Repetitive head trauma and apoE4 induce chronic cerebrovascular alterations that impair tau elimination from the brain.

Repetitive mild traumatic brain injuries (r-mTBI) sustained in the military or contact sports have been associated with the accumulation of extracellular tau in the brain, which may contribute to the pathogenesis of neurodegenerative tauopathies. The expression of the apolipoprotein E4 (apoE4) isoform has been associated with higher levels of tau in the brain, and worse clinical outcomes after r-mTBI, though the influence of apoE genotype on extracellular tau dynamics in the brain is poorly understood. We recently demonstrated that extracellular tau can be eliminated across blood-brain barrier (BBB), which is progressively impaired following r-mTBI. The current studies investigated the influence of repetitive mild TBI (r-mTBI) and apoE genotype on the elimination of extracellular solutes from the brain. Following intracortical injection of biotin-labeled tau into humanized apoE-Tr mice, the levels of exogenous tau residing in the brain of apoE4 mice were elevated compared to other isoforms, indicating reduced tau elimination. Additionally, we found exposure to r-mTBI increased tau residence in apoE2 mice, similar to our observations in E2FAD animals. Each of these findings may be the result of diminished tau efflux via LRP1 at the BBB, as LRP1 inhibition significantly reduced tau uptake in endothelial cells and decreased tau transit across an in vitro model of the BBB (basolateral-to-apical). Notably, we showed that injury and apoE status, (particularly apoE4) resulted in chronic alterations in BBB integrity, pericyte coverage, and AQP4 polarization. These aberrations coincided with an atypical reactive astrocytic gene signature indicative of diminished CSF-ISF exchange. Our work found that CSF movement was reduced in the chronic phase following r-mTBI (>18 months post injury) across all apoE genotypes. In summary, we show that apoE genotype strongly influences cerebrovascular homeostasis, which can lead to age-dependent deficiencies in the elimination of toxic proteins from the brain, like tau, particularly in the aftermath of head trauma.

Influence of traumatic brain injury on extracellular tau elimination at the blood-brain barrier.

Repetitive head trauma has been associated with the accumulation of tau species in the brain. Our prior work showed brain vascular mural cells contribute to tau processing in the brain, and that these cells progressively degenerate following repetitive mild traumatic brain injury (r-mTBI). The current studies investigated the role of the cerebrovasculature in the elimination of extracellular tau from the brain, and the influence of r-mTBI on these processes. Following intracranial injection of biotin-labeled tau, the levels of exogenous labeled tau residing in the brain were elevated in a mouse model of r-mTBI at 12 months post-injury compared to r-sham mice, indicating reduced tau elimination from the brain following head trauma. This may be the result of decreased caveolin-1 mediated tau efflux at the blood-brain barrier (BBB), as the caveolin inhibitor, methyl-ß-cyclodextrin, significantly reduced tau uptake in isolated cerebrovessels and significantly decreased the basolateral-to-apical transit of tau across an in vitro model of the BBB. Moreover, we found that the upstream regulator of endothelial caveolin-1, Mfsd2a, was elevated in r-mTBI cerebrovessels compared to r-sham, which coincided with a decreased expression of cerebrovascular caveolin-1 in the chronic phase following r-mTBI (> 3 months post-injury). Lastly, angiopoietin-1, a mural cell-derived protein governing endothelial Mfsd2a expression, was secreted from r-mTBI cerebrovessels to a greater extent than r-sham animals. Altogether, in the chronic phase post-injury, release of angiopoietin-1 from degenerating mural cells downregulates caveolin-1 expression in brain endothelia, resulting in decreased tau elimination across the BBB, which may describe the accumulation of tau species in the brain following head trauma.

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.

Impairment of cerebrovascular reactivity in response to hypercapnic challenge in a mouse model of repetitive mild traumatic brain injury.

Incidences of repetitive mild TBI (r-mTBI), like those sustained by contact sports athletes and military personnel, are thought to be a risk factor for development of neurodegenerative disorders. Those suffering from chronic TBI-related illness demonstrate deficits in cerebrovascular reactivity (CVR), the ability of the cerebral vasculature to respond to a vasoactive stimulus. CVR is thus an important measure of traumatic cerebral vascular injury (TCVI), and a possible in vivo endophenotype of TBI-related neuropathogenesis. We combined laser speckle imaging of CVR in response to hypercapnic challenge with neurobehavioral assessment of learning and memory, to investigate if decreased cerebrovascular responsiveness underlies impaired cognitive function in our mouse model of chronic r-mTBI. We demonstrate a profile of blunted hypercapnia-evoked CVR in the cortices of r-mTBI mice like that of human TBI, alongside sustained memory and learning impairment, without biochemical or immunohistopathological signs of cerebral vessel laminar or endothelium constituent loss. Transient decreased expression of alpha smooth muscle actin and platelet-derived growth factor receptor ß, indicative of TCVI, is obvious only at the time of the most pronounced CVR deficit. These findings implicate CVR as a valid preclinical measure of TCVI, perhaps useful for developing therapies targeting TCVI after recurrent mild head trauma.

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.

Sensitive Detection of Exosomal Proteins via a Compact Surface Plasmon Resonance Biosensor for Cancer Diagnosis.

Exosomes are small extracellular vesicles released by cells for cell-cell communication. They play important roles in cancer development, metastasis, and drug resistance. Exosomal proteins have been demonstrated by many studies as promising biomarkers for cancer screening, diagnosis, and monitoring. Among many detection techniques, surface plasmon resonance (SPR) is a highly sensitive, label-free, and real-time optical detection method. Commercial prism-based wavelength/angular-modulated SPR sensors afford high sensitivity and resolution, but their large footprint and high cost limit their adaptability for clinical settings. Recently, a nanoplasmonic exosome (nPLEX) assay was developed to detect exosomal proteins for ovarian cancer diagnosis. However, comparing with conventional SPR biosensors, the broad applications of nanoplasmonic biosensors are limited by the difficult and expensive fabrication of nanostructures. We have developed an intensity-modulated, compact SPR biosensor (25 cm × 10 cm × 25 cm) which uses a conventional SPR sensing mechanism and does not require nanostructure fabrication. Calibration from glycerol showed that the compact SPR biosensor offered sensitivity of 9.258 × 103%/RIU and resolution of 8.311 × 10-6 RIU. We have demonstrated the feasibility of the compact SPR biosensor in lung cancer diagnosis using exosomal epidermal growth factor receptor (EGFR) and programmed death-ligand 1 (PD-L1) as biomarkers. It detected a higher level of exosomal EGFR from A549 nonsmall cell lung cancer (NSCLC) cells than BEAS-2B normal cells. With human serum samples, the compact SPR biosensor detected similar levels of exosomal EGFR in NSCLC patients and normal controls, and higher expression of exosomal PD-L1 in NSCLC patients than normal controls. The compact SPR biosensor showed higher detection sensitivity than ELISA and similar sensing accuracy as ELISA. It is a simple and user-friendly sensing platform, which may serve as an in vitro diagnostic test for cancer.