Cerebral blood flow regulation and neurovascular dysfunction in Alzheimer disease.

Cerebral blood flow (CBF) regulation is essential for normal brain function. The mammalian brain has evolved a unique mechanism for CBF control known as neurovascular coupling. This mechanism ensures a rapid increase in the rate of CBF and oxygen delivery to activated brain structures. The neurovascular unit is composed of astrocytes, mural vascular smooth muscle cells and pericytes, and endothelia, and regulates neurovascular coupling. This Review article examines the cellular and molecular mechanisms within the neurovascular unit that contribute to CBF control, and neurovascular dysfunction in neurodegenerative disorders such as Alzheimer disease.

Investigating contactless high frequency ultrasound microbeam stimulation for determination of invasion potential of breast cancer cells.

In this article, we investigate the application of contactless high frequency ultrasound microbeam stimulation (HFUMS) for determining the invasion potential of breast cancer cells. In breast cancer patients, the finding of tumor metastasis significantly worsens the clinical prognosis. Thus, early determination of the potential of a tumor for invasion and metastasis would significantly impact decisions about aggressiveness of cancer treatment. Recent work suggests that invasive breast cancer cells (MDA-MB-231), but not weakly invasive breast cancer cells (MCF-7, SKBR3, and BT-474), display a number of neuronal characteristics, including expression of voltage-gated sodium channels. Since sodium channels are often co-expressed with calcium channels, this prompted us to test whether single-cell stimulation by a highly focused ultrasound microbeam would trigger Ca(2+) elevation, especially in highly invasive breast cancer cells. To calibrate the diameter of the microbeam ultrasound produced by a 200-MHz single element LiNbO3 transducer, we focused the beam on a wire target and performed a pulse-echo test. The width of the beam was ∼17 µm, appropriate for single cell stimulation. Membrane-permeant fluorescent Ca(2+) indicators were utilized to monitor Ca(2+) changes in the cells due to HFUMS. The cell response index (CRI), which is a composite parameter reflecting both Ca(2+) elevation and the fraction of responding cells elicited by HFUMS, was much greater in highly invasive breast cancer cells than in the weakly invasive breast cancer cells. The CRI of MDA-MB-231 cells depended on peak-to-peak amplitude of the voltage driving the transducer. These results suggest that HFUMS may serve as a novel tool to determine the invasion potential of breast cancer cells, and with further refinement may offer a rapid test for invasiveness of tumor biopsies in situ.

Role of the synaptobrevin C terminus in fusion pore formation.

Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets.

Anti-malaria drug artesunate prevents development of amyloid-ß pathology in mice by upregulating PICALM at the blood-brain barrier.

BACKGROUND: PICALM is one of the most significant susceptibility factors for Alzheimer's disease (AD). In humans and mice, PICALM is highly expressed in brain endothelium. PICALM endothelial levels are reduced in AD brains. PICALM controls several steps in Aß transcytosis across the blood-brain barrier (BBB). Its loss from brain endothelium in mice diminishes Aß clearance at the BBB, which worsens Aß pathology, but is reversible by endothelial PICALM re-expression. Thus, increasing PICALM at the BBB holds potential to slow down development of Aß pathology. METHODS: To identify a drug that could increase PICALM expression, we screened a library of 2007 FDA-approved drugs in HEK293t cells expressing luciferase driven by a human PICALM promoter, followed by a secondary mRNA screen in human Eahy926 endothelial cell line. In vivo studies with the lead hit were carried out in Picalm-deficient (Picalm+/-) mice, Picalm+/-; 5XFAD mice and Picalmlox/lox; Cdh5-Cre; 5XFAD mice with endothelial-specific Picalm knockout. We studied PICALM expression at the BBB, Aß pathology and clearance from brain to blood, cerebral blood flow (CBF) responses, BBB integrity and behavior. RESULTS: Our screen identified anti-malaria drug artesunate as the lead hit. Artesunate elevated PICALM mRNA and protein levels in Eahy926 endothelial cells and in vivo in brain capillaries of Picalm+/- mice by 2-3-fold. Artesunate treatment (32 mg/kg/day for 2 months) of 3-month old Picalm+/-; 5XFAD mice compared to vehicle increased brain capillary PICALM levels by 2-fold, and reduced Aß42 and Aß40 levels and Aß and thioflavin S-load in the cortex and hippocampus, and vascular Aß load by 34-51%. Artesunate also increased circulating Aß42 and Aß40 levels by 2-fold confirming accelerated Aß clearance from brain to blood. Consistent with reduced Aß pathology, treatment of Picalm+/-; 5XFAD mice with artesunate improved CBF responses, BBB integrity and behavior on novel object location and recognition, burrowing and nesting. Endothelial-specific knockout of PICALM abolished all beneficial effects of artesunate in 5XFAD mice indicating that endothelial PICALM is required for its therapeutic effects. CONCLUSIONS: Artesunate increases PICALM levels and Aß clearance at the BBB which prevents development of Aß pathology and functional deficits in mice and holds potential for translation to human AD.

Influenza virus-membrane fusion triggered by proton uncaging for single particle studies of fusion kinetics.

We report a method for studying membrane fusion, focusing on influenza virus fusion to lipid bilayers, which provides high temporal resolution through the rapid and coordinated initiation of individual virus fusion events. Each fusion event proceeds through a series of steps, much like multistep chemical reaction. Fusion is initiated by a rapid decrease in pH that accompanies the "uncaging" of an effector molecule from o-nitrobenzaldehyde, a photoisomerizable compound that releases a proton to the surrounding solution within microseconds of long-wave ultraviolet irradiation. In order to quantify pH values upon UV irradiation and uncaging, we introduce a simple silica nanoparticle pH sensor, useful for reporting the pH in homogeneous nanoliter volumes under conditions where traditional organic dye-type pH probes fail. Subsequent single-virion fusion events are monitored using total internal reflection fluorescence microscopy. Statistical analysis of these stochastic events uncovers kinetic information about the fusion reaction. This approach reveals that the kinetic parameters obtained from the data are sensitive to the rate at which protons are delivered to the bound viruses. Higher resolution measurements can enhance fundamental fusion studies and aid antiviral antifusogenic drug development.

3K3A-Activated Protein C Protects the Blood-Brain Barrier and Neurons From Accelerated Ischemic Injury Caused by Pericyte Deficiency in Mice.

Pericytes, mural cells of brain capillaries, maintain the blood-brain barrier (BBB), regulate cerebral blood flow (CBF), and protect neurons against ischemic damage. To further investigate the role of pericytes in ischemia, we induced stroke by 45-min transient middle cerebral artery occlusion (tMCAo) in 6-month-old pericyte-deficient Pdgfrb + ⁣/- mice and control Pdgfrb+/+ littermates. Compared to controls, Pdgfrb + ⁣/- mice showed a 26% greater loss of CBF during early reperfusion, and 40-50% increase in the infarct and edema volumes and motor neurological score 24 h after tMCAo. These changes were accompanied by 50% increase in both immunoglobulin G and fibrinogen pericapillary deposits in the ischemic cortex 8 h after tMCAo indicating an accelerated BBB breakdown, and 35 and 55% greater losses of pericyte coverage and number of degenerating neurons 24 h after tMCAo, respectively. Treatment of Pdgfrb + ⁣/- mice with 3K3A-activated protein C (APC), a cell-signaling analog of plasma protease APC, administered intravenously 10 min and 4 h after tMCAo normalized CBF during the early reperfusion phase and reduced infarct and edema volume and motor neurological score by 55-60%, with similar reductions in BBB breakdown and number of degenerating neurons. Our data suggest that pericyte deficiency results in greater brain injury, BBB breakdown, and neuronal degeneration in stroked mice and that 3K3A-APC protects the brain from accelerated injury caused by pericyte deficiency. These findings may have implications for treatment of ischemic brain injury in neurological conditions associated with pericyte loss such as those seen during normal aging and in neurodegenerative disorders such as Alzheimer's disease.

Blood-brain barrier link to human cognitive impairment and Alzheimer's Disease.

Vascular dysfunction is frequently seen in disorders associated with cognitive impairment, dementia and Alzheimer's disease (AD). Recent advances in neuroimaging and fluid biomarkers suggest that vascular dysfunction is not an innocent bystander only accompanying neuronal dysfunction. Loss of cerebrovascular integrity, often referred to as breakdown in the blood-brain barrier (BBB), has recently shown to be an early biomarker of human cognitive dysfunction and possibly underlying mechanism of age-related cognitive decline. Damage to the BBB may initiate or further invoke a range of tissue injuries causing synaptic and neuronal dysfunction and cognitive impairment that may contribute to AD. Therefore, better understanding of how vascular dysfunction caused by BBB breakdown interacts with amyloid-ß and tau AD biomarkers to confer cognitive impairment may lead to new ways of thinking about pathogenesis, and possibly treatment and prevention of early cognitive impairment, dementia and AD, for which we still do not have effective therapies.

Corrigendum: Acute ablation of cortical pericytes leads to rapid neurovascular uncoupling.

[This corrects the article DOI: 10.3389/fncel.2020.00027.].

Imaging subtle leaks in the blood-brain barrier in the aging human brain: potential pitfalls, challenges, and possible solutions.

Recent studies using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with gadolinium-based contrast agents (GBCA) have demonstrated subtle blood-brain barrier (BBB) leaks in the human brain during normal aging, in individuals with age-related cognitive dysfunction, genetic risk for Alzheimer's disease (AD), mild cognitive impairment, early AD, cerebral small vessel disease (SVD), and other neurodegenerative disorders. In these neurological conditions, the BBB leaks, quantified by the unidirectional BBB GBCA tracer's constant Ktrans maps, are typically orders of magnitude lower than in brain tumors, after stroke and/or during relapsing episodes of multiple sclerosis. This puts extra challenges for the DCE-MRI technique by pushing calculations towards its lower limits of detectability. In addition, presently, there are no standardized multivendor protocols or evidence of repeatability and reproducibility. Nevertheless, subtle BBB leaks may critically contribute to the pathophysiology of cognitive impairment and dementia associated with AD or SVD, and therefore, efforts to improve sensitivity of detection, reliability, and reproducibility are warranted. A larger number of participants scanned by different MR scanners at different clinical sites are sometimes required to detect differences in BBB integrity between control and at-risk groups, which impose additional challenges. Here, we focus on these new challenges and propose some approaches to normalize and harmonize DCE data between different scanners. In brief, we recommend specific regions to be used for the tracer's vascular input function and DCE data processing and how to find and correct negative Ktrans values that are physiologically impossible. We hope this information will prove helpful to new investigators wishing to study subtle BBB damage in neurovascular and neurodegenerative conditions and in the aging human brain.

A "multi-omics" analysis of blood-brain barrier and synaptic dysfunction in APOE4 mice.

Apolipoprotein E4 (APOE4), the main susceptibility gene for Alzheimer's disease, leads to blood-brain barrier (BBB) breakdown in humans and mice. Remarkably, BBB dysfunction predicts cognitive decline and precedes synaptic deficits in APOE4 human carriers. How APOE4 affects BBB and synaptic function at a molecular level, however, remains elusive. Using single-nucleus RNA-sequencing and phosphoproteome and proteome analysis, we show that APOE4 compared with APOE3 leads to an early disruption of the BBB transcriptome in 2-3-mo-old APOE4 knock-in mice, followed by dysregulation in protein signaling networks controlling cell junctions, cytoskeleton, clathrin-mediated transport, and translation in brain endothelium, as well as transcription and RNA splicing suggestive of DNA damage in pericytes. Changes in BBB signaling mechanisms paralleled an early, progressive BBB breakdown and loss of pericytes, which preceded postsynaptic interactome disruption and behavioral deficits that developed 2-5 mo later. Thus, dysregulated signaling mechanisms in endothelium and pericytes in APOE4 mice reflect a molecular signature of a progressive BBB failure preceding changes in synaptic function and behavior.

Endothelial LRP1 protects against neurodegeneration by blocking cyclophilin A.

The low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic and cell signaling transmembrane protein. Endothelial LRP1 clears proteinaceous toxins at the blood-brain barrier (BBB), regulates angiogenesis, and is increasingly reduced in Alzheimer's disease associated with BBB breakdown and neurodegeneration. Whether loss of endothelial LRP1 plays a direct causative role in BBB breakdown and neurodegenerative changes remains elusive. Here, we show that LRP1 inactivation from the mouse endothelium results in progressive BBB breakdown, followed by neuron loss and cognitive deficits, which is reversible by endothelial-specific LRP1 gene therapy. LRP1 endothelial knockout led to a self-autonomous activation of the cyclophilin A-matrix metalloproteinase-9 pathway in the endothelium, causing loss of tight junctions underlying structural BBB impairment. Cyclophilin A inhibition in mice with endothelial-specific LRP1 knockout restored BBB integrity and reversed and prevented neuronal loss and behavioral deficits. Thus, endothelial LRP1 protects against neurodegeneration by inhibiting cyclophilin A, which has implications for the pathophysiology and treatment of neurodegeneration linked to vascular dysfunction.

F-actin and myosin II accelerate catecholamine release from chromaffin granules.

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.

Channelrhodopsin Excitation Contracts Brain Pericytes and Reduces Blood Flow in the Aging Mouse Brain in vivo.

Brains depend on blood flow for the delivery of oxygen and nutrients essential for proper neuronal and synaptic functioning. French physiologist Rouget was the first to describe pericytes in 1873 as regularly arranged longitudinal amoeboid cells on capillaries that have a muscular coat, implying that these are contractile cells that regulate blood flow. Although there have been >30 publications from different groups, including our group, demonstrating that pericytes are contractile cells that can regulate hemodynamic responses in the brain, the role of pericytes in controlling cerebral blood flow (CBF) has not been confirmed by all studies. Moreover, recent studies using different optogenetic models to express light-sensitive channelrhodopsin-2 (ChR2) cation channels in pericytes were not conclusive; one, suggesting that pericytes expressing ChR2 do not contract after light stimulus, and the other, demonstrating contraction of pericytes expressing ChR2 after light stimulus. Since two-photon optogenetics provides a powerful tool to study mechanisms of blood flow regulation at the level of brain capillaries, we re-examined the contractility of brain pericytes in vivo using a new optogenetic model developed by crossing our new inducible pericyte-specific CreER mouse line with ChR2 mice. We induced expression of ChR2 in pericytes with tamoxifen, excited ChR2 by 488 nm light, and monitored pericyte contractility, brain capillary diameter changes, and red blood cell (RBC) velocity in aged mice by in vivo two-photon microscopy. Excitation of ChR2 resulted in pericyte contraction followed by constriction of the underlying capillary leading to approximately an 8% decrease (p = 0.006) in capillary diameter. ChR2 excitation in pericytes substantially reduced capillary RBC flow by 42% (p = 0.03) during the stimulation period compared to the velocity before stimulation. Our data suggests that pericytes contract in vivo and regulate capillary blood flow in the aging mouse brain. By extension, this might have implications for neurological disorders of the aging human brain associated with neurovascular dysfunction and pericyte loss such as stroke and Alzheimer's disease.

Acute Ablation of Cortical Pericytes Leads to Rapid Neurovascular Uncoupling.

Pericytes are perivascular mural cells that enwrap brain capillaries and maintain blood-brain barrier (BBB) integrity. Most studies suggest that pericytes regulate cerebral blood flow (CBF) and oxygen delivery to activated brain structures, known as neurovascular coupling. While we have previously shown that congenital loss of pericytes leads over time to aberrant hemodynamic responses, the effects of acute global pericyte loss on neurovascular coupling have not been studied. To address this, we used our recently reported inducible pericyte-specific Cre mouse line crossed to iDTR mice carrying Cre-dependent human diphtheria toxin (DT) receptor, which upon DT treatment leads to acute pericyte ablation. As expected, DT led to rapid progressive loss of pericyte coverage of cortical capillaries up to 50% at 3 days post-DT, which correlated with approximately 50% reductions in stimulus-induced CBF responses measured with laser doppler flowmetry (LDF) and/or intrinsic optical signal (IOS) imaging. Endothelial response to acetylcholine, microvascular density, and neuronal evoked membrane potential responses remained, however, unchanged, as well as arteriolar smooth muscle cell (SMC) coverage and functional responses to adenosine, as we previously reported. Together, these data suggest that neurovascular uncoupling in this model is driven by pericyte loss, but not other vascular deficits or neuronal dysfunction. These results further support the role of pericytes in CBF regulation and may have implications for neurological conditions associated with rapid pericyte loss such as hypoperfusion and stroke, as well as conditions where the exact time course of global regional pericyte loss is less clear, such as Alzheimer's disease (AD) and other neurogenerative disorders.

Improved surface-patterned platinum microelectrodes for the study of exocytotic events.

Surface-patterned platinum microelectrodes insulated with 300 nm thick fused silica were fabricated using contact photolithography. These electrodes exhibit low noise and were used for monitoring single vesicle exocytosis from chromaffin cells by constant potential amperometry as well as fast-scan cyclic voltammetry. Amperometric spike parameters were consistent with those obtained with conventional carbon fiber electrodes. Catecholamine voltammograms acquired with platinum electrodes exhibited redox peaks with full width at half-maximum of approximately 45 mV, much sharper than those of carbon fiber electrode recordings. The time course of voltammetrically measured release events was similar for platinum and carbon fiber electrodes. The fused-silica-insulated platinum electrodes could be cleaned and reused repetitively and allowed incorporation of micrometer precision surface-patterned poly-D-lysine. Poly-D-lysine-functionalized devices were applied to stimulate mast cells and record single release events without serotonin preloading. Microfabricated platinum electrodes are thus able to record single exocytotic events with high resolution and should be suitable for highly parallel electrode arrays allowing simultaneous measurements of single events from multiple cells.

Mitigating Antagonism between Transcription and Proliferation Allows Near-Deterministic Cellular Reprogramming.

Although cellular reprogramming enables the generation of new cell types for disease modeling and regenerative therapies, reprogramming remains a rare cellular event. By examining reprogramming of fibroblasts into motor neurons and multiple other somatic lineages, we find that epigenetic barriers to conversion can be overcome by endowing cells with the ability to mitigate an inherent antagonism between transcription and DNA replication. We show that transcription factor overexpression induces unusually high rates of transcription and that sustaining hypertranscription and transgene expression in hyperproliferative cells early in reprogramming is critical for successful lineage conversion. However, hypertranscription impedes DNA replication and cell proliferation, processes that facilitate reprogramming. We identify a chemical and genetic cocktail that dramatically increases the number of cells capable of simultaneous hypertranscription and hyperproliferation by activating topoisomerases. Further, we show that hypertranscribing, hyperproliferating cells reprogram at 100-fold higher, near-deterministic rates. Therefore, relaxing biophysical constraints overcomes molecular barriers to cellular reprogramming.

Pericyte loss leads to circulatory failure and pleiotrophin depletion causing neuron loss.

Pericytes are positioned between brain capillary endothelial cells, astrocytes and neurons. They degenerate in multiple neurological disorders. However, their role in the pathogenesis of these disorders remains debatable. Here we generate an inducible pericyte-specific Cre line and cross pericyte-specific Cre mice with iDTR mice carrying Cre-dependent human diphtheria toxin receptor. After pericyte ablation with diphtheria toxin, mice showed acute blood-brain barrier breakdown, severe loss of blood flow, and a rapid neuron loss that was associated with loss of pericyte-derived pleiotrophin (PTN), a neurotrophic growth factor. Intracerebroventricular PTN infusions prevented neuron loss in pericyte-ablated mice despite persistent circulatory changes. Silencing of pericyte-derived Ptn rendered neurons vulnerable to ischemic and excitotoxic injury. Our data demonstrate a rapid neurodegeneration cascade that links pericyte loss to acute circulatory collapse and loss of PTN neurotrophic support. These findings may have implications for the pathogenesis and treatment of neurological disorders that are associated with pericyte loss and/or neurovascular dysfunction.

The role of brain vasculature in neurodegenerative disorders.

Adequate supply of blood and structural and functional integrity of blood vessels are key to normal brain functioning. On the other hand, cerebral blood flow shortfalls and blood-brain barrier dysfunction are early findings in neurodegenerative disorders in humans and animal models. Here we first examine molecular definition of cerebral blood vessels, as well as pathways regulating cerebral blood flow and blood-brain barrier integrity. Then we examine the role of cerebral blood flow and blood-brain barrier in the pathogenesis of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis. We focus on Alzheimer's disease as a platform of our analysis because more is known about neurovascular dysfunction in this disease than in other neurodegenerative disorders. Finally, we propose a hypothetical model of Alzheimer's disease biomarkers to include brain vasculature as a factor contributing to the disease onset and progression, and we suggest a common pathway linking brain vascular contributions to neurodegeneration in multiple neurodegenerative disorders.