Mural cell dysfunction leads to altered cerebrovascular tau uptake following repetitive head trauma.

A pathological characteristic of repetitive traumatic brain injury (TBI) is the deposition of hyperphosphorylated and aggregated tau species in the brain and increased levels of extracellular monomeric tau are believed to play a role in the pathogenesis of neurodegenerative tauopathies. The pathways by which extracellular tau is eliminated from the brain, however, remains elusive. The purpose of this study was to examine tau uptake by cerebrovascular cells and the effect of TBI on these processes. We found monomeric tau interacts with brain vascular mural cells (pericytes and smooth muscle cells) to a greater extent than other cerebrovascular cells, indicating mural cells may contribute to the elimination of extracellular tau, as previously described for other solutes such as beta-amyloid. Consistent with other neurodegenerative disorders, we observed a progressive decline in cerebrovascular mural cell markers up to 12 months post-injury in a mouse model of repetitive mild TBI (r-mTBI) and human TBI brain specimens, when compared to control. These changes appear to reflect mural cell degeneration and not cellular loss as no difference in the mural cell population was observed between r-mTBI and r-sham animals as determined through flow cytometry. Moreover, freshly isolated r-mTBI cerebrovessels showed reduced tau uptake at 6 and 12 months post-injury compared to r-sham animals, which may be the result of diminished cerebrovascular endocytosis, as caveolin-1 levels were significantly decreased in mouse r-mTBI and human TBI cerebrovessels compared to their respective controls. Further emphasizing the interaction between mural cells and tau, similar reductions in mural cell markers, tau uptake, and caveolin-1 were observed in cerebrovessels from transgenic mural cell-depleted animals. In conclusion, our studies indicate repeated injuries to the brain causes chronic mural cell degeneration, reducing the caveolar-mediated uptake of tau by these cells. Alterations in tau uptake by vascular mural cells may contribute to tau deposition in the brain following head trauma and could represent a novel therapeutic target for TBI or other neurodegenerative disorders.

MMP9 modulation improves specific neurobehavioral deficits in a mouse model of Alzheimer's disease.

BACKGROUND: Matrix metallopeptidase 9 (MMP9) has been implicated in a variety of neurological disorders, including Alzheimer's disease (AD), where MMP9 levels are elevated in the brain and cerebrovasculature. Previously our group demonstrated apolipoprotein E4 (apoE4) was less efficient in regulating MMP9 activity in the brain than other apoE isoforms, and that MMP9 inhibition facilitated beta-amyloid (Aß) elimination across the blood-brain barrier (BBB) METHODS: In the current studies, we evaluated the impact of MMP9 modulation on Aß disposition and neurobehavior in AD using two approaches, (1) pharmacological inhibition of MMP9 with SB-3CT in apoE4 x AD (E4FAD) mice, and (2) gene deletion of MMP9 in AD mice (MMP9KO/5xFAD) RESULTS: Treatment with the MMP9 inhibitor SB-3CT in E4FAD mice led to reduced anxiety compared to placebo using the elevated plus maze. Deletion of the MMP9 gene in 5xFAD mice also reduced anxiety using the open field test, in addition to improving sociability and social recognition memory, particularly in male mice, as assessed through the three-chamber task, indicating certain behavioral alterations in AD may be mediated by MMP9. However, neither pharmacological inhibition of MMP9 or gene deletion of MMP9 affected spatial learning or memory in the AD animals, as determined through the radial arm water maze. Moreover, the effect of MMP9 modulation on AD neurobehavior was not due to changes in Aß disposition, as both brain and plasma Aß levels were unchanged in the SB-3CT-treated E4FAD animals and MMP9KO/AD mice compared to their respective controls. CONCLUSIONS: In total, while MMP9 inhibition did improve specific neurobehavioral deficits associated with AD, such as anxiety and social recognition memory, modulation of MMP9 did not alter spatial learning and memory or Aß tissue levels in AD animals. While targeting MMP9 may represent a therapeutic strategy to mitigate aspects of neurobehavioral decline in AD, further work is necessary to understand the nature of the relationship between MMP9 activity and neurological dysfunction.

APOE genotype dependent molecular abnormalities in the cerebrovasculature of Alzheimer's disease and age-matched non-demented brains.

Cerebrovascular dysfunction is a hallmark feature of Alzheimer's disease (AD). One of the greatest risk factors for AD is the apolipoprotein E4 (E4) allele. The APOE4 genotype has been shown to negatively impact vascular amyloid clearance, however, its direct influence on the molecular integrity of the cerebrovasculature compared to other APOE variants (APOE2 and APOE3) has been largely unexplored. To address this, we employed a 10-plex tandem isobaric mass tag approach in combination with an ultra-high pressure liquid chromatography MS/MS (Q-Exactive) method, to interrogate unbiased proteomic changes in cerebrovessels from AD and healthy control brains with different APOE genotypes. We first interrogated changes between healthy control cases to identify underlying genotype specific effects in cerebrovessels. EIF2 signaling, regulation of eIF4 and 70S6K signaling and mTOR signaling were the top significantly altered pathways in E4/E4 compared to E3/E3 cases. Oxidative phosphorylation, EIF2 signaling and mitochondrial dysfunction were the top significant pathways in E2E2 vs E3/E3cases. We also identified AD-dependent changes and their interactions with APOE genotype and found the highest number of significant proteins from this interaction was observed in the E3/E4 (192) and E4/E4 (189) cases. As above, EIF2, mTOR signaling and eIF4 and 70S6K signaling were the top three significantly altered pathways in E4 allele carriers (i.e. E3/E4 and E4/E4 genotypes). Of all the cerebrovascular cell-type specific markers identified in our proteomic analyses, endothelial cell, astrocyte, and smooth muscle cell specific protein markers were significantly altered in E3/E4 cases, while endothelial cells and astrocyte specific protein markers were altered in E4/E4 cases. These proteomic changes provide novel insights into the longstanding link between APOE4 and cerebrovascular dysfunction, implicating a role for impaired autophagy, ER stress, and mitochondrial bioenergetics. These APOE4 dependent changes we identified could provide novel cerebrovascular targets for developing disease modifying strategies to mitigate the effects of APOE4 genotype on AD pathogenesis.

Molecular Pathobiology of the Cerebrovasculature in Aging and in Alzheimers Disease Cases With Cerebral Amyloid Angiopathy.

Cerebrovascular dysfunction and cerebral amyloid angiopathy (CAA) are hallmark features of Alzheimer's disease (AD). Molecular damage to cerebrovessels in AD may result in alterations in vascular clearance mechanisms leading to amyloid deposition around blood vessels and diminished neurovascular-coupling. The sequelae of molecular events leading to these early pathogenic changes remains elusive. To address this, we conducted a comprehensive in-depth molecular characterization of the proteomic changes in enriched cerebrovessel fractions isolated from the inferior frontal gyrus of autopsy AD cases with low (85.5 ± 2.9 yrs) vs. high (81 ± 4.4 yrs) CAA score, aged-matched control (87.4 ± 1.5 yrs) and young healthy control (47 ± 3.3 yrs) cases. We employed a 10-plex tandem isobaric mass tag approach in combination with our ultra-high pressure liquid chromatography MS/MS (Q-Exactive) method. Enriched cerebrovascular fractions showed very high expression levels of proteins specific to endothelial cells, mural cells (pericytes and smooth muscle cells), and astrocytes. We observed 150 significantly regulated proteins in young vs. aged control cerebrovessels. The top pathways significantly modulated with aging included chemokine, reelin, HIF1α and synaptogenesis signaling pathways. There were 213 proteins significantly regulated in aged-matched control vs. high CAA cerebrovessels. The top three pathways significantly altered from this comparison were oxidative phosphorylation, Sirtuin signaling pathway and TCA cycle II. Comparison between low vs. high CAA cerebrovessels identified 84 significantly regulated proteins. Top three pathways significantly altered between low vs. high CAA cerebrovessels included TCA Cycle II, Oxidative phosphorylation and mitochondrial dysfunction. Notably, high CAA cases included more advanced AD pathology thus cerebrovascular effects may be driven by the severity of amyloid and Tangle pathology. These descriptive proteomic changes provide novel insights to explain the age-related and AD-related cerebrovascular changes contributing to AD pathogenesis. Particularly, disturbances in energy bioenergetics and mitochondrial biology rank among the top AD pathways altered in cerebrovessels. Targeting these failed mechanisms in endothelia and mural cells may provide novel disease modifying targets for developing therapeutic strategies against cerebrovascular deterioration and promoting cerebral perfusion in AD. Our future work will focus on interrogating and validating these novel targets and pathways and their functional significance.

Apolipoprotein E isoforms differentially regulate matrix metallopeptidase 9 function in Alzheimer's disease.

Apolipoprotein E (APOE) has been shown to influence amyloid-ß (Aß) clearance from the brain in an isoform-specific manner. Our prior work showed that Aß transit across the blood-brain-barrier was reduced by apoE4, compared to other apoE isoforms, due to elevated lipoprotein receptor shedding in brain endothelia. Recently, we demonstrated that matrix metallopeptidase 9 (MMP-9) induces lipoprotein receptor proteolysis in an apoE isoform-dependent manner, which impacts Aß elimination from the brain. The current studies interrogated the relationship between apoE and MMP-9 and found that apoE impacted proMMP-9 cellular secretion from brain endothelia (apoE2 < apoE3 = apoE4). In a cell-free assay, apoE dose-dependently reduced MMP-9 activity, with apoE4 showing a significantly weaker ability to inhibit MMP-9 function than apoE2 or apoE3. Finally, we observed elevated MMP-9 expression and activity in the cerebrovasculature of both human and animal AD brain specimens with an APOE4 genotype. Collectively, these findings suggest a role for apoE in regulating MMP-9 disposition and may describe the effect of apoE4 on Aß pathology in the AD brain.

Influence of Matrix Metallopeptidase 9 on Beta-Amyloid Elimination Across the Blood-Brain Barrier.

Lipoprotein receptor transport across the blood-brain barrier (BBB) mediates beta-amyloid (Aß) accumulation in the brain and may be a contributing factor in Alzheimer's disease (AD) pathogenesis. Lipoprotein receptors are susceptible to proteolytic shedding at the cell surface, which precludes the endocytic transport of ligands. A ligand that closely interacts with the lipoprotein receptors is apolipoprotein E (apoE), which exists as three isoforms (apoE2, apoE3, apoE4). Our prior work showed an inverse relationship between lipoprotein receptor shedding and Aß transport across the BBB, which was apoE-isoform dependent. To interrogate this further, the current studies investigated an enzyme implicated in lipoprotein receptor shedding, matrix metalloproteinase 9 (MMP9). Treatment with MMP9 dose-dependently elevated lipoprotein receptor shedding in brain endothelial cells and freshly isolated mouse cerebrovessels. Furthermore, treatment with a MMP9 inhibitor (SB-3CT) mitigated Aß-induced lipoprotein receptor shedding in brain endothelial cells and the brains of apoE4 animals. In terms of BBB transit, SB-3CT treatment increased the transport of Aß across an in vitro model of the BBB. In vivo, administration of SB-3CT to apoE4 animals significantly enhanced Aß clearance from the brain to the periphery following intracranial administration of Aß. The current studies show that MMP9 impacts lipoprotein receptor shedding and Aß transit across the BBB, in an apoE  isoform-specific manner. In total, MMP9 inhibition can facilitate Aß clearance across the BBB, which could be an effective approach to lowering Aß levels in the brain and mitigating the AD phenotype, particularly in subjects carrying the apoE4 allele.