang 1

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INTRODUCTION

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Glutamate (Glu) is the main excitatory neurotransmitter in the Central Nervous System

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(CNS) and exerts its functions through the activation of specific membrane receptors (GluRs)

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that are expressed in neurons and glia cells and are classified into two large gene families.

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The first one comprises homo or hetero-oligomeric ligand-gated ion channels known as

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ionotropic glutamate receptors (iGluRs). These cationic channels are permeable to Na+, K+

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and Ca2+ and have been classified based on their pharmacological, electrophysiological and

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molecular properties into: GRIA, GRIK, and GRIN receptors. In contrast, mGluRs belong to

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the G-protein coupled receptors superfamily and have been grouped in terms of its sequence

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similarity and their signaling properties into Group I (GRM1, GRM5), Group II (GRM2,

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GRM3) and Group III (GRM4, GRM6, GRM7, GRM8) Group I is primarily coupled to Gq,

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whereas Groups II and III are linked to Gi (1).

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A tight regulation of Glu extracellular levels depends on its removal from the synaptic cleft

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through a family of sodium-dependent excitatory amino acid transporters (EAATs) present

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in neurons and glia cells. Five subtypes of EAATs (1 to 5) have been described so far. Glial

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Glu uptake is the main mechanism that removes almost 80% of the neuronal released Glu

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and is crucial to avoid an excitotoxic death cascade (2). Glutamatergic transmission relies in

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an exquisite glia/neuronal coupling. Neuronal Glu availability is warranted by the so-called

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Glu/Glutamine shuttle: glial enriched EAAT1/GLAST and/or EAAT2/GLT-1 internalize

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synaptic released Glu which is rapidly metabolized to Gln via Gln synthetase and once

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accumulated exported to the vicinity of the presynaptic by the sodium-dependent neutral

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amino acids transporter 3 (SNAT3). Gln is then taken up (via SNAT2) by the presynaptic

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terminal that metabolizes it back to Glu, to be packed into synaptic vesicles completing the

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cycle (3). Disruption of this cycle compromises Glu-mediated neurotransmission and

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therefore the regulation of its components has attracted the attention of several research

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groups (4–6).

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Nitric Oxide (NO), a gas synthesized and released in response to GRIN activation in the

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cerebellar molecular layer, is produced by the action of neuronal nitric oxide synthase

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(nNOS), a Ca2+/calmodulin activated enzyme, on the synaptic terminals of the granule cells

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(parallel fibers) (7) and diffuses to the surrounding Bergmann glia in which it activates the



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soluble form of guanylate cyclase (8–10). Increased cGMP levels activate protein kinase G

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(PKG), known as the Ca2+/NO/cGMP/PKG signaling cascade (11).

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Bergmann glia cells are the most abundant non-neuronal cells in the entire cerebellum, their

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cell body resides in the Purkinje cell layer, and its ramifications span the three layers of the

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cerebellar cortex. These ramifications or fibers are used by granule cells to migrate to the

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granule cell layer in early stages of development (12, 13). Bergmann glia fibers completely

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encapsulate the synapses in the molecular layer (14). The most abundant glutamatergic

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synapse in the encephalon is the one established by the parallel fibers and the Purkinje cells

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in the cerebellar molecular layer. These synapses are of particular interest since it is a well-

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established example of a tripartite synapse due to the fact the Glu turnover depends on the

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activity of GLAST/EAAT1 expressed in Bergmann glia, and a functional coupling of parallel

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fiber/Purkinje cell/Bergmann glia has been described using a cerebellar slice preparation

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(15).

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The development of a primary culture of Bergmann glia cells (BGC) from avian cerebellum

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has allowed the characterization of the functional properties and regulation of

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GLAST/EAAT1 (16). A role for the Ca2+/NO/cGMP/PKG signaling cascade in the

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regulation of GLAST/EAAT1 has been reported by our group. A PKG-dependent increase

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in Glu uptake activity results in an augmentation of GLAST/EAAT1 transporters in the

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plasma membrane (17) favoring the notion of nNOS expression in the cultured cells, as has

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previously been suggested for rat cerebellum (18).

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In this context, the aim of this study was to demonstrate the expression and the Glu-dependent

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regulation of nNOS in cultured BGC. We report here the constitutive expression of nNOS,

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its Glu-dependent increase in protein steady state levels and function. Interestingly enough,

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Glu transporters are responsible for the Glu effect on nNOS.

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RESULTS AND DISCUSSION

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nNOS expression in cultured chick cerebellar BGC. The reported increase in cGMP in

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BGC after the activation of Glu receptors, the fact that exposure to a NO donor like sodium

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nitroprussiate (SNP) leads to an augmentation in [3H]-D-Aspartate uptake activity in BGC,

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and the reported expression of nNOS in rat cerebellar Bergmann glia (19) suggested that our


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primary culture express the neuronal isoform of NOS (17, 20). As well, nNOS expression

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has also been confirmed in oligodendrocytes, astrocytes, Müller cells, and glial cells of the

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spinal cord (21–25). The first approach was to perform immunocytochemical experiments,

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and as depicted in Figure 1, under basal conditions, a diffuse, but specific signal is detected.

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Interestingly a 15 min exposure to 1 mM Glu is sufficient to increase the cytoplasmatic signal

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(Figure 1, panel B). Several neuronal proteins have been identified in radial glia, such as

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GRIN (26–28), the YY1 transcription factor (29, 30), and many others. Therefore, it was not

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surprising to detect nNOS in our cultured cells. A plausible explanation for the radial glial

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expression of neuronal genes is their suggested neurogenic potential (31, 32). The fast

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increase in nNOS accumulation in cultured BGC, suggests that a post transcriptional event

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triggered by Glu is responsible for this up-regulation.

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Glu-Dependent nNOS augmentation in cultured BGC. In order to shed some light into

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the molecular mechanism(s) triggered by Glu that increases nNOS expression, we decided to

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characterize the time dependency of the excitatory amino acid effect. To this end, BGC were

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exposed to 1 mM Glu over a period of 60 min, and nNOS protein levels detected via Western

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blot analysis (Figure 2). As depicted in panel A of Figure 2, as early as 10 min of Glu

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exposure, results in a significant increase in nNOS, with a peak after 15 min, and a return to

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basal levels after 60 min. This effect is dose-dependent, with an EC50 of approximately 50

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M. These results further strengthen the post-transcriptional character of the Glu effect and

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also is indicative of a transporter-mediated effect since GLAST/EAAT1 KM has a value in

139

the M range (33). To test this latter possibility, BGC monolayers were exposed to 1 mM

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Glu in a Na+-free medium, and as expected, no increase in nNOS levels was found, since in

141

the absence of external Na+ (Figure 3 , panel A), GLAST/EAAT1 is not capable to transport

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the amino acid. In accordance to these results, the treatment with a fixed 1 mM D-Aspartate

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(Asp) concentration for different time periods reproduces the nNOS augmentation elicited

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by Glu in complete medium, favoring again, the notion that Glu transport triggers nNOS up-

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regulation (Figure 3, panel B). A pharmacological evidence for this interpretation was also

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sought. Remarkably enough, the competitive non-transportable EAATs blocker DL-threo-β-

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Benzyloxyaspartic acid (TBOA) completely inhibits the D-Asp effect. In the same vein, the

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GRIA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reduced only a small, non-



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significant fraction of the D-Asp response (Figure 4). Moreover, both the GRIA

150

noncompetitive antagonist MK801, and the selective GRIN antagonist D-AP5 did not

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prevent the D-Asp effect (Figure 5). Of relevance is to mention that preincubation with the

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sodium/calcium

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methanesulfonate (1:1), Carbamimidothioic acid (KBR7943) prevents the increase in nNOS

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protein levels triggered by D-Asp, suggesting the reversed function of NCX in response to

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the increased Na+entry through GLAST, as we have already reported (34, 35). Taken

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together, these results demonstrate that the Glu-dependent increase in nNOS protein levels is

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a GLAST/EAAT1 dependent effect.

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Over the last decade, the role of glial Glu transporters as signaling entities has been

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established (17, 35–38). Glia cells also express functional glutamatergic receptors of the

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ionotropic and metabotropic subtypes, so these cells have differential Glu-sensing molecules

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that regulate their function in response to neuronal activity. The abundance of plasma

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membrane Glu transporters, favor the notion of an important role of these molecules in

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glutamatergic signaling, although the presence of functional Glu receptors in these cells has

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been clearly demonstrated (39, 40).

exchanger

(NCX)

2-[4-[(4-nitrophenyl)methoxy]phenyl]ethyl

ester,

165 166

GLAST/EAAT1-mediated increase in nNOS half-life. In order to establish the molecular

167

mechanism of the Glu effect, experiments were carried out in the presence of the

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transcripcional inhibitor actinomycin D or the protein translation inhibitor cycloheximide

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(CHX) (Figure 6). Neither of these inhibitors blocks the D-Asp induced increase in nNOS

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protein levels, suggesting that an extended protein half-life would be responsible for

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augmented nNOS levels. To test this possibility, pulse-chase experiments were done using

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[35S]-Methionine. BGC monolayers were labeled for 60 min in methionine-free medium,

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washed, and then chased with complete medium for various time periods before nNOS was

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immunoprecipitated.

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immunoprecipitates, clearly demonstrate that the treatment with D-Asp during the chase time

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period, results in a higher amount of radiolabeled nNOS compared to control cells. The loss

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of radioactive nNOS is apparently monophasic and a first-order reaction with a half-life of

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approximately 30 min in control cells, while in D-Asp-exposed cells nNOS half -life is

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doubled (60 min, Figure 7). Increases in intracellular Ca2+ activates nNOS in a calmodulin

SDS-polyacrylamide

gel

electrophoresis


analysis

of

the

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dependent-manner, through the binding of the Ca2+/calmodulin (CaM) complex and the

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homodimerization of the enzyme (41). It is tempting to speculate that Ca2+ influx through

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NCX activated by Glu uptake in BGC (34, 35) , activates nNOS and also prevents its

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degradation. Covalent alterations in the nNOS substrate binding site leads to its

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ubiquitination by the C-terminus of Hsc70-interacting protein (CHIP), a ubiquitin-ligase of

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the E3 type that binds Hsp70 and Hsp90, resulting in its degradation (42). In support of our

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interpretation is the reported nNOS stabilization by Ca2+/CaM through Hsp90 (43).

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Furthermore, it has been demonstrated that inhibition of nNOS proteosomal degradation

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improves glucose transport (44), a feature in line with our previous findings linking Glu and

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glucose uptake in Bergmann glia (45).

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Nitric oxide production. Finally, in order to demonstrate the activity of nNOS and its

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plausible regulation through GLAST/EAAT1, we evaluated the amount of nitric oxide

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present in the culture media of cells exposed for different time periods to a fixed 1 mM D-

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Asp concentration. The Griess assay shows that 0.4 pg of nitric oxide is detected in control

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cells whereas in treated cultures a maximum increase of 0.8 pg of nitric oxide is detected

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after 15 min, the time in which we detected the higher amount of nNOS via Western blot

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(Figure 3, panel B). Therefore, it is quite possible that the reduced rate of nNOS degradation

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upon GLAST/EAAT1 activity is responsible for the increase in NO recorded.

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In summary, we report here the Bergmann glia expression of nNOS and its regulation through

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the activity of its most abundant and almost exclusive Glu transporter: GLAST/EAAT1. It

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quite possible that NO production in these cells has a dual function, in neighboring cerebellar

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granule cells could result in an increase in Ca2+ transients (46), favoring an augmentation of

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the amount of glutamate released in response to an action potential and by these means result

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in an strengthened synapse, while in Bergmann glia would increase Glu uptake in order to

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have a constant supply of the transmitter in the pre-synapsis (17, 47). A summary of previous

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findings and the ones described herein are depicted in Figure 8. Once released from the

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parallel fibers Glu activates its receptors in Purkinje cells. Glu is taken up from the synaptic

209

cleft through GLAST (33). The increase in [Na]i activates the Na+/K+ ATPse and most

210

possibly NCX1 (41) leading to the activation of the Ca2+/CaM -dependent protein kinase



211

(35), and the stabilization of nNOS. The resulting increase in NO activates the soluble form

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of guanylate cyclase in Bergmann glia cells that upregulates GLAST molecules at the plasma

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membrane and by these means augments the transport (17). Concomitantly, NO diffuses to

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the presynaptic neuron increasing Ca2+ entry and guanylate cyclase activity favoring Glu

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release (48).

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An increase in glucose entry is also expected to replenish ATP pools (45). It should be noted

217

that some of these findings have also been found in retina Müller glia cells (49), and in

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oligodendrocytes (50). Experiments in progress in our group are aimed towards a

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characterization of the signaling complexes formed in glia cells in an activity-dependent

220

manner that enable these cells to support sustained periods of glutamatergic transmission.

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METHODS

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Materials. Tissue culture reagents were obtained from GE Healthcare (Carlsbad, CA, USA).

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D-aspartate (D-Asp) and L-Glutamate (Glu) were obtained from Tocris-Cookson (St. Louis,

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MO USA). Polyclonal anti nNOS (Cat. sc-648) was purchased from Santa Cruz

226

Biotechnology. Horseradish goat anti-rabbit antibodies and the enhanced chemiluminescence

227

reagent (ECL) were obtained from Amersham Biosciences (Buckinghamshire, UK). Protein

228

A-Sepharose was obtained from GE Healthcare Cat. 28-9513-78). All other chemicals were

229

purchased from Sigma (St. Louis, MO, USA).

230 231

Primary Cultures. Cerebellar BGC were prepared from 14-day-old chick embryos as

232

previously described (51). Cell monolayers were maintained in DMEM containing 10 % fetal

233

bovine serum, 2 mM glutamine, and gentamicin (50 µg/mL), and used on the 4th to 7th day

234

after culture. Before any treatment, confluent monolayers were switched to non-serum

235

DMEM media containing 0.5 % bovine serum albumin (BSA) for 30 min and treated with

236

Glu or Asp added to culture medium for the indicated time periods. For cell immunostaining,

237

the dissociated cells were seeded on coverslips and fixed by exposure to ice-cold acetone for

238

10 min and air dried for 1 h. Cells were rinsed with phosphate-buffered saline (PBS) twice

239

and fixed for 10 min in 4% paraformaldehyde. Coverslips were rinsed twice with Tris-

240

buffered saline (TBS) and one more time with TBS/Tween 20 (0.05%). Non-specific binding

241

was prevented by incubation with 1% BSA in TBS (BSA/TBS) for 1 h. Cells were exposed


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to a 1:100 dilution of the primary antibody anti-nNOS, in BSA/TBS overnight at 4 °C,

243

followed by the incubation with the respective fluorescein-labeled goat anti-rabbit anti-sera

244

in BSA/TBS (1:500) for 2 h at room temperature. Preparations were mounted with

245

Fluoroshield/DAPI. Cell preparations were examined under a fluorescence microscopy

246

(Zeiss Axioskop 40 immunofluorescence microscope and the AxioVision software; Carl

247

Zeiss, Inc., Thornwood, NY).

248 249

SDS-PAGE and Western Blots. Cells from confluent monolayers were harvested with

250

phosphate-buffer saline (PBS) (10 mM K2HPO4/KH2PO4, 150 mM NaCl, pH 7.4) containing

251

phosphatase inhibitors (10 mM NaF, 1 mM Na2MoO4 and 1 mM Na3VO4). The cells were

252

lysed with RIPA buffer (50 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 1mM

253

phenylmethylsufonyl fluoride, 1mg/ml aprotinin, 1mg/ml leupeptin, 1% NP-40, 0.25%

254

sodium deoxycholate, 10 mM NaF, 1 mM Na2MoO4 and 1 mM Na3VO4 pH 7.4). Cell lysates

255

were denaturized in Laemmli’s sample buffer, and equal amount of proteins (50 g as

256

determined by the Bradford method) were resolved through 10% SDS-PAGE slab gels and

257

electroblotted to nitrocellulose membranes. Blots were stained with Ponceau S stain to

258

confirm that protein content was equal in all lanes. Membranes were soaked in PBS to

259

remove the Ponceau S and incubated in TBS containing 5% dried skimmed milk and 0.1%

260

Tween 20 for 60 min to block the excess of non-specific protein binding sites. Membranes

261

were then incubated overnight at 4˚C with the primary antibodies, followed by secondary

262

antibodies. Immunoreactive polypeptides were detected by chemiluminescence and the

263

densitometry analyses were performed with ImageJ software.

264 265

Metabolic Labeling and Immunoprecipitation. BGC were grown in 6-well plates and

266

incubated with 1.0 ml of methionine-free medium for 60 min, and then 80 Ci of [35S]-

267

methionine were added. Following the labeling period, protein synthesis was stopped with

268

300 g/L of cycloheximide for 15 min prior the treatment with 1 mM D-Asp for 60 min. At

269

appropriate time intervals, the chase was terminated by solubilization with 100 L of ice-

270

cold RIPA buffer. Cell debris was removed by centrifugation at 16,000 g in a microfuge for

271

10 min. nNOS was immunoadsorbed from the cell lysates with 5 L of anti-nNOS IgG and

272

5 L of protein A-Sepharose. Immune pellets were washed three times with TBS (50 mM



273

Tris and 140 Na2Cl) and bound antigen was eluted from the beads by boiling in SDS sample

274

buffer with 100 mM dithiothreitol. The purified proteins were resolved through 8% SDS-

275

polyacrylamide gels as described above.

276 277

Nitric Oxide release. NO production was determined by measuring the accumulation of

278

nitrite and nitrate as metabolites of Nitric Oxide released in the culture medium using a

279

colorimetric method with the Griess assay (Sigma).

280 281

ACKNOWLEDGEMENTS. This work was supported by grants from Conacyt-México

282

(255087), and by Soluciones para un México Verde S.A de C.V. R.T-F was supported by a

283

Conacyt-México PhD fellowship.

284

INDIVIDUAL AUTHOR CONTRIBUTIONS. Tiburcio-Félix R performed the

285

experiments and participated in the discussion of the results. Cisneros B contributed to the

286

design of the experiments and to the edition of the final manuscript. Hernández-Kelly LC

287

participated in the discussion, design and interpretation of the data, Hernández-Contreras A

288

contributed to the measurements of NO, Luna-Herrera J participated in the nNOS

289

immunodetection, Rea-Hernández I was involved in the nNOS half-time determination,

290

Jiménez-Aguilar R participated in the discussion of the data, Olivares-Bañuelos TN

291

contributed in the art work, the writing, editing and discussion of this contribution. Ortega A

292

designed the experiments, supervised the work and wrote this article.

293 294

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443

FIGURE LEGENDS

444

Figure 1. Immunofluorescence of nNOS in BGC cultures. Primary cultures of BGC were

445

incubated with anti-nNOS polyclonal antibodies (1:100 dilution).

446

secondary antibodies were used in a 1:500 dilution. Microphotographs of BGC shows anti-

447

nNOS (green) specificity, and DAPI stained nucleus (blue) in either control (cells without

448

Glu) or treated cells (Glu 1 mM for 15 min). Scale bar = 20 m.

Flourescein-coupled

449 450

Figure 2. Glutamate regulates nNOS protein levels in a time and dose-dependent

451

manner. (A) BGC monolayers were exposed to 1 mM glutamate (Glu) for the indicated time

452

periods (0-60 min 60 min), or (B) to increasing Glu concentrations for 15 min. Time zero

453

represents Glu unexposed BGC. At the indicated experimental condition, total extracts were

454

prepared and analyzed by Western blots with anti-nNOS or anti-actin antibodies (loading

455

control). A representative blot is presented on top of each graph. Three independent

456

experiments ± SD are graphed for each data point as nNOS/Actin % of control. Statistical

457

analysis was performed using a one-way ANOVA with a Dunnett’s multiple comparison test,

458

*p < 0.05.

459 460

Figure 3. Glu transporter-mediated regulation of nNOS expression in BGC. (A) BGC

461

monolayers were exposed to 1 mM glutamate (Glu) over a period of 60 minutes, in a Na+

462

free media. (B) BGC monolayers were exposed to 1 mM aspartate (Asp) over a period of 60

463

minutes in complete medium. At the indicated time points, total extracts were prepared and

464

nNOS protein levels analyzed via Western blots. A representative blot is presented on top of

465

each graph. Three independent experiments ± SD are graphed for each data point as

466

nNOS/Actin % of control. Statistical analysis was performed using a One-way ANOVA with

467

a Dunnett’s multiple comparison test, *p < 0.05.

468 469

Figure 4. Pharmacological characterization of the D-Asp-depedent nNOS expression.

470

BGC were pretreated with the EAAT blocker DL-threo- β-Benzyloxyaspartic acid (TBOA,

471

100 μM), or with GRIA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 μM)



472

prior to treatment with 1 mM Asp for 15 min. A representative blot of nNOS protein levels

473

is presented on top of each graph. Three independent experiments ± SD are graphed for each

474

data point as nNOS/Actin (% of control). Statistical analysis was performed using a One-way

475

ANOVA with a Dunnett’s multiple comparison test. *p < 0.05, ** p

Neuronal Nitric Oxide Synthase in Cultured Cerebellar Bergmann Glia

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