Calbindin-immunoreactive cholinergic neurones in the nucleus basalis of Meynert in Alzheimer-type dementia

Calbindin-immunoreactive cholinergic neurones in the nucleus basalis of Meynert in Alzheimer-type dementia

Brain Research, 499 (1989) 402-406 402 Elsevier BRES 23787 Caibindin-immunoreactive cholinergic neurones in the nucleus basalis of Meynert in Alzhe...

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Brain Research, 499 (1989) 402-406

402

Elsevier BRES 23787

Caibindin-immunoreactive cholinergic neurones in the nucleus basalis of Meynert in Alzheimer-type dementia Y. Ichimiya 1, P.C. Emson 1, C.Q. Mountjoy 3, D.E.M.

Lawson 2 and R. Iizuka 4

IMRC Group and 2Department of Cell Biology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge (U. K.), 3Department of Psychiatry, University of Cambridge, Cambridge (U.K.) and 4Department of Neuropsychiatry, Juntendo University, Tokyo (Japan) (Accepted 18 July 1989)

Key words." Calbindin, Cholinergic neuron; Nucleus basalis of Meynert; Alzheimer-type dementia

An antibody to the calcium binding protein, calbindin DasK (CaBP), was used to study the number and size of CaBP-immunoreactive neurones in the nucleus basalis of Meynert (nbM) of postmortem human brains from neurologically normal controls and from patients with neuropathoiogically diagnosed Alzheimer-type dementia (ATD). In controls, almost all the large neurones and their processes in the nbM were CaBP immunoreactive. Compared to neurologically normal controls the number of CaBP-immunoreactive neurones in the nbM in patients dying with ATD was significantly reduced and there was a clear loss of the majority of CaBP immunoreactive neurones. The few remaining nbM CaBP immunoreactive neurones in the ATD cases were smaller than those in the neurologically normal controls. Double-staining experiments revealed that many of the nbM CaBP-immunoreactive neurones contained choline acetyltransferase immunoreactivity, so that CaBP is an alternative marker for the nbM cholinergic neurones in the human fore-brain. These findings suggest that a disturbance in calcium homeostasis may be a possible factor contributing to the loss of these cholinergic/CaBP-containing neurones. Calcium ions are involved in a complex series of transport mechanisms, second messengers and ionchannels which are critical to neuronal function3'2°. Several calcium binding proteins which control intracellular Ca 2+ have been found in many tissues including the central nervous system s and some are believed to have a role in the maintenance of intracellular Ca 2+ homeostasis 3"s'2°. Calbindin D28K (CaBP), which was originally isolated from chick intestine2~, is one of these proteins. Subsequently two closely related CaBPs (27 and 29 kDa CaBP) have been identified in m a m m a l i a n brains 15"16As. CaBP-immunoreactive n e u r o n e s and processes have been found in the cerebellum (especially the Purkinje cells) la°, basal ganglia 6, hippocampus l° and cerebral cortex 5 of all m a m m a l i a n species examined. In the monkey brain, the large neurones of nucleus basalis of Meynert (nbM) are also CaBP immunoreactive4. Thus, because of the involvement of these neurones in the

pathology of Alzheimer-type dementia ( A T D ) 22, it was of interest to see if the n b M n e u r o n e s of the h u m a n brain were also CaBP immunoreactive. Although there have been m a n y reports of neuronal loss in the n b M of A T D 14, the cause of this loss (or cell shrinkage 17) in the n b M is still u n k n o w n . However, a reduction of the CaBP content in the brains of the patients with A T D has been reported 12 and a failure to maintain intracellular Ca 2+ homeostasis could lead to elevated intracellular Ca 2+ and result in neuronal death in ATD. Thus it was of considerable interest to establish whether the n b M neurones in h u m a n brain contain CaBP and if these are selectively damaged in ATD. In addition it was important to establish if CaBP is an alternative marker for the nucleus basalis cholinergic n e u r o n e s which are known to be damaged in A T D 22. Brains were obtained postmortem from 3 control patients who had no evidence of neurological or psychiatric disorder and from 5 clinically and neu-

Correspondence: P.C. Emson, MRC Group, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, CB2 4AT, U.K. 0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

403

Fig. 1. CaBP-immunoreactive nbM neurones in a neurologically normal control (A) and in a patient dying with ATD (B). x 19. High-power field (x47) of CaBP-immunoreactive neurones from a control case (C) and a serial section pretreated with CaBP (10"s M) before immunostaining with the anti-CaBP (D).

ropathologically diagnosed A T D patients. There was no statistically significant difference between control and A T D group with respect to age at death (control: mean 77.7 + 6.1 (S.D.) years, A T D : 82.8 + 7.3 (S.D.) years) or postmortem delay (control: 36.0 ___ 0.0 (S.D.) h; A T D : 41.2 + 15.3 (S.D.) h). The collection, storage and dissection of postmortem human brain has been previously described 2. Blocks of tissue, including the nbM, were taken from the basal forebrain of formalin-fixed brains 9. Prior to sectioning, the blocks were transferred to 30% sucrose (w/v) in 0.1 M phosphate-buffered saline (PBS) and stored at 4 °C until required. Twenty/~m sections were cut from the blocks on a freezing microtome and processed to visualize CaBP immunoreactivity using a peroxidase-antiperoxidase (PAP) technique 19. Anti-CaBP antibodies were raised in rabbits against purified chick intestinal CaBP. This antibody has been shown to recognise two calbindin forms (27 and 29 kDa) in rat TM and other species 15. The CaBP antibody was used at a final dilution of 1:500. For studies of the possible co-existence of CaBP and choline acetyltransferase (CHAT), calbindin immunoreactivity was detected

using the rabbit anti-CaBP primary antibody and a fluorescein isothiocyanate (FITC)-labelled sheep antirabbit IgG (1:100). After photography the coverslips were removed, sections washed in PBS and the sections then processed to visualize ChAT immunore-

cell size ~,~)

cell c o u n t s 100(3

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Fig. 2. Total number and mean size of CaBP-immunoreactive neurones in the nbM. C, controls; A , ATDs; bar = mean.

Significant differences were determined by Students t-test. The total number of cells counted and sized in the control brains (n = 3) was 2610, and in the Alzheimer cases (n = 5) was 1110.

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Fig. 3. Demonstration of the co-existence of CaBP and ChAT immunoreactivity in human nbM neurones. CaBP immunoreactivity neurones revealed by immunofluorescence (FITC) (A), are also positive for ChAT immunoreactivity (B). Arrows indicated strongly stained ChAT and CaBP-immunoreactive neurones (×200).

405 activity using a mouse monoclonal anti-ChAT (Boehringer) (2/~g/ml) and the PAP technique 19. Stained neurones were counted and their: areas measured with the aid of a Quantimet 720 image analyser (Cambridge Scientific Instruments Ltd.) as reported previously 13. The sections of the nbM for the image analyser were chosen from the sections through the mid portion of the nbM 9. Overlapping neurones were separated and unwanted features such as stained neurites were edited out by means of a light pen. Almost all of the large-sized neurones in the nbM of control cases were CaBP immunoreactive (Fig. 1A) and similarly stained neurones were found in the nbM of the ATD cases (Fig. 1B). However, both the numbers and mean size of CaBP-immunoreactive neurones in the nbM of ATD cases were significantly reduced compared to the control brains (Fig. 2). Although the brains used in this study all had a relatively long postmortem delay, the CaBP immunoreactivity was well preserved, so that some CaBP immunoreactive neurites as well as immunoreactive neurones were readily demonstrated in the nbM of control brains. The persistence of CaBP immunoreactivity even after significant postmortem delay was supported by our finding that the numbers of CaBP-immunoreactive neurones did not correlate with postmortem delay. Further, CaBP has been previously shown to be a remarkably stable protein being essentially unchanged even after several hours incubation at 37 °C in tissue homogenates TM. Our finding of CaBP in normal control human nbM is similar to the earlier observation of CaBP immunoreactive nbM neurones in monkey brains 4. The presence of CaBP immunoreactivity in the large nbM neurones which are known to be cholinergic 22 suggests that in these neurones CaBP may be an alternative marker for cholinergic neurones. When the anti-CaBP antibody used here was used for Western blot analysis of brain extracts it detected only the expected two mammalian calbindins 27 and 29 kDa CaBP 15. The antibody does not apparently cross-react with ChAT or acetylcholinesterase (ACHE) as it did not detect cross-reacting material separating at the position expected for ChAT (mol.wt. 70,000) or AChE (mol.wt. 80,000) on adenaturing polyacrylamide gels 15"as. Pre-absorption of the anti-CaBP antibody with authentic 27 kDa

CaBP at 10 -6 M blocked the staining of nbM neurones (Fig. 1D). Under the immunohistochemistry conditions used here the anti-CaBP antibody does not detect the related 29 kDa calbindin TM. Double'staining experiments (Fig. 3A,B) showed unequivocally that CaBP immunoreactivity co-exists with ChAT immunoreactivity in the nbM cholinergic neurones. Omission of the anti-ChAT monoclonal during the histochemistry procedure did not reveal any peroxidase-positive neurones indicating that there was no cross-reaction between the antibodies or antigens. These data show clearly that in the human nbM at least, CaBP is an alternative marker for the cholinergic neurones. As expected from earlier studies on ATD which have demonstrated a dramatic loss of cholinergic neurones and reduced ChAT activity9'14'22, there is also a substantial loss of CaBP containing/cholinergic neurones in ATD. It is also of interest to note that the few remaining CaBP immunoreactivity neurones are shrunken relative to the mean size of those from neurologically normal individuals. The extent of cell shrinkage is similar to that noted by Pearson et al. 17 in ATD, who suggested that there is only a shrinkage and not a loss of nbM neurones in ATD. However, our results using a much more robust immunohistochemical marker show that there is also a clear and substantial loss of CaBP-immunoreactive cholinergic neurones. It is not appropriate here to speculate in detail about the possible causes of degeneration of CaBP/ ChAT containing neurones in ATD, however, a reduction in cell CaBP content might make the CaBP containing nbM and cortical neurones more vulnerable to excitoxin damage or ischaemia TM. These observations do suggest that a detailed study of those factors controlling the expression of CaBP in nbM neurones would be worthwhile. We would like to thank Mr. R. Hills (MRC Brain Bank, Department of Psychiatry, University of Cambridge) for his assistance in the preparation of human brains, Mr. T. Buss for photographic work and Mrs. B.A. Waters for help in preparing the manuscript. Y.I. was a visiting research fellow from the Juntendo University School of Medicine, Tokyo, Japan. We are grateful to the Mental Health Foundation (U.K.) and Bayer A.G. (ER.G.) for supporting this work.

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