The β-secretase enzyme BACE1 as a therapeutic target for Alzheimer's disease

Amyloid plaques are defining histopathologic lesions in the brains of Alzheimer's disease (AD) patients and are composed of the amyloid-beta peptide, which is widely considered to play a critical role in the pathogenesis of AD. The β-secretase, or β-site amyloid precursor protein cleaving enzyme 1 (BACE1; also called Asp2, memapsin 2), is the enzyme that initiates the generation of amyloid beta. Consequently, BACE1 is an attractive drug target for lowering cerebral levels of amyloid beta for the treatment or prevention of AD. Much has been learned about BACE1 since its discovery over 10 years ago. In the present article, we review BACE1 properties and characteristics, cell biology, in vivo validation, substrates, therapeutic potential, and inhibitor drug development. Studies relating to the physiological functions of BACE1 and the promise of BACE1 inhibition for AD will also be discussed. We conclude that therapeutic inhibition of BACE1 should be efficacious for AD, although careful titration of the drug dose may be necessary to limit mechanism-based side effects

Melatonin Signal Transduction Pathways Require E-Box-Mediated Transcription of Per1 and Per2 to Reset the SCN Clock at Dusk.

Melatonin is released from the pineal gland into the circulatory system at night in the absence of light, acting as "hormone of darkness" to the brain and body. Melatonin also can regulate circadian phasing of the suprachiasmatic nucleus (SCN). During the day-to-night transition, melatonin exposure advances intrinsic SCN neural activity rhythms via the melatonin type-2 (MT2) receptor and downstream activation of protein kinase C (PKC). The effects of melatonin on SCN phasing have not been linked to daily changes in the expression of core genes that constitute the molecular framework of the circadian clock. Using real-time RT-PCR, we found that melatonin induces an increase in the expression of two clock genes, Period 1 (Per1) and Period 2 (Per2). This effect occurs at CT 10, when melatonin advances SCN phase, but not at CT 6, when it does not. Using anti-sense oligodeoxynucleotides (α ODNs) to Per 1 and Per 2, as well as to E-box enhancer sequences in the promoters of these genes, we show that their specific induction is necessary for the phase-altering effects of melatonin on SCN neural activity rhythms in the rat. These effects of melatonin on Per1 and Per2 were mediated by PKC. This is unlike day-active non-photic signals that reset the SCN clock by non-PCK signal transduction mechanisms and by decreasing Per1 expression. Rather, this finding extends roles for Per1 and Per2, which are critical to photic phase-resetting, to a nonphotic zeitgeber, melatonin, and suggest that the regulation of these clock gene transcripts is required for clock resetting by diverse regulatory cues

At CT 6, melatonin does not change the levels of <i>Per1</i> and <i>Per2</i> transcripts, although <i>Bmal1</i> is reduced at 120 min.

<p>Melatonin applied at CT 6 has no significant effect on the expression levels of <i>Per 1</i>, <i>Per2</i>, or <i>Bmal1</i> mRNA after 30 min <b>(A)</b>. After 120 min <b>(B)</b>, only <i>Bmal1</i> mRNA significantly decreases following initiation of melatonin treatment at CT 6. Data are shown as percent change of relative mRNA levels compared to control ± SEM, <i>n</i> = 3–9 /condition, p ≥ 0.05 (<i>Per 1</i>, <i>Per 2</i>), *p ≤ 0.05 (<i>Bmal1</i>), Student’s T-test.</p

E-box promoter motif is required for melatonin to shift SCN neuronal activity rhythms at CT 10.

<p><b>A)</b> The spontaneous electrical activity rhythm in SCN brain slices peaks at CT 6.38 ± 0.13 in controls (<i>n = 3</i>). The dotted line indicates the mean time-of-peak for untreated slices. Large, vertical boxes represent subjective night, CT 12–14. <b>B)</b> At CT 10, MEL (1 nM, 10 min) advances the electrical activity rhythm by 3.6 h ± 0.10 (<i>n = 3</i>). Arrow = time of melatonin treatment. <b>C)</b> E-box decoy ODN has no significant effect on the time-of-peak electrical activity (<i>n = 3</i>). Small box = duration of ODN exposure. <b>D)</b> The MEL-induced phase advance is blocked by the E-box decoy ODN (<i>n = 3</i>). <b>E)</b> Missense ODN has no effect on the MEL-induced advance in time-of-peak electrical activity (<i>n = 3</i>). <b>F)</b> Missense ODN does not block the MEL-induced phase advance at CT 10 (<i>n = 3</i>). <b>G)</b> Summary of the effects of ODN on MEL-induced phase advances at CT 10. **indicates statistically significant difference compared to controls (p ≤ 0.001) as determined by 1-way ANOVA with Tukey’s <i>post- hoc</i> analysis.</p

<i>Per1</i> and <i>Per2</i> αODN attenuate the expression of corresponding transcripts in the SCN.

<p>2-h incubation of SCN slices with αODN results in a 45% decrease in <i>Per1</i> transcripts <b>(A)</b> and a 60% decrease in <i>Per2</i> transcripts <b>(B)</b> 4 h after initiation of treatment with the corresponding αODN. No change in GAPDH mRNA was evident following either treatment, which was used as a normalization control.</p

The PKC inhibitor, chelerythrine chloride, blocks the increase of <i>Per1</i> and <i>Per2</i> mRNA induced by melatonin applied at CT 10.

<p>Pre-treatment with 0.25 mM of the PKC inhibitor, chelerythrine chloride, blocks the melatonin-induced increase in <i>Per1</i> <b>(A)</b> and <i>Per2</i> <b>(B)</b> transcripts after 120 min. Data are shown as percent change of relative mRNA levels compared to control ± SEM, <i>n</i> = 3/condition (** p ≤ 0.01, *p ≤ 0.05, 1-way ANOVA, Tukey’s <i>post-hoc</i> analysis). Controls were exposed to sham treatment lacking MEL. MEL = melatonin. CC = chelerythrine chloride.</p

E-box decoy blocks binding at E-box sites in SCN 2.2 cells.

<p>Electromobility shift assay of an E-box probe incubated with nuclear extracts of SCN 2.2 cells transfected with 1 μM E-box decoy or missense ODN. Media lane indicates non-transfected control. Arrow = retarded mobility of the E-box probe. This DNA-protein interaction is absent in SCN 2.2 cells transfected with the E-box decoy up to 24 h (<i>n = 3</i>).</p

At CT 10, melatonin induces of <i>Per1</i> and <i>Per2</i> transcription by 120 min.

<p><b>A)</b> qPCR amplification products migrate at the predicted size and are distinguishable on an 8% polyacrylamide gel stained with ethidium bromide (<i>Per1 =</i> 113 bp, <i>Per2</i> = 90 bp, <i>BMAL1</i> = 79 bp). <b>B)</b> Melatonin has no significant effect on the expression levels of <i>Per1</i>, <i>Per2</i>, or <i>Bmal1</i> mRNA 30 min following the initiation of treatment (p ≥ 0.05, Student’s T Test). <b>C)</b> Melatonin treatment significantly increases <i>Per1</i> and <i>Per2</i>, but not <i>Bmal1</i>, transcripts, at 120 min. Data are shown as percent change of relative mRNA levels compared to control ± SEM, <i>n</i> = 3-4/condition. ***p ≤ 0.001 (<i>Per1</i>), *p ≤ 0.05 (<i>Per2</i>), p ≥ 0.05 (<i>Bmal1</i>), Student’s T-test.</p

<i>Per1</i> is required for melatonin to alter the phase of SCN neuronal activity rhythms at CT 10.

<p><b>A)</b> The spontaneous electrical activity rhythm in SCN brain slices peaks at CT 6.38 ± 0.13 in controls. The dotted line indicates the mean time-of-peak for untreated slices. Long, vertical boxes represent subjective night, CT 12–14. <b>B)</b> At CT 10, MEL (1 nM, 10 min) advances the electrical activity rhythm by 3.6 h ± 0.10 (<i>n = 3</i>). Arrow = time of melatonin treatment. <b>C)</b> <i>Per1</i> αODN application from CT 8–10 has no significant effect on the time-of-peak electrical activity (<i>n = 3</i>). Small box = duration of ODN exposure. <b>D)</b> The MEL-induced phase advance is completely blocked by <i>Per1</i> αODN (<i>n = 3</i>). <b>E)</b> <i>Per1</i> missense ODN has no effect on the MEL-induced advance in time-of-peak electrical activity (<i>n = 3</i>). <b>F)</b> <i>Per1</i> missense ODN does not block the MEL-induced phase advance at CT 10. <b>G)</b> Summary of the effects of <i>Per1</i> ODN on MEL-induced phase advances at CT 10. **indicates statistically significant difference compared to controls (p ≤ 0.001) as determined by 1-way ANOVA with Tukey’s <i>post-hoc</i> analysis.</p