Changes in lipid metabolism track with the progression of neurofibrillary pathology in tauopathies.

BACKGROUND: Accumulation of tau leads to neuroinflammation and neuronal cell death in tauopathies, including Alzheimer's disease. As the disease progresses, there is a decline in brain energy metabolism. However, the role of tau protein in regulating lipid metabolism remains less characterized and poorly understood. METHODS: We used a transgenic rat model for tauopathy to reveal metabolic alterations induced by neurofibrillary pathology. Transgenic rats express a tau fragment truncated at the N- and C-terminals. For phenotypic profiling, we performed targeted metabolomic and lipidomic analysis of brain tissue, CSF, and plasma, based on the LC-MS platform. To monitor disease progression, we employed samples from transgenic and control rats aged 4, 6, 8, 10, 12, and 14 months. To study neuron-glia interplay in lipidome changes induced by pathological tau we used well well-established multicomponent cell model system. Univariate and multivariate statistical approaches were used for data evaluation. RESULTS: We showed that tau has an important role in the deregulation of lipid metabolism. In the lipidomic study, pathological tau was associated with higher production of lipids participating in protein fibrillization, membrane reorganization, and inflammation. Interestingly, significant changes have been found in the early stages of tauopathy before the formation of high-molecular-weight tau aggregates and neurofibrillary pathology. Increased secretion of pathological tau protein in vivo and in vitro induced upregulated production of phospholipids and sphingolipids and accumulation of lipid droplets in microglia. We also found that this process depended on the amount of extracellular tau. During the later stages of tauopathy, we found a connection between the transition of tau into an insoluble fraction and changes in brain metabolism. CONCLUSION: Our results revealed that lipid metabolism is significantly affected during different stages of tau pathology. Thus, our results demonstrate that the dysregulation of lipid composition by pathological tau disrupts the microenvironment, further contributing to the propagation of pathology.

Analog of kynurenic acid decreases tau pathology by modulating astrogliosis in rat model for tauopathy.

Kynurenines have immunomodulatory and neuroactive properties and can influence the central nervous system. Previous studies showed the involvement of the kynurenines in the pathogenesis and progression of neurodegenerative disease. In neurodegenerative disorders, including tauopathies, the tryptophan metabolism is shifted toward neurotoxic agents and the reduction of neuroprotectant products. Astrocyte-derived kynurenic acid serves as a neuroprotectant. However, systemic administration of kynurenic acid is not effective because of low permeability across the blood-brain barrier (BBB). We used a kynurenic acid analog with similar biological activity but higher brain permeability to overcome BBB limitations. In the present study, we used amide derivate of kynurenic acid N-(2-N, N-dimethylaminoethyl)- 4-oxo-1 H-quinoline-2-carboxamid (KYNA-1). We administered KYNA-1 for three months to tau transgenic rats SHR-24 and analyzed the effect on tau pathology and activation of glial cells. Primary glial cell cultures were applied to identify the mechanism of the KYNA-1 effect. KYNA-1 was not toxic to rats after chronic three-month administration. When chronically administered, KYNA-1 reduced hyperphosphorylation of insoluble tau in the brain of transgenic rats. Noteworthily, the plasma total tau was also reduced. We determined that the effect of KYNA-1 on tau pathology was induced through the modulation of glial activation. KYNA-1 inhibited LPS induced activation of astrocytes and induced transformation of microglia to M2 phenotype. We identified that the administration of KYNA-1 reduced tau hyperphosphorylation and neuroinflammation. KYNA-1 may serve as a promising treatment for tauopathies.

Pathophysiology of the choroid plexus in brain diseases.

The choroid plexus, located in the ventricular system of the central nervous system (CNS), obtains numerous roles critical for the proper development and operating of the CNS. The functions range from the best-known ones of the barrier and cerebrospinal fluid (CSF) producer, through participation in immune answer, 'nourishment, detoxification and reparation of the rest of the CNS. Increase number of studies point out the association between choroid plexus dysfunction, characterized by alterations in secretory, transport and barrier capabilities, and the broad spectrum of clinical conditions, as well as physiological aging. We present a brief overview of pathological states known or speculated to be connected to choroid plexus dysfunction, ranging from neurodevelopmental, to autoimmune and neurodegenerative diseases. We also cover the topic of choroid plexus tumors, as well explained involvement of the choroid plexus in pathogen invasion of the CNS, also referring to the currently actual SARS-CoV-2 infection. Finally, we have also touched conducted studies on the choroid plexus regenerative potential. With the information provided in the review we want to point out the importance and call for further research on the role of the choroid plexus in the sustainability of central nervous system health.

GM3 Ganglioside Linked to Neurofibrillary Pathology in a Transgenic Rat Model for Tauopathy.

Glycosphingolipids (GSLs) are amphipathic lipids composed of a sphingoid base and a fatty acyl attached to a saccharide moiety. GSLs play an important role in signal transduction, directing proteins within the membrane, cell recognition, and modulation of cell adhesion. Gangliosides and sulfatides belong to a group of acidic GSLs, and numerous studies report their involvement in neurodevelopment, aging, and neurodegeneration. In this study, we used an approach based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (HRMS/MS) to characterize the glycosphingolipid profile in rat brain tissue. Then, we screened characterized lipids aiming to identify changes in glycosphingolipid profiles in the normal aging process and tau pathology. Thorough screening of acidic glycosphingolipids in rat brain tissue revealed 117 ganglioside and 36 sulfatide species. Moreover, we found two ganglioside subclasses that were not previously characterized-GT1b-Ac2 and GQ1b-Ac2. The semi-targeted screening revealed significant changes in the levels of sulfatides and GM1a gangliosides during the aging process. In the transgenic SHR24 rat model for tauopathies, we found elevated levels of GM3 gangliosides which may indicate a higher rate of apoptotic processes.

Tau Protein and Its Role in Blood-Brain Barrier Dysfunction.

The blood-brain barrier (BBB) plays a crucial role in maintaining the specialized microenvironment of the central nervous system (CNS). In aging, the stability of the BBB declines and the permeability increases. The list of CNS pathologies involving BBB dysfunction is 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, neuronal dysfunction, and detrimental neuroinflammatory changes. Such processes have been implicated in different diseases, including vascular dementia, stroke, Alzheimer's disease (AD), Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, hypoxia, ischemia, and diabetes mellitus. The BBB damage is also observed in tauopathies that lack amyloid-ß overproduction, suggesting a role for tau in BBB damage. Tauopathies represent a heterogeneous group of around 20 different neurodegenerative diseases characterized by abnormal deposition of the MAPT in cells of the nervous system. Neuropathology of tauopathies is defined as intracellular accumulation of neurofibrillary tangles (NFTs) consisting of aggregated hyper- and abnormal phosphorylation of tau protein and neuroinflammation. Disruption of the BBB found in tauopathies is driven by chronic neuroinflammation. Production of pro-inflammatory signaling molecules such as cytokines, chemokines, and adhesion molecules by glial cells, neurons, and endothelial cells determine the integrity of the BBB and migration of immune cells into the brain. 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 BBB structural and functional changes.

Trafficking of immune cells across the blood-brain barrier is modulated by neurofibrillary pathology in tauopathies.

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.

Dynamics of Evans blue clearance from cerebrospinal fluid into meningeal lymphatic vessels and deep cervical lymph nodes.

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.

Changes of Cerebrospinal Fluid Peptides due to Tauopathy.

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.

Tauopathies - Focus on Changes at the Neurovascular Unit.

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-ß (Aß), 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.

Quantitative analysis of phenylalanine, tyrosine, tryptophan and kynurenine in rat model for tauopathies by ultra-high performance liquid chromatography with fluorescence and mass spectrometry detection.

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.

Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Homocysteine in Rat Plasma: Application to the Study of a Rat Model for Tauopathies.

Hyperhomocysteinemia is a common occurrence in many neurodegenerative diseases, including tauopathies. We developed and validated a simple and sensitive liquid chromatography-tandem mass spectrometry method for the analysis of homocysteine (Hcy) in rat plasma. Hcy was analyzed using ultra-performance liquid chromatography on a C8 column with detection by positive ESI tandem mass spectrometry. For optimal retention and separation, we used ion-pair reagent-heptafluorobutyric acid. The method utilizes heavy labeled internal standard and does not require any derivatization or extraction step. The procedure was validated in compliance with the European Medicines Agency guideline. The limit of detection was 0.15 µmol/L and the limit of quantification was 0.5 µmol/L. The method showed excellent linearity with regression coefficients higher than 0.99. The accuracy was in the range of 93-98%. The inter-day precision (n = 5 days), expressed as % relative standard deviation, was in the range 3-8%. Using this method, we analyzed plasma samples from two transgenic lines of the rat model for tauopathies.