Amyloid Triggers Extensive Cerebral Angiogenesis Causing Blood Brain Barrier Permeability and Hypervascularity in Alzheimer's Disease

Evidence of reduced blood-brain barrier (BBB) integrity preceding other Alzheimer's disease (AD) pathology provides a strong link between cerebrovascular angiopathy and AD. However, the “Vascular hypothesis”, holds that BBB leakiness in AD is likely due to hypoxia and neuroinflammation leading to vascular deterioration and apoptosis. We propose an alternative hypothesis: amyloidogenesis promotes extensive neoangiogenesis leading to increased vascular permeability and subsequent hypervascularization in AD. Cerebrovascular integrity was characterized in Tg2576 AD model mice that overexpress the human amyloid precursor protein (APP) containing the double missense mutations, APPsw, found in a Swedish family, that causes early-onset AD. The expression of tight junction (TJ) proteins, occludin and ZO-1, were examined in conjunction with markers of apoptosis and angiogenesis. In aged Tg2576 AD mice, a significant increase in the incidence of disrupted TJs, compared to age matched wild-type littermates and young mice of both genotypes, was directly linked to an increased microvascular density but not apoptosis, which strongly supports amyloidogenic triggered hypervascularity as the basis for BBB disruption. Hypervascularity in human patients was corroborated in a comparison of postmortem brain tissues from AD and controls. Our results demonstrate that amylodogenesis mediates BBB disruption and leakiness through promoting neoangiogenesis and hypervascularity, resulting in the redistribution of TJs that maintain the barrier and thus, provides a new paradigm for integrating vascular remodeling with the pathophysiology observed in AD. Thus the extensive angiogenesis identified in AD brain, exhibits parallels to the neovascularity evident in the pathophysiology of other diseases such as age-related macular degeneration

Angiogenesis not apoptosis induces alterations in tight junction immunoreactivity in Tg2576 mice.

<p>Representative confocal micrographs of TJs (ZO-1), double stained for markers of angiogenesis or apoptosis in aged wild-type and Tg2576 mice. All vessels stained for CD105 regardless of the TJ expression pattern. White arrowheads point to regions of TJ abnormality in the vasculature. Double staining of blood vessels with ZO-1 (red) and CD105 (green) in wild-type (A). and Tg2576 (B) neocortex. Double staining of vessels with ZO-1 (red) and caspase 3 (green) in wild-type (C) and Tg2576 (D) neocortex. Caspase-3 staining did not colocalize with ZO-1 staining indicating an absence of apoptosis in the vasculature. Results are representative of three separate experiments of three mice per group of brain tissues examined. Scale bar represents 20 µm.</p

Aged Tg2576 Mice have reduced tight function expression.

<p>The expression of occludin or ZO-1 were compared quantitatively between wild-type and Tg2576 mice in the neocortex (A and B) and hippocampus (C and D). (A) The percentage of cortical cerebral blood vessels with abnormal ZO-1 expression patterns was significantly higher in the aged Tg2576 mice compared to age-matched wild-type (***p<0.001). The incidence of ZO-1 disruption was also significantly higher in aged Tg2576 mice compared to young Tg2576 (Young wild-type, n = 4; Young Tg2576, n = 3; aged wild-type, n = 5; aged Tg2576, n = 4; *p<0.05) in the cortex. (B) Aged Tg2576 mice had significantly reduced occludin protein levels in the cortex compared to age-matched wild-type (n = 7, **p = 0.0072). (C) The percentage of hippocampal cerebral blood vessels with abnormal ZO-1 expression patterns was significantly higher in the aged Tg2576 mice compared to age-matched wild-type (***p<0.001). Similarly, the incidence of ZO-1 disruption was also significantly higher in aged Tg2576 mice compared to young Tg2576 (Young wild-type, n = 4; Young Tg2576, n = 3; aged wild-type, n = 5; aged Tg2576, n = 4; *p<0.05) in the hippocampus. (D) Aged Tg2576 mice had significantly reduced occludin protein levels in the hippocampus compared to age-matched wild-type (n = 7, **p = 0.0076). Values represent mean ± SEM.</p

Microvascular Density is increased in Aged Tg2576 and in Human patients with AD.

<p>The MVD, by CD105 staining, in the cerebrovasculature and CD105 protein expression were quantified in aged and young Tg2576 and wild-type. (A) Aged Tg2576 mice had a significantly higher MVD compared to age-matched wild-type (***p<0.001). Aged Tg2576 were had a significantly higher MVD compared to young Tg2576 (Aged wild-type, n = 5; aged Tg2576, n = 4; young wild-type, n = 4; young Tg2576, n = 3; *p<0.05). Although not significant, young Tg2576 mice trended to a higher average MVD compared to wild-type. (B) Aged Tg2576 mice had a significantly increased CD105 protein levels in the cortex compared to age-matched wild-type (wild-type, n = 5; Tg2576, n = 6; ***p<0.001). (C) Aged Tg2576 mice had a significantly increased CD105 protein levels in the hippocampus compared to age-matched wild-type (n = 7, *p<0.05). (D) The cortex of the AD patient had a significantly increased MVD, as measured by % area occupied by laminin staining, compared to the ND patient (n = 4, *p<0.05). (E) The hippocampus of the AD patient had a significantly increased MVD, as measured by % area occupied by laminin staining, compared to the ND patient (n = 4, ***p<0.001). Representative images of immunohistochemical staining for laminin in the cortex of the ND patient (F) and the AD patient (G). Scale bar represents 95 µm. Values represent mean ± SEM.</p

Tg2576 AD mice have cerebral tight junction pathology.

<p>Representative confocal micrographs of cerebral blood vessels from aged Tg2576 and wild-type mice immunolabeled for either occludin or ZO-1 (red) and counterstained for DNA (blue) with TOTO-3. Blood vessels, imaged in the neocortex and hippocampus, which exhibited strong, continuous and linear occludin (A and C) or ZO-1 (E and G) expression were considered normal, as demonstrated in the wild-type. Abnormal occludin (B and F) and ZO-1 (D and H) staining displayed punctate (white arrowheads), discontinuous or interrupted (hollow white arrows), as seen in the Tg2576 cerebrovasculature. Results are representative from three mice per group from three separate experiments. Scale bar represents 20 µm.</p

Major Histocompatibility Complex class I proteins are critical for maintaining neuronal structural complexity in the aging brain

Major histocompatibility complex class I (MHCI) proteins have been implicated in neuronal function through the modulation of neuritogenesis, synaptogenesis, synaptic plasticity, and memory consolidation during development. However, the involvement of MHCI in the aged brain is unclear. Here we demonstrate that MHCI deficiency results in significant dendritic atrophy along with an increase in thin dendritic spines and a reduction in stubby spines in the hippocampus of aged (12 month old) mice. Ultrastructural analyses revealed a decrease in spine head diameter and post synaptic density (PSD) area, as well as an increase in overall synapse density, and non-perforated, small spines. Interestingly, we found that the changes in synapse density and morphology appear relatively late (after the age of 6 months). Finally, we found a significant age dependent increase in the levels of the glutamate receptor, GluN2B in aged MHCI knockout mice, with no change in GluA2/3, VGluT1, PSD95 or synaptophysin. These results indicate that MHCI may be also be involved in maintaining brain integrity at post-developmental stages notably in the modulation of neuronal and spine morphology and synaptic function during non-pathological aging which could have significant implications for cognitive function.publishe