Putative glucosensing property in rat and human activated microglia

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Neurobiology of Disease 17 (2004) 1–9

Putative glucosensing property in rat and human activated microglia

D. Ramonet, M.J. Rodrıguez, M. Pugliese, and N. Mahy*

Unitat de Bioquımica, Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona,

08036 Barcelona, Spain

Received 16 May 2003; revised 30 October 2003; accepted 5 November 2003

Available online 17 July 2004

Microglial cells involved in the pathogenesis of many neurodegener-

ative diseases acquire the features of cytotoxic and phagocytic cells in

response to certain pathogens and inflammatory signals. KATP channels

are energy sensors of ATP availability that link the cell’s metabolic

state to its membrane excitability. In pancreatic beta cells, they

promote glucose-dependent insulin secretion, and in neurones,

hyperpolarization that protects against hypoxic damage. This study

analyses activated microglia in an in vivo rat neurodegenerative model

based on acute hippocampal glutamate receptor overactivation and in

postmortem samples from patients with Alzheimer’s disease. We

demonstrate that in activated microglia the KATP channel components

SUR-1 or SUR-2 are present together with glucokinase. Our results

indicate that, according to glucose availability, these channels may

modify microglia membrane potential. The functional relevance of

these channels is seen as a new mechanism modulating the effects of

external signals on microglia.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Neurodegenerative diseases; Alzheimer’s disease; Microglial

functions; Inflammation; ATP-sensitive potassium channel; Glucose

Introduction

Microglial cells proliferate and acquire the features of cytotoxic

and phagocytic cells in response to injury and immunological

challenges (Bauer et al., 2001; Gonzalez-Scarano and Baltuch,

1999; Lee et al., 2001). In disorders such as multiple sclerosis,

Parkinson’s disease (PD), and Alzheimer’s disease (AD), the

presence of activated microglia has been increasingly linked to

the neurodegenerative process (Gao et al., 2002; McGeer and

McGeer, 2000; Rogers and Shen, 2000). Thus, more precisely,

the microglia appears to be associated with progressive myelin

destruction (Bauer et al., 2001), involved in the last stage of the

pathogenetic process of PD (Banati et al., 1998; Hirsch et al.,

1999), and in the formation and maturation of plaques and

neuronal degeneration in AD (Gonzalez-Scarano and Baltuch,

0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2003.11.019

* Corresponding author. Unitat de Bioquımica, Institut d’Investigacions

Biomediques August Pi i Sunyer (IDIBAPS), Facultat de Medicina,

Universitat de Barcelona, C/Casanova 143, E-08036 Barcelona, Spain. Fax:

+34-93-403-58-82.

E-mail address: nmahy@ub.edu (N. Mahy).

Available online on ScienceDirect (www.sciencedirect.com.)

1999; Overmyer et al., 1999). Characterization of microglia in

animal models of neurodegeneration is crucial if we are to

understand the pathogenesis of the process and define the complex

cellular interplay taking place (Bernal et al., 2000b; Han et al.,

2002; Petegnief et al., 1999).

Microglial cultures widely used to investigate the mechanisms

and regulation that underlie their selective activated responses

have shown that rodent and human quiescent microglia lacks an

outward K+ current that appears when activated. Thus, despite

numerous studies describing K+ currents in microglia (Eder,

1998; Eder and Heinemann, 1996; Khanna et al., 2001), little

is known about which specific channels are expressed, and

whether they are involved in in vivo microglial activation or

functions. ATP-sensitive K+ channels (KATP) are found in a

diversity of tissues, including cardiac, skeletal and smooth

muscles, and the pancreatic beta cells. In these cells, KATP act

as energy sensors of ATP production dependent on glucokinase

(GK) and are thought to regulate various physiological functions

such as muscle contraction or insulin secretion by coupling cell

metabolism to membrane potential (Ashcroft, 1998). Similar

channels are widely expressed in various brain regions in which

they couple the electrical activity of the neurone to its metabolic

state (Levin, 2001; Levin et al., 2001; Liss and Roeper, 2001;

Melamed-Frank et al., 2001; Miki et al., 2001; Yamada et al.,

2001). For example, in substantia nigra pars reticulata, the region

with the highest density of KATP, reduction of cytosolic ATP of

GABAergic neurones opens these channels and hyperpolarizes

the membrane to avoid further seizure due to hypoxia (Yamada

and Inagaki, 2002; Yamada et al., 2001). This channel is an

octamer composed of two pore-forming subunits (Kir6.1 and 6.2)

and the sulfonylurea receptor (SUR-1 or SUR-2) that binds ATP

(Aguilar-Bryan et al., 1995; Inagaki et al., 1996; Isomoto et al.,

1996; Yamada et al., 1997).

The rapid activation of microglia in response to neuronal

damage requires the rapid availability of a large amount of

energy that may be critical in determining, at least in part, their

function in the pathogenetic process. On the basis of this

hypothesis and the suggested existence of an ATP-sensitive K+

channel in microglia (McLarnon et al., 2001), the present study

investigates whether activated microglia expresses GK, and if

KATP channels containing SUR-1 or SUR-2 colocalize within

these cells.

Activated microglia was investigated in an in vivo experi-

mental model of neurodegeneration and in postmortem tissue of

D. Ramonet et al. / Neurobiology of Disease 17 (2004) 1–92

patients with Alzheimer’s disease (AD) rich in plaques and

activated microglia (Overmyer et al., 1999). The experimental

model results from a stereotaxic microinjection of a-amino-3-

hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) into the

hippocampal formation (HF) previously described in our labora-

tory (Bernal et al., 2000a). This single AMPA injection induces a

neurodegenerative process that develops with astrogliosis, necrot-

ic neuronal death, and microglial activation. Here we report that

most rat and human-activated microglial cells were immuno-

positive for GK and KATP channel components, indicating that

intracellular ATP may modulate microglia membrane potential

through KATP channels. These results could indicate that micro-

glial signal transduction properties could be regulated by glucose

availability.

Materials and methods

Human samples

Human postmortem samples of cerebral cortex from patients

ranging in age from 72 to 86 years, with an established history of

Alzheimer’s disease (stage 6, n = 3), were obtained from our

local Neurological Tissue Bank (Serveis cientıfico-tecnics, Uni-

versitat de Barcelona, Spain) (Mahy, 1993) with the approval of

Fig. 1. AMPA-induced neurodegenerative process in the rat hippocampal format

staining of sham and (A2) AMPA-lesioned HF; note the massive AMPA-induced a

and (B2) AMPA-lesioned HF showing normal nuclei stained in blue and acidoph

showed quiescent astroglia. (C2) No GK immunolabelling was detected in the H

labelling of AMPA-lesioned rats, note the high reactivity of the astrocytes presentin

GK labelling in the same section showing some stained cells (arrowheads). (D3

arrowheads) while the typical astroglial shapes (solid arrowheads) did not presen

the appropriate medical ethics committee. Neuropathological

assessment was established by the Bank Neuropathologist accord-

ing to Braak and Newel criteria (Braak and Breaak, 1991; Newel

et al., 1999). Sex and age-matched controls with no known

history of neurological or psychiatric disorders were obtained

from the Bank and also included in the study (n = 3). Postmortem

delay ranged from 4 to 24 h and the time of storage of tissue

blocks before experiment ranged from 3 to 12 months. Paraffin

sections (7 Am) and 250-mg portions of the frozen human

prefrontal cortex samples were obtained from the corresponding

blocks.

Animals and stereotaxic procedure

Adult male Sprague–Dawley rats (body weight: 250–300 g

at the beginning of the study) were used for experiments

conducted in line with European standards (86/609/EU). The

Ethical Committee of the University of Barcelona approved

procedures. All efforts were made to minimize animal suffering

and to use only the number of animals needed for reliable

scientific data.

Rats were anaesthetized with equitesin (a mixture of chloral

hydrate and sodium pentobarbitone; 0.3 ml/100 g body weight, ip)

and placed in a stereotaxic frame (David Kopf, Carnegie Medicin,

Sweden) with the incisor bar set at �3.3 mm. Unilateral microin-

ion (HF). (A1) Low magnification picture of a representative cresyl violet

lteration of CA1, CA3 layers, and dentate gyrus. (B1) VAF staining of sham

ilic necrotic neurones in red. (C1) GFAP immunolabelling of sham animals

F of sham rats. (C3) Merge of C1 and C2. (D1) Illustrative image of GFAP

g hypertrophy, hyperplasia, and GFAP immunoreactivity (arrowheads). (D2)

) Merge of D1 and D2: most of GK labelling was outside astrocytes (thin

t GK labelling. Scale bars: A–B, 2 mm; C–D, 40 Am.

Fig. 2. Expression of GK in the hippocampal formation (HF) of sham and AMPA-lesioned rats. (A1) Illustrative image of OX42 labelling in the HF of sham rats

showing ramified resting microglia (arrowheads). (A2) No GK labelling was detected in the HF of sham rats. (A3) Merge of A1 and A2. (B1) GK positive

immunoreactivity (arrowheads) was found in the ventromedial hypothalamic and (B2) dorsomedial hypothalamic nuclei of the same section. (C1)

Representative image of reactive microglia in the HF of AMPA-lesioned rats (arrowheads). (C2) GK labelling in the same section showing some stained cells

(arrows). (C3) Merge of C1 and C2 where GK and OX42 labelling is coincident (arrowheads); note that all GK immuno labelling localized in microglia (small

arrows). (D) 3D reconstruction of a microglial cell with GK labelling inside. (E) GK-Western blot of sham and AMPA-lesioned HF. Homogenates were

normalized for protein content quantified by Bradford’s method and 50 Ag of protein was applied in each gel lane. Each lane corresponds to a different rat and

the quantification corresponds to three independent Western blot experiments. Arrowheads show GK position (*P < 0.05, Student’s t test,). (F) Detail of OX42

and GK labelling of a reactive microglial cell showing orthogonal cross-sectional views. The yz and xz planes are indicated with yellow lines, and the xy plane

corresponds to the middle of the projection. In each cross-section, the GK signal is surrounded by the OX42 one (arrowheads and arrows). Except for F, scale

bars: 40 Am; F scale bar: 10 Am.

D. Ramonet et al. / Neurobiology of Disease 17 (2004) 1–9 3

D. Ramonet et al. / Neurobiology of Disease 17 (2004) 1–94

jection into the dorsal hippocampus was performed at the coor-

dinates: 3.3 mm caudal, 2.2 mm lateral to bregma, and 2.9 mm

ventral from dura (Paxinos and Watson, 1986). Each rat received a

single 0.5-Al injection of 50 mM phosphate-buffered saline (PBS;

pH 7.4) (n = 8) or of 5.4 mM AMPA dissolved in PBS (n = 11).

This AMPA dose was the same as in previous studies (Petegnief et

al., 1999; Rodrıguez et al., 2000). Fifteen days later, animals were

separated at random into two sets: one group (n = 4 sham + 6

AMPA) was transcardiacally perfused with 4% paraformaldehyde

and their brains were removed and frozen for histological studies.

Fig. 3. Expression of SUR isoforms in AMPA-lesioned rat hippocampal formati

showing ameboid activated microglia at the lesioned HF (arrowhead). (A2) S

(arrowheads). (A3) Merge of A1 and A2: most of SUR-1 labelling coincides w

neurones (solid arrowhead). (B) 3D reconstruction of a typical microcyte verifies

localization. (C) Illustrative Western blot with anti-SUR-1 antibody in human hea

compatible with the glycosilated species of SUR-1. (D1) OX42 labelling of a repre

2 labelling in the same section. (D3) Merge of D1 and D2 showed that most SUR-2

to hippocampal neurones. (E) 3D reconstruction of a reactive microglial cell verifi

with anti-SUR-2 antibody in human heart and rat hippocampal extracts revealing

The other group (n = 4 sham + 5 AMPA) was decapitated and their

dorsal hippocampus was microdissected and homogenized for

biochemical assays.

Materials

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

Antihuman h-amyloid monoclonal antibody directed against the

amino acids 8–17 came from Dako (Glostrup, Denmark). Mouse

monoclonal antibody anti-rat CD11b, clone MRC OX-42, was

on (HF). (A1) Representative confocal OX-42 immunofluorescence image

UR-1 immunolabelling in the same section showing some stained cells

ith OX42 (thin arrowhead); when not, it may correspond to hippocampal

that SUR-1 staining is present in the microglia in a membrane-compatible

rt and rat hippocampal extracts revealing one band at range 160–190 kDa

sentative lesioned HF showing activated microglia (arrowheads). (D2) SUR-

labelling coincides with OX-42 (thin arrowheads); when not, it may belong

es that SUR-2 staining is present inside the cell. (F) Illustrative Western blot

the expected band at 140 kDa. Scale bars: 40 Am.

Fig. 4. Expression of GK in the frontal cortex of postmortem samples of AD patients. (A) Double labelling immunohistochemistry of h-amyloid and GFAP of

the prefrontal cortex from a patient with AD stage VI disease, demonstrating the laminar distribution of h-amyloid protein. Layers V and VI show intensely

stained deposits with a central, compact amyloid core. Note the spatial relationship between GFAP-positive hypertrophic astrocytes (brown, DAB) and neuritic

plaques (black, Ni-DAB) in the insert. (B) HLA-DR immunohistochemistry (red, AEC) of a control frontal cortex sample, hematoxylin counterstained.

Microglia recognizable by characteristic square-shaped nuclei (thin arrowhead) presents faint HLA-DR signal, revealing low reactivity. (C) HLA-DR

immunohistochemistry (red, AEC) of AD frontal cortex sample, hematoxylin counterstained. Some reactive microglia is also recognizable by characteristic

square-shaped nuclei (thin arrowhead, insert). (D) GK immunohistochemistry (red, AEC) of a control frontal cortex sample, hematoxylin counterstained. Some

microglia is recognizable by characteristic square-shaped nuclei (thin arrowhead), but no GK signalling is present. (E) An adjacent section with GK

immunostaining (red, AEC); apart from some vessel walls (asterisk), specific GK labelling is close to square-shape nuclei (thin arrowhead, inset). (F) 3D

reconstruction of a reactive microglial cell with GK labelling inside. (G1) Representative Western blots of AD and control frontal cortex samples with anti-

HLADR and anti-GK antibodies. Protein (50 Ag) quantified by Bradford was used by lane. Both HLA-DR bands were detected at 27 and 36 kDa; nominal GK

weight is 50 kDa. (G2) Quantification of HLA-DR (n = 3 control, n = 3 AD; error bar = standard deviation). (G3) Quantification of GK (n = 3 control, n = 3

AD; error bar = standard deviation). (H1) HLA-DR fluorescent immunolabelling of reactive microglia (thin arrowheads) in an AD frontal cortex sample. (H2)

Glucokinase immunoreactivity in the same section showing some stained cells (arrows); note that the weak non-quenched autofluorescence is easily

distinguishable by their shape (asterisks). (H3) Merge of E1 and E2 where yellow shows colocalization between HLA-DR and GK. Scale bars A, B, C, D and E:

100 Am, inserts 10 Am; H1– 3: 40 Am.

D. Ramonet et al. / Neurobiology of Disease 17 (2004) 1–9 5

D. Ramonet et al. / Neurobiology of Disease 17 (2004) 1–96

purchased from Serotec (Serotec Ltd, Oxford, UK); mouse

monoclonal antihuman MHC II (HLA-DR), clone BRA-30, was

from Neomarkers (Fremont, CA). Mouse monoclonal anti-rat

fibrillary acidic protein (GFAP), clone G-A-5, was from Sigma.

Rabbit polyclonal antihuman glucokinase (GK) (fragment 318–

405), goat polyclonal antihuman SUR-1 (fragment C-16), and

antihuman SUR-2 (fragment C-15) were purchased from Santa

Cruz Biotech. (Santa Cruz, CA). Additional secondary antibodies,

reagents, and chemical compounds were purchased from Santa

Cruz Biotech, Sigma, or Jackson Immunoresearch (West Grove,

PA).

Immunohistochemistry and immunofluorescence

Cryostat serial sections (12 Am) were obtained from the rat

dorsal hippocampus (�3.3 mm to bregma). Paraffin sections (7

Am) were obtained from bank blocks of the human cortex and

paraffin was removed immediately before use.

In human samples, immunohistochemistry was carried out by

the avidin–biotin–peroxidase method with different antibodies

separately. HLA-DR antibody (diluted 1:100) was used to immu-

nostain specifically activated microglia (Mattlace et al., 1990),

anti-GFAP antibody (diluted 1:200) to stain astrocytes, GK was

detected with the antihuman GK antibody (diluted 1:200), and

antihuman h-amyloid antibody (diluted 1:50) was used to visu-

alize plaques and confirm AD diagnostic. Each bound antibody

was detected with the standard ABC method. Development was

carried out using 3,3V-diaminobenzidine (DAB) or 3-amino-9-

ethylcarbazole (AEC) as peroxidase substrate. Adjacent sections

were processed for Nissl standard staining with cresyl violet or

Vanadium acid fucsin (VAF) staining to visualize necrotic acido-

philic neurones.

To ensure labelling of h-amyloid, a pretreatment with 98–

100% formic acid for 5 min was performed. Some sections were

double-labelled for h-amyloid and anti-GFAP. In these cases, h-amyloid protein was visualized first using a mixture of DAB and

nickel chloride to give a black reaction product, and the GFAP

antibody was developed with DAB alone to give a brown reaction

product.

Double indirect immunofluorescence of human sections was

performed as follows: mouse anti-HLA-DR (1:100) was com-

bined with rabbit anti-GK (1:200) and labelled with TRITC goat

anti-mouse IgG (1:64) and FITC donkey anti-rabbit IgG (1:200).

For rat sections, mouse anti-OX42 (1:100) or anti-GFAP antibody

(1:200) was combined with rabbit anti-GK and labelled with

FITC sheep anti-mouse IgG (1:64) and Cy3 goat anti-rabbit

mouse IgG (1:50).

Fig. 5. Expression of SUR isoforms in the frontal cortex samples of AD patien

microglia is shown. (A2) SUR-1 immunolabelling in the same section showing som

human AD reactive microglia (thin arrowheads). T lymphocytes also labelled by th

quenched autofluorescence artifacts are marked with asterisks. (C2) SUR-1 immuno

Merge of C1 and C2; yellow shows colocalization between HLA-DR and SUR-1

with SUR-1 (solid arrowhead). (D) 3D reconstruction of a reactive microglial cell w

with anti-SUR-1 antibody in human heart and AD cortex extracts revealing a band

tissue. Arrowhead points to a faint HLA-DR signal that could be isolated activated

of B1 and B2. Note the colocalization of both signals in the same structure. (F1) Re

the frontal cortex of an AD patient. (F2) SUR-2 labelling in the same section sh

reactive microglia stained in yellow when HLA-DR and SUR-2 colocalize, note th

and that other cell types present only anti-SUR-2 immunoreactivity (small arrow

Illustrative Western blot with anti-SUR-2 antibody in human heart (control) and AD

Mouse anti-HLA-DR (1:100) or anti-OX42 (1:100) was also

combined with goat anti-SUR-1 (1:200) or anti-SUR-2 (1:200) and

labelled with TRITC donkey anti-goat IgG (1:200) and FITC sheep

anti-mouse IgG (1:64). Incubations with mouse or goat IgG as

primary bodies were used for negative controls.

Human brain autofluorescent lipofuscin artifacts were reduced

to near background levels by immersing the slides before montage

in 70% ethanol supplemented with 0.1% Sudan black B, as

described in Baschong et al., 2001.

For 3D reconstruction, confocal laser scanning microscopy was

performed using a standard Leica TCS system. After filtration

through a noise removal filter, isosurfaces were calculated from

confocal 3D image stacks. Standard extended focus projections

were used as a guide for the manual selection of the appropriate

value for each fluorophorus. The resulting isosurfaces were pro-

jected together in the same axis with the color corresponding to

their labelling.

Western blot analysis

All isolated rat hippocampus and small portions (around 250

mg) of frozen precortex of healthy (n = 3) and AD (n = 3)

patients were manually homogenized in 1:10 (w/v) cold 50 mM

Tris–HCl, pH 7.6, 1 mM PMSF, 2 mM EDTA. Protein was

determined by the Bradford method (Bradford, 1976). Western

blot analysis was performed as described elsewhere with the

antibodies against GK (1:1000), SUR-1 (1:2000), SUR-2

(1:2000), or human HLA-DR (1:2000). The immunocomplexes

were detected using Lumi-Light (Roche Diagnostics, Mannheim,

Germany) enhanced chemoluminescence and films were devel-

oped, scanned, and analyzed by computer-assisted image analysis

(Scion Image, Scion Co., MD).

Results

Activated microglia in rat hippocampal lesion

As previously described, 15 days after acute AMPA adminis-

tration in the rat HF, a neurodegenerative process characterized by

a severe necrotic neuronal death and an astroglial reaction was

evidenced by Nissl and VAF stainings (Figs. 1A1,2 and B1,2) and

GFAP immunohistochemistry (Figs. 1C1 and D1).

Confocal microscopy of OX42 antibody that labels microglia

cells revealed the presence of a significant microglial reaction

(Figs. 2A1 and C1). HF of the sham group was not labelled

with the anti-GK antibody (Fig. 2A2), and as expected, abun-

dant positive GK neurones in the ventromedial and dorsomedial

ts. (A1) HLA-DR immunolabelling of human control tissue. No reactive

e faint signals. (A3) Merge of A1 and A2. (C1) HLA-DR immunolabelling of

e antibody are distinguishable by their round shape (solid arrowhead). Non-

labelling in the same section showing some stained cells (arrowheads). (C3)

(thin arrowhead); note that HLA-DR-positive lymphocytes are not labelled

ith SUR-1 labelling in most of their membrane. (E) Illustrative Western blot

in 160–190-kDa range. (B1) HLA-DR immunolabelling of human control

microglia. (B2) SUR-2A/B immunolabelling in the same section. (B3) Merge

active microglia (thin arrowheads) and T lymphocytes (solid arrowheads) in

owing some stained cells (arrowheads). (F3) Merge of F1 and F2 results in

at lymphocytes are not labelled with anti-SUR-2 antibody (solid arrowhead)

). (G) 3D reconstruction of a reactive microglia with SUR-2 labelling. (H)

cortex extracts revealing the expected bands at 140 kDa. Scale bars: 40 Am.

D. Ramonet et al. / Neurobiology of Disease 17 (2004) 1–9 7

hypothalamic nuclei of control animals were detected (Fig.

2B1–2). Anti-GK and OX42 double immunohistochemical la-

belling revealed a high expression of glucokinase in most

reactive microglia (Fig. 2C1–3), but not in quiescent one (Fig.

2A1–3). 3D reconstruction revealed a GK localization restricted

to cytoplasm (Fig. 2D) also evidenced in orthogonal cross-

sectional views (Fig. 2F). No labelling was observed in astro-

cytes (Figs. 1C1–3 and D1–3). Quantification of GK immuno-

blots of sham and AMPA-lesioned hippocampi showed a

significant 4-fold increased expression (6748 F 3844 vs.

27100 F 2066) (Fig. 2E).

Specificity of SUR-1 and SUR-2 antibodies was assessed in

human heart and rat brain homogenates by Western blot analysis

showing bands (Figs. 3C and F) at the expected molecular

D. Ramonet et al. / Neurobiology of Disease 17 (2004) 1–98

weights. Double immunohistochemical labelling with anti-SUR-1

or anti-SUR-2 antibodies combined with OX42 indicated that

most reactive microglia expressed SUR-1 (Fig. 3A1–3) or SUR-2

(Fig. 3D1–3). Reactive microglia was also identified by their

ameboid morphology. SUR-1 and SUR-2 also labelled some

neurones (Figs. 3A1–3 and D1–3). 3D reconstruction demonstrat-

ed SUR-1 and SUR-2 widespread membrane distribution (Figs.

3B and E). SUR-1 and SUR-2 immunoblot values of AMPA-

lesioned hippocampi were 115% and 323% of sham values,

respectively (raw data not shown).

Activated microglia in Alzheimer’s patients

GK, SUR-1, and SUR-2 immunoreactivities were also studied

in the prefrontal cortex of Alzheimer’s and normal healthy patients.

Prefrontal cortex of AD patients revealed densely packed h-amyloid deposits arranged in a typical laminar distribution associ-

ated with astrogliosis with an increased number of astrocytic

processes, enlargement of processes, and soma hypertrophy and

microgliosis (Fig. 4A). The external astroglial layer did not present

h-amyloid deposits, and the Layer I contained numerous diffuse

plaques. Layers II and III presented numerous diffuse plaques and

few h-amyloid deposits with a condensed core (neuritic plaques).

Layers V and VI had a much greater density of neuritic plaques,

also evident at the bottom portion of Layer VI (grey–white matter

junction). h-Amyloid deposits were also found in the white matter

underlying the cortex.

After Sudan-black treatment to reduce autofluorescent arti-

facts, HLA-DR and anti-GK double immunofluorescent labelling

revealed a high GK expression in most reactive microglia (Fig.

4H1–3). Immunolocalization of each antibody in control and AD

patients with the avidin–biotin–peroxidase method gave similar

results and confirmed the confocal microscopy data (Figs. 4B–

E). Widespread homogenous distribution of GK was shown by

3D reconstruction (Fig. 4F). Correlation between HLA-DR and

GK immunoblot quantifications of prefrontal cortex homoge-

nates of healthy (n = 3) and AD patients (n = 3) resulted highly

significant values (r = 0.51; P < 0.05) (Fig. 4G1–3).

Specificity of SUR-1 and SUR-2 antibodies was assessed in

human heart and brain homogenates by Western blot analysis

showing bands at the expected molecular weights (Figs. 5E and

H). SUR-1 and SUR-2 immunoblot values of the cortex of AD

patients were 223% and 124% of healthy patients values,

respectively (raw data not shown). Confocal microscopy with

anti-SUR-1 or anti-SUR-2 and anti-HLA-DR to label microglia,

also identified by its ameboid morphology, indicated that some

75% of human reactive microglia expressed SUR-1 (Figs. 5

A1–3 and C1–3) and some 40% expressed SUR-2 (Figs. 5B1–3

and F1– 3). SUR-2 also labelled some neurones (Fig. 5F3).

Widespread homogenous membrane distribution of SUR-1 or

SUR-2 was shown by 3D reconstruction (Figs. 5D and G).

Discussion

The present study demonstrates that activated microglia

characterized in a rat model of neurodegeneration and in

postmortem samples of AD patients strongly expresses a KATP

channel similar to the one described in cardiac and muscular

tissues, neurones, and pancreatic beta cells. We choose to work

with this in vivo model and AD patients because both are on-

going processes that develop with a progressive neuronal

death, astrogliosis, and microgliosis. The localization of SUR-

1 or SUR-2 in activated microglia, but not in the quiescent

one, may explain the reported presence of an outward K+

current in cultured activated microglia (Eder, 1998; Eder and

Heinemann, 1996). SUR-1 and SUR-2 detected in rat and

human activated microglia suggest that both are needed be-

cause their activities may be regulated differently (Liss et al.,

1999). The presence of activated microglia may also explain

part of the increased Kir6.1 and Kir6.2 expression described in

brain hypoxia, which avoids further neuronal damage (Yamada

et al., 2001). Along with a significant neuronal loss, preser-

vation of KATP channels has been described in the hippocam-

pus of Alzheimer’s patients (Xu et al., 2002). This may be due

to their increased microglial expression that would compensate

for neuronal disappearance.

Voltage- and calcium-dependent potassium channels have been

proposed to participate in the control of several functions exerted

by microglia, such as scavenging and neuroprotective actions

(Eder, 1998; Khanna et al., 2001). For example in culture, blockade

of K+ channels inhibits the respiratory burst and the morphological

changes associated with microglia activation (Khanna et al., 2001).

In this study, we provide evidence of the presence of the ATP-

binding subunits SUR-1 and SUR-2 in most activated microcytes;

the same for GK expression. None of the three signals was detected

in astrocytes. The presence of GK provides activated microglia

with a putative energy-sensing capability not explored till now. The

(patho)physiological effects of this coupling of cell metabolism to

membrane potential is not at all trivial. Like glucosensing neurones

use glucose as a signalling molecule to regulate firing and

transmitter release, GK might be a key element for controlling

microglial activation and secretion according to glucose availabil-

ity. However, besides a direct role in the regulation of membrane

potential and cell volume, KATP channels might also participate

indirectly in ion transport through other channels, exchangers, or

pumps.

Microglial functions rely on a diversity of external signals

such as neurotransmitters, trophic factors, cytokines, membrane-

bound ligands etc., which interact with their constitutive or

inducible receptors (see review Aloisi, 2001). Activation of KATP

channels associated with GK expression results in a general

membrane hyperpolarization that could modulate the microglial

effects of each signal. For example, TNFalpha actions through

activation of K+ outward currents in murine and human microglia

(McLarnon et al., 2001) are different when KATP outward currents

are generated. If this is true, the effects of an external stimulus on

microglia are likely to be modulated at each moment by the

number and degree of activated KATP channels, which in turn

would depend on GK activity and glucose availability. The

crosstalk between glucose availability and the external stimuli

to adjust at any moment the microglial functions could be

proposed as a general mechanism that could help integrate the

cellular response to the tissue status.

In conclusion, our results demonstrate that during neurodegen-

eration, GK and KATP channels comprising SUR-1 or SUR-2 are

expressed in in vivo activated rat and human microglia, in which

the channels may exert control on the functions of microglia

according to energy availability. Further studies on their relation-

ship with phagocytic and apoptotic microglial actions are needed to

understand whether these channels are good targets for therapeutic

control of microglia activation.

D. Ramonet et al. / Neurobiology of Disease 17 (2004) 1–9 9

Acknowledgments

The authors thank Dr. F. Michetti of the Rome Catholic

University, Italy, for the gift of the h-anti-amyloid antibody, Dr. F.

Bernal for his helpful suggestions and are also grateful to the

Serveis Cientıfico-Tecnics of the Universitat de Barcelona (Seccio

d’Imatges and Unitat de Banc de Teixits Neurologics). Supported

by V-2003-REDG167 and SGRC00380.

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