Prog. Nemo-Psychophormocol 6 Bml. Psychiot. 1985, Vol. 9. pp. 625-628 Printed in Great Britain All nghts reserved.

0278-5846/95 $0.00 + .50 Copyright0 1985 Pergamon Press Ltd.

ROLE OF THE SENILE PLAQUE IN NEUROPEPTIDE DEFICITS OF ALZHFJMER’S DISEASE

HENRIK K. KUIMALA and J. THOMAS HUTTON

Dept. of Med. & Surg. Neurol., Texas Tech University Health Sciences Center, Lubbock, Texas, USA

(Final form, July 1985)

Abstract

Kulmala, Henrik K. and J. Thomas Hutton: Role of the senile plaque in neuropeptide deficits of Alzheimer's disease. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1985,z (5/6): 625-628

1. Senile plaques participate in a cropping of the dendritic tree of enkephalinergic den- tate granule cells and hl and subicular pyramidal cells.

2. Somatostatin-containing pyramidal neurons are lost in Alzheimer's disease, whereas non- pyramidal somatostatin neurons are less affected. Fibers containing somatostntin pene- trate immature, but not end-stage senile plaques.

3. The senile plaque may precipitate much of the hippocampal denervation seen in Alzheimer's disease.

Keywords: Alzheimer's disease, enkephalin, entorhinal cortex, hippocsmpus, somatostatin

Abbreviations: Alzheimer's disease (AD); enkephalin-like immunoreactivity (ELI); hematoxylin and eosin (H&E); peroxidase-antiperoxidase (PAP); senile plaque (SP); somatostatin-like immunoreactivity (SLI)

Introduction

The etiology of Alzheimer's disease (AD), which accounts for the majority of the dementias seen in the elderly, is unknown (Ball 1982). The clinical diagnosis of AD must be confirmed pathologically by the presence of high concentrations of neurofibrillary tangles and senile plaques (SP's) within the cerebral cortex (Ball 1982; Hyman et al. 1984). Indeed, the num- bers of plaques and tangles correlate well with the degree of dementia. Therefore, these abnormalities would seem important in the etiology of AD. The aim of the present study was to determine if the SP has an effect on hippocampal neurons containing either somatostatin- or enkephalin-like immunoreactivity (SLI and ELI, respectively).

Methods

Patient population. Postmortem brain tissue was obtained within 2.5 to 16 hr after death from 7 control subjects and from 13 patients with AD (including 2 also showing multi-infarct pathology). The pathological diagnosis was made by a neuropathologist (Dr. M.J. Ball) after examination of sections through the left hippocampus. The anterior third of the right hip- pocampal formation, usually with the adjacent entorhinal cortex, was fixed by immersion in ice-cold buffered 4% paraformaldehyde for 48-72 hr, the stored at least 12 hr in 5% sucrose in phosphate buffer. Sections, 40 microns thick, were cut on a Jung C02-freezing microtome and collected into ice-cold phosphate buffer.

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626 H. K. Kulmala and J. T. Hutton

Antisera. Antisera directed against enkephalin (gift from Dr. R.J. Miller, Univ. of Chmnd somatostatin (Immune Nuclear, Stillwater, MN) were utilized. Control sections were incubated with antibody diluent (phosphate-buffered saline containing 0.3% Triton X- 100) or with primary antiserum previously preabsorbed for 12-24 hr in solution at 4 degrees C with synthetic peptide (Sigma, St. Louis, MO; 100 0x 850 micrograms/ml). Only specific staining is described herein. The peroxidase-antiperoxidase (PAP) procedure was employed utilizing secondary sheep anti-rabbit antibodies (Antibodies, Inc., Davis, CA) and rabbit PAP (Cappel, Cochranville, PA).

Experimental procedure. Nonspecific binding sites and endogenous peroxidase activity were eliminated as previously described (Kulmala 1985a). Following 12 hr incubation at 4 degrees C with primary antiserum diluted 1:kOO - 1:2000, sections were washed with buffer, incubated for 1 hr at room temperature with secondary antiserum (1:lOO - l:kOO), washed, and incubated with rabbit PAP (1:200 - 1:500) for 1 hr at room temperature. Sections then were washed with Tris buffer (Trizma 7.6; Sigma) and incubated for lo-30 min in a fresh disminobenzidine solution (50 mg/lOO ml Tris) containing 0.001% hydrogen peroxide. Sections were washed and mounted from Tris:water (1:4) onto gelatin-coated slides. Some nonimmune-stained sections also were slide mounted. These latter sections and alternate PAP-stained sections were dried overnight and stained with hematoxylin and eosin (H&E) prior to dehydration and coverslipping. All other sections were dried, dehydrated, and coverslipped.

Data analysis. Sections were examined on an Olympus light microscope. The localization of SLI- or ELI-containing neurons was determined in several sections from each case and was compared with the distribution of SP's in the H&E counterstained sections. The H&E staining did not affect immunostaining and the PAP staining did not affect H&E staining of SP's.

Results

Enkephalin-like immunoreactivity (ELI)

In several of our AD cases, some ELI-containing dentate granule cell dendrites were seen to penetrate SP's. Interestingly, many more ELI-containing neurites were noted in areas of the molecular layer not containing such SP's. Some dendrites appeared to stop just prior to penetrating an SP. As previously described (Kulmala 1985a), numerous pyramidal cells in hl and subiculum contained ELI. Some of these neurons previously were seen to contain neuro- fibrillary tangles (Kulmala 1985b). We noted the frequent presence of SP's in close apposi- tion to the soma of ELI-containing pyramidal cells.

Somatostatin-like immunoreactivity (SLI)

Neurons containing SLI were seen only in the few specimens obtained soon after death, both in control hippocampi and in the brains of subjects with AD. Unfortunately, the entorhinal cortex was lost from the earliest postmortem control case. Numerous cells in the white mat- ter below the entorhinal cortex contained SLI. These cells were of several different shapes and were found both in control and AD brains. Similarly, nonpyramidal cells in several entorhinal cortical layers also contained SLI. Only one SLI-positive pyramidal neuron, found in layer II, was seen in any of the sections through an AD entorhinal cortex. Lightly stained SLI-containing pyramidal cells were noted in the entorhinal cortex of the control case obtained after the second shortest postmortem delay interval.

Fibers positive for SLI were noted throughout the entorhinal cortex. Some of these fibers were seen to run perpendicular to the outer cortical surface, then to bifurcate in the outer layers and run long distances parallel to the surface. In the cases with AD, SLI-containing fibers were seen to penetrate immature SP's, as seen in H&E counterstained sections. Many of these SLI-positive fibers appeared grossly swollen. There was an absence of such fibers in, or near, end-stage plaques.

Discussion

Senile plaques (SP's)

The formation of the SP is a dynamic process. Plaques are classified according to their relative content of smyloid and abnormal neurites as Ilimmature", tlmaturew, or "end-stage"

Alzheimer's senile plaques and neuropeptides 627

(Wisniewski and Merz 1983). The actual agent leading to the formation of SP's is unknown. In support of the pathoclisis hypothesis of Vogt and Vogt (1937), it has been suggested that the basal forebrain cholinergic neurons are affected by some pathological process and res- pond with abnormal changes in their cortical axons, leading to the develoment of SP's (Struble et al. 1982). These SP's subsequently damage other neuronal systems. However, despite the considerable loss of cerebral cholinergic innervation in the end stages of AD, immature and mature SP's, rather than end-stage SP's, predominate. If cholinergic neurons were active in the formation of SP's, one would expect to see many more end-stage SP's than the few that are present in AD (Wisniewski and Merz 1983).

Neuropeptides and pathoclisis

The results of this and one previous study (Kulmala 1985b) do not support a role for neuropeptides in the pathoclisis hypothesis. The intrinsic neurotransmitter did not appear to be the deciding factor in whether pyramidal neurons contain neurofibrillary tangles. Furthermore, some SLI-containing neurons, but not others, are destroyed by AD. The dif- ferentiation is on the basis of neuronal type, that is, pyramidal output neurons are lost, whereas interneurons are spared. It is possible that this differentiation is on the basis of cholinergic innervation - that SLI-positive pyramidal,cells are so innervated, but in- terneurons are not. However, it is just as likely that the differentiation is determined by neuronal type and connections. The pyramidal output neurons in layers II and IV of entorhi- nal cortex are especially damaged in AD (Hyman et al. 1984). At least some of these cells normally contain SLI. Such SLI-positive neurons might project to the dentate gyrus. The loss of these cells then would reflect a retrograde effect due to axonal damage in SP's in the dentate molecular layer.

While few SLI-positive pyramidal neurons are found in the AD entorhinal cortex, many SLI- containing fibers are present. Some of these fibers are seen to penetrate immature SP's, but not mature SP's. This would suggest that such fibers originate from nonpyramidal neu- rons and that such neurites are lost as SP's mature. That such cells are not lost in AD may reflect an anatomical difference between pyramidal and nonpyramidal cells. For instance, a neuron making extensive axonal projections would not be as affected by the loss of a collateral as a cell giving rise to a single or restricted projection. However, the loss of an axon collateral or denclritic fiber would affect neurotransmission within such nonpyramidal cells, leading to a further isolation of the hippocsmpus.

The presence of a neuronal element within an SP does not ensure the loss of such a fiber. Some apparently normal ELI-positive granule cell processes are found in SP's. However, many SP's do not contain such elements, despite being localized to a region that should contain such fibers. Thus, it is probable that a considerable cropping of the dendritic tree of granule cells exists, presumably caused by SP's. Similarly, within the subiculum and hl, some SP's are seen in close apposition to the soma of ELI-containing pyramidal cells in the region where the dendrites achieve considerable collateralization. That no ELI- positive processe are seen in these SP's may reflect a cropping of such dendritic trees.

Conclusion

The loss of somatostatin in AD has not been reposted to be as great as the loss of cholinergic neurons (Davies and Terry 1981). However, it now appears that this loss reflects an almost absolute loss of a subset of SLI-containing neurons. We are left with the impression that it is the localization of senile plaques which determines the specific neuronal and neurotransmitter deficits present in AD. This would imply that the treatment of this disorder should concentrate on limiting the development of the senile plaque.

This work was supported in part by grants PG21, MRC(C); AGNS 03047, NIA; 858, OMIIF to Dr. M.J. Ball and the University Hospital Research Trust F'und and Tarbox Parkinson's Disease Institute of Texas Tech University School of Medicine to HKK. The authors acknowledge the neuropathological expertise of Dr. M.J. Ball.

628 H. K. Kulmala and J. T. Hutton

References

BALL, M. J. (1982) Alzheimer's disease: the mystery of senile dementia. Mod. Med. Can. 37: 1137-1143.

DAVIES, P. and TERRY, R. D. (1981) Cortical somatostatin-like immunoreactivity in cases of Alzheimer's disease and senile dementia of the Alzheimer type. Neurobiol. Aging 2: g-14.

HYMAN, B. T., VAN HOESEN, G. W., DAMASIO, A. R., BARNES, C. L. (1984) Alzheimer's disease: Cell specific pathology isolates the hippocampal formation. Science 225: 1168-1170.

KULMALA, H. K. (1985a) Immunocytochemical localization of enkephalin-i%e immunoreactivity in neurons of human hippocampal formation: Effects of aging and Alzheimer's disease. Neuropath. Appl. Neurobiol. 11: 105-115.

KULMALA, H. K. (1985b) Some enkephalin or VIP-immunoreactive hippocampal pyramidal neurons contain neurofibrillary tangles in the brains of aged humans and persons with Alzheimer's disease. Neurochem. Path. 3: 41-51.

STRUBLE, R. G., CORK, L. C., WHITEHOUSE, P. J., and PRICE, D. L. (1982) Cholinergic innervation in neuritic plaques. Science 216: 413-415.

VOGT, C. and VOGT, 0. (1937) S't 1 z und Wesen der Krankheiten im Lichte der topostischen Hirnforschung und der Variierens der Tiere. J. Psychol. Neural. (Leipzig) 9: 237-457.

WISNIEWSKI, H. M. and MERZ, G. S. (1983) Neuritic and amyloid plaques in senile dementia of the Alzheimer type. In: Banbury Report 15: Biological Aspects of Alzheimer's Disease, pp. 145-153. Cold Spring Harbor Laboratory, New York.

Inquiries and reprint requests should be addressed to:

Dr. Henrik K. Kulmala Department of Pharmacology Northeastern Ohio Universities College of Medicine 4209 State Route 44 Rootstown, OH, USA 44272