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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: [email protected] (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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