THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE

163 lYWUNOHlSTOCHEMlCAL AND ULTRASTRCUTURAL STUDIES OF GRANULOVACUOLAR

DEGENERATION. K. Okaaoto, S. Hirai, Il. Yamaguchi, K. lshiguro. and

1. Takataaar. Department of Neurology, Gunma University School of

Medicine. and *Geriatrics Research Institute and Hospital, Naebashi,

Gunma 371, Japan.

The granulovacuolar degeneration (GVD) manifests as saall inclusions,

3 to 5~1 diaaeter spherical vacuoles containing centrally-located ar-

gentophilic and hematoxyphilic granules. which are found aost comaonly

in the hippocampal pyraaidal neurons of Alzheiaer type dementia (ATO).

However, previous light and electron aicroscopic examinations give few

clues as to the origin of these structural changes.

Materials and methods: Well-fixed autopsied samples of hippocaapus

from 4 patients of ATD were examined by light and electron microscopy.

We also exaained GVD by iaaunohistocheaical stainings with several

antibodies and by analytical electron microscopy.

Results: Iaaunohistocheaical examinations disclosed that the majority

of the granules of GVD in our 4 patients showed positive reactions for

anti-ubiquitin antisera. Tubulin-like iaaunoreactivitv was detected

in soae granules by Q- and ,@tubulin imunostainings, but it was weak

and similar to that shown in the neuronal cytoplasm and processes.

Other antibodies, including neurofilaaent. tau, PHF and actin. failed

to stain the granules of GVD. We observed a variety of granulovacuo-

lar aorphologies by electron aicroscopy. Based on electron aicroscop-

ic observations, morphogenesis of GVD is considered to be as follows:

slight-to-aoderate amounts of electron-dense material appear in the

cytoplasm at the early stage, and are then surrounded and demarcated

by a two-layered aeabrane (probably from smooth endoplasaic reticulum)

, following which some inner material is digested to becoae floccular

and liquid-like materials while undigested material reaains as coarse

electron-dense granules. Analytical electron microscopy disclosed that the granules in GVD contained soae aluainua.

In conclusion: Granulovacuoles are considered to be an age-related

special type of autophagosoae.

164

COMPARATIVE STUDY OF GRANULOVACUOLAR DEGENE- RATION IN NEURODEGENERATIVE DISEASES, U. Liibke, M. Mercken, M. Vandermeeren, C. Ceuterick, E. Van Mechelen, J.-J. Martin, P. Cras, Born-Bunge Foundation, University of Antwerp, Wilrijk (Belgium) and Innogenetics, N.V., Ghent (Belgium).

Granulovacuolar degeneration (GVD) consists of small spherical vacuoles containing an argentophilic granule, which are found most frequently in hippocampal pyramidal neurons. It was recently reported that the microtubular associated protein r, which is the main component of neurofibrillary tangles (NFT), is sequestered in GVD. It has therefore been suggested that GVD may be an intermediary in the formation of NFT. In an effort to define the biochemical composition of GVD, we have studied formalin fixed brain tissue of Alzheimer disease, progressive supranuclear palsy (PSP), Parkinson dementia complex and amyotrophic lateral sclerosis of Guam, Down syndrome and age matched controls. GVD was found predominantly in hippocampal neurons. In PSP, however, numerous GVD containing neurons were found in the pontine reticular formation. We found no immunoreaction of GVD with one polyclonal and 5 well characterized monoclonal antibodies directed to normal and abnormally phosphorylated 1. Antibodies to Tdid immunostain neuronal granules of different sizes in perinuclear and peripheral cytoplasmic locations. Also, abnormal filaments, sometimes outlining the GVD, and NFT in different stages of development were labelled. In contrast, immunoreactivity of variable intensity was present with antibodies to neurofilaments and ubiquitin. The granule was also labelled by immunogold with an antibody lo neurofilaments. Our results suggest that GVD in neurodegenerative disease and normal aging accumulates neurofilaments and ubiquitinated proteins, but does not sequester T protein

165 ALZHEIMER'S AND NORMAL BRAINS CONTAIN INHIBITORS OF ANTAGONIST BINDING TO THE MUSCARINIC ACETYLCHOLINE RECEPTOR, W.H. Prey II, M.E. Wiebenga, C.R. Emory, S. Saxena and G.D. Tollefson. Depts

of Psychiatry and Neurology, Ramsey Clinic/St. Paul-Ramsey Medical Center and University of Minnesota, St. Paul, MN 55101, U.S.A.

The 100,000 x g soluble fractions from homogenates of Alzheimer's disease (AD) and normal frontal cortex gray matter wgre examined for the presence3of endogenous inhibitors of ( H)-quinuclidinyl benzilate ( H-QNB) bind+ to the muscarinic acetylcholine receptor (mAChR). H-QNB binding to mAChR from normal human brain was significantly more inhibited by AD than normal supernatant fractions when 12 matched pairs of AD and normal brains were compared (p=.O36). Scatchard analysis revealed a statistically significant reduction in receptor affinity (K

P 39 to 111 PM) and3maximum binding

(Bw 1.5 to 0.9 fmo esfmg protein) of H-QNB in the presence of the soluble inhibitor of AD brain. Membrane dialysis of the supernatant fractions using

SpectrafPor 3 reveals the presence of a low molecular weight (MW) soluble inhibitor (c 3,500 daltons). Chromatography on Sephadex G-10 indicates the MW of this inhibitor is between 182 and 1,350. It was not destroyed bg acetylcholinesterase and was stable to both 1N HCl and heat (80 C for 5 min). It was also stable to 0.1 N NaOH for 10 min at 24'C. Partial purification of the low MW inhibitor was obtained from frontal cortex by homogenization and 5 min incubation in hot (8O'C) 1N acetic acid, centrifugations to obtain a 100,000 x g supernatant, dialysis against water through SpectraiPor 3 and lyophilization. The inhibitor binds to CM Acti-disk but not PEI Acti-disk, indicating it has a positive charge. It is not likely to be a divalent cation since EDTA does not eliminate inhibition and it elutes well before the salt peak on Sephadex G-10. B-mercaptoethanol (3OmM) was required in the assay for maximal inhibitor activity. The activity of the low MU inhibitor is three times greater in AD (5 cases, mean age of 81 yrs.) than normal (4 cases, mean age of 86 yrs.) brain (p< .OOl) Chromatography of the 100,000 x g supernatant fractions of AD

and normal human brain on either Sephadex G-50 or Sephadex G-15 reveals an inhibitor between 25,000 and 67,000 daltons which is mnre prominent in AD than normal brain. This is the first report of endogenous inhibitors in human brain of ligand binding to the mAChR and of increased inhibitor activity in AD.

166 SYNAPTOPAYSIN EXPRESSIVITY OF TBE CORTICAL NEOROPIL IN PRIMARY NEURODEGENERATIVE AND VASCULAR DEMENTIA S.- S. Zhanx*z, K. Beyreuther2 and A. P. Schmitt= XInstitute for Neuropathology and Wenter for Mole- cular Biology, University of Beidelberg; Germany The morphologic correlates of organic dementia in primary neurodegenerative and cerebra-vascular dis- orders are atill unclear. In dementia of Alzheimer type (AD) it is doubtful whether plagues and tangles may be regarded aa causers of the mental impairment or only as markers of the disease. In vascular dementia (VD), the significance of multiple infarct6 (-MID-) or myelin atrophy for dementia hae also remained a matter of debate. Finally, the reasons for dementia in Auntington's (ED) and Parkineon'e disease (PD) are still enigmatic. Recent observations seem to point to the fact that the structural causes of organic demen- tia might be more succesefully searched at the synap- tic level. Immunocytochemical markers have proved to be useful tools to ae~ese the synaptic density in the cerebral grey matter-In order to find out whether sy- napae changee may be an important factor in the ori- gin of organic dementia in general and not only pecu- liar to AD, we examined the cortical synaptophysin immunoreactivity not only in 17 cases of AD. but also in 1 case of rapid dementia with extensive amyloid pathology, 5 cases of ED, 11 of PD (5 with dementia), and 14 of VD, including hypertensive encephalopathy (HE) ~common and Binswanger type) (n=9). cerebral thromboangiitia obliterans (CTOPO) (n=3), "MID" (n=l), and mixed (BE and AD, n=l). 16 brains of non-demented served as controls. A reduction of the cortical naptophysin

=Y- expressivity was found in all caees with

dementia. The overall reduction in HD (10.4%) almost matched the one in AD (10.9%). followed by VD (9.8%) and Parkinaoniem with dementia (8.2%, without 5.3%). In the VD group, the highest reduction was found in CT0 (10.9%, rest 8.6%) being as severe a~ in AD.Our data euggest that independent of amyloid and tangle pathology. changes at the synapse level, finally re- sulting in a numerical decline of synapees may be a common, possibly primary event in the pathogenesis of organic dementia.