Invest Clin 64(4): 505 - 512, 2023 https://doi.org/10.54817/IC.v64n4a7

Corresponding autor. Jesús Alberto Mosquera-Sulbarán. Instituto de Investigaciones Clínicas “Dr. Américo Negrette”,

Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela. E-mail: mosquera99ve@yahoo.com

Nuclear and cytoplasmic expressions

of the receptor for advanced glycation

end products (RAGE) in the rat central

nervous system.

Jesús Mosquera-Sulbarán1, Adriana Pedreáñez2, Yenddy Carrero1

and Catherina Peña1

1 Instituto de Investigaciones Clínicas “Dr. Américo Negrette”, Facultad de Medicina,

Universidad del Zulia, Maracaibo, Venezuela.

2 Cátedra de Inmunología, Escuela de Bioanálisis, Facultad de Medicina, Universidad

del Zulia, Maracaibo, Venezuela.

Keywords: RAGE; ligands; nucleus; cerebral cortex; cerebellum.

Abstract. The receptor for advanced glycation end products (RAGE) is a

transmembrane protein involved in the induction of inflammatory processes

and oxidative stress after interacting with its ligands on the cell surface. Lo-

calization on the cell surface is necessary for interaction with the ligands. This

study aimed to determine the expression of RAGE in different parts of the nor-

mal rat brain and cerebellum using the immunofluorescence technique. Sev-

eral cerebral cortex layers (molecular/granular layers: M/GL; pyramidal layer:

PL) and the hypothalamus were analyzed, as well as the molecular layer (CML)

and the granular layer (CGL) of the cerebellum. Cells with RAGE-positive nu-

clei were generally observed in the brain’s cerebral cortex and cerebellum. In

the M/GL, cells with different degrees of positivity in the nucleus and cyto-

plasm accompanied by RAGE-positive material in the adjacent extracellular

space were observed, and RAGE-positive material in the neuropile. Pyramidal

neurons presenting various degrees of nuclear RAGE-positive material budding

and cells with different degrees of nuclear and cytoplasmic positivity were ob-

served in PL. The hypothalamus showed a high number of cells with RAGE-

positive granules adjacent to the nucleus and in the cytoplasm; nuclei remained

negative. Many positive nuclei were observed in CML; they were scarce in CGL.

These data suggest the storage of RAGE at the nuclear and cytoplasmic levels

in healthy rats and hypothesize the possible translocation of this molecule to

the cell surface in pathological conditions.

506 Mosquera-Sulbarán et al.

Investigación Clínica 64(4): 2023

Expresión nuclear y citoplasmática del receptor para

compuestos de glicosilación avanzada en el sistema nervioso

central de la rata.

Invest Clin 2023; 64 (4): 505 – 512

Palabras clave: RAGE; ligandos; núcleo; corteza cerebral; cerebelo.

Resumen. El receptor para compuestos de glicosilación avanzada (RAGE)

es una proteína transmembrana involucrada en la inducción de procesos infla-

matorios y en el estrés oxidativo después de su interacción con sus ligandos en

la superficie celular. La localización de este receptor en la superficie celular es

necesaria para su interacción con sus ligandos. El objetivo de este estudio fue

determinar la expresión de RAGE en las diferentes partes del cerebro y cerebelo

de la rata normal. Mediante la utilización de técnicas de inmunofluorescencia

se analizaron varias capas de la corteza cerebral (capas molecular/granular:

CM/G; capa piramidal: CP) y el hipotálamo. Las capas molecular (CMC) y la

capa granular (CGC) del cerebelo fueron también analizadas. Se observaron

células con el núcleo positivo para RAGE tanto en cerebro como cerebelo.

En CM/G se apreciaron células con diversos grados de positividad para RAGE

acompañadas de material positivo para RAGE en el espacio extracelular adya-

cente y en la neuropila. En la CP se observaron neuronas piramidales presen-

tando diversos grados de gemación de material nuclear positivo para RAGE y

diversas células con diferentes grados de positividad nuclear y citoplasmática.

En el hipotálamo se apreciaron gran número de células expresando gránulos

positivos a RAGE tanto adyacente al núcleo como en el citoplasma; el núcleo

permaneció negativo. Alto número de núcleos positivos se apreciaron en la

capa CMC a diferencia de la capa CGC del cerebelo. Estos hallazgos sugieren

el almacenamiento del RAGE en el núcleo y en el citoplasma en la rata normal

e hipotetizan una posible translocación de esta molécula a la superficie celular

en condiciones patológicas.

Received: 26-11-2023 Accepted: 06-08-2023

INTRODUCTION

The receptor for advanced glycation end

products (RAGE) is a transmembrane protein

and a multireceptor belonging to the immu-

noglobulin superfamily, expressed on the cell

surface and capable of binding to various li-

gands, inducing cell activation, cell dysfunc-

tion, and tissue damage 1, 2. RAGE has been

implicated in various pathophysiological pro-

cesses such as neurodegenerative diseases

and infectious processes 1-3. After RAGE binds

to its ligand, pro-inflammatory processes in-

crease with the induction of pro-inflammato-

ry cytokines and oxidative stress, determin-

ing a vicious circle of inflammation mediated

by the overexpression of the nuclear tran-

scription factor kB (NF-kB) that leads to cell

damage 1,3. Traditionally, the interaction of

RAGE with its ligands at the cell surface level

inducing intracellular signals leading to pro-

inflammatory processes has been reported 4,

RAGE in rat cerebrum and cerebellum 507

Vol. 64(4): 505 - 512, 2023

5. The interaction of RAGE with its ligands in

the central nervous system is of paramount

importance in the induction of neurodegen-

erative diseases 3, 6-8; however, there is little in-

formation about the location of RAGE in cen-

tral nervous system cells in non-pathological

conditions. The present study is focused on

determining by immunohistochemical meth-

ods the location of RAGE in the rat central

nervous system under healthy conditions.

MATERIAL AND METHODS

This study used healthy male Sprague-

Dawley rats (weight 150 to 200 g) (N=10).

All rats had unlimited access to tap water

and food. All animals were euthanized, and

samples from rat cerebrum (including the hy-

pothalamus) and cerebellum were embedded

in Tissue-Tek (Miles, Inc, Diagnostic Division,

Kankakee, Illinois, United States), frozen in

acetone and dry ice, and stored at -70 ºC un-

til use. Cryostat sections (4 µm) from sam-

ples were treated with a rabbit anti-rat RAGE

antibody (5µg/mL: ab3611; Abcam, Cam-

bridge, United Kingdom) to analyze RAGE

brain expression. Rabbit immunoglobulin G

(IgG) in tissues was localized by a secondary

rhodamine-conjugated goat anti-rabbit IgG

at 5µg/mL (Sigma-Aldrich, St. Louis, Mis-

souri, USA). Antibody against a nonrelevant

antigen or normal rabbit serum was used as

the negative control. Sections were mounted

in a solution of p-phenylenediamine in phos-

phate-buffered saline‒glycerol and viewed un-

der an epifluorescent microscope (Axioskop,

Zeiss, Wetzlar, Germany). Positive cells were

expressed as the number of cells per 0.0625

µ2 from 20 randomly selected fields of the

brain or cerebellum. Experiments were per-

formed according to the ethical guidelines of

the committee of bioethical and biosecurity

of FONACIT (Caracas, Venezuela) following

the Guide for the Care and Use of Labora-

tory Animals (National Institutes of Health

Publication No. 8023, revised 1978) and the

committee of bioethics of the Universidad del

Zulia School of Medicine.

Statistical analysis was performed us-

ing GraphPad Prism, version 7.0 (GraphPad

Software, San Diego, USA). Measurement

data with normal distribution is represented

as mean ± standard deviation. For continu-

ous variables that were normally distributed,

differences between groups were determined

by ANOVA and the posttest of Bonferroni. A

p-value < 0.05 was considered to be statisti-

cally significant.

RESULTS

The analysis of different rat cerebrum

and cerebellum areas showed high reactivity

to the anti-RAGE antibody in several areas. At

the level of the cerebral cortex, positive cells

were observed in the molecular/granular lay-

er (M/GL) and the pyramidal layer (PL). Posi-

tive cells were seen in the cerebellum in the

molecular layer (CML) and less frequently in

the cerebellar granular layer (CGL). A high

number of positive cells was observed in the

hypothalamus (Fig. 1). Histological analysis

showed numerous cells with high nuclear

reactivity to the anti-RAGE antibody in M/

GL (Fig. 2). Likewise, cells with negative or

scarcely positive nuclei were found accompa-

nied by cytoplasmic and adjacent extracellu-

lar positive reactivity to RAGE, simulating a

comet.

Interestingly, extracellular RAGE-positive

areas without cell presence were observed (Fig.

3). Pyramidal neurons with highly positive nu-

clei and positive glial cells were observed in PL

(Figs. 4 and 5). RAGE-positive nuclei present-

ing structures resembling nuclear buds in pyra-

midal neurons were observed (Fig. 5). The cells

of the hypothalamus showed a high frequency

of cytoplasmic RAGE-positive granules but no

nuclear positivity (Figs. 1 and 6).

Nuclear expression of RAGE in the cere-

bellum was observed mainly in the CML, with

a low frequency of positive nuclei in CGL;

however, some cells showed granular cyto-

plasmic positivity in this layer. Purkinje cells

were found to be negative (Figs. 1 and 7).

508 Mosquera-Sulbarán et al.

Investigación Clínica 64(4): 2023

DISCUSSION

In this study, the expression of RAGE

in the rat central nervous system was mainly

limited to the cell nucleus and cytoplasm.

Functionally, RAGE is expressed on the cell

surface, where it interacts with various li-

gands to activate intracellular pathways that

produce a pro-inflammatory and oxidative

stress state 1-3, 6-8.

The presence of this receptor within the

cell nucleus observed in this study suggests a

nuclear function or represents a storage site

for a subsequent trajectory of this receptor

from the nucleus to the cell surface to exert

its functions. Previous studies have shown the

passage of intranuclear molecules to the cy-

toplasm 9-13, suggesting a possible cell surface

expression pathway for RAGE. In this regard,

the immunohistochemical findings of this re-

port show cerebral cells showing decreased

expression of nuclear RAGE accompanied by

increased cytoplasm expression and adjacent

extracellular RAGE-positive material. In ad-

dition, pyramidal neurons showed budding

of RAGE-positive nuclear material, and cells

with cytoplasm RAGE-positive granules were

observed, suggesting a possible nuclear-to-

cytoplasmic pathway.

The presence of RAGE in the nucleus

suggests its nuclear localization prior to its

synthesis, possibly as a storage site, as oc-

curs with the non-histone chromosomal pro-

teins “high mobility group” (HMG) that are

present in the cell nucleus bound to DNA 10,

12 and perform functions such as determi-

nation of nucleosomal structure and stabil-

ity, and binding of transcription factors to

their cognate DNA sequences 14. HMG can

be localized in the nucleus, cytoplasm, and

the extracellular space during some patho-

logical processes where it can interact with

Fig. 1. Expression of the receptor for advanced glyca-

tion end products (RAGE) in the rat central

nervous system. High nuclear expression of

RAGE was observed in the molecular/granular

layer of cerebral cortex and in the molecular la-

yer of the cerebellum. Cells from the hypotha-

lamus did not express nuclear RAGE but a high

number expressed cytoplasmic RAGE in a gra-

nular form. M/GL: molecular/granular layer;

PL: pyramidal layer; CGL: cerebellar granular

layer; CML: cerebellar molecular layer; HIPO:

hippocampus. * p<0.01 vs. PL, CGL, CML.

Fig. 2. Expression of the receptor for advanced

glycation end products (RAGE) in the mole-

cular/granular layer of the cerebral cortex.

A) Panoramic view where a large number of

positive nuclei can be observed. B) Detail.

Arrows: positive nuclei. Cross arrows: nega-

tive nuclei. Original magnification: A: x600;

B: x1000.

RAGE in rat cerebrum and cerebellum 509

Vol. 64(4): 505 - 512, 2023

Fig. 3. Expression of the receptor for advanced

glycation end products (RAGE) in the mo-

lecular/granular layer of the cerebral cor-

tex. A and B) Cells with different degrees

of nuclear and cytoplasmic positivity to

RAGE (arrows). RAGE-reactive extracellu-

lar material is seen in the neuropile (cros-

sed arrow). C) Detail of cell showing weak

nuclear RAGE positivity and high positivity

for cytoplasmic. Note the presence of RA-

GE-positive material adjacent to the cell.

N: nucleus; C: cytoplasm; E: extracellular

space. Original magnification: A and B:

x600; C: x1000.

Fig. 4. Expression of the receptor for advanced glyca-

tion end products (RAGE) in the pyramidal

layer of the cerebral cortex. Pyramidal neu-

rons with high nuclear positivity for RAGE are

appreciated. Arrows: Positive neurons. Cross

arrow: probably a positive glial cell nucleus.

Original magnification: x1000.

Fig. 5. Expression of the receptor for advanced glyca-

tion end products (RAGE) in the pyramidal

layer of the cerebral cortex. A) Nuclear RAGE-

positive pyramidal neurons showing nuclear

budding (small arrows). Cross arrow: positive

neuron without nuclear budding. Thick arrow:

pyramidal neuron dendrite. B) Nuclear RAGE-

positive pyramidal neuron showing nuclear

budding (small arrow). Cross arrow: positive

glial cell nucleus. Thick arrow: neuron axon.

Original magnification: x1000.

510 Mosquera-Sulbarán et al.

Investigación Clínica 64(4): 2023

RAGE 9, 11, 13. There is no information avail-

able on the presence of RAGE in the nucle-

us, and possibly the expression of nuclear

RAGE on the cell surface obeys mechanisms

similar to those of HMG. Binding to DNA

may represent the storage mechanism of

RAGE in the nucleus. In this regard, RAGE’s

ability to bind to DNA 15 and its role in par-

ticipating in DNA double-strand repair pro-

cesses 16 has been reported. Another point

of analysis is the intranuclear role of RAGE

and HMG since the latter represents one of

the RAGE’s ligands 17.

Fig. 6. Expression of the receptor for advanced

glycation end products (RAGE) in hip-

pocampus. A) Overview of hypothalamic

cells, the majority of which are positi-

ve for cytoplasmic granular expression

of RAGE. B) Hippocampal cells presen-

ting positive RAGE granules adjacent to

the nucleus or in the cytoplasm (arrows).

Original magnification: A: x200; B:x600.

Fig. 7. Expression of the receptor for advanced

glycation end products (RAGE) in the ce-

rebellum of normal rats. A) High number

of cells expressing RAGE-positive nuclei

(arrow) in the molecular layer, compared

to low number in the granular layer. Arrow:

positive nucleus. Cross arrow: negative nu-

cleus. B) Scarce presence of RAGE-positive

nuclei (thick arrows) and RAGE-positive

granules (small arrows) in cells of the gra-

nular layer. C) Scarce presence of RAGE-

positive nuclei and RAGE-positive granules

(small arrows) in the granular layer. Negati-

vity was observed in Purkinje cells (arrows).

Cerebellar molecular layer: CML. Cerebellar

Granular layer: CGL. A and B: x200; C: x600.

RAGE in rat cerebrum and cerebellum 511

Vol. 64(4): 505 - 512, 2023

The expression of RAGE in the central

nervous system makes this tissue vulner-

able to inflammatory processes. The role

of RAGE in neurodegenerative processes,

neuroinflammation, Parkinson’s, and Al-

zheimer’s diseases, among other encepha-

lopathies, has been reported 1, 3, 18, 19. Cere-

bral and cerebellar nuclear and cytoplasmic

RAGE could play a role in these patholo-

gies. Perhaps the factors that induce those

pathologies induce the passage of nuclear

and cytoplasmic RAGE to the cell surface.

In conclusion, this report demon-

strates the presence of RAGE as a nuclear

protein and its cytoplasmic expression in

the cerebrum and cerebellum of normal

rats. These data highlight possible stud-

ies on the translocation of RAGE from the

nucleus to the cell surface, on nuclear func-

tions, and on the interaction of RAGE with

HMG in the nucleus.

Conflict of interest

Authors report no conflict of interest

Funding statement

This investigation has no financial sup-

port.

Author’s ORCID

• Jesús A Mosquera-Sulbarán (JMS):

0000-0002-1496-5511

• Adriana Pedreáñez (AP):

0000-0002-3937-0469

• Yenddy Carrero (YC):

0000-0003-4050-4468

• Caterina Peña (CP):

0009-0009-2824-2566

Author Contributions

JMS: conceptualization, methodology,

data curation, writing - original draft, writ-

ing - review & editing. AP: methodology,

software, formal analysis, writing - review &

editing. YC: resources, conceptualization,

methodology, writing - review & editing. CP:

methodology, software, formal analysis.

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