Amyloid-β and APP Deficiencies Cause Severe Cerebrovascular Defects: Important Work for an Old Villain

<div><p>Alzheimer’s disease (AD) is marked by neuritic plaques that contain insoluble deposits of amyloid-β (Aβ), yet the physiological function of this peptide has remained unclear for more than two decades. Using genetics and pharmacology we have established that Aβ plays an important role in regulating capillary bed density within the brain, a function that is distinct from other cleavage products of amyloid precursor protein (APP). APP-deficient zebrafish had fewer cerebrovascular branches and shorter vessels in the hindbrain than wild-type embryos; this phenotype was rescued by treatment with human Aβ peptide, but not a smaller APP fragment called p3. Similar vascular defects were seen in zebrafish treated with a β-secretase inhibitor (BSI) that blocked endogenous Aβ production. BSI-induced vascular defects were also improved by treatment with human Aβ, but not p3. Our results demonstrate a direct correlation between extracellular levels of Aβ and cerebrovascular density in the developing hindbrain. These findings may be relevant to AD etiology where high levels of Aβ in the brain parenchyma precede the development of neuritic plaques and dense aberrantly-branched blood vessel networks that appear between them. The ability of Aβ to modify blood vessels may coordinate capillary density with local metabolic activity, which could explain the evolutionary conservation of this peptide from lobe-finned fish to man.</p> </div

Cerebrovascular defects in APP-deficient zebrafish embryos.

<p>(A) Dark field (top) and fluorescence (bottom) images of a control transgenic embryo at 3 dpf shows vascular structures dues to EGFP expression in endothelial cells. (B) Confocal image (projected stack) of cerebrovascular structures in the head of the fish in <i>A</i>. (C) Dark field (top) and fluorescence (bottom) images of a zAPP-MO embryo at 3 dpf. (D) Confocal image (projected stack) of cerebrovascular structures in the head of the fish in <i>C</i>. (E) Dark field (top) and fluorescence (bottom) images of a ctrl-MO embryo at 3 dpf. (F) Confocal image (projected stack) of cerebrovascular structures in the head of the fish in <i>E</i>. (G) Graph showing the number of CtA branches in control (N = 30), zAPP-MO (N = 15), and ctrl-MO (N = 15) zebrafish at 3 dpf (***, <i>P</i> < 8.9e-16). (H) Mean CtA branch lengths in control (N = 28), zAPP-MO (N = 14), and ctrl-MO (N = 8) embryos at 3 dpf (***, P < 9.8e-23); scale bars = 100 μm.</p

Aβ-deficiency induced by BSI-treatment caused vascular defects that were rescued by Aβ, but not p3.

<p>(A) Confocal image of cerebrovascular structures in an untreated control zebrafish at 3 dpf (control). (B) Vascular structures in a BSI-treated embryo at 3 dpf (BSI). (C) Cerebrovascular structures in a BSI-treated embryo that was treated with Aβ (BSI+Aβ) showed rescue of the vascular defects. (D) Vascular defects in a BSI-treated embryo were not rescued by p3 (BSI+p3). (E) Graph of CtA branch numbers in control (N = 30), BSI (N = 29), BSI+Aβ (N = 8), and Aβ+p3(N = 10) embryos at 3 dpf. Differences between control and BSI were significant (<i>P</i> < 0.0006), but there was no significant difference between BSI+Aβ and control embryos. BSI+p3 embryos had significantly fewer branches than control embryos (<i>P</i> < 3.0e-7). (F) Graph of mean CtA branch lengths in control (N = 28), BSI (N = 21), BSI+Aβ (N = 8), and BSI+p3(N = 10) embryos at 3 dpf. Differences between control and BSI were significant (<i>P</i> < 3.0e-13), but there was no significant difference between BSI+Aβ and control embryos. BSI+p3 embryos had significantly shorter vessel lengths than control embryos (<i>P</i> < 3.6e-13).</p

Aβ rescued vascular defects in APP-deficient (zAPP-MO) zebrafish embryos at 3 dpf.

<p>(A) Confocal image (projected stack) of a control zebrafish embryo at 3 dpf. (B) Comparable image of a zAPP-MO embryo at 3 dpf. (C) Cerebrovascular structures of an Aβ-treated zAPP-MO were similar to non-injected controls. (D) p3 treatment did not rescue vascular defects in zAPP-MO embryos. (E) Graph of CtA branch numbers in embryos in the control (N = 30), zAPP-MO (N = 15), Aβ-treated zAPP-MO (N = 15), and p3-treated zAPP-MO (N = 15) embryos at 3 dpf. Differences between control and zAPP-MO were significant (<i>P</i> < 8.9e-16), but there were no significant differences between Aβ-treated zAPP-MO and control or ctrl-MO. p3-treated zAPP-MO had significantly fewer branches than control embryos (<i>P</i> < 8.7e-14). (F) Graph of mean CtA branch lengths in control (N = 28), zAPP-MO (N = 14), Aβ-treated zAPP-MO (N = 10), and p3-treated zAPP-MO (N = 10) embryos at 3 dpf. Differences between control and zAPP-MO were significant (<i>P</i> < 9.8e-23), but there were no significant differences between Aβ-treated zAPP-MO and control or ctrl-MO. p3-treated zAPP-MO had significantly shorter vessel lengths than control embryos (<i>P</i> < 1.3e-15).</p

Schematic of APP processing that produces Aβ and p3 peptides.

<p>APP is initially cleaved by either α-secretase or β-secretases to yield a C87 in the alpha pathway, or C99 in the beta pathway, respectively. These cleavage events also produce an extracellular soluble APP (sAPP) fragment, from the amino terminus, that is slightly longer with α-secretase cleavage. The C87 and C99 fragments are subsequently cleaved within the transmembrane domains (tm) by γ-secretase to produce p3 and Aβ peptides, respectively. Both γ-secretase events produce an Aβ-intracellular-domain (AICD) fragment that is entirely cytosolic. Variability of γ-secretase cleavage on C99 produces Aβ fragments from 39–43 amino acids - only Aβ(1–42) is shown – and similar variability with C87 cleavage.</p

Alzheimer’s-Related Peptide Amyloid-β Plays a Conserved Role in Angiogenesis

<div><p>Alzheimer’s disease research has been at an impasse in recent years with lingering questions about the involvement of Amyloid-β (Aβ). Early versions of the amyloid hypothesis considered Aβ something of an undesirable byproduct of APP processing that wreaks havoc on the human neocortex, yet evolutionary conservation - over three hundred million years - indicates this peptide plays an important biological role in survival and reproductive fitness. Here we describe how Aβ regulates blood vessel branching in tissues as varied as human umbilical vein and zebrafish hindbrain. High physiological concentrations of Aβ monomer induced angiogenesis by a conserved mechanism that blocks γ-secretase processing of a Notch intermediate, NEXT, and reduces the expression of downstream Notch target genes. Our findings allude to an integration of signaling pathways that utilize γ-secretase activity, which may have significant implications for our understanding of Alzheimer’s pathogenesis vis-à-vis vascular changes that set the stage for ensuing neurodegeneration.</p> </div

Analysis of CtA blood vessels in 3 dpf embryos.

<p>A) Digitized image of the regions highlighted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039598#pone-0039598-g003" target="_blank">figure 3</a> in a control (untreated) 3 dpf embryo. B) CtA branching in GSI-treated (10 µg/mL) embryo at 3 dpf. C) CtA branching in an embryo treated with 15 µg/mL Aβ. D) CtA branching in an embryo treated with 25 µg/mL Aβ at 3 dpf. E) RevAβ did not significantly increase CtA branching. F) Scatter plot of CtA branches in all embryos in each condition (open circle). In the case of multiple values at given value for each condition, the circles were slightly offset. The mean for each group is shown as a filled circle and error bars represent standard errors of the mean. A dashed line indicates the mean value of the controls for comparison across the other treatments. Asterisks indicate significant differences with the untreated control group (*** = p<0.001; * = p<0.05 one-way ANOVA with Bonferroni correction). Scale bar  = 25 µm, for A–E. (CtA – cerebral artery, CCtA – common CtA, PCeV – posterior cerebral vein, PHBC - posterior hindbrain channel).</p

Morphology and vascular imaging of GFP-expressing zebrafish embryos.

<p>A) Projection image of a 3 dpf embryo using images captured by confocal microscopy. B, C) Comparable projection of a 3 dpf embryo treated with Aβ (25 µg/mL) and Rev Aβ, respectively. Boxes indicate the region analyzed for CtA branching, shown in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039598#pone-0039598-g004" target="_blank">figure 4</a>. Scale bar  = 100 µm, for A–C.</p

Proposed mechanism for hypervascularization in response to high concentrations of Aβ.

<p>High levels of Aβ (green helix) develop in the brain prior to the onset of Alzheimer’s pathology (top left). Longer forms of Aβ have more hydrophobic amino acid residues (gold) on remnant transmembrane domain, thereby increasing transient insertion (downward arrow) into the plasma membranes of nearby cells including endothelial cells (red). Elevated tissue levels of Aβ cause an accumulation of Aβ on the surface of the cell (several helices). Some peptide enters the endosome pathway (left). The amino termini of inserted Aβ peptides are identical to the Nicastrin-binding region of the APP intermediate C99 (not shown) and transiently bind to the γ-secretase complex (two sided arrow). Competition for Nicastrin binding and γ-secretase access reduces the processing of other substrates such as NEXT (shown). Interference with NEXT processing inhibits the production of NICD, which de-represses transcription factors such as Hes-1 and Hey-1. GSI has a similar effect. De-repression of NICD targets causes endothelial shaft cells to adopt tip cell morphology (upper right). Tip cells lead to formation of new blood vessels, and cycling of this process (looped arrow) over years and decades leads to hypervascularization (top right). This process may also occur with other extracellular cleavage (ex) products of γ-secretase (shown) creating feedback that tempers γ-secretase activity.</p

Aβ monomer induces tip cell formation in HUVEC.

<p>A) HUVEC plated on a 3-D matrix spontaneously formed vessel-like tube structures after 4 h. Tip cells are indicated with arrows. B) Tip cell frequency was significantly higher in cultures treated with GSI (p = 4.6e−29). C) Cultures treated with 55 nM Aβ did not have significantly more tips than controls, but cultures treated with 225 nM Aβ (D) had significantly more tip cells (p = 2.3e−29). E) Treatment with reverse Aβ42-1 did not increase tip cell frequency. F) Histogram of mean tip cells in all cultures (*** = p<0.001 vs. control, one-way ANOVA with Bonferroni correction). Scale bar  = 250 µm, for A–E.</p