Nicotinic acetylcholine receptor immunohistochemistry in Alzheimer's disease and dementia with Lewy bodies: differential neuronal and astroglial pathology

Journal of the Neurological Sciences 225 (2004) 39 – 49 www.elsevier.com/locate/jns

Nicotinic acetylcholine receptor immunohistochemistry in Alzheimer’s disease and dementia with Lewy bodies: differential neuronal and astroglial pathology Thanasak Teaktonga, Alison J. Grahama, Jennifer A. Courta, Robert H. Perryb, Evelyn Jarosb, Mary Johnsona, Ros Halla, Elaine K. Perrya,* a

Centre Development in Clinical Brain Aging, MRC Building, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, NE4 6BE, UK b Department of Neuropathology, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE, UK Received 15 July 2003; received in revised form 21 June 2004; accepted 22 June 2004 Available online 11 September 2004

Abstract Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB) are common forms of dementia in the elderly. The neuropathology of AD and DLB is related to cholinergic dysfunctions, and both a4 and a7 nicotinic acetylcholine receptor (nAChR) subunits are decreased in several brain areas in both diseases. In this immunohistochemical study, we compared neuronal and astroglial a4 and a7 subunits in AD, DLB and age-matched controls in the hippocampal formation. The numbers of a4 reactive neurons were decreased in layer 3 of the entorhinal cortex of AD and DLB, whereas those of a7 reactive neurons were decreased in layer 2 of the subiculum of AD and DLB and in layer 3 of the entorhinal cortex of DLB. In contrast, the intensity of a7 reactive neuropil was significantly higher in AD than in controls or DLB in a number of areas of the hippocampus (CA3/4 and stratum granulosum), subiculum and entorhinal cortex. An increase in a7 immunoreactivity in AD was also associated with astrocytes. The number of astrocytes double-labelled with a7 and glial fibrillary acidic protein (GFAP) antibodies was increased in most areas of the hippocampus and entorhinal cortex in AD compared with controls and DLB. Increased astrocyte a7 nAChRs in AD may be associated with inflammatory mechanisms related to degenerative processes specific to this disease. D 2004 Elsevier B.V. All rights reserved. Keywords: Cholinergic receptors; Dementia; Neuroglia; Limbic system; Parahippocampal gyrus

1. Introduction Nicotinic acetylcholine receptors (nAChRs) are ligandgated ion channels composed of different subunits assembled in pentameric structure [1]. Neuronal nAChRs are divided into two principal subtypes based on their subunit composition. Heteromeric nAChRs consist of combinations of a (a2–a6) and h (h2–h4) subunits, whereas homomeric nAChRs consist of a7, a8 or a9

* Corresponding author. Tel.: +44 191 4444416; fax: +44 191 4444402. E-mail address: [email protected] (E.K. Perry). 0022-510X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2004.06.015

subunits [2,3]. Neuronal nAChRs are involved in a range of cerebral functions including attention [4], learning and memory [5]. A loss of nAChRs subunits is associated with diseases such as Alzheimer’s disease (AD), dementia with Lewy bodies (DLB), Parkinson’s disease and schizophrenia [6,7]. AD is the most common form of dementia in elderly and is characterized by cognitive impairment and progressive memory loss. Beta-amyloid deposition and senile plaques formation together with neurofibrillary tangles are the major histopathological features of AD [8]. Cholinergic functions are impaired and brain nAChR binding in AD is decreased predominantly in the neo- and archi-cortices [9]. Reductions in a3, a4, and a7, but not h2 measured by Western blot

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analysis have been reported in the cortex and hippocampus [10,11]. Reduced numbers and intensity of a4, a7 and h2 immunoreactive (IR) neurons have been observed in the frontal cortex in AD [12,13]. DLB is characterized by a-synuclein positive Lewy bodies (LB) and neurites in the cerebral cortex and brainstem [14]. Clinical features include visual hallucinations, parkinsonism, and fluctuating cognition [14]. [3H]Nicotine binding is decreased in the parietal cortex [15], basal ganglia and hippocampus [16,17], and [3H]epibatidine binding is decreased in the frontal cortex [18]. Reduced [125I]a-bungarotoxin binding has also been reported in the frontal cortex [18] and in the reticular nucleus of the thalamus [19] but not in the temporal cortex [20]. In a pilot study, Sparks et al. [13] reported reduced a4 and h2 subunit immunoreactivity in the brains of patients with AD-type dementia in combination with Lewy bodies, but to date, there has been no detailed immunohistochemical investigation of nAChR subunits in DLB. The present immunohistochemical study investigates whether a4 and a7 subunits are similarly altered in AD and DLB compared with controls. Assessment was made in the hippocampus and entorhinal cortex, brain areas which are severely affected in AD and DLB pathology.

stained with von Braunmqhl silver. The National Institute on Aging and Reagan Institute (NIA–RI) guideline [22] was used based on Braak staging [23] and tangles stained with tau-2 immunohistochemistry and Palmgren silver. Consensus DLB diagnostic criteria [14] were used based on cortical scores of Lewy bodies counts of neocortical stained with asynuclein immunohistochemistry. The neuropathological characteristics of the AD and DLB cases are summarized in Table 1. Mean postmortem delays of control, AD and DLB cases were 46.0F18.9, 58.8F24.5 and 41.2F33.4 h and mean fixation time were 8.3F12.1, 2.8F1.2 and 3.3F2.4 months, respectively. Although postmortem delay tended to be longer in the AD group and the control group had a longer fixation time than the other two groups, there appeared to be no relationship between postmortem delay and fixation time and a4 or a7 immunoreactivity in the human brain [24]. At autopsy, the right hemisphere was fixed in 10% buffered formalin and blocks of hippocampus and adjacent cortex were embedded in paraffin. Sections of 10-Am thickness were cut with a microtome and mounted onto Vectabondk reagent (Vector Laboratories, Peterborough, UK) treated slides. 2.2. Immunohistochemistry

2. Materials and methods 2.1. Tissue preparation Autopsy samples of the temporal cortex were obtained from 6 cases of AD (age 83.7F2.7 years), 6 cases of DLB (age 76.5F3.1 years) and 4 age-matched controls (age 82.5F9.5 years). None of the controls smoked tobacco or had any history of a neurological or psychiatric disease or significant neuropathological abnormality. AD and DLB were diagnosed according to neuropathological examinations and clinical assessments. The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) guideline [21] was used based on scores of neocortical neuritic plaques

For nAChR immunohistochemistry, monoclonal antibodies against the a4 [mAb 299 (Cambridge Bioscience, Cambridge, UK)] and a7 nAChR subunits [mAb 306 (Cambridge Bioscience)] of the nAChR were used. Characterization and specifity of the monoclonal antibodies have been described in detail elsewhere (for mAb 299, see Refs. [25–27]; for mAb 306, see Refs. [28–30]). The a7 antibody specifically binds to a sequence in the cytoplasmic loop [30] and will not therefore cross react with the recently described acetylcholine binding protein which shares homology with extracellular portion of the a7 nAChR subunit [31]. Sections were deparaffinized in histological clearing agent [Histocleark II (Raymond A Lamb, East Sussex,

Table 1 Pathological classifications of the AD and DLB cases Diagnosis

Age (years)

CERAD

NIA–RI

DLB type

BRAAK stage

AD AD AD AD AD AD DLB DLB DLB DLB DLB DLB

79 85 84 84 87 83 75 78 71 77 79 79

definite AD definite AD and vascular lesions definite AD and vascular lesions definite AD and vascular lesions definite AD definite AD and vascular lesions definite AD and PD-related changes probable AD and PD-related changes definite AD and PD-related changes definite AD and PD-related changes definite AD and PD-related changes definite AD and PD-related changes

high likelihood of AD intermediate likelihood of AD high likelihood of AD high likelihood of AD high likelihood of AD intermediate likelihood of AD low likelihood of AD no AD intermediate likelihood of AD intermediate likelihood of AD low to intermediate likelihood of AD low likelihood of AD

no LB disease no LB disease no LB disease no LB disease no LB disease no LB disease neocortical DLB limbic DLB neocortical DLB diffuse DLB diffuse DLB neocortical DLB

6 5 5 6 5 4 2 0 3 3 2 2

T. Teaktong et al. / Journal of the Neurological Sciences 225 (2004) 39–49 Table 2 Number of a4 immunoreactive neuronal cell bodies Brain area

Controla (n=4)

Pyramidal cell layer CA1 2.5 (1.75) CA2 3.5 (1.75) CA3 3 (0.75) CA4 3 (0) Subiculum Layer 2 2.5 (1) Layer 3 2.5 (1) Entorhinal cortex Layer 2 3 (0.75) Layer 3 3 (0.75)

ADa (n=6)

DLBa (n=6)

2 3 3 3

2.5 (1.25) 2.5 (1) 3 (0.25) 3 (0)

(1.25) (1.5) (0.25) (0.25)

2 (0.5) 2 (1)

2 (2) 2 (1)

2.5 (1.25) 1.5 (1)*

2 (1.25) 1 (1)*

a

Data are expressed as median and interquartile range. * pb0.05; compared to control.

41

Vector Novared substrate kit (Vector Laboratories) was used instead of glucose–glucose oxidase/nickel-DAB. After the visualization of immunoreactivity by incubation with Vector Novared for 5 min, sections were rinsed in PBS for 5 min, followed by incubation with PBS overnight at RT. Sections were rinsed in PBS (35 min), then incubated with PBS containing 10% blotting substrate solution for 30 min at RT and immunostained for glial fibrillary acidic protein [GFAP (DAKO, Cambridgeshire, UK)] (diluted 1:4000) for 1 h at RT, followed by biotinylated goat anti-rabbit IgG (diluted 1:200) for 30 min at RT, ABC (diluted 1:50) for 30 min at RT. Vector SG substrate kit (Vector Laboratories) was used as second chromogen. Sections were then dehydrated in acetone (30 s), cleared in histoclear (230 s) and mounted using DPX.

UK), rehydrated through methanol (BDH Laboratory Supplies, Dorset, UK) to distilled water and then endogenous peroxidase was quenched with 0.9% hydrogen peroxide in distilled water (30 min, RT), followed by a short rinse in distilled water. Antigens were unmasked by microwaving in 0.01 M citrate buffer (pH 6.0) for 10 min. Subsequently, sections were rinsed in distilled water (25 min), and then in phosphate-buffered saline (PBS) (pH 7.4) for 5 min, followed by incubation with PBS containing 2% rabbit serum in the case of a4 subunit or horse serum in the case of a7 subunit (30 min, RT) to eliminate nonspecific binding. The sections were then incubated with primary antibodies mAb 299 (diluted 1:8000 in PBS containing 1% bovine serum albumin (BSA) or mAb 306 (diluted 1:4000 in PBS containing 1% BSA) for 1 h at RT. The sections were then rinsed thoroughly with PBS (35 min) followed by incubation (30 min, RT) with a biotinylated rabbit antirat IgG (Vector Laboratories) in the case of mAb 299 or a biotinylated horse anti-mouse IgG (Vector Laboratories) in the case of mAb 306 (both were diluted 1:200 in the corresponding blocking serum. For visualization of antigen– antibody reaction, the sections were rinsed in PBS and then incubated with the Vectastain Elite avidin-biotinylated enzyme complex [ABC (Vector Laboratories)] (diluted 1:50 in PBS) for 30 min at RT, followed by rinsing in PBS (25 min), then in acetate buffer (pH 6.0) for 5 min and incubation with the glucose–glucose oxidase/nickelenhanced 3,3V-diaminobenzidine (DAB) for 10 min. Sections were counterstained with methyl green (2 min) followed by dehydration in acetone (30 s), cleared in Histoclear (230 s) and mounted using DPX (Raymond A Lamb). 2.3. Double a7 and GFAP immunohistochemical labelling Immunohistochemistry of a7 subunit was first performed using the procedure described above, except that nonspecific binding was blocked using BM chemiluminescence blotting substrate solution (Roche Products, Hertfordshire, UK) diluted 1:10 in PBS in place of normal serum and

Fig. 1. a4 Immunoreactivity in entorhinal cortex layer 3 of control (A), Alzheimer (B) and dementia with Lewy bodies cases (C). Numbers of a4 immunoreactive neurons were significantly decreased in AD and DLB compared with controls. Scale bars, 20 Am.

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2.4. Semiquantitative assessment of immunoreactivity Assessments were made in each area using the light microscope at 20 magnification. One section per subject was assessed for each receptor antibody. The section was sampled from the same coronal level of the hippocampus and entorhinal cortex which was at the level of the lateral geniculate. The validity of the assessment procedure was established with independent assessment by two investigators blinded to diagnostic category who obtained values which were highly correlated (Pearson correlation ranges from 0.669 to 0.883; pb0.01). The IR neurones were considered as the neurons contain 50% or more IR granules in the neuronal cytoplasm. The number of IR neurons was determined in a4- and a7-immunostained sections and was graded from 0 to 4: 0=no IR neurons, 1=occasional (b5), 2=moderate (5–10), 3=large (11–15), 4=very large (N15) numbers of positive neurons per optical field. The number of IR cell processes (neurites and astrocytic cell processes connect to neuronal and astrocytic cell bodies) was similarly graded from 0 to 4: 0=no IR

processes, 1=occasional (b5) IR processes, 2=moderate numbers (5–10) of IR fibres throughout optical field, 3=large numbers (11–15) of IR fibres throughout optical field, 4=dense (N15) fibres throughout optical field. Neuropil was defined as the materials including part of neurons, dendrites and astrocytic processes outside of the cell bodies and blood vessels. The intensity of IR neuropil was graded from 0 to 4: 0=none, 1=faint, 2=moderate, 3=strong, 4=intense. The number of IR astrocytes was determined on a7 immunostained sections and sections double-labeled with antibodies against the a7 subunit and GFAP and graded from 0 to 4: 0=no IR astrocytes, 1=occasional (b5) IR astrocytes, 2=moderate (5–10) numbers of IR astrocytes, 3=large (11–15) numbers of IR astrocytes, 4=numerous (N15) IR astrocytes. Three layers of subiculum and entorhinal cortex were delineated using the subdivision of Amarel and Insausti [32]. For subiculum, layer 1 corresponds to the superficial molecular layer, layer 2 to the larger external layer near the molecular layer and layer 3 to the smaller internal layer adjacent to white matter. For entorhinal cortex, layer 1

Fig. 2. a7 Immunoreactivity in subiculum layer 2 (A–C), entorhinal cortex layer 3 (D–F) of controls (A and D), Alzheimer (B and E) and dementia with Lewy bodies cases (C and F). In comparison with control, numbers of a7 immunoreactive neurons were significantly decreased in AD and DLB in subiculum layer 2 and those were significantly decreased in DLB in entorhinal cortex layer 3. Scale bars, 20 Am.

T. Teaktong et al. / Journal of the Neurological Sciences 225 (2004) 39–49

43

processes was moderate in CA1, CA2, CA3, CA4, layers 2 and 3 of the subiculum and layers 2 and 3 of the entorhinal cortex of control and DLB group. Despite the lack of statistically significant difference between the groups, there was in the AD group a trend towards a lower number of reactive cell processes compared to the two other groups. Furthermore, reactive cell processes were not found in the areas of alveus of AD and DLB groups, stratum oriens of the AD group, stratum granulosum of AD and DLB groups, stratum lacunosum of the AD group and layer 1 of the subiculum and entorhinal cortex of the AD group (data not shown).

corresponds to the superficial acellular layer, layer 2 to the layer of islands of small pyramidal cells and layer 3 to the layers of medium and large pyramidal cells extending below layer 2 to the white matter. 2.5. Statistical methods SPSS statistic program package for windows version 10.1 was employed to analyse differences in the numbers of a4 and a7 IR neuronal cell, and cell processes, intensity of a4 and a7 IR neuronal cells, and neuropil and numbers of a7, GFAP IR and double-labelled astrocytes between groups. Group differences of those parameters were evaluated by the Mann–Whitney test.

3.2. a7 Immunoreactivity A large number of a7 IR neuronal cell bodies were found in CA2, CA3 and CA4 and no significant differences between the groups were observed in these hippocampal areas. A significant decrease in the number of reactive neurons was apparent in layer 2 of the subiculum in AD and DLB groups (Fig. 2A–C) and in layer 3 of the entorhinal cortex in the DLB group when compared with the control group (Fig. 2D–F). There was in addition a non-significant trend for a decline in the numbers of a7 IR neuronal cell bodies in CA1 and layer 3 of the subiculum in AD and in layer 2 in DLB groups compared with controls (Table 3). Controls showed faint or no neuropil IR in all areas of the hippocampus and entorhinal cortex. In AD, there was a higher intensity of IR neuropil compared to controls in most areas of the hippocampus and entorhinal cortex. Significant

3. Results 3.1. a4 Immunoreactivity The numbers of reactive neuronal cell bodies in the control group were greatest in CA1, CA2, CA3 and CA4 of the hippocampal formation, layers 2 and 3 of the subiculum and layer 2 of the entorhinal cortex. Significant decreases in numbers of a4 immunoreactive neurons in AD and DLB groups were found in layer 3 of the entorhinal cortex when compared with controls ( pb0.05) (Table 2; Fig. 1A–C). The areas of alveus, stratum oriens, stratum granulosum, stratum lacunosum, layer 1 of the subiculum and layer 1 of the entorhinal cortex in controls had no or few a4 reactive cell processes. The number of a4 reactive cell

Table 3 Number of a7 immunoreactive neuronal cell bodies and intensity of a7 immunoreactive neuropil Brain area

Alveus Stratum oriens Pyramidal cell layer CA1 CA2 CA3 CA4 Stratum granulosum Stratum moleculare Stratum lacunosum Stratum radiatum Subiculum Layer 1 Layer 2 Layer 3 Entorhinal cortex Layer 1 Layer 2 Layer 3 a

Neuronal cell bodies

Neuropil

Controla (n=4)

ADa (n=6)

DLBa (n=6)

Controla (n=4)

ADa (n=6)

DLBa (n=6)

– –

– –

– –

0.5 (1.75) 0 (0.75)

2 (2.25) 1 (0.5)

1 (1.5) 0 (0.25)

(1.25) (1.25) (1) (0.25)

1 (1.25) 2 (1.5) 2 (1.5) 2.5 (1.5) – – – –

0 0 0 0 0 0 0 0

0 (0.5) 1 (1.25) 1 (0.25)* 2 (1.25)* 1 (1)* 0.5 (1.25) 1 (0.5) 0 (0.5)

0 0 0 0 0 0 0 0

– 2.5 (1) 2.5 (1)

– 1 (1)* 1.5 (1.5)

– 1 (0.5)* 2 (1.25)

0.5 (1) 0 (0) 0 (0)

2 (1.25)* 1 (1.25) 1 (0.5)*

1 (1.25) 0 (0.25) 0 (0.25)

– 2.5 (2.5) 2 (0.75)

– 2 (1.25) 1 (1.25)

– 1 (1.5) 1 (1.25)*

0 (0.75) 0 (0) 0 (0)

1.5 (1)* 1 (0.5)* 1 (1.25)

0 (1.25) 0 (0.5) 0 (1.25)

2 3 3 3 – – – –

(1.5) (0.75) (1.5) (0.75)

0 2 3 3 – – – –

Data are expressed as median and interquartile range. * pb0.05; compared to control.

(0.75) (0.75) (0.75) (0.75) (0) (0.75) (0.75) (0)

(0) (0.25) (0.25) (1) (1) (0.25) (0.25) (0.25)

44

T. Teaktong et al. / Journal of the Neurological Sciences 225 (2004) 39–49

increases were observed in the CA3, CA4, stratum granulosum, layers 1 and 3 of the subiculum and layers 1 and 2 of the entorhinal cortex of the AD group (Table 3). Fig. 3B demonstrates the increase of neuropil immunoreactivity in AD in CA4. There were no changes in reactive neuropil in the DLB group compared with the control group (Table 3; Fig. 3A and C). The number of a7 reactive astrocytes was markedly increased in all areas of the hippocampus and entorhinal cortex in AD brains. The increases in reactive astrocytes in stratum oriens, CA1, CA2, CA3, CA4, stratum granulosum, stratum moleculare, stratum lacunosum, stratum radiatum, layers 1, 2 and 3 of the subiculum and layers 1, 2 and 3 of the entorhinal cortex of the AD group were statistically

significant when compared to the DLB group and controls (Table 4). Increased astrocytic a7 immunoreactivity was not observed in the DLB compared to control group (Table 4; Fig. 4A–I). 3.3. GFAP immunoreactivity GFAP-positive astrocytes were found in all areas of the hippocampus and entorhinal cortex with a high density in CA4 and layer 3 of the entorhinal cortex in all three groups. Total numbers of GFAP-positive astrocytes were similar in controls and DLB in all areas of the hippocampus and entorhinal cortex (Table 4). In AD, the number of GFAPpositive astrocyte was higher than controls in most areas of the hippocampus and entorhinal cortex. Significant increases were found in CA1, CA3, stratum radiatum and layer 3 of the subiculum in the AD group compared with controls (Table 4). 3.4. Double labelling of a7 and GFAP-reactive astrocytes Most areas in the hippocampus and entorhinal cortex of all three groups had both single GFAP- and double a7 and GFAP-reactive astrocytes. However, significantly higher in number of double-labelled astrocytes was observed in most areas in the AD group compared with controls and DLB. Fig. 5 illustrates the increase of double-labelled astrocytes in CA3 and entorhinal cortex layer 2 in AD compared to control and DLB, respectively. Between control and DLB groups, no difference in number of double-labelled astrocytes was observed (Table 4).

4. Discussion 4.1. nAChR abnormalities in Alzheimer’s disease

Fig. 3. a7 Immunoreactive neuropil in CA4 of control (A), Alzheimer (B) and dementia with Lewy bodies case (C). Intensity of a7 immunoreactive neuropil in AD was significantly increased compared with controls and DLB cases. Scale bars, 20 Am.

In the present study, the most dramatic difference in a4 and a7 subunit expression in the hippocampal formation in AD compared to controls and DLB was the increase in a7 expression associated with astrocytes. Wevers et al. [12] also noted a7 expression in astrocytes in the frontal cortex in AD, in contrast to age-matched controls, however, the extent of a7 immunoreactivity on astrocytes in the frontal cortex was not as widespread as that reported here. The upregulation of mRNA for a7 observed by Hellstrom-Lindahl et al. [33] in the hippocampus, but not in the temporal cortex or the cerebellum, is consistent with increased astrocytic a7 subunit expression in the brain area most affected by pathology and inflammation in AD. We also observed that numbers of a4 and a7 nAChR reactive neurons were moderately decreased in some areas of the hippocampus and entorhinal cortex of the AD group compared to age-matched controls. The a4

T. Teaktong et al. / Journal of the Neurological Sciences 225 (2004) 39–49

45

Table 4 Number of a7-, GFAP-positive and double-labeled astrocytes Brain area

a7-Positive astrocytes a

Alveus Stratum oriens Pyramidal cell layer CA1 CA2 CA3 CA4 Stratum granulosum Stratum moleculare Stratum lacunosum Stratum radiatum Subiculum Layer 1 Layer 2 Layer 3 Entorhinal cortex Layer 1 Layer 2 Layer 3

a

GFAP-positive astrocytes a

a

a

Double-labelled astrocytes

Control (n=4)

AD (n=6)

DLB (n=6)

Control (n=4)

AD (n=3)

DLB (n=3)

Controla (n=4)

ADa (n=3)

DLBa (n=6)

1 (0.75) 0.5 (1)

2 (1) 2 (1.25)*

1 (0.5) 0 (1.25)

2 (0.75) 1.5 (1.5)

2 (1) 2 (2)

2 (2) 2 (0)

1 (0) 0.5 (1)

1 (1) 1 (1)

1 (1) 1 (1)

0 (0.75) 0 (0.75) 0 (0.75) 0.5 (1.75) 0 (0.75) 0 (1.5) 0 (0.75) 0 (0.75)

2 (1.25)* 2 (1.25)* 2 (2)* 3.5 (2)* 2 (0)** 3 (0.5)* 2 (1.25)* 2 (1)*

0 (0.25) 0 (0.25) 0 (1.25) 1 (2) 0.5 (1) 1 (1.5) 0 (1.25) 0 (0.25)

1.5 (1.75) 1 (3) 1 (2.25) 3 (0.75) 1 (0.75) 1 (1.5) 1 (0.75) 1 (1.5)

3 3 4 3 2 3 2 3

1 1 2 3 1 1 2 1

0 0 0 1 0 0 0 0

2 2 3 3 2 2 2 2

0 0 0 0 0 0 0 0

1 (1.5) 0 (0) 0 (0)

2.5 (1.25)* 2 (1.25)** 3 (1)**

1.5 (2) 0 (0.25) 1 (1.5)

2 (1.5) 1 (1.5) 1 (0.75)

1 (1) 2 (0) 2 (1)*

2 (1) 2 (2) 2 (3)

1 (1.5) 0 (0.75) 0 (0)

1 (0) 2 (1)* 2 (0)*

1 (1) 0 (1) 0 (2)

1 (0.75) 0 (0) 0 (0.75)

2 (0.25)** 1 (0)** 2 (1)**

1 (1.25) 0 (0.25) 1 (2.25)

2 (0.75) 1 (0.75) 2 (1.5)

1 (0) 2 (3) 3 (1)

1 (2) 2 (1) 3 (1)

1 (0.75) 0 (0) 0.5 (1)

1 (0) 2 (1)* 2 (0)*

1 (0) 0 (1) 1 (1)

(1)* (2) (1)* (1) (2) (0) (2) (1)*

a

(2) (1) (2) (1) (1) (2) (1) (1)

(0.75) (1.5) (1.5) (0.75) (0.75) (0.75) (0.75) (0.75)

(0)* (1) (1)* (1)* (1)* (1)* (1)* (1)*

(1) (0) (2) (3) (1) (1) (1) (1)

a

Data are expressed as median and interquartile range. * pb0.05. ** pb0.01; compared to control.

subunit was significantly decreased in layer 3 of the entorhinal cortex in AD, whereas a7 subunit was decreased in layer 2 of the subiculum. These findings are consistent with loss of a4 and a7 reactive neurons in AD in the neocortex [12,13]. Our results are also in accordance with radioligand binding studies which consistently indicate a significant decrease in nAChRs with high affinity for agonists in the hippocampus and entorhinal cortex in AD [9], and with the reported reduction in a4 protein expression in the hippocampus in AD [11]. The increase in a7 immunoreactivity in neuropil concurrent with increased a-reactive astrocytes were seen in AD compared to controls and DLB. It is likely that astrocytic processes contribute to increased a7 IR neuropil. Reduced a-bungarotoxin binding and a7 protein expression in the neocortex and hippocampus in AD is a less robust finding than the decline in high affinity nicotine binding [9], despite indications that the total numbers of neurons expressing a7 receptors in the neocortex are equally reduced compared to a4 expressing neurons [13]. The present findings of increased a7 immunoreactive astrocytes in conjunction with reduced numbers of a7 neurons in AD may be a possible explanation for this inconsistency. 4.2. nAChR abnormalities in dementia with Lewy bodies In the present immunohistochemical study, we also found that neuronal a4 subunit expression was signifi-

cantly decreased in layer 3 of the entorhinal cortex and neuronal a7 subunit expression declined significantly in layer 2 of the subiculum compared with age-matched controls. These changes were similar to those observed in AD. In contrast to AD, the proportion of astrocytes that expressed a7 was low in DLB and similar to age-matched controls. In DLB, although LB and LB-related neurites occur in the presence of concomitant neuropathological features of AD in the hippocampal formation [34], it is generally confined to h-amyloid deposits (although not as extensive as in AD) and with few neurofibrillary tangles [35]. The difference in astrocytic a7 nAChR expression between AD and DLB may reflect differences in pathological mechanisms. 4.3. Expression of a7 in astrocytes in Alzheimer’s disease Astrogliosis is involved in the pathology of AD. Astrocytes induce neuronal cells damage [36,37] and may promote amyloid plaque maturation [38,39]. In this study, the total population of GFAP immunoreactive astrocytes was significantly increased in CA1, CA3, stratum radiatum and layer 3 of the subiculum in the AD group and a higher percentage of a7 IR astrocytes was found in most areas of the hippocampus and entorhinal cortex. These results suggest that: (1) as previously established, astrocytes are associated with AD pathology, (2) astrocytes in AD have greater expression of a7 nAChR subunits than control and DLB, (3) not all astrocytes have a7 immunoreactivity and (4) astrocytes

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T. Teaktong et al. / Journal of the Neurological Sciences 225 (2004) 39–49

Fig. 4. a7 Immunoreactivity in stratum granulosum (A–C), subiculum layer 3 (D–F) and entorhinal cortex layer 1 (G–I) of controls (A, D and G), Alzheimer (B, E and H) and dementia with Lewy bodies cases (C, F and I). In AD, a7-positive astrocytes (arrows) were significantly increased. GL, granule cell layer of the dentate gyrus. Scale bars, 20 Am.

which are involved in AD may be those which express a7 nAChR subunit. In comparison to the large numbers of a7 reactive astrocytes found in many areas of the hippocampus and entorhinal cortex of AD, no a4 reactive astrocytes were found in the hippocampal formation and adjacent cortex. a7 nAChR is homomeric ligand gated ion channel which displays highly selective permeability to calcium and a7 nAChR activation causes an increase in intracellular calcium [40,41]. In addition, stimulation of a-bungarotoxin sensitive nAChR on astrocytes has been shown to increase intracellular calcium released from intracellular stores [42]. Elevated expression of a7 nAChR on astrocytes may lead to abnormally high levels of intracellular calcium and this in turn may initiate a number of inflammatory cascades. One such mechanism could involve the activation of calcium-dependent astrocytic NOS with a consequent increase in nitric oxide production. Beta-amyloid induces inflammatory cytokines interleukin-1h (IL-1h) and tumor necrosis factor-a (TNF-a) [43,44] in monocytes and microglial cells. These cytokines induce nitric oxide production and release from astrocytes [45,46]. The astrocytic expression of the calcium-dependent NOS enzymes is reported to be

increased in both the archi- and neocortices in AD [36,47]. Furthermore, h-amyloid can induce superoxide anion (O2! ) production in AD [48] and co-production of NO can promote neuronal cell death by generation of the neurotoxic substance peroxynitrite [49]. Peroxynitrite can induce apoptosis [50] via oxidation of neuronal synaptosomal membrane protein [51], membrane lipid peroxidation [52], mitochondrial and nuclear DNA damage [53]. Our results suggest that over-expression of a7 subunits on astrocytes may be linked to AD pathology by elevating astrocytic intracellular calcium induced neuronal cell death. Further studies are required to explore the potential contribution of a7 nAChR to inflammatory mechanisms in astrocytes and to investigate how these receptors may provide a possible therapeutic target for AD.

Acknowledgements This study was funded by Janssen Pharmaceutical Research Foundation. We would like to thank Mrs. Jean Dawes for demographic data of patients. We also thank

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Fig. 5. a7 and GFAP double immunolabelling in CA3 (A–C) and entorhinal cortex layer 2 (D–F) of controls (A and D), Alzheimer (B and E) and dementia with Lewy bodies cases (C and F). Red and blue/gray stainings show a7 and GFAP immunoreactivity, respectively. Both a7 and GFAP immunoreactivity were increased in Alzheimer’s disease but not dementia with Lewy bodies compared with controls. Arrows indicate increase of double-labelled astrocytes in AD. Scale bars, 20 Am.

Prof. Mondhon Sanguansermsri (President of Naresuan University, Thailand) for his great support of this study.

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