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The blood-brain barrier plays a crucial role in maintaining the specialized microenvironment of the central nervous system. In aging, there is a decline in the stability of the blood-brain barrier leading to increased permeability. The list of central nervous system pathologies involving blood-brain barrier dysfunction is growing. The opening of the blood-brain barrier and subsequent infiltration of serum components to the brain can lead to a host of processes resulting in progressive synaptic, neuronal dysfunction, and detrimental neuroinflammatory changes. Such processes have been implicated in different diseases, including vascular dementia, stroke, Alzheimer´s disease, Parkinson´s disease, multiple sclerosis, amyotrophic lateral sclerosis, hypoxia, ischemia and diabetes mellitus. The blood-brain barrier damage is also observed in tauopathies that lack As over-production, suggesting a role for tau in blood-brain barrier damage. Tauopathies represent a heterogeneous group of around twenty different neurodegenerative diseases characterized by abnormal deposition of the microtubule-associated protein tau in cells of the nervous system. Neuropathology of tauopathies is defined as intracellular accumulation of neurofibrillary tangles consisting of aggregated hyper- and abnormal phosphorylation of tau protein and neuroinflammation. Chronic neuroinflammation in tauopathies is driven by glial cells that potentially trigger the disruption of the blood-brain barrier. Pro-inflammatory signaling molecules such as cytokines, chemokines and adhesion molecules produced by glial cells, neurons and endothelial cells, in general, cooperate to determine the integrity of the blood-brain barrier by influencing vascular permeability, enhancing the migration of immune cells and altering transport systems. The inflammatory processes promote structural changes in capillaries such as fragmentation, thickening, atrophy of pericytes, accumulation of laminin in the basement membrane and increased permeability of blood vessels to plasma proteins. Here we summarize the knowledge about the role of tau protein in blood-brain barrier structural and functional changes.

Tauopathies represent a heterogeneous group of neurodegenerative disorders characterized by abnormal deposition of the hyperphosphorylated microtubule-associated protein tau. Chronic neuroinflammation in tauopathies is driven by glial cells that potentially trigger the disruption of the blood-brain barrier (BBB). Pro-inflammatory signaling molecules such as cytokines, chemokines and adhesion molecules produced by glial cells, neurons and endothelial cells, in general, cooperate to determine the integrity of BBB by influencing vascular permeability, enhancing migration of immune cells and altering transport systems. We considered the effect of tau about vascular permeability of peripheral blood cells in vitro and in vivo using primary rat BBB model and transgenic rat model expressing misfolded truncated protein tau. Immunohistochemistry, electron microscopy and transcriptomic analysis were employed to characterize the structural and functional changes in BBB manifested by neurofibrillary pathology in a transgenic model. Our results show that misfolded protein tau ultimately modifies the endothelial properties of BBB, facilitating blood-to-brain cell transmigration. Our results suggest that the increased diapedesis of peripheral cells across the BBB, in response to tau protein, could be mediated by the increased expression of endothelial signaling molecules, namely ICAM-1, VCAM-1, and selectins. We suggest that the compensation of BBB in the diseased brain represents a crucial factor in neurodegeneration of human tauopathies.

Objectives Recently, it has been confirmed, that excess fluid and waste products from the brain are drained into the cerebrospinal fluid (CSF) and afterwards cleared via the olfactory route and/or lymphatic vessels in the brain dura and corresponding extracranial lymphatic structures. Therefore, the aim of present study was to monitor time-dependent uptake of Evans blue (EB) tracer from subarachnoid space into the meningeal lymphatic vessels and extracranial lymph nodes in rats during 3 hours-12 days. Methods EB was injected into the cisterna magna of anesthetized rats and after required survival, plasma, brain dura matter and corresponding lymph nodes (cervical, thoracic and lumbar) were dissected and processed for lymphatic vessels analyses using immunofluorescence and immunohistochemistry. Furthermore, we have used sensitive ultra-high-performance liquid chromatography (UHPLC) method for the determination of EB concentrations in selected samples. Results Using a combination of imaging methods, we have detected two different types of the vascular structures in the brain dura and in deep cervical lymph nodes. The blood vessels, which were RECA-1 + positive and the lymphatic-like vessels, expressing bright intense red fluorescence of EB tracer. Subsequently, using UHPLC with UV detection, we have quantified the EB concentration in positive structures by 3 hours up to 12 days after tracer delivery. A significant increase of EB concentration was detected in deep cervical lymph nodes already at 3 hours with a peak at 1 day that decreased to about one-tenth of its peak value by 12 days. Similar pattern was detected in brain dura. On the contrary, the brain tissue and plasma were almost negative for EB tracer during all tested time periods. Conclusion Our results demonstrate the dynamic changes of EB in meningeal lymphatic vessels and in deep cervical lymph nodes, thus recapitulating the downstream outflow of intracisternally injected tracer during 3 hours-12 days via dura mater lymphatic vessels towards corresponding extracranial draining system, particularly the deep cervical lymph nodes.

Alzheimer’s disease (AD) and progressive supranuclear palsy are two common neurodegenerative tauopathies, and the most common cause of progressive brain dementia in elderly affecting more than 35 million people. The tauopathies are characterized by abnormal deposition of microtubule associated protein tau into intracellular neurofibrillary tangles composed mainly of the hyperphosphorylated form of the protein. The diagnosis of tauopathies is based on the presence of clinical features and pathological changes. Over the last decade, there has been an intensive search for novel biochemical markers for clinical diagnosis of AD and other tauopathies. In the present study, we used transgenic rat model for tauopathy expressing human truncated tau protein (aa 151–391/4R) to analyze the cerebrospinal fluid (CSF) peptidome using liquid chromatography – matrix assisted laser desorption/ionization mass spectrometry (LC-MALDI TOF/TOF). From 345 peptides, we identified a total of 175 proteins. Among them, 17 proteins were significantly altered in the CSF of transgenic rats. The following proteins were elevated in the CSF of transgenic rats when compared to the control animals: neurofilament light and medium chain, apolipoprotein E, gamma-synuclein, chromogranin A, reticulon-4, secretogranin-2, calsyntein-1 and -3, endothelin-3, neuroendocrine protein B72A, alpha-1-macroglobulin, and augurin. Interestingly most of the identified proteins were previously linked to AD and other tauopathies, indicating the significance of transgenic animals in biomarker validation.

Blood-brain barrier changes are an integral part of many neurodegenerative diseases. Evans blue is an intravital dye that binds to albumin and can be therefore used to monitor extravasation of this plasma protein across the blood-brain barrier in animal models of neurodegeneration. To monitor extravasation of albumin across the blood-brain barrier, we developed and validated an ultra-high performance liquid chromatography method for the analysis of Evans blue in rat plasma and brain samples. Analyte was separated on ACQUITY UPLC BEH C18 column (2.1 mm x 50 mm) using a 5 minute gradient run and detected by an UV detector. The limits of quantification (LOQ) were 10 µg/ml in plasma and 0.5 µg/g in brain samples. The limits of detection (LOD) were 1 µg/ml in plasma and 0.015 µg/g in brain samples. The method showed excellent linearity with regression coefficients higher than 0.99. The accuracy was within the range of 91-105%. The inter-day precision was in the range of 1.3-8%. The benefit of using UPLC is selectivity, short analysis period and thus, a very good sample throughput. Using this method, we analysed albumin extravasation across the blood-brain barrier in transgenic rat model for tauopathy SHR-72 and age-matched control animals.

In the past, the blood-brain barrier (BBB) has been characterized mainly as a layer of endothelial cells forming the vessel/capillary wall of the brain. More recently, the BBB is considered to be a part of a highly dynamic and interactive system called the neurovascular unit (NVU), consisting of vascular cells, glial cells, and neurons. The list of central nervous system (CNS) pathologies involving BBB dysfunction is rapidly growing. The opening of the BBB and subsequent infiltration of serum components to the brain can lead to a host of processes resulting in progressive synaptic and neuronal dysfunction and loss. Such processes have been implicated in different diseases, including vascular dementias, stroke, Alzheimer´s disease (AD), Parkinson´s disease, multiple sclerosis, amyotrophic lateral sclerosis, hypoxia, ischemia, and diabetes mellitus. Tauopathies represent a heterogeneous group of around 20 different neurodegenerative diseases characterized by abnormal deposition of microtubule-associated protein tau in cells of the nervous system. Increased microvascular permeability has been more typically related to cerebrovascular deposition of amyloid-β (Aβ), but in contrast very little is known about the connection between functional impairment of the BBB and the misfolded tau proteins. Here, we review what is known about tauopathies, the BBB, and the NVU.

We developed and validated a simple and sensitive ultra-high performance liquid chromatography (UHPLC) method for the analysis of phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and kynurenine (Kyn) in rat plasma. Analytes were separated on Acquity UPLC HSS T3 column (2.1 mm×50 mm, 1.8 μm particle size) using a 4 min ammonium acetate (pH 5) gradient and detected by fluorescence and positive ESI mass spectrometry. Sample preparation involved dilution of plasma, deproteinization by trichloroacetic acid and centrifugation. The procedure was validated in compliance with the FDA guideline. The limits of quantification (LOQ) were 0.3 μM for Kyn and from 1.5 to 3 μM for Phe, Tyr, Trp. The method showed excellent linearity with regression coefficients higher than 0.99. The accuracy was within the range of 86-108%. The inter-day precision (n=5 days), expressed as % RSD, was in the range 1-13%. The benefit of using UHPLC is a short analysis period and thus, a very good sample throughput. Using this method, we analyzed plasma samples and detected significant changes of Kyn and Phe in transgenic rat model for tauopathies.