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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 9, 2000 as doi:10.1096/fj.00-0528fje. |
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Mayo Clinic Jacksonville, Jacksonville, Florida 32224, USA
2Correspondence: Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA. E-mail: Eckman{at}mayo.edu
SPECIFIC AIMS
Thedeposition of Aß in the form of senile plaques in sufficient quantity is a pathological hallmark of Alzheimer’s disease. In this study we examine whether the phosphatidyl-inositol kinase inhibitor wortmannin can reduce Aß accumulation both in vitro and in vivo.
PRINCIPAL FINDINGS
1. Wortmannin reduces extracellular Aß accumulation in cell
culture
In cell-based screens, we identified wortmannin, a specific
inhibitor of phosphatidyl-inositol kinases, as an Aß-lowering agent.
Wortmannin treatment of CHO 2B7 cells results in a dose-dependent
reduction in Aß40 and Aß42. Both cell viability assays and washout
experiments indicate that this reduction in Aß accumulation is not
simply due to a toxic effect of the drug.
2. Wortmannin treatment reduces sAPP
and increases
carboxyl-terminal fragments of ßAPP
Aß is formed by the proteolytic cleavage of a larger precursor
molecule, referred to as ßAPP. Large secreted derivatives (sAPP) are
normally produced and secreted into the conditioned medium, whereas
carboxyl-terminal fragments of ßAPP (CTFs), some of which contain the
entire Aß sequence, are found within the cell. Consistent with a
previous report by Petanseska and Gandy, who used a different cell
line, we observed a reduction in the amount of sAPP in the medium
and an increase in the levels of CTFs in the cell after wortmannin
treatment. We interpret these data to suggest that wortmannin may be
influencing Aß accumulation in the extracellular milieu by altering
trafficking of the ßAPP. For example, the observed phenomenon would
be consistent with the sequestration of ßAPP in an intracellular
compartment where the secretase enzymes were still able to cleave, but
where the metabolites failed to be secreted. This is consistent with
the hypothesis advanced by Petanceska and Gandy and with findings by
others that wortmannin can influence trafficking of several other
proteins at different steps.
3. Twice-daily chronic oral administration of wortmannin results in
a decrease in Aß accumulation in young, nondepositing Tg2576 mice
Tg2576 mice, which express the familial AD-linked Swedish
mutation (ßAPPK670N/M671L) under control of the prion
promoter, accumulate extracellular Aß with increasing age and develop
plaques associated with dystrophic neurites and prominent gliosis.
One-month-old mice were dosed orally twice daily for a period of 4
months. At the conclusion of the experiment, brains were harvested and
analyzed for Aß accumulation by formic acid extraction and sandwich
enzyme-linked immunoassay (ELISA) as described previously. A
significant, nonoverlapping 52 ± 12% reduction in Aß40 and a
44 ± 7% reduction in Aß42 were observed in the treated group
when compared to the controls (P<0.05, Mann-Whitney).
4. Chronic oral wortmannin treatment results in a reduction in the
number and area occupied by senile plaques, one of the pathological
hallmarks of Alzheimer’s disease
To determine the effect of wortmannin on senile plaque formation,
we examined the effects of chronic oral treatment on Aß accumulation
in a second group of animals beginning at 4 months of age (still prior
to detectable plaque formation) and concluding 4.5 months later, at an
age where plaques become apparent. One-half of the brain was processed
for Aß quantitation by ELISA and one-half was prepared for
immunohistochemical analysis. Wortmannin treatment again resulted in a
significant nonoverlapping reduction in Aß40 (42±9%) and Aß42
(39±15%) in the older group of animals (P<0.05,
Mann-Whitney). The degree of reduction was such that the drug-treated
mice in the second treatment group, which were killed at 8.5 months of
age, had levels of Aß40 and Aß42 similar to those of the control
mice in the first experiment, which were killed at 
5 months of
age. Immunohistochemical analyses of the brains from these animals
showed that wortmannin treatment resulted in a significant,
nonoverlapping 52 ± 13% reduction in the number of plaques as
assessed with antibodies specific to Aß. Image analysis showed that
the total area occupied by these plaques was also reduced by 44 ±
14% in the drug-treated mice (Fig. 1
).
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CONCLUSIONS
Collectively, these data indicate that wortmannin treatment causes
a significant reduction in extracellular Aß accumulation in
vitro and a significant reduction in Aß accumulation, senile
plaque number, and the area occupied by senile plaques in
vivo in the Tg2576 animal model. Although more work is necessary
to determine the mechanism by which wortmannin reduces Aß
accumulation, the data presented in this manuscript, coupled with that
shown previously by Petanceska and Gandy, indicate that wortmannin may
be acting by influencing trafficking of ßAPP and/or its metabolites,
resulting in decreased secretion of the peptide. Identification of the
specific pathway(s) for these wortmannin-induced changes may yield
additional information regarding the processes involved in Aß
accumulation and may possibly generate novel therapeutic targets
(Fig. 2
).
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Recently, Schenk et al. and Lim et al. have reported significant reductions in plaque number and Aß accumulation in the brains of transgenic mice through either immunization with aggregated Aß1-42 or with high doses of ibuprofen, respectively. Coupled with the current study, these findings indicate that reductions in Aß accumulation and senile plaques can be observed in experimental animal models by targeting what appear to be different processes. The identification of multiple possible therapeutic targets such as these is important, as any one or more may have undesired side effects.
Although it is tempting to speculate that all of these strategies may be therapeutic, it is important to keep in mind that though there is considerable evidence arguing for a critical role for Aß in the etiology and pathogenesis of AD, the proof is not definitive. The ultimate test of the therapeutic usefulness of this or any other compound will be whether or not patients demonstrate a significant behavioral improvement and whether any side effects are tolerable. To this end, it is important to strive toward the development of animal models, or perhaps behavioral tests in the current models, that faithfully recapitulate at least some of the behavioral disturbances observed in AD.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0528fje To cite this article, use (November 9, 2000) FASEB J. 10.1096/fj.00-0528fje 
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