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Deficiency of a cholinergic differentiating factor in fibroblasts of patients with Alzheimer's disease.

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BRIEF COMMUNICATIONS
Deficiency of a Cholinergic
Differentiating Factor in
Fibroblasts oCPatients
with Alzheimer’s Disease
John A. Kessler, M D
Skin fibroblasts from patients with Alzheimer’s disease
and from apparently normal control subjects were compared for their production of a cholinergic differentiating factor. The factor’s activity was assayed by measuring the induction of choline acetyltransferase (CAT)
activity in cultured sympathetic neurons. Culture medium conditioned by exposure to normal human fibroblasts induced substantial levels of CAT activity in
sympathetic neurons, indicating that human fibroblasts
produce a soluble factor that promotes cholinergic expression. In contrast, medium conditioned by Alzheimer
fibroblasts induced only about one-third as much CAT
activity, a highly significant reduction ( p < 0.01).These
observations suggest that Alzheimer fibroblasts may be
deficient in their secretion of a cholinergic factor and
raise the possibility that the pathophysiology of the disease is related to a defect in the release of this factor.
The fibroblast abnormalities suggest that Alzheimer’s
disease may be a systemic disease involving nonneuronal
cells that are outside as well as within the brain.
Kessler JA: Deficiency of a cholinergic
differentiating factor in fibroblasts of patients
with Alzheimer’s disease.
Ann Neurol 2195-98, 1987
The cause and pathophysiology of Alzheimer’s disease
remain obscure. Most knowledge of the disease process has been acquired from autopsy material, and in
vivo biochemical approaches have been limited by the
unavailability of living brain tissue and the lack of an
animal model of the disease. Although the clinical and
pathological findings in Alzheimer’s disease are restricted to the brain, recent studies have reported abnormalities attributed to the disorder in a variety of
peripheral tissues, including blood cells and fibroblasts
f2, 3, 11, 12, 14, IS}. If the underlying cellular deficits
in Alzheimer’s disease are reflected in peripheral tissues, the ready availability of such tissues would greatly
facilitate study of the molecular basis of the disorder.
From the Departments of Neurology and Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461.
Received Oct 24, 1985, and in revised form Mar 17 and May 16,
1986. Accepted for publication May 20, 1986.
Address reprint requests to Dr. Kessler.
One hypothesis regarding Alzheimer’s disease is
that the neuronal abnormalities result from a deficient
secretion of one or more neuronotrophic factors by
nonneuronal cells in the brain. Peripheral nonneuronal
cells such as fibroblasts also secrete factors that stimulate brain neurons, which suggests that peripheral tissues produce factors similar to those that normally act
on the neurons of the central nervous system [b, 10,
131. Since fibroblasts from patients with Alzheimer’s
disease are accessible, we sought to determine whether
fibroblast secretion of a cholinergic-stimulating factor
{G, 131 is altered in the disease state.
Materials and Methods
Culture and Treatment
Sympathetic Neurons
Dissociated sympathetic neurons from the neonatal rat
superior cervical ganglion were cultured, as previously described [9], in a medium consisting of F12 and Eagle’s minimal essential medium (EMEM) (75:25) with 10% heatinactivated fetal calf serum, nerve growth factor (100 nglml),
penicillin (50 unidml), and streptomycin (50 pglml). Cultures were maintained at 37°C in a 95% air to 5% COz
atmosphere at nearly 100% relative humidity. Ganglion nonneuronal cells were eliminated by treatment with cytosine
arabinoside (5 x lO-‘M) on days 1 and 3 of culture. Cultures were treated three times weekly, starting on day 4, with
50% fibroblast-conditioned medium. Control (untreated)
cultures were fed with 50% fresh fibroblast growth medium.
O n day 18, neuron numbers were determined by counting,
as described previously 193, and the cultures were harvested
for measurement of choline acetyltransferase (CAT) and tyrosine hydroxylase (TH) activities, as described previously
[7]. Neuronal counts and all biochemical measurements
were done on a coded (“blind”) basis.
Fibroblast Cultures
Some fibroblasts were obtained from the Institute for Medical Research (IMR), Camden, NJ. The fibroblasts from 8
patients with Alzheimer’s disease (AG6262, AG6263,
AG5809, AG4400, AG5770, AG4401, GM0364A,
AG58 10) and the fibroblasts from 8 age-matched, apparently
normal control subjects (GM3658, GM4260, GM0288A,
GM2623A, GM1681, GM0967B, GM3525, AG2261A)
were grown in flasks containing F12 and EMEM (50:50)
with 10% fetal calf serum. After the cells achieved confluency, the medium that was removed during feeding three
times weekly was used as conditioned medium after being
filtered through a 0.22-p filter (Millipore). Fibroblasts were
passaged every 2 to 3 weeks. Passage numbers used in these
studies ranged between 3 and 8.
Additional fibroblasts were obtained from skin biopsies
taken over the deltoid muscle in 17 patients with a clinical
diagnosis of Alzheimer’s disease and in 17 age-matched control subjects (whenever possible from the spouse of the patient). (This increased the sample size to 25.) All diagnoses
were made prior to the start of this study in patients being
followed by the Long-Term Care Gerontology Center of
Albert Einstein College of Medicine, and the diagnostic cri-
95
teria for Alzheimer’s disease of the Diagnostic and Statistical
Manual 111 (DSM 111) were met in each patient 111. Since
severely demented patients were not deemed competent to
give informed consent for a biopsy, skin samples were obtained only from patients with mild to moderate dementia.
The biopsies were cultured as explants in EME,M with 15%
fetal calf serum to allow fibroblasts to propagate as a monolayer in the flask. The fibroblasts were then trypsinized into
culture flasks and handled in the same manner as that for the
IMR fibroblasts. The fibroblasts were mycoplasma-free. Fibroblast doubling time and viability (trypan-blue exclusion)
were determined by the methods of Freshney 51.
Biochemical Methods
CAT and TH activities were measured as described previously [4, 71.
Results
Using the fibroblast cultures obtained from the IMR,
the results were as follows. After two weeks of treatment with culture medium removed during feeding of
the fibroblasts (conditioned medium ICM]), the sympathetic neurons were examined for content of the
cholinergic biosynthetic enzyme CAT. Control cultures treated with fresh (nonconditioned) fibroblast
growth medium contained only low levels of CAT activity (0.57 pmole product per neuron per hour
Ippnh)), as reported previously 17, 81. Treatment with
normal human fibroblast CM increased CAT activity
to 7.48 ppnh, a twelve-fold increase. Thus normal human fibroblasts produced a factor that stimulated cholinergic function in rat neurons. By contrast, treatment
with Alzheimer fibroblast CM increased CAT activity
to only 2.50 ppnh, less than one-third of the value for
normal fibroblasts.
Because the number of Alzheimer fibroblast lines
available from the IMR is limited, skin biopsies were
obtained from 17 patients with a clinical diagnosis of
Alzheimer’s disease and from 17 age-matched con-
trols, increasing the sample size to 25 for each group.
Cultures of the fibroblasts were used to condition medium, which was tested for the presence of cholinergicinducing activity (Table 1). Treatment of sympathetic
neurons with normal human fibroblast CM significantly increased CAT activity to 6.82 ppnh. However,
treatment with Alzheimer fibroblast CM increased
CAT activity to only 2.60 ppnh, 38% of the control
value. By contrast, TH activity was not signlficantly
altered by the treatments, indicating the health of the
cultures. Moreover, neuron numbers were unchanged
by the treatments, indicating that differences in CAT
activity did not reflect differential survival of neurons.
CAT activity in the Alzheimer group significantly differed from that of the control group: p < 0.01, using a
two-tailed t test.
The diminished induction of CAT activity by Alzheimer fibroblast CM suggested that Alzheimer fibroblasts released less cholinergic promoting factor than
did normal fibroblasts. It was also possible, however,
that Alzheimer fibroblasts produced an inhibitor that
prevents induction of CAT activity. To test this hypothesis, the effects of treating with a mixture of Alzheimer CM and normal fibroblast CM were examined
(Table 2). If Alzheimer fibroblast CM contained an
inhibitor of CAT induction, then mixing should inhibit
the stimulation of CAT by normal fibroblast CM.
Treatment with control fibroblast CM alone increased
CAT activity to 5.12 ppnh, while treatment with Alzheimer fibroblast CM increased CAT activity to only
1.64 ppnh. Treatment with a mixture of both conditioned media increased CAT activity to 6.57 ppnh,
approximately the sum of the individual treatments
(Table 2). Thus it is unlikely that the Alzheimer
fibroblast CM contained an inhibitor of CAT induction.
The Alzheimer and control fibroblast populations
did not differ significantly in age or gender (Table 3).
Tabie 1. Eflects of Treatment of Cultured Sympathetic Neurons with Fibroblast Conditioned Medium
Fibroblast Linea
~~~
CAT Activityb
TH Activityc
0.57
2
0.05
3.1
* 0.16
9,090 k 370
6.82
?
1.21
2.7
?
0.22
9,030 k 335
2.60
2
0.23d
3.0
5
0.21
9,120 2 490
~
Untreated medium
(n = 8)
Normal fibroblast CM
(n = 25)
Alzheimer fibroblast CM
(n = 25)
~~
Neuron No.
~
See text for explanation of methods.
“A minimum of SIX cultures was assayed to obtain a mean value for each fibroblast line. The mean values for each line were averaged to obtain
the mean values for the control and Alzheirner groups.
bValues for CAT activity are expressed as mean pmole product per neuron per hour.
‘Values for TH activity are expressed as the mean fmole product per neuron per minute.
dDiffers from control at p < 0.01.
CM = conditioned medium; CAT = choline acetyluansferase; TH = ryrosine hydroxylase
96 Annals of Neurologv Vol 21 N o
1 January 1987
of Mixing Alzheimer and Control Fibroblast Conditioned Media"
Table 2. Eflects
Mediumb
~~~~~~~~~
A.
B.
C.
D.
~
~~
~
~
~
CAT Activity'
Neuron No.
5.12 2
1.64 -t
6.57
0.54 t
9,720
9,850 ?
9,610 ?
9,940 2
* 360
~
Normal fibroblast CM
Alzheimer fibroblast CM
Both Normal and Alzheimer fibroblast CM
Nonconditioned fibroblast medium
*
1.1
0.3
0.9
0.3
290
320
360
'Dissociated sympathetic neurons were treated three times weekly starting on day 4 with either (A) 30% normal fibroblast CM, (B) 30%
Alzheimer fibroblast CM, or (C) both. Other control cultures (D) were treated with nonconditioned fibroblast medium. On day 18, neuron
numbers were determined by counting as previously described 191 and the cultures were harvested for measurement of CAT activity.
'Pooled from 10 normal fibroblast lines and from 10 Alzheimer fibroblast lines.
'Values are expressed as mean pmole product per neuron per hour (n = 8).
CM = conditioned medium; CAT = choline acetyltransferase.
Table 3. Comparison of Alzheimer and Normal Fibroblast Cultures
Culture
Donor Age"
MaledFernales
Doubling Timeb
Viability'
Control
67.8 t 2.1
(Range 43-80)
68.6 k 2.2
(Range 47-83)
13/12
4.8
k
0.3
97.7
12/13
4.6
?
0.3
97.1
Alzheimer
-t
0.8
0.7
"Expressed as years f SEM.
'Mean doubling times {Sj are expressed in days
SEM.
'Expressed as mean percentage -t SEM of cells excluding trypan blue after trypsinization and removal of the cells from the culture flasks.
*
Moreover, there was no correlation between age or
gender and level of CAT-promoting activity. There
was no difference in passage numbers between the two
groups, and there was no correlation between passage
number and CAT-promoting activity. Further, the
Alzheimer and control fibroblasts appeared identical
morphologically. Nevertheless, to exclude the possibility that fibroblast viability of the two groups differed,
fibroblast doubling times and trypan-blue exclusion
were examined; no difference was found between the
two groups (Table 3). Control fibroblast numbers
doubled within 4.8 days, while Altheimer fibroblast
numbers doubled in 4.6 days. After trypsinization of
the cells from their flasks, more than 97% of both
control and Alzheimer fibroblasts excluded trypan
blue.
Discussion
Our observations indicate that normal human fibroblasts, like rodent fibroblasts {13}, release a soluble factor that stimulates cholinergic function in sympathetic
neurons. Moreover, release of the factor by cultured
Altheimer fibroblasts is deficient. It is not yet clear
whether the reduced release reflects diminished synthesis of the factor, deficient secretory mechanisms, or
release of a modified molecule with less activity. In any
case, the fibroblast abnormalities indicate that Alzheimer's disease may be a systemic disease involving
nonneuronal cells that are outside of as well as within
the brain. In turn, this raises the possibility that the
primary defect in the disease may lie within nonneuronal cells andor the extracellular matrix, and that
the neuronal abnormalities may result secondarily
from deficiencies in release of neuronotrophic factors
by nonneuronal cells. Clearly, this suggests that treatment with the appropriate factors might prevent
neuronal deterioration in afflicted subjects.
Although the Alzheimer fibroblasts in this study
produced uniformly low levels of the cholinergic factor, there was greater variability among control subjects, with values for some normal subjects overlapping those of the Alzheimer group (see Table 1). The
significance of low values for some apparently normal
fibroblasts is unknown. It is possible that low production of the CAT-promoting protein is a risk factor for
Alzheimer's disease, but that additional factors influence the development of the clinical syndrome. It is
also possible, of course, that some control subjects
with low values are destined to develop the disease.
Alternatively, release of the factor by fibroblasts may
be imperfectly correlated with levels of the factor released in the brain. Although the difference between
the normal and Altheimer cell lines in this study is
large and highly significant (p < O.Ol), it will be necessary to study fibroblasts from patients with other
neurodegenerative disorders to validate the hypothesis
Brief Communication: Kessler: Deficient Cholinergic Differentiating Factor in AD
97
that deficient release of this cholinergic factor is specifically linked to Alzheimer’s disease.
Supported by NIH Grant NS 20778 and by grants from the March
of Dimes and United Cerebral Palsy Foundation.
Protocols for these studies were approved by the Albert Einstein
College of Medicine Human Experimentation Committee.
I would like to acknowledge the superb technical assistance of Ms
Julie Hyman and Ms Kathryn Sweeney. I thank Drs Jim Goldman
and Alcmene Chalazonitis for critically reviewing the manuscript.
References
1. American Psychiatric Association Task Force on Nomenclature
and Statistics: Diagnostic and Statistical Manual of Mental Disorders, 111. Washington, DC,American Psychiatric Association,
1980, pp 124-126
2. Andia-Waltenbaugh AM, Puck IT:Alzheimer’s disease: furrher
evidence of a microtubular defect. J Cell Biol 75:279a, 1977
3. Diamond JM, Matsuyama S S , Meier K,Jarvik LF.Elevation of
erythrocyte counter transport rates in Alzheimer’s dementia.
New Eng J Med 309:1061-1062, 1983
4. Fonnum F Radiochemical microassays for the determination of
choline acetyluansferase and acetylcholinesterase activities.
Biochem J 115:465-472, 1969
5. Freshney IU:Culture of Animal Cells: A Manual of Basic Technique. New York, A. R. Liss, 1982, pp 207-215
6. Giess MC, Weber MJ: Acetylcholine metabolism in rat spinal
cord cultures: regulation by a factor involved in the determination of the neurotransmitter phenotype of sympathetic neurons.
J Neurosci 4:1442-1452, 1984
I . Kessler JA: Environmental co-regulation of substance P,
somatostatin, and neurotransmitter synthesizing enzymes in cultured sympathetic neurons. Brain Res 321:155-159, 1984
8. Kessler JA: Non-neuronal cell conditioned medium stimulates
peptidergic expression in sympathetic and sensory neurons in
vitro. Dev Biol 106:61-69, 1984
9. Kessler JA: Differential regulation of peptide and catecholamine characters in cultured sympathetic neurons. Neuroscience 155327439, 1985
10. Kessler JA: Differential regulation of cholinergic and peptidergic development in the rat strianun in culture. Dev Biol
113:77-89, 1986
11. Khansari N, Whitten HD, Chow YK, Fudenberg HH: Immunological dysfunction in Alzheimer’s disease. J Neuroimmunol 7:729-785, 1985
12. Nordenson I, Adolfson R, Beckman G, et al: Chromosomal
abnormality in dementia of Alzheimer type. Lancet 1:481-482,
1980
13. Patterson PH, Chun LLY: The induction of acetylcholine synthesis in primary cultures of dissociated Sympathetic neurons. 1.
Effects of conditioned medium. Dev Biol 56:263-280, 1977
14. Peterson C, Gibson GE, Blass JP: Altered calcium uptake in
cultured skin fibroblasts from patients with Alzheimer’s disease.
N Engl J Med 312:1063-1065, 1985
15. Sims NR, Finegan JM, Blass JP: Altered glucose metabolism in
fibroblasts from patients with Alzheimer’s disease. N Engl J
Med 313:683-684, 1985
7
Caloric-Induced
Nystagmus with Isoelectric
Electroencephalogram
Manmohan Nayyar, MD, Robert J. Strobos, MD,
Brij M. Singh, MD, Marie Brown-Wagner, MD,
and Anthony Pucillo, MD
Caloric vestibular testing induced nystagmus in a patient with an isoelectric electroencephalogram after cardiopulmonary arrest. This has been demonstrated previously in patients in a chronic persistent vegetative state
with intact brainstem reflexes, but never in a patient
with an isoelectric electroencephalogram. Animal studies indicate that the quick phase of nystagmus and horizontal saccades are generated in the paramedian pontine
reticular formation. The present case supports the conclusion that caloric-induced nystagmus originates in the
brainstem in rudimentary form.
Nayyar M, Strobos RJ, Singh BM, Brown-Wagner M,
Pucillo A: Caloric-induced nystagmus
with isoelectric electroencephalogram.
Ann Neurol 21:98-100, 1987
It has been repeatedly stated that cold caloric stimulation produces tonic conjugate deviation of the eyes
toward the irrigated ear in patients in coma with intact
brainstem function. If caloric stimulation evokes nystagmus, it has been presumed that cerebral connections are intact and that the impairment of consciousness is either extremely mild or that the “coma” is
psychogenic 11, 101.We report what we believe is the
first description of nystagmus induced by caloric stimulation in neocortical death.
Case Report
A 50-year-old white man, a heavy smoker with a history of
hypertension, had complained of anginal pains for the past
10 years. On October 13, 1985, the pain became more severe and several hours later he was found unconscious. After
arrival of the emergency medical service, it took 45 minutes
to resuscitate him from cardiopulmonary arrest. Ventricular
fibrillation was converted to atrial fibrillation with cardioversion.
On admission, blood pressure was 92 mm Hg palpable;
pulse rate was 88 per minute; respiration was spontaneous,
requiring intermittent assistance; and temperature was normal. An electrocardiogram showed atrial fibrillation with a
right bundle branch block and absent Q waves. Results of
From the Department of Neurology, New York Medical College,
Valhalla, NY 10595.
Received Apr 21, 1986, and in revised form June 3. Accepted for
publication June 8, 1986.
Address reprint requests to Dr Strobos.
98
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factors, patients, differentiation, deficiency, disease, alzheimers, fibroblasts, cholinergic
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