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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 6, 2001 as doi:10.1096/fj.00-0719fje. |
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Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan
3Correspondence: Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan. E-mail: tnabeshi{at}med.nagoya-u.ac.jp
SPECIFIC AIMS
In this study, we investigated the hypothesis that a chronic intracerebroventricular (i.c.v.) infusion of amyloid ß-peptide (Aß) into rat can stimulate nitric oxide (NO) production, which may contribute to neurotoxicity in vivo. We also addressed whether inappropriate production of NO is involved in the functional consequences of Aß-induced brain dysfunction: the cholinergic impairment and the memory deficit.
PRINCIPAL FINDINGS
1. Chronic i.c.v. infusion of Aß1–40 induces expression of the
inducible nitric oxide synthase (iNOS) mRNA in rat hippocampus
The i.c.v. infusion of Aß1–40 (300 pmol/day) induced a
time-dependent expression of iNOS mRNA as analyzed by reverse
transcription-polymerase chain reaction (RT-PCR). In naive rats and
control rats infused with Aß40–1, the specific bands of iNOS cDNAs
at 234 bp were almost undetectable whereas in Aß1–40-infused rats
there were specific bands, as expected (Fig. 1A
). The specific bands of iNOS cDNAs visualized by agarose
gel electrophoresis were quantified relative to that of ß-actin. One
day after the start of Aß infusion, an increase in iNOS gene
expression was observed in both Aß1–40-infused rats and the
Aß40–1-infused control group. On days 3 and 5 after Aß infusion, a
significant increase in iNOS expression was evidenced in rats receiving
Aß1–40, indicating that infusion of the active peptide induced iNOS
expression in vivo. On day 7, expression of iNOS returned to the basal
level (Fig. 1B
).
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The nitrite and nitrate levels in the hippocampus were also increased on day 3 and day 5 after Aß infusion and correlated well with the iNOS mRNA expression data. Measurement of NOS activity on days 3 and 5 after Aß infusion further revealed a pronounced increase in Ca2+-independent, but not Ca2+-dependent, NOS activity in Aß1–40-infused rats when compared with control rats. Since iNOS activity is Ca2+ independent, these findings indicated that iNOS is continuously active to produce a large amount of NO. Immunohistochemical staining with mouse monoclonal antibody against iNOS also identified the localization in the dentate gyrus and, to a lesser extent, in the CA1 subfield of iNOS-immunoreactive cells. Double immunostaining with specific antibodies against iNOS and markers for microglia and astrocytes further revealed that iNOS was expressed in both microglia and astrocytes.
2. Treatment with iNOS inhibitors prevents an impairment of
nicotine-evoked acetylcholine release in the brain of
Aß-infused rats
It is likely that the overproduction of NO is involved in
Aß-induced brain dysfunction. First, we examined the effects of
various Aß fragments on nicotine evoked-acetylcholine (ACh) release
in vivo by a microdialysis technique. Continuous infusion of neurotoxic
Aß fragments, including Aß1–42, Aß1–40 at 300 pmol/day and
Aß25–35 at 3 nmol/day, for 10 days resulted in a significant
attenuation in nicotine-evoked ACh release as compared with naive rats
or rats receiving either vehicle or the nontoxic reverse fragment
Aß40–1, indicating that Aß impaired nicotine-evoked ACh release.
To assess the involvement of NO in the impairment of the
nicotine-evoked ACh release after Aß infusion, rats were injected
daily with a single dose of the iNOS inhibitor aminoguanidine [AG, 100
mg/kg, intraperitoneal (i.p.)], S-methylisothiourea (SMT, 10 mg/kg,
i.p.), or the neuronal NOS (nNOS) inhibitor 7-nitroindazole (7-NI, 30
mg/kg, i.p.) for 10 days. Daily treatment with AG or SMT recovered the
impairment of nicotine-evoked ACh release without affecting the basal
release. Coadministration of L-arginine (200 mg/kg, i.p.), a NO
precursor, eliminated the recovery of nicotine-evoked ACh release
induced by AG, thus providing evidence that iNOS inhibitors are able to
ameliorate the Aß-induced impairment of nicotine-evoked ACh release
in vivo. The nNOS inhibitor 7-NI failed to prevent the
impairment of nicotine-evoked ACh release.
3. The iNOS inhibitor AG prevents memory impairment induced by Aß
The effect of Aß1–40 on spatial reference and working memory
was assessed with a radial eight-arm maze. Working memory error was
measured by counting the number of entries into an arm containing food
but previously visited whereas reference memory error was assessed by
the entry into unbaited arms. The time spent in the maze to collect
baits was also measured as an index of locomotor function.
An ANOVA with repeated measures of total number of errors among the
three groups revealed significant effects of group
(F(2,21)=6.431; P<0.01),
trial (F(4,84)=54.514;
P<0.0001), but not group by trial interaction
(F(8,84)=0.735; P=0.66).
Post hoc analysis with the Bonferroni test indicated that Aß1–40
significantly impaired spatial memory formation (P<0.01),
which was prevented by treatment with AG (P<0.005)
(Fig. 2A
). No difference in time spent per trial in the arms was
found among groups (Fig. 2B
), indicating that the effects of
Aß1–40 and AG on maze performance are not due to alteration of
locomotor function.
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To elucidate which memory category was impaired by Aß, total memory
errors were divided into working (Fig. 2C
) and reference
(Fig. 2D
) memory errors. Repeated-measure ANOVA indicated
significant effects of group on both working memory
(F(8,81)=4.481; P<0.05)
and reference memory (F(8,81)=6.834;
P<0.01, Fig. 2D
) errors. Post hoc analysis with
the Bonferroni test showed that working (P<0.05) and
reference (P<0.05) memory formation was significantly
impaired by Aß1–40 and that AG significantly prevented Aß-induced
impairment of working (P<0.05) and reference memory
(P<0.002).
CONCLUSIONS AND SIGNIFICANCE
Here we have demonstrated that continuous infusion of Aß1–40 into rat cerebral ventricle induced a time-dependent expression of iNOS in the dentate gyrus and, to a lesser extent, in the CA1 subfield of the hippocampus. The expression of iNOS in both microglia and astrocytes after Aß infusion suggests that iNOS is induced in non-neuronal cells and subsequently activates microglia and/or astrocytes. After the iNOS expression, Ca2+-independent NOS activity and NO production in the hippocampus also increased in the same pattern, further supporting the finding that Aß stimulates NO production.
Previously we had demonstrated that infusion of Aß1–40 impairs nicotine-evoked ACh and dopamine release in the frontal cortex/hippocampus and the striatum, respectively. In the present study, infusion of the various Aß fragments 1–40, 1–42, or 25–35 also impaired nicotine-evoked ACh release., thus confirming that Aß impairs signal transduction via the nicotinic ACh receptors. It has been reported that Aß binds to scavenger receptors on microglia and may induce cell death by generating free radicals, including NO. Evidence is accumulating that an inappropriate formation of NO synthesized by iNOS may cause neurotoxicity. In our experiments, iNOS inhibitors such as AG and SMT reversed the impairment of nicotine-evoked ACh release, implicating the involvement of NO in the Aß-induced dysfunction of nicotinic ACh receptor signaling and the ameliorating effects of iNOS inhibitors. In contrast to iNOS inhibitors, the nNOS inhibitor 7-NI failed to recover nicotine-evoked ACh release. Although only one nNOS inhibitor was used, the result seems to be consistent with the fact that Ca2+-dependent activities did not change. Accordingly, it is unlikely that NO synthesized by nNOS participates in the Aß-induced cholinergic dysfunction under our experimental conditions. The mechanism of NO involvement in this regard needs to be defined; however, the formation of peroxynitrite and/or the nitration of synaptic proteins may affect signal transduction pathways of cellular regulation.
It has been believed that cholinergic dysfunction is one of the primary causes of cognitive deficits in Alzheimer’s disease patients. We have shown here that continuous i.c.v. infusion of Aß1–40 impaired the spatial reference and working memory performance in the radial eight-arm maze. It is interesting that AG, an iNOS inhibitor that is able to recover the impairment in nicotine-evoked ACh release, also exerts an ameliorating effect on memory performance. Thus, it may be suggested that NO synthesized by iNOS contributes to the toxic effect of Aß on brain function.
In conclusion, we suggest that chronic i.c.v. infusion of Aß1–40
induces iNOS expression in vivo that produces neurotoxic levels of NO
and results in the cholinergic signaling dysfunction and memory
impairment. Treatment with iNOS inhibitors can ameliorate cholinergic
dysfunction and the memory performance, thus implying a therapeutic
strategy for Alzheimer’s disease (Fig. 3
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0719fje ; to cite this
article, use FASEB J. (April 6, 2001) 10.1096/fj.00-0719fje 
2 These authors contributed equally to this work.
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