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* Department of Medical Chemistry, University of Szeged;
Department of Comparative Physiology, University of Szeged;
Department of Analytical and Inorganic Chemistry, University of Szeged;
Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences;
|| Department of Biology, Juhász Gyula College, University of Szeged; and
¶ Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary
1Correspondence: Department of Medical Chemistry, University of Szeged, Dóm Square 8, Szeged 6720, Hungary. E-mail: szegv{at}mdche.szote.u-szeged.hu
SPECIFIC AIMS
The central cause of Alzheimer’s disease (AD) is widely believed to be the aggregation of amyloid proteins (mainly Aßbeta;1–42); thus small, peptide-based aggregation inhibitor molecules [ßbeta;-sheet-breakers (BSBs)] may represent a promising, novel treatment of the disease. Here we investigated the biological effects of an endogenous tetrapeptide, endomorphin-2 (YPFF, End-2), the structure of which shows high similarity to Leu-Pro-Phe-Phe-Asp (LPFFD), a well-known BSB peptide, on the Aßbeta;1–42 induced biological effects and on the aggregation process by using transmission electron microscopy (TEM), quasielastic light-scattering (QLS), tritium labeled End-2 binding assay, circular dichroism (CD), a cell-viability assay, and in vitro and in vivo electrophysiology.
PRINCIPAL FINDINGS
1. End-2 binds to Aßbeta; but does not interfere with the aggregation of Aßbeta;1–42
Experiments were carried out by using Aßbeta;1–42, LPFFD, and End-2 synthesized in house.
For the TEM experiments, 10–4 M Aßbeta;1–42 containing solutions were incubated for 5 days either alone or with 5 x 10–4 M End-2. Electromicrographs were made immediately after dissolving Aßbeta;1–42 (0 h) and after incubation periods of 1 and 5 days. A few protofibrils and spherical oligomers could be observed at day 0 in either solution (Fig. 1
, A and B, top). After 1 day of incubation, both samples contained long, mature, smooth amyloid fibrils emanating from the seeding centers without any observable difference between the two time-matched different samples (Fig. 1, A
and B
, middle). Although, after 5 days of incubation period, the concentration of fibrils increased further, the End-2 containing solution resembled the control one. Our observations by electron microscopy indicate that the tetrapeptide did not affect the fibrillogenesis of Aßbeta;1–42 (Fig. 1, A
and B
, bottom). Binding assay carried out with tritium labeled End-2 showed that the tetrapeptide binds to aggregated Aßbeta;1–42 in a reversible manner. The particle size distribution of Aßbeta; and End-2 containing solution did not differ from that of Aßbeta; alone after 48 h, as revealed by QLS. In contrast, LPFFD shifted the distribution toward oligomers and dimers. CD measurements proved that End-2 and Aßbeta;1–42 forms a complex in the initial state of the aggregation process, although the aggregation kinetics remained unchanged.
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2. End-2 reduces the neurotoxicity of Aßbeta;1–42 in a cell-viability assay
We investigated whether End-2 protects against Aßbeta;1–42 induced neurotoxicity by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) cell viability assay on differentiated SH-SY5Y cells. Because End-2 acts on µ-opioid receptor, a synthetic peptide agonist of the same receptor, [D-Ala (2), N-Me-Phe (4), Gly (5)-ol]-enkephalin (DAMGO), was used as control substance. Data were analyzed by using ANOVA with post hoc Bonferroni test.
Both End-2 and DAMGO were applied in 1, 10, 100, and 200 µM, either alone or in a mixture with 10–5 M Aßbeta;1–42. Application of 10–5 M Aßbeta;1–42 resulted in a decrease of cell viability. Reduction of MTT into formazan in the SH-SY5Y cell line decreased to 58 ± 8% (n=21), as described previously. DAMGO, applied in gradually increasing concentrations, was unable to interfere with Aßbeta;1–42 induced decrease of MTT reduction. In contrast, End-2 could significantly attenuate the inhibition of MTT reduction of Aßbeta;1–42 in a dose-dependent manner. The effect was significant, when the tetrapeptide was applied in 100 and in 200 µM concentration with 10–5 M Aßbeta;1–42 (77±7 and 79±10%, respectively, P<0.01, n=21). DAMGO and End-2 did not affect cell viability alone at the concentrations used.
3. End-2 prevents the Aßbeta;1–42 induced field excitatory postsynaptic potential attenuation in vitro
Coronal brain slices from the primary motor cortices of 20- to 30-day-old Wistar rats were placed in a recording chamber and were perfused continuously with artificial cerebrospinal fluid. The amplitude of the initial negative component of the field excitatory postsynaptic potential (fEPSP) evoked by stimuli applied through horizontally displaced electrodes in layers II/III was recorded. Statistical significance was determined by means of Student’s t test. The applied 10–5 M Aßbeta;1–42 decreased the amplitude to 58.9 ± 10% (P<0.05, n=15), while End-2 successfully prevented the attenuation (92.8±16%, n=6). In contrast, application of Aßbeta;1–42 and DAMGO containing mixture (1:5 M) resulted in significant fEPSP amplitude attenuation (57±14%, n=8, P<0.05). End-2 and DAMGO in a concentration of 5 x 10–5 M did not alter the measured fEPSPs (100.3±17.6%, n=13 and 91.7±11.4%, n=14, respectively).
4. End-2 protects against the hyperexcitability caused by Aßbeta;1–42 in vivo
NMDA-evoked neuronal firing was registered from the CA1 region of Wistar rats weighting 300–360 g by means of extracellular single-unit recordings combined with microiontophoresis. NMDA was ejected every minute; the statistical evaluations were performed by using the total number of spikes evoked during each epoch of excitation. To elucidate the mechanism of protection of End-2, two kinds of application were used: 1) ejection of Aßbeta;1–42 and End-2 or Aßbeta;1–42 and DAMGO containing mixture (5x10–5 M and 2.5x10–4 M, respectively), and 2) ejection of 2.5 x 10–4 M End-2/DAMGO right before 5 x 10–5 M Aßbeta;1–42 application (coiontophoresis; Fig. 2
).
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Aßbeta;1–42 ejection alone induced a massive enhancement of neuronal excitability (269±16%; P<0.05; n=14). The coiontophoretic application of End-2 and Aßbeta;1–42 resulted in a similar increase of firing (348±65%, P<0.05; n=7). In contrast, ejection of Aßbeta;1–42 and End-2 containing solution did not increase the NMDA-evoked responses (115±21%, n=6). DAMGO applied either coiontophoretically or in a mixture with Aßbeta;1–42 emerged in hyperexcitability similar to Aßbeta;1–42 induced responses (238±37%, P<0.05, n=6 and 269±37%, P<0.05, n=6, respectively). End-2 and DAMGO alone increased the NMDA-elicited neuronal firing in a less pronounced manner (162±14%, P<0.05, n=9 and 167±5%, P<0.05, n=5).
CONCLUSIONS
Our results show that an endogenous tetrapeptide, End-2, protects neurons against Aßbeta;1–42 induced effects Fig. 3
. The mechanism of protection is not mediated by the µ-opioid receptor. Data from TEM, QLS, binding assay, and CD measurements suggest that the tetrapeptide is capable of binding to the surface of Aßbeta; assemblies without interfering in the kinetics of fibrilogenesis. It is widely accepted that protective substances that bind to aggregated Aßbeta; have a BSB effect. We report for the first time that a neuroprotective, Aßbeta;1–42 binding small molecule does not arrest fibril formation. These results are in accord with the findings that brain structures with high End-2 content are unaffected both in AD patients and in transgenic animals. However, the hippocampal region and the neocortex, which do not show End-2 like immunoreactivity, are severely damaged in the disease, suggesting a possible role for the endogenous tetrapeptide in the area-selective neuron-loss seen in AD. Unfortunately, approaches aimed to increase End-2 concentration in the brain presumably would not offer rational treatment of AD patients. The activation of µ-opioid receptors increases the net excitability in the CA1 region and may result in focal epileptic seizures. Therefore, the sequence of End-2 is what may be exploited as a lead sequence for drug development in the treatment of AD.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4891fje
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0.05).