Beneficial Effects of Estrogen in a Mouse Model of Cerebrovascular Insufficiency

BACKGROUND: The M(5) muscarinic acetylcholine receptor is known to play a crucial role in mediating acetylcholine dependent dilation of cerebral blood vessels. Previously, we reported that male M(5) muscarinic acetylcholine knockout mice (M5R(-/-) mice) suffer from a constitutive constriction of cerebral arteries, reduced cerebral blood flow, dendritic atrophy, and short-term memory loss, without necrosis and/or inflammation in the brain. METHODOLOGY/PRINCIPAL FINDINGS: We employed the Magnetic Resonance Angiography to study the area of the basilar artery in male and female M5R(-/-) mice. Here we show that female M5R(-/-) mice did not show the reduction in vascular area observed in male M5R(-/-) mice. However, ovariectomized female M5R(-/-) mice displayed phenotypic changes similar to male M5R(-/-) mice, strongly suggesting that estrogen plays a key role in the observed gender differences. We found that 17beta-estradiol (E2) induced nitric oxide release and ERK activation in a conditional immortalized mouse brain cerebrovascular endothelial cell line. Agonists of ERalpha, ERbeta, and GPR30 promoted ERK activation in this cell line. Moreover, in vivo magnetic resonance imaging studies showed that the cross section of the basilar artery was restored to normal in male M5R(-/-) mice treated with E2. Treatment with E2 also improved the performance of male M5R(-/-) mice in a cognitive test and reduced the atrophy of neural dendrites in the cerebral cortex and hippocampus. M5R(-/-) mice also showed astrocyte swelling in cortex and hippocampus using the three-dimensional reconstruction of electron microscope images. This phenotype was reversed by E2 treatment, similar to the observed deficits in dendrite morphology and the number of synapses. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that M5R(-/-) mice represent an excellent novel model system to study the beneficial effects of estrogen on cerebrovascular function and cognition. E2 may offer new therapeutic perspectives for the treatment of cerebrovascular insufficiency related memory dysfunction

Loss of M5 muscarinic acetylcholine receptors leads to cerebrovascular and neuronal abnormalities and cognitive deficits in mice.

The M5 muscarinic acetylcholine receptor (M5R) has been shown to play a crucial role in mediating acetylcholine-dependent dilation of cerebral blood vessels. We show that male M5R-/- mice displayed constitutive constriction of cerebral arteries using magnetic resonance angiography in vivo. Male M5R-/- mice exhibited a significantly reduced cerebral blood flow (CBF) in the cerebral cortex, hippocampus, basal ganglia, and thalamus. Cortical and hippocampal pyramidal neurons from M5R-/- mice showed neuronal atrophy. Hippocampus-dependent spatial and nonspatial memory was also impaired in M5R-/- mice. In M5R-/- mice, CA3 pyramidal cells displayed a significantly attenuated frequency of the spontaneous postsynaptic current and long-term potentiation was significantly impaired at the mossy fiber-CA3 synapse. Our findings suggest that impaired M5R signaling may play a role in the pathophysiology of cerebrovascular deficits. The M5 receptor may represent an attractive novel therapeutic target to ameliorate memory deficits caused by impaired cerebrovascular function

TMEM30A is a candidate interacting partner for the β-carboxyl-terminal fragment of amyloid-β precursor protein in endosomes

<div><p>Although the aggregation of amyloid-β peptide (Aβ) clearly plays a central role in the pathogenesis of Alzheimer’s disease (AD), endosomal traffic dysfunction is considered to precede Aβ aggregation and trigger AD pathogenesis. A body of evidence suggests that the β-carboxyl-terminal fragment (βCTF) of amyloid-β precursor protein (APP), which is the direct precursor of Aβ, accumulates in endosomes and causes vesicular traffic impairment. However, the mechanism underlying this impairment remains unclear. Here we identified TMEM30A as a candidate partner for βCTF. TMEM30A is a subcomponent of lipid flippase that translocates phospholipids from the outer to the inner leaflet of the lipid bilayer. TMEM30A physically interacts with βCTF in endosomes and may impair vesicular traffic, leading to abnormally enlarged endosomes. APP traffic is also concomitantly impaired, resulting in the accumulation of APP-CTFs, including βCTF. In addition, we found that expressed BACE1 accumulated in enlarged endosomes and increased Aβ production. Our data suggested that TMEM30A is involved in βCTF-dependent endosome abnormalities that are related to Aβ overproduction.</p></div

Intracellular interaction between βCTF and TMEM30A.

<p>A: Schematic depiction of APP-CTFs and their specific antibodies. TM; Transmembrane domain. B: COS-7 cells were transfected with APP and CFP-TMEM30A, untagged TMEM30A, or BACE1. To segregate β1- and β11-CTFs, immunoblot analysis was performed using high-resolution electrophoresis. β1CTF was detected by 82E1, which is specific for β1 cleaved end. Because BACE1 activity competes with α-secretase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200988#pone.0200988.ref032" target="_blank">32</a>], α′CTF appeared to be derived from α-secretase cleavage. C, D: COS-7 cells were transfected with APP and the wild-type or KKLN mutant of CFP-TMEM30A. (C) Coimmunoprecipitation analysis of COS-7 cell lysates using control mouse IgG or GFP antibody to precipitate CFP-TMEM30A. (D) Immunofluorescence analysis. Cells were labeled with APPC15 antibody (green). Scale bar: 20 μm. E: Coimmunoprecipitation analysis of COS-7 cells that were transfected with artificial βCTF, SC100, and wild-type or KKLN mutant of CFP-TMEM30A using control mouse IgG or GFP antibody.</p

Direct interaction of βCTF with the extracellular region of TMEM30A.

<p>GST pulldown assay. (A) Upper panel: schematic view of recombinant TMEM30A extracellular region (67–323 AA) fused with GST (G-TmEx). Lower panel: Purified proteins were shown by Coomassie brilliant blue staining. (B) Lysate of COS-7 cells transfected with SC100 or (C) synthetic Aβ40 (1 μM) were incubated with recombinant GST or G-TmEx prebound to GSH-Sepharose.</p

Coexpression of TMEM30A and APP decreased sAPPβ production but increased FL-APP and APP-CTFs.

<p>A, B: COS-7 cells were transfected with APP and the wild-type or KKLN mutant of TMEM30A or BACE1. After 24-h transfection, the medium was replaced, and the cells were further incubated for 24 h. Immunoblot analyses of cell lysate (A) and medium (B) are shown. C–H: Quantitative analyses are shown as follows and compared with control: (C) FL-APP, (D) αCTF, (E) βCTF, (F) sAPPα, (G) sAPPβ, and (H) Aβ [independent experiments were performed four times (<i>n</i> = 4), mean ± SEM, *<i>P</i> < 0.05, **<i>P</i> < 0.01; N.S., no significant difference].</p

TMEM30A interacted with APP at the APP N-terminal domain and Aβ N-terminal sequence.

<p>A: Schematic depiction of deletion mutants of APP-Venus used in this study. TM; Transmembrane domain. B: COS-7 cells were transfected with mCherry-TMEM30A and the wild-type or deletion mutant of APP-Venus. After 24 h, the medium was replaced with a fresh medium containing a γ-secretase inhibitor (10 μM DAPT) and further incubated for 24 h. (B) Coimmunoprecipitation analysis of COS-7 cell lysates using control mouse IgG or GFP antibody (3E6) to precipitate APP-Venus. The arrowhead, arrow, and asterisks represent mature, immature mCherry-TMEM30A, and deletion mutants of APP-Venus, respectively.</p

Coexpression of TMEM30A and APP induces enlarged endosomes.

<p>A, B: COS-7 cells were transfected with APP-Venus and mCherry-TMEM30A. (A) Coexpression of APP and TMEM30A resulted in the redistribution of these proteins in enlarged vesicles. Scale bar: 20 μm. (B) The distribution of APP-containing vesicles in transfected cells classified by their size. The average number was evaluated by eight fields (×100, 8–11 cells), with counting in each experiment [independent experiment performed thrice (<i>n</i> = 3), mean ± SEM, *<i>P</i> < 0.05, ***<i>P</i> < 0.001]. Auto-thresholded images were processed using Image J. (C) Upper panel: COS-7 cells were transfected with mCherry-TMEM30A. Cells were labeled with anti-Rab5 antibody (green). Lower panel: COS-7 cells were transfected with APP-Venus and CFP-TMEM30A. Cells were labeled with early endosome marker, anti-Rab5 (red). Scale bar: 20 μm.</p

Effect of BACE1 expression in APP-TMEM30A transfected cells.

<p>A–C: COS-7 cells were transfected with CFP-TMEM30A and APP. After 24 h of transfection, cells were further transfected with BACE1. After 24 h of BACE1 transfection, the medium was refreshed, and cells were further incubated for 24 h. (A) Immunofluorescence analysis. Cells were labeled with anti-APP C-terminal antibody [mC99 (70–80)] and anti-BACE1 C-terminal antibody. Scale bar: 20 μm. (B) Quantification analysis of sAPPβ levels in the medium by immunoblotting (independent experiments were performed thrice (n = 3), mean ± SEM; *P < 0.05; N.S., no significant difference). (C) Quantification of secreted Aβ by ELISA (n = 3, mean ± SEM).</p