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Alterations in the transmembrane potential of synaptosomes prepared from Alzheimer's disease brains
FOURTH INTERNATIONAL
CONFERENCE
ON ALZHEIMER’S
did not show any differences between centenarians and younger donors. The proliferative response to IGF-I, IGF-II, several growth factors (FGF, EGF, 20% FBS) was similar in the two groups. Cumulative population doublings (CPD) of fibroblasts from
centenarians agrees with the linear regression analysis between CPD max and donor age. Our data represent the first study on growth properties of cells from centenarians and suggest that this population might have peculiar features at cellular level showing proliferative capacity comparable with those of younger donors.
51 ALTERATIONS IN THE TRANSMEMBRANE POTENTIAL OF SYNAPTOSOMES PREPARED FROM ALZHEIMBR’S DISEASE BRAINS. A. S. Bloom. B. Bunch. L. Lamer% K. Nicholson and P. G Antuono. Dept. Pharmacology anh?oxicology and Dept. of Neurology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. Increases in the fluidity of brain synaptic plasma membranes have been observed in Alzheimer’s disease. An important function of the neuronal membrane is to maintain a potential difference across it.
In order to find a
model for determining functional implications of altered neuronal membrane fluidity in AD, we have measured changes in transmembrane potenrial difference using synaptosomes prepared from brains of persons dying with AD and age matched controls. Transmembrane potential was estimated by measuring the accumulation of tetraphenylphosphonium (l?P+), a cationic probe that passively equilibrates across the membrane depending upon the membrane potential. TPP+ accumulation is increased when the synaptosome becomes hyperpolarized and is decreased when the membrane potential becomes less negative. Synaptosomes were prepared from frontal, temporal, parictal and occipital cortex. TPP+ accumulation was significantly increased in synaptosomes prepared from the temporal and parietal cortex of AD brains when compared to the same regions of control brains indicating that they were more hvperpolarized. AD synaptosomes from these TeEions were also more resistani*to depolarization by-in&easing concentrations of K+ when compared to controls. Similar differences were not seen in svnautosomes oreuared from
and occipital cortex. Taken as a whole, thisdab suggest; that there are differences in the ability of synaptosomes prepared from AD brains to
frontal
DISEASE
s13
53 ADAPTATION OF THE MORRIS WATER MAZE FOR THE AGING DOG. R.G. White. M. J. Russell, and S. M. Shirley Depts. of Anesthesiology and Otolaryngology, Davis California University of Calif, Davis 95616. The Morris water maze has been used extensively to test rodents for spatial memory loss Canines spontaneously develop amyloid plaques identical to seen in Alzheimer’s patients. and as a result have been suggested as a potential model of Alzheimer’s disease. As part of an ongoing effort to develop behavioral tests corresponding to the deficits found in Alzheimer’s disease in this species we have examined fifty dogs with an adapted Morris water maze. The dogs varied in age from 2- I7 years (mean of 8.8) and were examined on two separate occasions The initial test consisted of two successive trials daily on three consecutive days. The second test was done in exactly the same manner. but consisted of only two trials in a single day Before testing each dog received a complete neuralgic and sensory-motor examination Animals with visual or motor deficits were excluded. Prior to testing each animal was acclimated lo the water in the test apparatus. The acclimation Procedure consisted of the handler entering the pool with the dog, walking next to the animal
for two
minutes
then leading
platform
The acclimation
apparatus
was a round
submerged 36in
visual
were begun
pool
by placing
did not complete
significant behavior
learning
television
5 min. the trial of variance The results
over daily trials.
the one year absence
plastic
floating
of testing
beads to
at one of two
a stopwatch
was started
the attempted
was terminated
indicated
the
animal
The resultant
learning that there
retained
to
entering
for this test. lfthe
to determine
a
maze testing
The time from
and the animals
with
platform
plastic
The water
When
measurement
The
(Doughboy)
near the side of the pool
was stopped
was the outcome
after a one year interval.
during
with
platform.
release into the pool
the watch
by an analysis
pool
was a round
was covered
an animal upon
the task within
data were analyzed retention
surface
by an overhead
the platform
to the platform
The island
of a submerged
part of the test trial
above ground
of the submerged
Immediately
and the was monitored raise itselfon
the pool
The pool location
start location.
24A. diameter
within
in diameter
prevent trials
island
it to the placement
was not considered
and was
specific
learned
(PC0 001)
maintain a aansmembrane potential when compared to age matched contmls. This difference was only seen in those regions most affected by AD and may be due in part 10 alterations in K+ channel function. (Supported in part by medical research funds from the Department of Veterans Affairs and an Alzheimer’s Association/Ladies Auxiliary of the Fleet Reserve Association Research Grant.)
54
52 APOLIPOPROTEIN E AND BETA AMYLOlD(llA4)IMMUNOREACTIVE RAQUE.S IN THE AGED MONKEY CORTEX Elliott I. Mufson’~‘, William C. Benzing’~‘, Dwine E. Emerich’, Jobn R. Sklek Jr.‘, John Morrison’. and Jeffrey H. .md Rush Alleimr’s Disease Ceoterf Rush Presbyterian-St.Luke’s Med. Ctr., IL 60612, ‘Cytothempeutics Inc. Providence RI, ‘Dept. Neumsci., Chicago Med. Sch., Chicago IL, ‘Dept. Neumsci., ML. Sii Sch. Med., NY USA. Tbe lpolipoprotein E type 4 allele (ApoEd) is gmetically associated with the. common lute onset familial and sporadic forms of Alzheimer’s disease (AD). Over rqnserdstioo of tbe ApoE allele is rrsaiated with inc& nmyloid II-peptide (BA4) plaque deposition in the cerebral cortex in Inte-oa~ AD. The linkage of ApoE with 8A4 may be important since it b&s been suggested that 8A4 wumul&ion is P primary event underlying the degezlentive process seen in AD. Aged Rhesus monkeys develop E-amyloid containing senile plaques in areas which display extensive amyloid &ology in AD. Thus, aged &eys provide I valluble nonbumsn primate model for AD-like pathology, especially for investigntions examining the prowssea underlying tbe formation In tbe p-t stiy, we examined tbe relationship of amyloidswtaining plaques.
Kordower’.‘.Dept. NeumlogicaiSciences’
Chicago.
between ApoE md amyloid (8M)sontnining
profiles witbin the cerebral cortex of seven
young (7-10 yr), middle aged (18 md 21 yr) Pnd 10 aged (25-32 yr) Rbssus monkeys. Following pamfomuldehyde fixation, bssue fmm tbe temporal lobe wns cut at 40&m on P sliding t&zing mi+ome aad adjacent sections were immuaobistochemically stained either singly for EA4 (R1282), ApoE (Cbemicon, CA) or concurrently for each protein. We found ApoE-immunoreactive plaques scattered witbin tbe temporal naocortex, plnlimbic cortex IS well as the amygtia md to s lesser degree the bippwzmpw of aged Rhesus monkeys. Within the cortex ApoE and tlA4-immunoructive pl4ues were found primarily within layers III-V. Although virmnlly all ApoE-immuaonxctive plaques codistributed with 8-amyloid in a namer similar to that reported in AD. we otied many more lkmyloid pmitive/ApoE immunoneg~tive plaquea within the aged monkey cortex. Tbis relationship was most pmmwnced in the hippwampw where virhlslly all aged monkeys displayed numerous 8A4, hut few, ifsny, ApoE-immunoreactivepl4ues. These observnticms suggest that 8A4 deposition occurs prior to ApoE expression in tbe aging monkey. ApoE and BAl-immunorepctivity was not iwencinted with sonata within the temporal cortex amygtianln. or bippocampus in ally monkeys regardless of age. The p-t observ&ion tbnt ApoE and 8-amyloid are differeutinlly expressed in the aged monkey cortex provides a model for fuhln studies kwd at clnrifyiig tbe interactions between these proteins in nomul aging and pathologic conditions includiig AD.
BRAIN AGINGINNONHUMANPRIMATES: A SIMILARPROCESS TO HUMANS? C.I. Fern&de.& L. Alvarez M.A. Robinson; R. Ma&s; O.Go&er H. Molina. International Center of Neurologic Restoration. Ave 25No. 1580Le/ 158~ 160. Cubana&. Playa.Havana, Cuba. Aged monkeys mimic the declineinthe ability to remember certain types of information during “normal aging” in many humans older 65 years old. The aim of our study was to evaluate this process in monkeys (Maca~a sp. and Papio sp.) through spontaneous motor activity recording learningmemory capabilities: CT scan and inmunolo&xl status using animals oidifferent ages. The datacomparison between humans and monkeys will permit us assess the application of Neurotrophic Factors (e.g. NGF) as potentml therapy in age-related neurodegerative disorders like AIzheimer’s disease. Briefly our results indicate that; 1) Learning, retention and chaise reaction time (DNMS task) suffer modifications agedependent sinlila+ to those ocurring in older/demented humans: 2) Aged monkeys exlubit cortical atrophy. low tisular density and dilated lateral ventricles with an age-dependent progression in all species; 3) Aged monkeys motor behavior resemble some old human features (perseverance, stereotype) in spite of specie-specific differences; 4) The inmunological status in old humans and monkeys is similar in age terms. The above cited structural and neurophysiological similarities between humans and nomhuman prinlates support the selection of monkeys as model of brain agjng to preclinical evaluation of neurorestaurative procedures.
CONFERENCE
ON ALZHEIMER’S
did not show any differences between centenarians and younger donors. The proliferative response to IGF-I, IGF-II, several growth factors (FGF, EGF, 20% FBS) was similar in the two groups. Cumulative population doublings (CPD) of fibroblasts from
centenarians agrees with the linear regression analysis between CPD max and donor age. Our data represent the first study on growth properties of cells from centenarians and suggest that this population might have peculiar features at cellular level showing proliferative capacity comparable with those of younger donors.
51 ALTERATIONS IN THE TRANSMEMBRANE POTENTIAL OF SYNAPTOSOMES PREPARED FROM ALZHEIMBR’S DISEASE BRAINS. A. S. Bloom. B. Bunch. L. Lamer% K. Nicholson and P. G Antuono. Dept. Pharmacology anh?oxicology and Dept. of Neurology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. Increases in the fluidity of brain synaptic plasma membranes have been observed in Alzheimer’s disease. An important function of the neuronal membrane is to maintain a potential difference across it.
In order to find a
model for determining functional implications of altered neuronal membrane fluidity in AD, we have measured changes in transmembrane potenrial difference using synaptosomes prepared from brains of persons dying with AD and age matched controls. Transmembrane potential was estimated by measuring the accumulation of tetraphenylphosphonium (l?P+), a cationic probe that passively equilibrates across the membrane depending upon the membrane potential. TPP+ accumulation is increased when the synaptosome becomes hyperpolarized and is decreased when the membrane potential becomes less negative. Synaptosomes were prepared from frontal, temporal, parictal and occipital cortex. TPP+ accumulation was significantly increased in synaptosomes prepared from the temporal and parietal cortex of AD brains when compared to the same regions of control brains indicating that they were more hvperpolarized. AD synaptosomes from these TeEions were also more resistani*to depolarization by-in&easing concentrations of K+ when compared to controls. Similar differences were not seen in svnautosomes oreuared from
and occipital cortex. Taken as a whole, thisdab suggest; that there are differences in the ability of synaptosomes prepared from AD brains to
frontal
DISEASE
s13
53 ADAPTATION OF THE MORRIS WATER MAZE FOR THE AGING DOG. R.G. White. M. J. Russell, and S. M. Shirley Depts. of Anesthesiology and Otolaryngology, Davis California University of Calif, Davis 95616. The Morris water maze has been used extensively to test rodents for spatial memory loss Canines spontaneously develop amyloid plaques identical to seen in Alzheimer’s patients. and as a result have been suggested as a potential model of Alzheimer’s disease. As part of an ongoing effort to develop behavioral tests corresponding to the deficits found in Alzheimer’s disease in this species we have examined fifty dogs with an adapted Morris water maze. The dogs varied in age from 2- I7 years (mean of 8.8) and were examined on two separate occasions The initial test consisted of two successive trials daily on three consecutive days. The second test was done in exactly the same manner. but consisted of only two trials in a single day Before testing each dog received a complete neuralgic and sensory-motor examination Animals with visual or motor deficits were excluded. Prior to testing each animal was acclimated lo the water in the test apparatus. The acclimation Procedure consisted of the handler entering the pool with the dog, walking next to the animal
for two
minutes
then leading
platform
The acclimation
apparatus
was a round
submerged 36in
visual
were begun
pool
by placing
did not complete
significant behavior
learning
television
5 min. the trial of variance The results
over daily trials.
the one year absence
plastic
floating
of testing
beads to
at one of two
a stopwatch
was started
the attempted
was terminated
indicated
the
animal
The resultant
learning that there
retained
to
entering
for this test. lfthe
to determine
a
maze testing
The time from
and the animals
with
platform
plastic
The water
When
measurement
The
(Doughboy)
near the side of the pool
was stopped
was the outcome
after a one year interval.
during
with
platform.
release into the pool
the watch
by an analysis
pool
was a round
was covered
an animal upon
the task within
data were analyzed retention
surface
by an overhead
the platform
to the platform
The island
of a submerged
part of the test trial
above ground
of the submerged
Immediately
and the was monitored raise itselfon
the pool
The pool location
start location.
24A. diameter
within
in diameter
prevent trials
island
it to the placement
was not considered
and was
specific
learned
(PC0 001)
maintain a aansmembrane potential when compared to age matched contmls. This difference was only seen in those regions most affected by AD and may be due in part 10 alterations in K+ channel function. (Supported in part by medical research funds from the Department of Veterans Affairs and an Alzheimer’s Association/Ladies Auxiliary of the Fleet Reserve Association Research Grant.)
54
52 APOLIPOPROTEIN E AND BETA AMYLOlD(llA4)IMMUNOREACTIVE RAQUE.S IN THE AGED MONKEY CORTEX Elliott I. Mufson’~‘, William C. Benzing’~‘, Dwine E. Emerich’, Jobn R. Sklek Jr.‘, John Morrison’. and Jeffrey H. .md Rush Alleimr’s Disease Ceoterf Rush Presbyterian-St.Luke’s Med. Ctr., IL 60612, ‘Cytothempeutics Inc. Providence RI, ‘Dept. Neumsci., Chicago Med. Sch., Chicago IL, ‘Dept. Neumsci., ML. Sii Sch. Med., NY USA. Tbe lpolipoprotein E type 4 allele (ApoEd) is gmetically associated with the. common lute onset familial and sporadic forms of Alzheimer’s disease (AD). Over rqnserdstioo of tbe ApoE allele is rrsaiated with inc& nmyloid II-peptide (BA4) plaque deposition in the cerebral cortex in Inte-oa~ AD. The linkage of ApoE with 8A4 may be important since it b&s been suggested that 8A4 wumul&ion is P primary event underlying the degezlentive process seen in AD. Aged Rhesus monkeys develop E-amyloid containing senile plaques in areas which display extensive amyloid &ology in AD. Thus, aged &eys provide I valluble nonbumsn primate model for AD-like pathology, especially for investigntions examining the prowssea underlying tbe formation In tbe p-t stiy, we examined tbe relationship of amyloidswtaining plaques.
Kordower’.‘.Dept. NeumlogicaiSciences’
Chicago.
between ApoE md amyloid (8M)sontnining
profiles witbin the cerebral cortex of seven
young (7-10 yr), middle aged (18 md 21 yr) Pnd 10 aged (25-32 yr) Rbssus monkeys. Following pamfomuldehyde fixation, bssue fmm tbe temporal lobe wns cut at 40&m on P sliding t&zing mi+ome aad adjacent sections were immuaobistochemically stained either singly for EA4 (R1282), ApoE (Cbemicon, CA) or concurrently for each protein. We found ApoE-immunoreactive plaques scattered witbin tbe temporal naocortex, plnlimbic cortex IS well as the amygtia md to s lesser degree the bippwzmpw of aged Rhesus monkeys. Within the cortex ApoE and tlA4-immunoructive pl4ues were found primarily within layers III-V. Although virmnlly all ApoE-immuaonxctive plaques codistributed with 8-amyloid in a namer similar to that reported in AD. we otied many more lkmyloid pmitive/ApoE immunoneg~tive plaquea within the aged monkey cortex. Tbis relationship was most pmmwnced in the hippwampw where virhlslly all aged monkeys displayed numerous 8A4, hut few, ifsny, ApoE-immunoreactivepl4ues. These observnticms suggest that 8A4 deposition occurs prior to ApoE expression in tbe aging monkey. ApoE and BAl-immunorepctivity was not iwencinted with sonata within the temporal cortex amygtianln. or bippocampus in ally monkeys regardless of age. The p-t observ&ion tbnt ApoE and 8-amyloid are differeutinlly expressed in the aged monkey cortex provides a model for fuhln studies kwd at clnrifyiig tbe interactions between these proteins in nomul aging and pathologic conditions includiig AD.
BRAIN AGINGINNONHUMANPRIMATES: A SIMILARPROCESS TO HUMANS? C.I. Fern&de.& L. Alvarez M.A. Robinson; R. Ma&s; O.Go&er H. Molina. International Center of Neurologic Restoration. Ave 25No. 1580Le/ 158~ 160. Cubana&. Playa.Havana, Cuba. Aged monkeys mimic the declineinthe ability to remember certain types of information during “normal aging” in many humans older 65 years old. The aim of our study was to evaluate this process in monkeys (Maca~a sp. and Papio sp.) through spontaneous motor activity recording learningmemory capabilities: CT scan and inmunolo&xl status using animals oidifferent ages. The datacomparison between humans and monkeys will permit us assess the application of Neurotrophic Factors (e.g. NGF) as potentml therapy in age-related neurodegerative disorders like AIzheimer’s disease. Briefly our results indicate that; 1) Learning, retention and chaise reaction time (DNMS task) suffer modifications agedependent sinlila+ to those ocurring in older/demented humans: 2) Aged monkeys exlubit cortical atrophy. low tisular density and dilated lateral ventricles with an age-dependent progression in all species; 3) Aged monkeys motor behavior resemble some old human features (perseverance, stereotype) in spite of specie-specific differences; 4) The inmunological status in old humans and monkeys is similar in age terms. The above cited structural and neurophysiological similarities between humans and nomhuman prinlates support the selection of monkeys as model of brain agjng to preclinical evaluation of neurorestaurative procedures.