Neurotrophic factor therapy of Alzheimer's disease

FOURTH INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE

the end of weeks 1,2, and 3 of haloperidol treatment. Baseline behavior ratings (BEHAVE-AD)indicated that 97% of patients had activity disturbances, 97% had aggressiveness, 62% had anxieties and phobias, 62% had paranoid and delusional ideation, 59% had diurnal rhythm disturbances, 28% had affective disturbances, and 17% had hallucinations. Although no significant correlations were apparent between clinical improvement (% change BEHAVE-AD) and blood levels of haloperidol, a good response (2 30% improvement) was observed in 55% of the patients who entered the study, At the end of the third week of treatment, these patients had plasma haloperidol levels ranging from 0.3 to 1.8 &ml and red blood cell haloperidol levels ranging from 0.3 to 1.9 ng/ml. Future studies with slightly higher and lower dosages of haloperidol will be used to further define the optimal blood level range for maximal treatment benefit.

induce hypettrophy of the surviving cholinergic neurons and not a replacement therapy. By attenuating the rate of degeneration and improving the function of the remaining choliuergic cells, it is expected to ameliorate the symptomatic behavioral

changes

which

arc due to the cholinergic

deficit.

Limited

clinical

trials

have been initiated with encouraging result. Biotechuically engineered pluripotent growth factors or combinations of factors, possibly including the NGF relatives neurotrophin-3 and neurotrophiu-4/5, may be necessary to obtain optimal behavioral effects.

POSTER SESSION III APP Biology

335 EXPERIMENTAL

TROPHIC

PHENOTYPE

THERAPY.

PROTECTION

OF

CHOLlNERGlC

337

ANDCORTICALSYNAPTICREMODELLING.

A. Claudia

Cuello,

Department

of Pharmacology&Therapeutics,

McGill

University,

Montreal,

AGGREGATION OF ENDOGENOUS SECRETED AMYLOID BETA PEPTIDE (Ap) INTO SDS-STABLE MULTIMERS. M.B. Podlisny, E. Koo,

Quebec,

Canada. NGF phenotype

and some in

Experimental beyond

evidence

would

choline

infarctions. neocortex.

Thus.

cholinergic

microscopic

could

in CNS,

of cortically

NGF

injured

(1992)

results

Natl.

an

functional treated therapy

A.C.,

(1993)

Acad.

Sci.,

with

high

cortical

demonstrates

remodelling”

in the

These

a doseand

unilateral

of the

number

of presynaptic NGF.

injury. actions

provokes activity

analysis

and behavioural

cortical

boutons.

These

changes

observed

observations

will

be

rn AD.

Progr. USA.,

Neuroscience, 57(l): 21-40; Garofalo, Neurol., (in press). (1993)

image

increase

and hypertrophy

animals

NGF

of rats bearing with

cholinergic

can have

enzymatic

by a “synaptic

in

CNS following

factors

Thus,

studies combined

of a possible

Cuello, Proc.

cortex

are accompanied

therapy

to protect animals

neurotrophic

phenotype.

the remarkable

in the context

adult

acetyltransferase

and synapses

explain

References: L. &al.,

changes

of

that

in the remaining

varicosities

changes

indicate

of choline

uptake

Electron

have been shown

neurons

of cbolinergic

up-regulation

that these biochemical

discussed

neurotrophins forebrain

the maintenance

dependent affinity

some

Br.

Res.,

98: 265-277;

Garofalo,

89: 2639-2643;

Garofalo,

L.

A.C.,

and Cuello,

L. etal.,

(1994)

Expl

Acknowledeements: Medical Research Council of Canada (MRC)

336 NEUROTROPHIC FACTOR THERAPY OF ALZHEIMER’S DISEASE. F. Hefti. Dept. of Neuroscience, Genentech. Inc., South San Francisco, CA 94080 Neurotrophic factors are important for development and maintenance of function of the adult nervous system. Administration of neurotrophic factors can attenuate age-related and experimentally induced degeneration and behavioral deficits in experimental animals, suggesting that these molecules may become important in the treatment of neurodegeneration. Among the best characterized neurotrophtc factors are NGF, which may be useful in the treatment of peripheral sensory neuropathy and Alzheimer’s disease, and CNTF, which may be beneficial in ALS. Effective neumtroohic factors have recently been discovered for douaminereic neurons and motorue~rons. Various strategiescan be pursued to develop thera&eutically aoolicable molecules which mimic neurotrouhic factor actions or stimulate their production or receptor mechanisms. In Alaheimer’s disease, there is region-specific loss of neuronal populations, particularly in areas connected to the hippocampus. Strongly affected are hippo&pal inputs from the entorhinal cortex, the cholinereic basal forebrain and the noradtenereic lccus cceruleus. The extent of the chol6ergic deficit, which is one of the earlTestchanges in AD, correlates well with the degree of memory impairment observed in individual patients. NGF is necessary for normal development of forebrain cholinergic neurons and these cells respond to NGF not only during development but throughout their entire lifespan. There am deIlcits in cholinergic systems in mutant mice lackiug functioual NGF genes. Animal experiments indicate that NGF preventscholinergic degeneration asauciated withnormalagingor withneuronalinjury.Jnadultrats withtransections of theaepto-hippccampal pathways,NGF administration

reverses degenerative changes of the choliuergic celI bodies. NGF treatment also iucreases the function of presynaptic cholinergic terminals, prevents postsynaptic cholinergic receptor supersensitivity, and improves the behavioral performance of partially lesioned or selected behaviorally impaired aged rats. Similar findings have been obtained in primates. The cause of the cholmergic decline in AD remains to be identified. NGF levels am normalin AD brains,however,anyotherstepin NGF relatedmechanisms couldbe compromised. The animalstudies suggest that NGF administration will

counteract cholinergic atrophy, irrespective of the actual cause. Thus, administration of NGF to AD patients represents a pharmacologic attempt to

S. Squazzo, D.B. Teplow and D.J. Selkoe. Center for Neurologic Diseases, Harvard Med. Sch. & Brigham & Women’s Hosp., Boston, MA 02115 USA. Extensive studies with synthetic Ag peptides have demonstrated that 1) peptide length and concentration are critical to the aggregation state of Ag; 2) the association of exogenous proteins or metals can affect the aggregation of Ag; and 3) aggregated Ag, but not soluble monomeric Ag. can be directly and/or indirectly toxic to neurons in culture. Because soluble Ag is constitutively secreted by normal cells in viva and in vim , and because aggregated, polymeric Ag is deposited in the cortex and blood vessels in AD, we looked for evidence of aggregation of endogenous secreted Ag under physiological conditions in culture. CHO cells transfected with PAP451 (either wildtype or mutant) that expressed high levels of gAPP were metabolically labeled overnight. The conditioned media (CM) were then immuno recipitated with Rl280 (raised to synthetic Agt.40) and electrophoresed in d DS-tncme ‘. gels. Autoradiography revealed the expected intense bands at 4 kD (A@ and 3 kD (p3), plus weaker bands at -6, 8 and 12 kD. All of these bands were also immunoprecipitated by other Afi antisera and were fully absorbable by synthetic Ag peptide. Antisera to 8APP regions flanking A8 did not precipitate these bands. 3H-phenylalanine radiosequencing of the gel-purified 8 kD band showed phenylalanines at positions 4, 19 and 20, directly confirming its identity as an Ag multimer (presumably a dimer). Additional phenylalanine peaks consistent with an Ag species beginning at arg 5 were also found in this band. To examine the possibility that the Ag aggregation occurred during immunoprecipitation or sample boiling in SDS, the precipitated samples were eluted from Protein A-sepharose with glycine and electrophoresed without heating; in this case, the same multimers were observed. Moreover, the same immunoprecipitation protocol failed to precipitate mummers of either synthetic Agt.40 [at a concentration (5 nM) similar to that of endogenous Ap] or endogenous A!.3from high Ag-secreting 293 cells transfected with gAPPg95. Prolonged incubation of CHO75I CM at 37’ led to increased aggregation of endogenous Ag. In conclusion, SDS-stable multimers of endogenous Ag form spontaneously in tissue culture media. This system can be used to examine factors which regulate aggregation of heterogeneous Ag peptides secreted by living cells at nanomolar concentrations, thus avoiding the concerns of studies of a single Ag synthetic peptide of defined length incubated at high doses under non-physiological conditions.

338 THE INHIBITORY EFFECT OF /3-AWYLOID PEPTIDE ON RAT BRAIN NAJCA EXCHANGE. Anfan Wu and Robert A. Colvin. Dept. Biological Sciences, Ohio University College of Osteopathic Medicine, Athens, OH 45701 USA. These studies were performed to determine the effect of addition of Alaheimer’s disease fl-amyloid peptide (#-AP) on Nat/Cal+ exchange activity. disease (AD) is characterized by certain Alzheimerrs

neuropathological lesions, including deposition of @AP, however, its pathogenic role is still not clear. Na+/Ca'+ exchange activity was measured in plasma membrane vesicles (PWV) purified from rat brain. The effect of synthetic peptide ~+IP~~ and scrambled (#AP,) were examined. PMV were preincubated with each peptide for 15 minutes(37°C) in buffer containing 137 arM NaCl, 10 mW HEPES(pH 7.4), and protease inhibitors. Ca'+ uptake was initiated by diluting PWv 20 fold with buffer containing either 137 mW NaCl or 137 mW cholinecl and %aCl. Caz* uptake was terminated by addition of 5 IUW LaCl, and rapid filtration. S-J%%35 inhibited Nai*-dependent Cat* uptake with an ICso of 800