<em>In Vivo</em> Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an ...

University of Tokyo

[Involvement of ouabain (digitalis-like factor) in the pathogenesis of hypertension and hypertensive target-organ damage].

The metabolism of amyloid beta peptide (A beta) in the brain is crucial to the pathogenesis of Alzheimer disease. A body of evidence suggests that A beta is actively transported from brain parenchyma to blood across the blood-brain barrier (BBB), although the precise mechanism remains unclear. To unravel the cellular and molecular mechanism of A beta transport across the BBB, we established a new in vitro model of the initial internalization step of A beta transport using TR-BBB cells, a conditionally immortalized endothelial cell line from rat brain. We show that TR-BBB cells rapidly internalize A beta through a receptor-mediated mechanism. We also provide evidence that A beta internalization is mediated by LRP1 (low density lipoprotein receptor-related protein 1), since administration of LRP1 antagonist, receptor-associated protein, neutralizing antibody, or small interference RNAs all reduced A beta uptake. Despite the requirement of LRP1-dependent internalization, A beta does not directly bind to LRP1 in an in vitro binding assay. Unlike TR-BBB cells, mouse embryonic fibroblasts endogenously expressing functional LRP1 and exhibiting the authentic LRP1-mediated endocytosis (e.g. of tissue plasminogen activator) did not show rapid A beta uptake. Based on these data, we propose that the rapid LRP1-dependent internalization of A beta occurs under the BBB-specific cellular context and that TR-BBB is a useful tool for analyzing the molecular mechanism of the rapid transport of A beta across BBB.

The low density lipoprotein receptor-related protein 1 mediates uptake of amyloid beta peptides in an in vitro model of the blood-brain barrier cells.

Amyloid beta (Abeta) immunotherapy is emerging as a promising disease-modifying therapy for Alzheimer's disease, although the precise mechanisms whereby anti-Abeta antibodies act against amyloid deposition and cognitive deficits remain elusive. To test the "peripheral sink" theory, which postulates that the effects of anti-Abeta antibodies in the systemic circulation are to promote the Abeta efflux from brain to blood, we studied the clearance of (125)I-Abeta(1-40) microinjected into mouse brains after intraperitoneal administration of an anti-Abeta monoclonal antibody 266. (125)I-Abeta(1-40) was rapidly eliminated from brains with a half-life of approximately 30 min in control mice, whereas 266 significantly retarded the elimination of Abeta, presumably due to formation of Abeta-antibody complex in brains. Administration of 266 to APP transgenic mice increased the levels of monomer Abeta species in an antibody-bound form, without affecting that of total Abeta. We propose a novel mechanism of Abeta immunotherapy by the class of anti-Abeta antibodies that preferentially bind soluble Abeta, i.e., intracerebral, rather than peripheral, sequestration of soluble, monomer form of Abeta, thereby preventing the accumulation of multimeric toxic Abeta species in brains.

Abeta immunotherapy: intracerebral sequestration of Abeta by an anti-Abeta monoclonal antibody 266 with high affinity to soluble Abeta.

Halichlorine, isolated from a marine sponge Halichondria okadai Kadota, has a unique structure and its physiological activity is virtually unknown. In the present study, we investigated the direct effect of halichlorine on vascular contractility. In endothelium-denuded rat aorta, while the treatment of halichlorine (0.01-10microM) did not induce vascular contraction, halichlorine (0.01-10microM) dose-dependently inhibited both the steady-state precontractions induced by high K(+) (65.4mM) and phenylephrine (1microM). The vasodilator effect of halichlorine (10microM) on high K(+) (65.4mM)-induced contraction was more potent than that on phenylephrine (1microM)-induced contraction (65.4mM high K(+): 72.7+/-3.4%; 1microM phenylephrine: 34.7+/-2.3%). To investigate the mechanism underlying the suppressive effect of halichlorine on vascular contractility, we examined the effect of halichlorine on intracellular Ca(2+) concentration in vascular smooth muscle with a fluorescent Ca(2+) indicator, fura-2. Treatment of halichlorine (10microM) significantly inhibited the sustained [Ca(2+)](i) elevation induced by high K(+) (65.4mM) (45.3+/-5.5%). Furthermore, current measurements by whole-cell mode patch-clamp recording in rat aortic smooth muscle cells (A7r5 cells) demonstrated that halichlorine (10microM) decreased the current density of the L-type Ca(2+) channel (peak Ca(2+)-channel current densities: -2.09+/-0.27pA/pF for control; -0.58+/-0.07pA/pF for halichlorine). These results suggest that halichlorine inhibits L-type Ca(2+) channels in vascular smooth muscle cells, which inhibit intracellular Ca(2+) influx, and then reduce vascular contractions.

Halichlorine is a novel L-type Ca(2+) channel inhibitor isolated from the marine sponge Halichondria okadai Kadota.

Halichlorine is a marine alkaloid isolated from the marine sponge Halichondria okadai KADOTA, and its pathophysiological effect on vascular cells remains unknown. Here, we examined the anti-atherosclerosis activity of halichlorine on endothelial cells by assessing the expression of adhesion molecules. In bovine aortic endothelial cells (BAECs), pretreatment with halichlorine (10 microM, 2 h) inhibited lipopolysaccharide (LPS) (3 microg/ml, 3 h)-induced mRNA expressions of vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM-1), and E-selectin. Consistently, pretreatment with halichlorine (10 microM, 2 h) reduced LPS (3 microg/ml)-induced monocyte (U937) adhesion to endothelial monolayer. To investigate the mechanism underlying this phenomenon, we examined the effect of halichlorine on nuclear factor-kappaB (NF-kappaB) activity in endothelial cells. Treatment with LPS (3 microg/ml) for 1 h increased the ratio of cells showing NF-kappaB p65 translocation from cytosol to nucleus. Pretreatment with halichlorine (10 microM, 2 h) significantly inhibited the LPS-induced NF-kappaB p65 translocation. Finally, we examined the cytotoxicity of halichlorine on endothelial cells and found that halichlorine (10 microM, 24 - 48 h) did not influence BAECs proliferation and viability. Herein, we provided the first evidence that halichlorine inhibits LPS-induced NF-kappaB activation, which results in the suppression of VCAM-1, ICAM-1, and E-selectin gene expression and monocyte-adhesion to endothelial cells.

Halichlorine reduces monocyte adhesion to endothelium through the suppression of nuclear factor-kappaB activation.

CD2-associated protein (CD2AP) is an SH3-containing scaffold adaptor protein which regulates the actin cytoskeleton. Recently, CD2AP was identified as a genetic risk factor for Alzheimer's disease (AD) by several genome-wide association studies. One of the hallmarks of AD is the accumulation of aggregated forms of Amyloid-β (Aβ) in the brain. In humans, CD2AP AD susceptibility locus (rs9349407) is associated with an increased plaque burden. Aβ production is highly regulated by endocytosis and is influenced by lysosomal function. Lysosomal trafficking is influenced by CD2AP. In this study, we decreased CD2AP levels in N2a neuroblastoma cultures and PS1APP mice and analyzed Aβ levels and plaque burden.

Effects of CD2-associated protein deficiency on amyloid-β in neuroblastoma cells and in an APP transgenic mouse model.

Intracellular accumulation of tau as neurofibrillary tangles (NFTs) is the hallmark of Alzheimer's disease (AD) as well as in other tauopathies. Tau is present not only in the cytoplasm but also in the extracellular space such as cerebrospinal fluid (CSF) and brain interstitial fluid (ISF). Although clearance is one critical parameter leading to such intracellular/extracellular tau accumulation, in vivo turnover of tau has not been well characterized. The current study has attempted to precisely determine in vivo turnover rates of tau utilizing tet-off regulatable mice. In particular, we assessed intracellular tau and extracellular tau, soluble tau, insoluble tau and phosphorylated tau at certain sites utilizing a combination of in vivo microdialysis, biochemical analysis and specific ELISAs recognizing each species. To examine the effect of a tauopathy-associated mutation on tau clearance, half-lives of various tau species were compared between the mice with a FTDP-17 mutation that induces β-sheet formation, ΔK280 mutation (pro-aggregant mice) and control mice with additional β-sheet breaking mutations (anti-aggregant mice).

Analysis of in vivo turnover of tau in a mouse model of tauopathy.

Tau is a microtubule binding protein highly abundant in cytoplasm of neurons. However now there is compelling evidence that it is also physiologically released in the extracellular space. Tau is detectable in culture media of various cells, cerebrospinal fluid (CSF), and brain interstitial fluid (ISF). Being able to measure extracellular tau levels in vivo would deep our understanding on the underlying mechanisms of physiological/pathological tau release from neurons. To meet this need, this chapter describes an in vivo microdialysis protocol and materials for the measurement of extracellular tau in ISF from freely moving mice.

In Vivo Microdialysis of Brain Interstitial Fluid for the Determination of Extracellular Tau Levels.

APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease. ApoE4 increases brain amyloid-β pathology relative to other ApoE isoforms. However, whether APOE independently influences tau pathology, the other major proteinopathy of Alzheimer disease and other tauopathies, or tau-mediated neurodegeneration, is not clear. By generating P301S tau transgenic mice on either a human ApoE knock-in (KI) or ApoE knockout (KO) background, here we show that P301S/E4 mice have significantly higher tau levels in the brain and a greater extent of somatodendritic tau redistribution by three months of age compared with P301S/E2, P301S/E3, and P301S/EKO mice. By nine months of age, P301S mice with different ApoE genotypes display distinct phosphorylated tau protein (p-tau) staining patterns. P301S/E4 mice develop markedly more brain atrophy and neuroinflammation than P301S/E2 and P301S/E3 mice, whereas P301S/EKO mice are largely protected from these changes. In vitro, E4-expressing microglia exhibit higher innate immune reactivity after lipopolysaccharide treatment. Co-culturing P301S tau-expressing neurons with E4-expressing mixed glia results in a significantly higher level of tumour-necrosis factor-α (TNF-α) secretion and markedly reduced neuronal viability compared with neuron/E2 and neuron/E3 co-cultures. Neurons co-cultured with EKO glia showed the greatest viability with the lowest level of secreted TNF-α. Treatment of P301S neurons with recombinant ApoE (E2, E3, E4) also leads to some neuronal damage and death compared with the absence of ApoE, with ApoE4 exacerbating the effect. In individuals with a sporadic primary tauopathy, the presence of an ε4 allele is associated with more severe regional neurodegeneration. In individuals who are positive for amyloid-β pathology with symptomatic Alzheimer disease who usually have tau pathology, ε4-carriers demonstrate greater rates of disease progression. Our results demonstrate that ApoE affects tau pathogenesis, neuroinflammation, and tau-mediated neurodegeneration independently of amyloid-β pathology. ApoE4 exerts a 'toxic' gain of function whereas the absence of ApoE is protective.

ApoE4 markedly exacerbates tau-mediated neurodegeneration in a mouse model of tauopathy.

The pathological aggregation of tau protein is a hallmark of a set of neurodegenerative diseases collectively referred to as tauopathies. Tau aggregates independently in each neuron, but this aggregation can also occur in a non-cell autonomous manner in which aggregated tau is transmitted from one cell to another. Such trans-cellular propagation is initiated by the uptake of extracellular tau, which then seeds soluble tau in the recipient cells to spread the tau pathology. Accumulating evidence has demonstrated that tau is not only present in the cytoplasm of neurons but also actively released into the extracellular space. This finding has led to the idea that extracellular tau could be a novel therapeutic target to halt the propagation of tau pathology. From this perspective, the present review article focuses on recent advances in understanding the mechanisms regulating the levels of extracellular tau and discusses the role of such mechanisms in the propagation of tau pathology.

Extracellular Tau and Its Potential Role in the Propagation of Tau Pathology.

α-Synuclein is a presynaptic protein abundant in the cytoplasmic compartment of neurons, whereas its presence in the extracellular space has also been observed under physiological conditions. Extracellular α-synuclein has pathological significance, exhibiting cellular toxicity and impairment of synaptic transmission. Notably, misfolded α-synuclein drives the cell-to-cell propagation of pathology via the extracellular space. However, the primary mechanism that regulates the extracellular levels of α-synuclein remains to be determined.

Kaoru Yamada

Department of Neuropathology,
Graduate School of Medicine,
Department of Neuropathology, Graduate School of Medicine

University of Tokyo

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

Kaoru Yamada

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neuropathology,Graduate School of Medicine,Department of Neuropathology, Graduate School of Medicine

University of Tokyo

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

Department of Neuropathology,
Graduate School of Medicine,
Department of Neuropathology, Graduate School of Medicine