Cytochalasins useful in providing protection against nerve cell injury associated with neurodegenerative disorders

The present invention relates to novel therapeutic uses of certain compounds to protect nerve cells from injury and death. The compounds include cytochalasin D and related analogs, and cytochalasin E and related analogs.

FIELD OF THE INVENTION 
The present invention relates to a novel therapeutic use of certain 
compounds to protect nerve cells from injury and death. The compounds 
include cytochalasin D and related analogs, and cytochalasin E and related 
analogs. 
BACKGROUND OF THE INVENTION 
Nerve cell injury and death leads to a number of. neurodegenerative 
disorders such as Alzheimer's disease and stroke. 
The leading cause of dementia and the fourth leading cause of death in the 
developed world is Alzheimer's disease which afflicts an estimated 10% of 
the population over 65 years of age in the United States. Alzheimer's 
disease imposes a tremendous financial burden on afflicted individuals 
because they require prolonged care. 
Affected individuals are at first forgetful. As this progressive disorder 
gradually worsens, these affected individuals, although able to recall 
occurrences in the distant past, are unable to remember recent events. 
Subsequently, speech, the ability to calculate, visuospatial orientation, 
judgement, and social behavior become progressively abnormal. Eventually, 
profound dementia sets in and frequently the individual dies of 
superimposed infections. The duration of diseases ranges from 3 to 10 
years. 
The diagnosis of Alzheimer's disease is usually made on the basis of 
clinical history, neurological examination and laboratory tests that help 
to exclude other disorders, some of which are potentially treatable. 
Unfortunately, other than direct examination of brain tissue obtained by 
cerebral biopsy or at autopsy, no tests to establish a diagnosis of 
Alzheimer's disease presently exist. 
At autopsy, the brains of individuals with Alzheimer's disease are usually 
slightly smaller than normal for their age. Microscopic examination 
discloses four characteristic pathological features that are essential for 
the diagnosis of Alzheimer's disease: neurofibrillary tangles, loss of 
specific population of nerve cells, senile plaques and deposits of 
amyloid. 
Neurofibrillary tangles, that is, fibrillar inclusions within cell bodies 
of affected neurons, consist of abnormal filaments thought to be derived 
in part from cytoskeletal elements normally present in nerve cells. 
Neurofibrillary tangles consist of abnormal accumulations of cytoskeletal 
and other proteins whereas senile plaques consist of aggregates of amyloid 
.beta.-peptide (A.beta.). Major components of neurofibrillary tangles are 
the microtubule-associated protein tau, and ubiquitin, a "heat-shock" 
protein involved in targeting proteins for proteolytic degradation. 
Postranslational alterations in tau such as phosphorylation, and 
disassociation of tau from microtubules may promote the assembly of tau 
into the abnormal straight and paired helical filaments that characterize 
neurofibrillary tangles. Although the mechanism leading to neurofibrillary 
degeneration is not clear, several observations suggest a role for 
dysregulation of neuronal calcium homeostasis with resultant elevations of 
intracellular free calcium concentration, Ca.sup.2+ !.sub.i. Among the 
evidence supporting this hypothesis is: experimentally induced elevations 
of Ca.sup.2+ !.sub.i in hippocampal neurons (in vitro and in vivo) can 
elicit antigenic, biochemical and ultrastructural changes in cytoskeletal 
proteins (tau and spectrin) similar to those seen in neurofibrillary 
tangles; neurons vulnerable to neurofibrillary degeneration bear high 
levels of glutamate receptors; aggregated A.beta. can be neurotoxic and 
can render neurons vulnerable to excitotoxicity by a mechanism that 
involves destabilization of Ca.sup.2+ !.sub.i homeostasis. In addition, 
studies indicate that mitogen-activated protein (MAP) kinases can 
phosphorylate tau in a manner very similar to that observed in the paired 
helical filaments of Alzheimer's disease, and MAP kinases are known to be 
activated glutamate and elevation of Ca.sup.2+ !.sub.i. 
Degeneration and death of certain populations of nerve cells occur in 
certain brainstem nuclei, the basal forebrain, the amygdala, the 
hippocampus and neocortex. In the brain, specific populations of nerve 
cells use specific neurotransmitters. Also, neurochemical studies have 
shown that the brains of individuals with Alzheimer's disease exhibit a 
selective reduction in markers for certain neurotransmitter systems. 
The third characteristic brain abnormality associated with Alzheimer's 
disease is the presence of abundant senile plaques, composed of several 
elements: abnormal neurites (enlarged filament-containing axons and 
terminals), extracellular amyloid fibrils and non-neuronal reactive cells. 
The presence of plaques correlates with the presence of dementia and with 
the severity of loss of certain neurotransmitter markers, particularly 
cholinergic enzymes. 
Localized in plaques and around cerebral blood vessels, amyloid is composed 
of a 4-kilodalton protein designated amyloid .beta.-peptide. The amyloid 
.beta.-peptide is a 40-42 amino acid peptide arising from a much larger 
membrane-spanning .beta.-amyloid precursor protein (695-770 amino acids) 
which is a transmembrane glycoprotein that accumulates as diffuse 
(unaggregated) and compact (aggregated) plaques in the brain of victims of 
Alzheimer's disease. The diffuse plaques are not associated with neuronal 
pathology, whereas compact A.beta. is surrounded by degenerative neurites 
with characteristic cytoskeletal pathology. Cell culture studies have 
shown that A.beta. can be directly neurotoxic, and can render neurons 
vulnerable to excitotoxicity and oxidative injury. The mechanism of 
A.beta. toxicity is related to its secondary structure and appears to 
involve free radical-mediated damage to the plasma membrane and disruption 
of cellular calcium homeostasis resulting in elevated rest Ca.sup.2+ 
!.sub.i and increased Ca.sup.2+ !.sub.i responses to depolarization and 
excitatory amino acids. 
The major pathway for .beta.-amyloid precursor protein (.beta.APP) 
metabolism involves an enzymatic cleavage within the A.beta. sequence and 
obviates deposition of amyloidogenic A.beta.. On the other hand, A.beta. 
is released from brain cells at low levels and is present in the 
cerebrospinal fluid at nanomolar concentrations indicating an alternative 
processing pathway of .beta.APP. A cleavage of .beta.APP at the N-terminus 
of A.beta. leaves behind a C-terminal fragment of .beta.APP which contains 
potentially amyloidogenic A.beta.. Some cases of inherited Alzheimer's 
disease have been linked to mutations in .beta.APP which may alter 
processing of .beta.APP in a way that leads to increased production of AS. 
The link between altered metabolism of .beta.APP and neuronal injury in 
Alzheimer's disease is supported by studies showing that synthetic A.beta. 
peptides can be directly neurotoxic to primary cultures of hippocampal and 
cortical neurons, and can render neurons vulnerable to glutamate 
excitotoxicity, glucose deprivation, and oxidative injury. The 
neurotoxicity of A.beta. is dependent upon its ability to form aggregates 
which accumulate at plasma membranes and disrupt cellular calcium 
homeostasis. The mechanism whereby A.beta. disrupts calcium regulation at 
the plasma membrane may involve the peptide forming calcium-conducting 
pores. 
Calcium influx through glutamate receptors and voltage dependent channels 
mediates an array of functional and structural responses in neurons. 
However, unrestrained calcium influx can injure and kill neurons; such 
calcium overload can be induced by the excitatory transmitter glutamate 
and Alzheimer amyloid .beta.-peptide, and is therefore implicated in both 
acute and chronic neurodegenerative conditions. Actin microfilaments are a 
major cytoskeletal element whose polymerization state is highly sensitive 
to calcium. Several key adaptive physiological processes in the brain, 
including neurotransmitter release, postsynaptic signalling and regulation 
of neurite outgrowth and synaptogenesis during development are triggered 
by calcium influx. However, excessive calcium influx plays a primary role 
in excitotoxicity, a form of neuronal injury resulting from 
overstimulation of glutamate receptors, and which is believed operative in 
a variety of both acute (e.g., stroke and traumatic brain injury) and 
chronic (e.g., Alzheimer's and Huntington's diseases) neurodegenrative 
conditions. In Alzheimer's disease the 40-42 amino acid amyloid 
.beta.-peptide which forms insoluble plaques in the brain may kill neurons 
by inducing Ca.sup.2+ influx and increasing sensitivity to 
excitotoxicity. 
Actin and tubulin are two major structural proteins in neurons which 
polymerize to form microfilaments and microtubules, respectively. 
Microfilaments and microtubules are highly dynamic structures exquisitely 
sensitive to environmental stimuli that elevate Ca.sup.2+ !.sub.i, such 
as glutamate. 
Stroke is our nation's third leading killer and the number one cause of 
adult disability. One-half million people suffer a stroke each year. A 
stroke is the result of a sudden decrease in the flow of blood to an area 
of the brain. When blood cannot reach the brain, brain cells become 
deprived of oxygen and die. Consequently, functions normally controlled by 
the damaged brain area become impaired. For example, paralysis of certain 
body parts may occur. The interruption in blood flow can be due to 
blockage from a blood clot or narrowed artery in the head or neck or to 
the bursting of an artery in the brain. 
Nerve cell injury and death is also responsible for other neurodegenerative 
disorders such as Down's syndrome, amyotrophic lateral sclerosis, 
Parkinson's disease, Huntington's disease, Cerebral ischemia, cerebral 
infarction, cerebral vasospasm, hypoglycemia, cardiac arrest, status 
epilepticus, perinatal asphyxia, anoxia, pulmonary surgery, or cerebral 
trauma. 
SUMMARY OF THE INVENTION 
The present invention relates to a novel therapeutic use of certain 
compounds to protect nerve cells from injury and death. The compounds 
include cytochalasin D and related analogs, and cytochalasin E and related 
analogs. More particularly, the present invention concerns a method for 
reducing adverse effects of a neurodegenerative disorder comprising: 
administering to a patient a therapeutically effective amount of at least 
one compound selected from the group consisting of compounds represented 
by Formulas (I)-(II) and their pharmaceutically acceptable salts: 
##STR1## 
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 
represent hydrogen, C.sub.1 to C.sub.6 alkyl or hydroxy, or where R.sub.3 
and R.sub.4 together represent a carbonyl group; or 
##STR2## 
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, 
R.sub.8, and R.sub.9 represent hydrogen, C.sub.1 to C.sub.6 alkyl, 
hydroxy, or OAC, or where R.sub.3 and R.sub.4 together represent a 
carbonyl group. 
The present invention also relates to a pharmaceutical composition 
comprising at least one compound selected from the group consisting of 
compounds represented by Formulas (I)-(II) and their pharmaceutically 
acceptable salts: 
##STR3## 
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.7 and R.sub.7 
represent hydrogen, C.sub.1 to C.sub.6 alkyl or hydroxy, or where R.sub.3 
and R.sub.4 together represent a carbonyl group; or 
##STR4## 
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, 
R.sub.8, and R.sub.9 represent hydrogen, C.sub.1 to C.sub.6 alkyl, 
hydroxy, or OAC, or where R.sub.3 and R.sub.4 together represent a 
carbonyl group. 
Neurodegenerative disorders treatable by the present method include, but 
are not limited to, Alzheimer's disease, Down's syndrome, amyotrophic 
lateral sclerosis, Parkinson's disease, Huntington's disease, cerebral 
ischemia, cerebral infarction, thromboembolic and hemorrhagic stroke, 
cerebral vasospasm, hypoglycemia, cardiac arrest, status epilepticus, 
perinatal asphyxia, anoxia, pulmonary surgery, or cerebral trauma. 
The present invention also relates to a method of attenuating intracellular 
calcium levels in mammalian nerve cells of a patient in need of therapy 
for amyloid-.beta. peptide toxicity associated with neurodegenerative 
disorders which comprises administration to a mammal in need of such 
therapy an effective amount of a compound which attenuates intracellular 
calcium levels in the mammalian nerve cell. 
Other features of the invention will become apparent in the course of the 
following description of the exemplary embodiments which are given for 
illustration of the invention and are not intended to be limiting thereof.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is based, in part, on the inventors' surprising and 
unexpected discovery of a method for reducing adverse effects of a 
neurodegenerative disorder comprising: administering to a patient a 
therapeutically effective amount of at least one compound selected from 
the group consisting of compounds represented by Formulas (I)-(II) and 
their pharmaceutically acceptable salts: 
##STR5## 
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 
represent hydrogen, C.sub.1 to C.sub.6 alkyl or hydroxy, or where R.sub.3 
and R.sub.4 together represent a carbonyl group; or 
##STR6## 
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, 
R.sub.8, and R.sub.9 represent hydrogen, C.sub.1 to C.sub.6 alkyl, 
hydroxy, or OAC, or where R.sub.3 and R.sub.4 together represent a 
carbonyl group. 
The present invention is also based, in part, on the inventors' surprising 
and unexpected discovery of a method of attenuating intracellular calcium 
levels in mammalian nerve cells of a patient in need of therapy for 
amyloid-.beta. peptide toxicity associated with neurodegenerative 
disorders which comprises administration to a mammal in need of such 
therapy an effective amount of a compound which attenuates intracellular 
calcium levels in the mammalian nerve cell. 
Neurodegenerative disorders treatable by the present method include, but 
are not limited to, Alzheimer's disease, Down's syndrome, amyotrophic 
lateral sclerosis, Parkinson's disease, Huntington's disease, cerebral 
ischemia, cerebral infarction, thromboembolic and hemorrhagic stroke, 
cerebral vasospasm, hypoglycemia, cardiac arrest, status epilepticus, 
perinatal asphyxia, anoxia, pulmonary surgery, or cerebral trauma. 
A preferred compound represented by Formula (I) is cytochalasin D and is 
sold commercially by Sigma Chemical Company under that name. Cytochalasin 
D has the following structural formula: 
##STR7## 
A preferred compound represented by Formula (II) is cytochalasin E and is 
sold commercially by Sigma Chemical Company under that name. Cytochalasin 
E has the following structural formula: 
##STR8## 
The compounds for use in the present invention can be administered as a 
pharmaceutical composition. The pharmaceutical compositions used in the 
methods of this invention for administration to animals and humans 
comprise an active agent in combination with a pharmaceutical carrier or 
excipient acceptable for delivery of the compounds to neurons. 
The pharmaceutical compositions can be in the form of tablets (including 
lozenges and granules), dragees, capsules, pills, ampoules or 
suppositories comprising a compound of the invention. "Pharmaceutical 
composition" means physically discrete coherent portions suitable for 
medical administration. "Pharmaceutical composition in dosage unit form" 
means physically discrete coherent units suitable for medical 
administration, each containing a daily dose or a multiple (up to four 
times) or a sub-multiple (down to a fortieth) of a daily dose of the 
active compound of the invention in association with a carrier and/or 
enclosed within an envelope. Whether the composition contains a daily 
dose, or for example, a half, a third or a quarter of a daily dose will 
depend on whether the pharmaceutical composition is to be administered 
once or, for example, twice, three times or four times a day, 
respectively. 
Advantageously, the compositions are formulated as dosage units, each unit 
being adapted to supply a fixed dose of active ingredients. Tablets, 
coated tablets, capsules, ampoules and suppositories are examples of 
preferred dosage forms according to the invention. It is only necessary 
that the active ingredient constitute an effective amount, i.e., such that 
a suitable effective dosage will be consistent with the dosage form 
employed in single or multiple unit doses. The exact individual dosages, 
as well as daily dosages, are determined according to standard medical 
principles under the direction of a physician or veterinarian. 
The active agents can also be administered as suspensions, solutions and 
emulsions of the active compound in aqueous or non-aqueous diluents, 
syrups, granulates or powders. Diluents that can be used in pharmaceutical 
compositions (e.g., granulates) containing the active compound adapted to 
be formed into tablets, dragees, capsules and pills include the following: 
(a) fillers and extenders, e.g., starch, sugars, mannitol and silicic 
acid; (b) binding agents, e.g., carboxymethyl cellulose and other 
cellulose derivatives, alginates, gelatine and polyvinyl pyrrolidone; (c) 
moisturizing agents, e.g., glycerol; (d) disintegrating agents, e.g., 
agar-agar, calcium carbonate and sodium bicarbonate; (e) agents for 
retarding dissolution, e.g., paraffin; (f) resorption accelerators, e.g., 
quaternary ammonium compounds; (g) surface active agents, e.g., cetyl 
alcohol, glycerol monostearate; (h) adsorptive carriers, e.g., kaolin and 
bentonite; (i) lubricants, e.g., talc, calcium and magnesium stearate and 
solid polyethylene glycols. 
The tablets, dragees, capsules and pills comprising the active agent can 
have the customary coatings, envelopes and protective matrices, which may 
contain opacifiers. They can be so constituted that they release the 
active ingredient only or preferably in a particular part of the 
intestinal tract, possibly over a period of time. The coatings, envelopes 
and protective matrices may be made, for example, from polymeric 
substances or waxes. 
The active ingredient can also be made up in microencapsulated form 
together, with one or several of the above-mentioned diluents. 
The diluents to be used in pharmaceutical compositions adapted to be formed 
into suppositories can, for example, be the usual water-soluble diluents, 
such as polyethylene glycols and fats (e.g., cocoa oil and high esters, 
(e.g., C.sub.14 -alcohol with C.sub.16 -fatty acid) or mixtures of these 
diluents. 
The pharmaceutical compositions which are solutions and emulsions can, for 
example, contain the customary diluents (with, of course, the 
above-mentioned exclusion of solvents having a molecular weight below 200, 
in the presence of a surface-active agent), such as diluents, dissolving 
agents and emulsifiers. Specific non-limiting examples of such diluents 
are water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl 
acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene 
glycol, dimethylformamide, oils (for example, ground nut oil, glycerol, 
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of 
sorbitol or mixtures thereof. 
For parenteral administration, solutions and suspensions should be sterile, 
e.g., water or arachis oil contained in ampoules and, if appropriate, 
blood-isotonic. 
The pharmaceutical compositions which are suspensions can contain the usual 
diluents, such as liquid diluents, e.g., water, ethyl alcohol, propylene 
glycol, surface active agents (e.g., ethoxylated isostearyl alcohols, 
polyoxyethylene sorbitols and sorbitan esters), polycrystalline cellulose, 
aluminum methahydroxide, agar-agar and tragacanth, or mixtures thereof. 
The pharmaceutical compositions can also contain bulking agents and 
preservatives, as well as perfumes and flavoring additions (e.g., 
peppermint oil and eucalyptus oil, and sweetening agents, (e.g., saccharin 
and aspartame). 
The pharmaceutical compositions will generally contain from about 0.0001 to 
90 wt. %, preferably about 0.01 to 10 wt. % of the active ingredient by 
weight of the total composition. In addition to the active agent, the 
pharmaceutical compositions and medicaments can also contain other 
pharmaceutically active compounds. 
Any diluent in the pharmaceutical compositions of the present invention may 
be any of those mentioned above in relation to the pharmaceutical 
compositions. Such compositions may include solvents of molecular weight 
less than 200 as the sole diluent. 
The active compound is administered perorally, parenterally (for example, 
intramuscularly, intraperitoneally, subcutaneously, transdermally or 
intravenously), rectally or locally, preferably orally or parenterally, 
especially perlingually, or intravenously. 
The dosage rate, e.g., 0.0001 to 20 mg/kg of body weight, will be a 
function of the nature and body weight of the human or animal subject to 
be treated, the individual reaction of this subject to the treatment, type 
of formulation in which the active ingredient is administered, the mode in 
which the administration is carried out and the point in the progress of 
the disease or interval at which it is to be administered. Thus, it may in 
some case suffice to use less than a minimum dosage rate, while other 
cases an upper limit must be exceeded to achieve the desired results. 
Where larger amounts are administered, it may be advisable to divide these 
into several individual administrations over the course of the day. 
EXAMPLE 1 
(hippocampal cell culture and experimental treatments) 
Dissociated embryonic rat hippocampal cell cultures were established and 
maintained. All procedures conformed with the NIH Guide for the Care and 
Use of Laboratory Animals and were approved by the University of Kentucky 
Animal Care and Use Committee. Cultures were maintained on a 
polyethyleneimine-coated substrate in plastic 35-mm dishes or 96 wall 
plates (for cell survival studies) or glass-bottom 35-mm dishes (for 
Ca.sup.2+ !.sub.i imaging studies). The cell density was .sup..about. 
70-100 cells/mm.sup.2. Cells were maintained in Eagle's minimum essential 
medium supplemented 10% with fetal bovine serum and containing 20 mM 
sodium pyruvate. All experiments were performed in 6-10-day-old cultures, 
a time at which neurons exhibit calcium responses to glutamate mediated by 
both NMDA and .alpha.-amino-3-hydroxy-5-methylisoxazole-4-propionic acid 
(AMPA)/kainate receptors, and are vulnerable to excitotoxicity and A.beta. 
toxicity. A.beta.25-35 (Bachem, lot ZJ744) and A.beta.1-40 (Bachem, lot 
ZK600) were stored in the lyophilized form, and stocks were prepared 
immediately before use by dissolving the peptide at a concentration of 1 
mM in sterile distilled water. Preliminary characterization of the 
aggregation kinetics and neurotoxicity profiles of each of these peptides 
showed that the peptides aggregated rapidly when placed in culture medium 
and progressively killed neurons over a 48-h period when added to cultures 
in a soluble form. Glutamate and colchicine (Sigma) were prepared as 
200-500x stocks in saline. Cytochalasins D and E (Sigma) and calcium 
ionophore 4-bromo-A23187 (Calbiochem) were prepared as 500x stocks in 
dimethyl sulfoxide. Vitamin E (.alpha.-tocopherol; Sigma) was prepared as 
a 500x stock in ethanol. Nordihydroguaiaretic acid and FeSo.sub.4 were 
prepared as 500x stocks in sterile water. 
EXAMPLE 2 
(analysis of neuronal survival) 
Neuronal survival was quantified by counting viable neurons in the same 
microscope fields (10x objective) immediately before experimental 
treatment and at time points after treatment. In addition, cells were 
grown in 96-well plates and Alamar blue fluorescence (Alamar Laboratories) 
was quantified by using a fluorescence plate reader. Alamar Blue is a 
non-fluorescent substrate that, after reduction by cell metabolites, 
becomes fluorescent. Usually neurons that died in the time intervals 
(20-48 h) between examination points were absent. Viability of the 
remaining neurons was assessed by morphological criteria. Neutrons with 
intact neurites of uniform diameter and a soma with a smooth, round 
appearance were considered viable, whereas neurons with fragmented 
neurites and a vacuolated or swollen soma were considered nonviable. 
Survival values were expressed as percentages of the initial number of 
neurons present before experimental treatment. Statistical comparisons 
were made using ANOVA and Scheffe post-hoc tests for pairwise comparisons. 
TABLE 1 
______________________________________ 
Effects of cytochalasin D and colchicine on 
neurotoxicity of glutamate and A.beta. 
Treatment Condition 
Neuron Survival (% of control) 
Alamar Blue Assay Cell Counts 
______________________________________ 
Vehicle (DMSO) 100 .+-. 2.4 
100 .+-. 3.6 
10 nM Cytochalasin D 
97 .+-. 7.8 
89 .+-. 2.4 
100 nM Cytochalasin D 
91 .+-. 5.9 
87 .+-. 2.2 
100 nM Colchicine 40 .+-. 5.2.sup.b 
85 .+-. 1.9.sup.a 
100 .mu.M Glutamate 
18 .+-. 5.4.sup.b 
15 .+-. 3.3.sup.b 
100 .mu.M Glutamate + 10 nM CYD 
43 .+-. 6.7.sup.d 
54 .+-. 7.4.sup.c 
100 .mu.M Glutamate + 100 nM CYD 
42 .+-. 4.2.sup.d 
45 .+-. 9.8.sup.d 
100 .mu.M Glutamate + 100 nM Colch 
12 .+-. 2.2.sup.b 
24 .+-. 7.4 
50 .mu.M A.beta. 47 .+-. 3.5.sup.b 
n.d. 
50 .mu.M A.beta. + 10 nM Cytochalasin D 
55 .+-. 2.5 
n.d. 
50 .mu.M A.beta. + 100 nM Cytochalasin D 
74 .+-. 1.3.sup.e 
n.d. 
50 .mu.M A.beta. + 100 nM Colchicine 
23 .+-. 2.8 
n.d. 
20 .mu.M Glutamate 74 .+-. 0.7.sup.b 
n.d. 
50 .mu.M A.beta. + 20 .mu.M Glutamate 
24 .+-. 0.8.sup.f 
n.d. 
50 .mu.M A.beta. + 20 .mu.M Glut .+-. 
63 .+-. 2.5.sup.g 
n.d. 
100 nM CyD 
______________________________________ 
Values represent the means and SEM of 4 separate cultures (cell counts) or 
4 culture wells (Alamar blue assay). .sup.a p&lt;0.05 compared to control 
value. .sup.b p&lt;0.001 compound to control value. .sup.c p&lt;0.01 compared to 
value for glutamate-treated cultures. .sup.d p&lt;0.005 compared to value for 
glutamate-treated cultures. .sup.e p&lt;0.001 compared to value for 
A.beta.-treated cultures. .sup.f p&lt;0.001 compared to value for cultures 
treated with 20 .mu.M glutamate. .sup.g p&lt;0.001 compared to cultures 
treated with 50 .mu.M A.beta.+20 .mu.M glutamate. n.d. not determined. 
At concentrations known to depolymerize actin, cytochalasin D alone (1-100 
nM) had no significant effect on neuronal survival, while colchicine (100 
nM) reduced survival (Table 1). Although cytochalasin D did not adversely 
affect neuronal survival, it did inhibit growth cone motility. Neuronal 
survival in cultures exposed to 100 .mu.M glutamate for 24 hr was reduced 
to less than 20%. Glutamate neurotoxicity was significantly attenuated in 
cultures pretreated for 1 hr with cytochalasin D, whereas colchicine was 
ineffective (Table 1). Neuronal survival was reduced in less than 50% of 
control values in cultures exposed for 24 hr to 50 .mu.M A.beta.25-35 
(Table 1). A.beta. neurotoxicity was essentially eliminated in parallel 
culture cotreated with 100 nM cytochalasin D, whereas colchicine 
exacerbated A.beta. toxicity (Table 1). Cytochalasin E(10-100 nM), another 
microfilament-disrupting agent, also protected cultural hippocampal 
neurons against the toxicities of glutamate and A.beta.. 
EXAMPLE 3 
(measurement of Ca.sup.2+ !.sub.i 
Fluorescence ratio imaging of the Ca.sup.2+ indicator dye fura-2 was used 
to quantify Ca.sup.2+ !.sub.i in neuronal somata. Cells were incubated 
for 30-40 min in the presence of 2 .mu.M acetoxymethyl ester form of the 
Ca.sup.2+ !.sub.i indicator dye fura-2 and were then washed twice (2 
ml/wash) with fresh medium and allowed to incubate at least 40 min before 
imaging. Immediately before imaging, the normal culture medium was 
replaced with Hanks' balanced saline solution (GIBCO) containing 10 mM 
HEPES buffer and 10 mM of glucose. Cells were imaged using a Zeiss 
Attofluor system with a oil objective or Quantex system with a 40x oil 
objective. The ratio of the fluorescence emission using two different 
excitation wavelengths (334 and 380 nm) was used to determine Ca.sup.2+ 
!.sub.i. The system was calibrated using solutions containing either no 
Ca.sup.2+ or a saturating level of Ca.sup.2+ (1 mM) according to the 
following formula: Ca.sup.2+ !.sub.i =K.sub.D (R-R.sub.min) / (R.sub.max 
-R)! (F.sub.0 /F.sub.S). Values represent the average Ca.sup.2+ !.sub.i 
in the neuronal cell body. Experimental treatments were added to the 
bathing medium by dilution from 100-500x stocks. 
EXAMPLE 4 
(localization of phalloidin with fluorescence microscopy 
Hippocampal cells were exposed to cytochalasin D and then fixed for 30 min 
in 4% paraformaldehyde/phosphate-buffered saline. Cell membranes were 
permeabilized by incubating for 5 min in a solution of 0.2% Triton X-100 
in phosphate-buffered saline. Cells were then incubated for 30 min in PBS 
containing 0.005 U/ml fluorescein-phalloidin (Molecular Probes). Cells 
were rinsed in water and mounted in Vectashield antifade solution (Vector 
Laboratories) . Fluorescence images were acquired using a Sarastro 2000 
confocal laser scanning microscope with a 60x oil-immersion objective. All 
images were acquired using identical settings for excitation intensity and 
detector gain. 
EXAMPLE 5 
Fura-2 calcium imaging technology revealed that cytochalasin D attenuated 
Ca.sup.2+ !.sub.i responses to glutamate, A.beta., and membrane 
depolarization (FIG. 1). In control cultures, 50 .mu.M glutamate induced a 
rapid approximately five-fold increase in neuronal Ca.sup.2+ !.sub.i. In 
contrast, the Ca.sup.2+ !.sub.i response to glutamate in neurons 
pretreated with 100 nM cytochalasin D for 1 hr was reduced by 
approximately 30% (FIG. 1A). The neuronal Ca.sup.2+ !.sub.i response to 
glutamate was greatly enhanced in cultures pretreated with A.beta. for 3 
hr; Ca.sup.2+ !.sub.i rapidly rose to well over 1 .mu.M compared to an 
increase to approximately 600 nM in neurons in untreated control cultures. 
Cytochalasin D completely blocked potentiation of Ca.sup.2+ !.sub.i 
response to glutamate in A.beta.-pretreated culture (FIG. 1B). 
Cytochalasin D pretreatment also suppressed K.sup.+ -induced elevation of 
Ca.sup.2+ !.sub.i (FIG. 1C). Colchicine alone caused an elevation of rest 
Ca.sup.2+ !.sub.i and did not attenuate Ca.sup.2+ !.sub.i response to 
glutamate or A.beta. (data not shown). Taken together with the previous 
patch clamp studies of Rosenmund and Westbrook and Johnson and Byerly, 
these data indicate that actin depolymerization reduces Ca.sup.2+ influx 
induced by glutamate and membrane depolarization. Moreover, actin 
depolymerization abrogates the Ca.sup.2+ !.sub.i -destabilizing actin of 
A.beta.. The suppressive effect of cytochalasin D on glutamate-induced 
elevation of Ca.sup.2+ !.sub.i was more pronounced with increasing 
concentrations of glutamate (FIG. 1D). Whereas the reduction in peak 
Ca.sup.2+ !.sub.i response was only approximately 60 nM in neurons 
exposed to 10 .mu.M glutamate, the reductions were approximately 170 and 
460 nM in neurons exposed to 50 and 100 .mu.M glutamate, respectively 
(FIG. 1D). 
EXAMPLE 6 
Kainate Lesion Paradigm 
A kainate lesion paradigm was employed in adult rats to determine whether 
cytochalasin D was also excitoprotective. Twenty-four adult male 
Sprague-Dawley rats (250-300 g) were divided into the indicated 
experimental groups (4-5 rats/group). Kainate (0.5 82 g/0.5 ml) was 
injected stereotaxically into region CA1 of the right hippocampus of 
anesthetized rats. Cytochalasin D (the indicated concentration in 1 .mu.l 
of saline-1% DMSO) or vehicle was infused into the right lateral ventricle 
during a 5-10 min period immediately following kainate injection. Rats 
were killed 48 hr later and perfused transcardially with 4% 
paraformaldehyde. Brain sections (30 .mu.m) were stained with cresyl 
violet and stained neurons in three adjacent 40X microscope fields of 
hippocampal region CA3 were counted (5 sections/rat) . The neuronal counts 
were performed in sections removed approximately 200-400 .mu.m from the 
injection site. This method results in highly reproducible, 75-99%, 
neuronal loss in the CA3 region in over 99% of the rats injected with 
kainate (calculations based on 302 rats receiving intra-hippocampal 
kainate). Stereotaxic injection of a convulsant dose of kainate into 
region CA1 of the hippocampus resulted in selective damage to neurons in 
region CA3 evident 48 hr following administration (FIG. 2). Infusion of 
cytochalasin D into the lateral ventricles immediately following injection 
of kainate resulted in a highly significant reduction in neuronal injury 
to CA3 neurons which was related to the dose of cytochalasin D (FIG. 2). 
Approximately 80% of the neurons were killed by kainate in control animals 
receiving an intracerebroventricular infusion of saline immediately 
following kainate administration. 
The present data indicate that the cytoskeleton plays an active role in 
modulating potentially neurotoxic elevations of Ca.sup.2+ !.sub.i and 
demonstrates that compounds that affect actin polymerization prove useful 
in alleviating neuronal injury and death in a variety of neurodegenerative 
conditions. 
EXAMPLE 7 
Intraventricular infusion of cytochalasin D immediately following injection 
of kainate resulted in a highly significant reduction in neuronal injury 
to CA3 neurons which was related to the dose of cytochalasin D (FIG. 3). 
Only 20% of the neurons were damaged by kainate in rats receiving 0.1 or 
1.00 .mu.g of cytochalasin D. 
EXAMPLE 8 
Cytochalasin D (10-1000 nM) caused a concentration-dependent reduction in 
phalloidin staining in neurons and loss of actin stress fibers in 
astrocytes, demonstrating its predicted disruptive effect on 
microfilaments (FIG. 4). Exposure of cultured neurons to increasing 
concentrations of glutamate also resulted in a concentration-dependent 
reduction in levels of phalloidin fluorescence (FIG. 4B). Glutamate did 
not reduce phalloidin staining in cultures incubated in Ca.sup.2+ -free 
medium indicating that Ca.sup.2+ influx was required for the 
actin-depolymerzing action of glutamate (FIG. 4B). 
EXAMPLE 9 
Glutamate caused a dose-dependent reduction in neuronal survival during a 
20-h exposure period with a maximum killing of over 80% of the neurons 
with a glutamate concentration of 100 .mu.M and an ED.sub.50 of 
approximately 35 .mu.M (FIG. 5A). Exposure of cultures to cytochalasin D 
(1-1000 nM) prior to exposure to glutamate resulted in a highly 
significant dose-dependent increase in neuronal survival compared to 
parallel control cultures exposed to glutamate (FIG. 5A). The 
concentrations of cytochalasin D that were most effective in protecting 
neurons against glutamate toxicity were 10 and 100 nM, with higher 
concentrations not providing a further increase in neuronal survival. The 
protection against glutamate neurotoxicity afforded by cytochalasin D was 
most striking in cultures exposed to submaximally toxic concentrations of 
glutamate (e.g., 50 .mu.M; FIG. 5B). Whereas 50 .mu.M glutamate killed 
over 70% of the neurons in control cultures, fewer than 10% were killed in 
cultures pretreated with 100 nM cytochalasin D and then exposed to 50 
.mu.M glutamate (FIG. 5B). Cytochalasin D (10-100 nM) alone had no 
significant effect on neuronal survival during a 20-h exposure period 
(FIG. 5B). 
EXAMPLE 10 
Cytochalasin E also afforded significant protection against glutamate 
toxicity (FIG. 6A). The microtubule-disrupting agent colchicine did not 
protect neurons against glutamate toxicity (FIG. 6A), indicating that 
cytoskeletal disruption, in general, is not excitoprotective. If actin 
depolymerization was excitoprotective, then exposure of neurons to an 
agent that blocks microfilament depolymerization should increase 
vulnerability to glutamate toxicity. In order to test this prediction 
cultures were preincubated for 3 h in the presence of 10 .mu.M 
jasplakinolide, a cyclic peptide which promotes actin polymerization, and 
then exposed to glutamate. Glutamate neurotoxicity was significantly 
potentiated in cultures treated with jasplakinolide (FIG. 6B), indicating 
that neurons are more vulnerable to excitotoxicity under conditions in 
which actin depolymerization does not occur. 
EXAMPLE 11 
Exposure of hippocampal cultures to increasing concentrations of A23187 
resulted in a concentration-dependent reduction in neuronal survival 
(100-1000 nM) with essentially all neurons being killed by 1 .mu.M A23187 
(FIG. 7). The concentration-response curve for A23187 neurotoxicity in 
cultures pretreated with 100 nM cytochalasin D was essentially identical 
to that in control cultures, indicating that actin depolymerization was 
ineffective in protecting neurons against a Ca.sup.2+ mediated insult 
that does not involve Ca.sup.2+ influx through endogenous plasma membrane 
ion channels. 
EXAMPLE 12 
Exposure of hippocampal cultures to increasing concentrations of FeSo.sub.4 
(10-100 nM) resulted in a concentration-dependent loss of neurons (FIG. 
8). Neuronal loss induced by iron was not significantly altered in 
cultures pre-treated with 100 nM cytochalasin D, suggesting that the 
mechanism whereby cytochalasins protected against excitotoxicity did not 
involve an antioxidant effect. In contrast to cytochalasin D, two 
well-known antioxidants, vitamin E and nordihydro-guaiaretic acid 
completely blocked the neurotoxicity of iron (FIG. 8). 
EXAMPLE 13 
In order to determine whether glutamate-induced disruption of actin 
filaments was involved in reducing Ca.sup.2+ !.sub.i responses to 
glutamate, cultures were pretreated with the actin filament-stabilizing 
agent jasplakinolide and then exposed to glutamate. Glutamate-induced 
elevation of Ca.sup.2+ !.sub.i was potentiated in neurons pretreated with 
jasplakinolide compared to control cultures (FIG. 9) 
EXAMPLE 14 
In contrast to its ability to suppress glutamate-induced elevation of 
Ca.sup.2+ !.sub.i, cytochalasin D did not attenuate the Ca.sup.2+ 
!.sub.i response to 500 nm calcium ionophore A23187 (FIG. 10), suggesting 
that actin depolymerization specifically affects influx through plasma 
membrane channels as opposed to enhancement of Ca.sup.2+ extrusion or 
buffering. Colchicine (100 nM) did not attenuate Ca.sup.2+ !.sub.i 
responses to glutamate. 
EXAMPLE 15 
Cells were exposed to 100 nM cytochalasin D for 1 h, membranes were 
permeabilized, and then cells were stained with fluorescein-labeled 
phalloidin. Cells were examined by confocal laser scanning microscopy and 
images of optical sections through neurons are shown in FIG. 11. In 
control cultures not exposed to cytochalasin D, intense phalloidin 
fluorescence was present in neurons where it appeared to be concentrated 
in the vicinity of the plasma membrane. In addition, growth cones stained 
intensely with phalloidin. Cytochalasin D caused a pronounced reduction in 
phalloidin staining in neurons, indicating loss of actin filaments (FIG. 
11). 
EXAMPLE 16 
Exposure of cultured rat hippocampal neurons to increasing concentrations 
of A.beta.25-35 resulted in a concentration-dependent reduction in 
neuronal survival during a 24-h incubation period (FIG. 12A). A.beta. 
neurotoxicity was significantly attenuated in parallel cultures pretreated 
with 100 nM cytochalasin D. The amount of protection against A.beta. 
neurotoxicity conferred by cytochalasin D was related to the concentration 
of cytochalasin D used. Significant protection occurred with 10 and 100 nM 
cytochalasin (FIG. 12B). 
EXAMPLE 17 
Exposure of neurons to both A.beta. and glutamate resulted in significantly 
more neuronal death than in cultures exposed to either insult alone (FIG. 
13). Cytochalasin D attenuated significantly neurotoxicity induced by 
combined exposure to A.beta. and 20 .mu.M glutamate (FIG. 13). 
EXAMPLE 18 
Cytochalasin E, another member of the cytochalasin family that selectively 
disrupts actin, attenuated A.beta. neurotoxicity significantly (FIG. 14). 
In contrast, the microtubule-disrupting agent colchicine, at a 
concentration known to disrupt microtubules, did not protect neurons 
against A.beta. toxicity (FIG. 14). 
EXAMPLE 19 
Exposure of hippocampal cultures to increasing concentrations of hydrogen 
peroxide resulted in a concentration-dependent reduction in neuronal 
survival during a 20-h exposure period (FIG. 15). Neuronal killing by 
hydrogen peroxide was not altered in cultures pretreated with 100 nM 
cytochalasin D, suggesting that this cytochalasin had little or no 
antioxidant activity. The neurotoxicity of hydrogen peroxide was largely 
prevented by pretreating cultures with the antioxidant vitamin E (FIG. 
15), indicating that the toxicity of hydrogen peroxide was mediated 
largely by reactive oxygen species. 
EXAMPLE 20 
In control cultures, rest Ca.sup.2+ !.sub.i was .about.100 nM (FIG. 16A). 
Exposure of hippocampal cultures to 50 .mu.M A.beta. resulted in a 
significant increase in rest Ca.sup.2+ !.sub.i to .about.280 nM after 3 h 
of exposure and .about.440 nM after 6 h (FIG. 16A) In parallel cultures 
pretreated with 100 nM cytochalasin D for 1 h and then exposed to 50 .mu.M 
A.beta. for 3 and 6 h., the rest Ca.sup.2+ !.sub.i was not significantly 
elevated (FIG. 16A). We therefore determined whether cytochalasin D would 
affect the enhancement of Ca.sup.2+ !.sub.i responses to glutamate in 
neurons pretreated with A.beta.. As expected, the neuronal Ca.sup.2+ 
!.sub.i response to glutamate (50 .mu.M) was greatly enhanced in cultures 
pretreated with A.beta. for 3 h; Ca.sup.2+ !.sub.i rose rapidly to &gt;1,500 
nM compared with an increase to .about.600 nM in neurons in untreated 
control cultures (FIG. 16B) Cytochalasin D suppressed the A.beta.-induced 
enhancement of the Ca.sup.2+ !.sub.i response to glutamate. In contrast 
to its ability to suppress Ca.sup.2+ !.sub.i responses to A.beta. and 
glutamate, cytochalasin D did not attenuate the Ca.sup.2+ !.sub.i 
response to 500 nM calcium ionophore A23187 (data not shown), suggesting 
that the major action of cytochalasin D was a reduction in calcium influx 
rather than an enhancement of calcium extrusion or buffering. DETAILED 
DESCRIPTION OF THE DRAWINGS 
FIG. 1. Cytochalasin D attenuates Ca.sup.2+ !.sub.i represents a 
glutamate, A.beta., and membrane depolarization in cultured hippocampal 
neurons. 
(A) Cultures were pretreated for 1 hr with 100 nM cytochalasin D and then 
Ca.sup.2+ !.sub.i in neurons was monitored prior to and following 
exposure to 50 .mu.M glutamate. The records represent the mean Ca.sup.2+ 
!.sub.i in 8-12 neurons. Similar results were obtained in 4 separate 
experiments. 
(B) A.beta.-induced potentiation of Ca.sup.2+ !.sub.i response to 
glutamate is abrogated by cytochalasin D. Cultures were left untreated 
(Control) or were exposed to 20 .mu.M A.beta.25-35 for 6 hr or 
cytochalasin D plus A.beta.25-35 for 6 hr. The Ca.sup.2+ !.sub.i was then 
monitored prior to following exposure to 50 .mu.M glutamate. Values 
represent the means of 8-14 neurons. Similar results were obtained in 3 
separate experiments. 
(C) Cultures were pretreated for 3 hr with 50 .mu.M A.beta. alone or in 
combination with 100 nM cytochalasin D. The Ca.sup.2+ !.sub.i in neurons 
was then monitored prior to and following exposure to 50 mM KCl. Values 
represent the mean of 14-23 neurons. Similar results were obtained in a 
separate experiment. 
(D) Cultures were pretreated for 1 h with 100 nM cytochalasin D. The 
Ca.sup.2+ !.sub.i in neurons was measured immediately prior to, and at 5 
min following, exposure to the indicated concentrations of glutamate. 
Values represent the means and SEM of determinations made in 17-26 
neurons. *P&lt;0.05 (10 .mu.M glutamate), P&lt;0.02 (50 .mu.M glutamate), P&lt;0.01 
(100 .mu.AM glutamate) compared to corresponding control values. 
FIG. 2. Cytochalasin D protects hippocampal neurons against kainate 
toxicity in vivo. 
Cresyl violet-stained coronal sections from brains of rats administered 
kainate alone (left) of kainate plus cytochalasin D (CyD; right). Note 
damage to CA3 neurons induced by kainate, and marked reduction in the 
damage in the rat receiving cytochalasin D (arrows). Lower micrographs are 
high magnification of a region of CA3 from the sections shown in the upper 
panels. 
FIG. 3. Counts of viable neurons were made in region CA3 of Sham-operated 
rats, and rats infused intraventricularly with vehicle or increasing 
concentration of cytochalasin D prior to unilateral kainate injection into 
region CA1 of the hippocampus. Neuronal survival in the kainate injected 
hippocampus is expressed as a percentage of neurons in the contralateral 
hippocampus. Values represent the mean and SEM (4 or 5 rats/group). 
*p&lt;0.01 compared to value for sham rats. **p&lt;0.01 compared to value for 
vehicle-infused rats. (ANOVA with Scheffe's post-hoc test). 
FIG. 4. (A) Micrographs are confocal laser scanning microscope images of 
cultured hippocampal cells stained with fluorescein-labeled phalloidin. 
Cells were exposed for 3 h to either vehicle (control; left) or 100 nM 
cytochalasin D (right) prior to staining with phalloidin. The top 
micrographs show several neurons (arrowheads point to neuronal cell 
bodies); note reduction in phalloidin fluorescence in cytochalasin-treated 
neurons. The bottom micrographs show astrocytes; note intense staining of 
stress fibers in control astrocytes (e.g., arrowhead) and loss of stress 
fibers and cell rounding in cytochalasin-treated astrocytes. 
(B) Cytochalasin D and glutamate cause a reduction in neuronal phalloidin 
fluorescence. Parallel cultures were exposed to vehicle (control), 
cytochalasin D (10, 100, and 1000 nM), or glutamate (50, 100, and 200 
.mu.M) for 3 h. Additional cultures were incubated in Ca.sup.2+ -free 
medium (no added Ca.sup.2+ plus 1 mM ETGA) and exposed to cytochalasin D 
or glutamate for 3 h. Cells were then fixed and stained with 
fluorescein-labeled phalloidin. Values represent the mean fluorescence 
intensity per neuronal cell body (.+-.SEM; n=15-18). The reduction in 
phalloidin fluorescence in neurons exposed to increasing concentrations of 
cytochalasin D and glutamate were highly significant (P&lt;0.0001 by ANOVA). 
FIG. 5. Cultures were exposed to the indicated treatments for 20 h. 
Cytochalasin D was added to cultures 30 min prior to exposure to 
glutamate, and control cultures were exposed to 0.2% dimethylsulfoxide. 
(A) Neuronal survival was quantified in cultures that had been pretreated 
with the indicated concentrations of cytochalasin D and then exposed to 50 
.mu.M glutamate for 20 h. Values represent the means and SEM of 
determinations made in four separate cultures. The overall effect of 
cytochalasin D in increasing neuronal survival was highly significant 
(P&lt;0.0001 by ANOVA) . Pairwise statistical comparisons of 
cytochalasin-treated cultures with the control survival (Scheffe's test) 
indicated 1 nM cytochalasin D, P&lt;0.05; 10 nM cytochalasin D, P&lt;0.001; 100 
and 1000 nM cytochalasin D, P&lt;0.0001. 
(B) Neuronal survival was quantified in cultures that had been pretreated 
with either vehicle (control) or 100 nM cytochalasin D and then exposed to 
the indicated concentrations of glutamate for 20 h. Values represent the 
means and SEM of determinations made in four separate cultures. Neuronal 
survival was significantly greater in cultures pretreated with 
cytochalasin D and exposed to 10 .mu.M glutamate (P&lt;0.05), 50 .mu.M 
glutamate (P&lt;0.001), and 100 .mu.M glutamate (P&lt;0.02). ANOVA with 
Scheffe's post hoc test. 
FIG. 6. (A) Cultures were pretreated with 100 nM cytochalasin D (CyD), 100 
nM cytochalasin E (CyE), or 100 nM colchicine (Colch). Cultures were then 
exposed to 50 .mu.M glutamate for 20 h. Values for neuronal survival 
represent the means and SEM of determinations made in four separate 
cultures. *P&lt;0.001 compared to control value. **P&lt;0.001 compared to value 
for cultures exposed to glutamate alone. 
(B) Cultures were pretreated for 1 h with 5 .mu.M jasplakinolide or vehicle 
and then exposed for 20 h to the indicated concentrations of glutamate. 
Values represent the means and SEM of determinations made in four separate 
cultures. *P&lt;0.01 compared to value for cultures exposed to 20 .mu.M 
glutamate alone. **P&lt;0.05 compared to cultures exposed to 50 .mu.M 
glutamate alone. 
FIG. 7. Cultures were pretreated for 1 h with 0.2% dimethylsulfoxide 
(control) or 100 nM cytochalasin D. Calcium ionophore A23187 was then 
added at the indicated concentrations and neuronal survival was assessed 8 
h later. Values represent the means and SEM of determinations made in four 
separate cultures. 
FIG. 8. Cultures were preincubated 1 h in the presence of 0.5% 
dimethylsulfoxide (control), 100 nM cytochalasin D, 50 .mu.g/ml vitamin E 
(VitE), or 2 .mu.M nordihydroguairetic acid (NDGA). Cultures were then 
exposed for 6 h to the indicated concentrations of FeSO.sub.4 and neuronal 
survival was quantified. Values represent the means and SEM of 
determinations made in four separate cultures. *P&lt;0.001 compared to value 
for cultures exposed to 100 .mu.M FeSO.sub.4 alone. 
FIG. 9. Cultures were pretreated for 3 h with vehicle (control) or 10 .mu.M 
jasplakinolide. The Ca.sup.2+ !.sub.i was monitored prior to and 
following exposure to 50 .mu.M glutamate. Values represent the mean of 
determinations made in 10-12 neurons. Similar results were obtained in two 
additional experiments. 
FIG. 10. Cytochalasin D does not affect Ca.sup.2+ !.sub.i responses to 
calcium ionophore. Cultures were pretreated with 100 nM cytochalasin D or 
vehicle (control). The Ca.sup.2+ !.sub.i in neurons was then monitored 
prior to and following exposure to 500 nM calcium ionophore A23187. Values 
represent the mean of 12-20 neurons. Similar results were obtained in a 
separate experiment. 
FIG. 11. Micrographs are confocal laser scanning microscope images of 
cultured hippocampal cells stained with fluorescein-labeled phalloidin. 
Cells were exposed for 1 h to either vehicle (Control) or 100 nM 
cytochalasin D before fixation and staining with FITC-phalloidin. Each 
panel shows several neurons. Note intense fluorescence in peripheral 
regions of cell bodies (e.g., arrowheads) and in growth cones (arrow) in 
control cultures and loss of fluorescence in cytochalasin-treated neurons 
(arrowheads point to edge of neuronal cell bodies). 
FIG. 12. (A) Cultures were pretreated for 1 h with vehicle 0.2% dimethyl 
sulfoxide (Control) or 100 nM cytochalasin D (CyD) and were exposed to the 
indicated concentrations of A.beta.25-35 for 48 h. Data are mean and SEM 
values of determinations made in four separate cultures. Neuronal survival 
was significantly increased in cytochalasin D-treated cultures exposed to 
20 .mu.M A.beta. (p&lt;0.05) and 50 .mu.M A.beta. (p&lt;0.01). 
(B) Cultures were pretreated for 1 h with the indicated concentrations of 
cytochalasin D and then exposed to 50 .mu.M A.beta.25-35; parallel 
cultures were exposed to cytochalasin D alone (Control) . Data are mean 
and SEM values of determinations made in four separate cultures. Neuronal 
survival was significantly increased in A.beta.-treated cultures exposed 
to 10 or 100 nM cytochalasin D (p&lt;0.01). 
FIG. 13. Cultures were exposed to 50 .mu.M A.beta. alone; 20 .mu.glutamate 
alone; 50 .mu.M A.beta. plus 20 .mu.M glutamate; or 100 nM cytochalasin D 
plus 50 .mu.M A.beta. plus 20 .mu.M glutamate. Neuronal survival was 
assessed 48 h after treatment. Data are mean and SEM values of 
determinations made in four separate cultures. *p&lt;0.05, compared with 
value for cultures exposed to A.beta. alone; and p&lt;0.01, compared with 
cultures exposed to glutamate alone. **p&lt;0.01, compared with value for 
cultures exposed to A.beta. plus glutamate. 
FIG. 14. Cultures were exposed to 100 nM colchicine; 50 .mu.M A.beta.; 100 
nM colchicine plus 50 .mu.M A.beta.; 100 nM cytochalasin E (CyE) plus 
A.beta. (colchicine and cytochalasin E were added 1 h before A.beta.); 
neuronal survival was assessed 48 h later. Data are mean and SEM values of 
determinations made in four separate cultures. *p&lt;0.01, compared with 
values for control or colchicine alone. **p&lt;0.01, compared with value for 
cultures exposed to A.beta. alone. 
FIG. 15. Cytochalasin D does not protect neurons against hydrogen peroxide 
toxicity. Cultures were pretreated for 1 h with vehicle (Control), 100 nM 
cytochalasin D (CyD), or 50 .mu.g/ml vitamin E (vE) and then exposed for 
20 h to the indicated concentrations of hydrogen peroxide. Data are mean 
and SEM values of determinations made in four separate cultures. 
FIG. 16. Cytochalasin D attenuates A.beta.-induced elevation of rest 
Ca.sup.2+ !.sub.i and potentiation of Ca.sup.2+ !.sub.i response to 
glutamate in cultured hippocampal neurons. 
(A) Cultures were exposed to the indicated treatments and Ca.sup.2+ 
!.sub.i was determined in neurons at 3 and 6 h after treatment. CyD, 
cytochalasin D (100 nM; added 1h before exposure to A.beta.); Abeta (50 
.mu.M A.beta.25-35). Data are mean and SEM values of determinations made 
in 17-24 neurons in three separate cultures per time point. 
(B) A.beta.-induced potentiation of Ca.sup.2+ !.sub.i response to 
glutamate is abrogated by cytochalasin D. Cultures were left untreated 
(Control) or were exposed to 20 .mu.M A.beta.25-35 for 6 h or cytochalasin 
D plus A.beta.25-35 for 6 h. The Ca.sup.2+ !.sub.i was then monitored 
before and after exposure to 50 .mu.M glutamate (glutamate was added at 
the time point indicated by the arrow). Data are mean values of eight to 
14 neurons. Similar results were obtained in three separate experiments. 
The purpose of the above description and examples is to illustrate some 
embodiments of the present invention without implying any limitation. It 
will be apparent to those of skill in the art that various modifications 
and variations may be made to the composition and method of the present 
invention without departing from the spirit or scope of the invention. All 
patents and publications cited in the bibliographic citation below are 
incorporated by reference in their entireties. 
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