Targeting the Vasculature in Alzheimer's Disease and Vascular Cognitive Impairment

Resources

Barry Festoff

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Philip Gorelick

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Steven Greenberg

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Constantino Iadecola

Benigni A, Corna D, Zoja C, et al. Disruption of the Ang II type 1 receptor promotes longevity in mice. J. Clin. Invest. 2009;119(3):524-30.

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Kejal Kantarci

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Sperling R, Salloway S, Brooks DJ, et al. Amyloid-related imaging abnormalities in patients with Alzheimer's disease treated with bapineuzumab: a retrospective analysis. Lancet Neurol. 2012;11(3):241-9.

Vincent Marchesi

Buée L, Hof PR, Bouras C, et al. Pathological alterations of the cerebral microvasculature in Alzheimer's disease and related dementing disorders. Acta. Neuropathol. 1994;87(5):469-80.

Merlini M, Meyer EP, Ulmann-Schuler A, Nitsch RM. Vascular β-amyloid and early astrocyte alterations impair cerebrovascular function and cerebral metabolism in transgenic arcAβ mice. Acta. Neuropathol. 2011;122(3):293-311. [Epub 2011 Jun 19.]

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Suvorava T, Kojda G. Reactive oxygen species as cardiovascular mediators: lessons from endothelial-specific protein overexpression mouse models. Biochim. Biophys. Acta. 2009;1787(7):802-10. [Epub 2009 Apr 23.]

Wang J, Xiong S, Xie C, et al. Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer's disease. J. Neurochem. 2005;93(4):953-62.

Bruce Reed

Chui HC, Zheng L, Reed BR, et al. Vascular risk factors and Alzheimer's disease: are these risk factors for plaques and tangles or for concomitant vascular pathology that increases the likelihood of dementia? An evidence-based review. Alzheimers Res. Ther. 2012;4(1):1.

Debette S, Bis JC, Fornage M, et al. Genome-wide association studies of MRI-defined brain infarcts: meta-analysis from the CHARGE Consortium. Stroke 2010;41(2):210-7.

Godin O, Tzourio C, Maillard P, et al. Apolipoprotein E genotype is related to progression of white matter lesion load. Stroke 2009t;40(10):3186-90.

Mungas D, Jagust WJ, Reed BR, et al. MRI predictors of cognition in subcortical ischemic vascular disease and Alzheimer's disease. Neurology 2001;57(12):2229-35.

Nordal RA, Wong CS. Molecular targets in radiation-induced blood–brain barrier disruption. Int. J. Radiat. Oncol. Biol. Phys. 2005;62(1):279-87.

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Gustavo Román

Ancoli-Israel S, Palmer BW, Cooke JR, et al. Cognitive effects of treating obstructive sleep apnea in Alzheimer's disease: a randomized controlled study. J. Am. Geriatr. Soc. 2008;56(11):2076-81. [Epub 2008 Sep 15.]

Bader GG, Turesson K, Wallin A. Sleep-related breathing and movement disorders in healthy elderly and demented subjects. Dementia 1996;7(5):279-87.

Bassetti CL, Milanova M, Gugger M. Sleep-disordered breathing and acute ischemic stroke: diagnosis, risk factors, treatment, evolution, and long-term clinical outcome. Stroke 2006;37(4):967-72. [Epub 2006 Mar 16.]

Canessa N, Castronovo V, Cappa SF, et al. Obstructive sleep apnea: brain structural changes and neurocognitive function before and after treatment. Am. J. Respir. Crit. Care Med. 2011;183(10):1419-26.

Canessa N, Ferini-Strambi L. Sleep-disordered breathing and cognitive decline in older adults. JAMA 2011;306(6):654-5.

Erkinjuntti T, Partinen M, Sulkava R, et al. Sleep apnea in multiinfarct dementia and Alzheimer's disease. Sleep 1987;10(5):419-25.

Harbison J, Gibson GJ, Birchall D, et al. White matter disease and sleep-disordered breathing after acute stroke. Neurology 2003;61(7):959-63.

Joo EY, Tae WS, Lee MJ, et al. Reduced brain gray matter concentration in patients with obstructive sleep apnea syndrome. Sleep 2010;33(2):235-41.

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Chris Schaffer

Farkas E, Luiten PG. Cerebral microvascular pathology in aging and Alzheimer's disease. Prog. Neurobiol. 2001;64(6):575-611.

Nishimura N, Schaffer CB, Friedman B, et al. Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke. Nat. Methods 2006;3(2):99-108.

Sidney Strickland

Cortes-Canteli M, Paul J, Norris EH, Bronstein R, Ahn HJ, Zamolodchikov D, Bhuvanendran S, Fenz KM, Strickland S. Fibrinogen and beta-amyloid association alters thrombosis and fibrinolysis: a possible contributing factor to Alzheimer's disease. Neuron 2010;66(5):695-709.

Paul J, Strickland S, Melchor JP. Fibrin deposition accelerates neurovascular damage and neuroinflammation in mouse models of Alzheimer's disease. J. Exp. Med. 2007;204(8):1999-2008.

Zamolodchikov D, Strickland S. Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin. Blood 2012;119(14):3342-51.

Cheryl Wellington

Besler C, Heinrich K, Rohrer L, et al. Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in patients with coronary artery disease. J. Clin. Invest. 2011;121(7):2693-708.

Hirsch-Reinshagen V, Maia LF, Burgess BL, et al. The absence of ABCA1 decreases soluble ApoE levels but does not diminish amyloid deposition in two murine models of Alzheimer disease. J. Biol. Chem. 2005;280(52):43243-56.

Khera AV, Cuchel M, de la Llera-Moya M, et al. Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis. N. Engl. J. Med. 2011;364(2):127-35.

Lefterov I, Fitz NF, Cronican AA, et al. Apolipoprotein A-I deficiency increases cerebral amyloid angiopathy and cognitive deficits in APP/PS1DeltaE9 mice. J. Biol. Chem. 2010;285(47):36945-57.

Stukas S, May S, Wilkinson A, et al. The LXR agonist GW3965 increases apoA-I protein levels in the central nervous system independent of ABCA1. Biochim. Biophys. Acta. 2012;1821(3):536-46.

Wahrle SE, Jiang H, Parsadanian M, et al. Overexpression of ABCA1 reduces amyloid deposition in the PDAPP mouse model of Alzheimer disease. J. Clin. Invest. 2008 Feb;118(2):671-82.

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When Alois Alzheimer first described the "peculiar" condition we today know as Alzheimer's disease (AD), he could not have predicted the large amount of attention it would attract from generations of scientists. Despite this worldwide research effort, AD remains a devastating disorder that has generated as many questions as it has answers. Are the classic "senile plaques" and "neurofibrillary tangles" sufficient to define the full range of pathology that exists in the brains of millions of AD sufferers? What additional biomarkers could support a mechanism to explain the breadth of cognitive outcomes in AD? What mechanistic parallels in AD can inform us about other types of cognitive impairment?

In the last half-century scientists have addressed these questions and others through careful examination of the blood–brain relationship. The brain, engulfed in miles of deep penetrating arteries, capillaries, and veins, has a rich vascular architecture that creates an environment where neurons are highly susceptible to vascular insult. Multiple lines of evidence support the role of neurovascular mechanisms as a major factor in AD pathogenesis. And although scientists have yet to determine whether the vascular dysfunction leads to neuronal damage or whether the vascular pathology stems from the neurodegenerative process, there are results from human imaging, mouse models, and postmortem studies that support a critical role for the vascular dysfunction in AD. Affirming the correlation is the fact that the elderly have the highest rates of AD and other dementias as well as the highest rates of vascular-associated illnesses. Therefore, as this population grows so does the need to identify effective therapeutic targets that will address the complexity of these interactions. At the Targeting the Vasculature in Alzheimer's Disease and Vascular Cognitive Impairment symposium scientists from academia and industry met to share their knowledge on clinical and translational approaches to the study of vascular contributions to AD and to other forms of cognitive decline.

In his meeting introduction, Howard Fillit, from the Alzheimer's Drug Discovery Foundation, noted that although scientists first described blood vessel damage in the AD brain more than 100 years ago, this particular aspect of AD pathology has not always received the same attention as have other neurodegenerative descriptors since that time. Sharing an historical perspective on some of the earliest depictions of vascular involvement and explaining some of the developments in the field since then, Fillit reminded the audience that while more work on vasculature and AD are needed, current findings have far progressed from the superficial depictions of 100 years ago. Studies using electron microscopy show marked capillary damage in AD patient tissue and show that fibrillary plaques may in fact flow out of blood vessels. Evidence from studies using immunohistochemical stains for capillaries, as well as PET and FDG neuroimaging studies also show significant capillary-vessel interactions. Work in animal models reveals perivascular deposits of amyloid in the brain, suggesting a critical role for the blood brain barrier. Finally, clinical evidence reveals increasingly high comorbidity rates of older patients with diabetes and/or hypertension and AD—supporting a role of neurovascular dysfunction as a contributor to cognitive decline.

Speakers:
Philip Gorelick, Hauenstein Neuroscience Center
Costantino Iadecola, Weill Cornell Medical College
Vincent T. Marchesi, Yale University
Steven Greenberg, Harvard Medical School
Bruce Reed, University of California, Davis Alzheimer's Research Center

Highlights

• A guidance statement on neurovascular contributions to cognitive impairment includes treatment of blood pressure and other cardiovascular risk factors and lifestyle changes that could reduce the probability of cognitive impairment or dementia and cardiovascular disease.
• Oxidative stress contributes to the dysfunction of cerebral blood vessels via amyloid-beta (Aβ) activation of NADPH oxidase.
• Somatic mutations may yield mutant proteins that contribute to blood vessel damage.
• Multimodal imaging studies can be used to define causal pathways between small vessel disease and neuropathology.
• Elderly individuals with VCI show direct and detectable cognitive changes, even without the presence of Aβ.

Review of cerebrovascular disease in AD

Philip Gorelick from the Hauenstein Neuroscience Center kicked off the discussion on neurovascular contributions to cognitive impairment by reviewing the development of a guidance statement on this issue from the American Heart Association and the American Stroke Association. Gorelick noted that in the 1980s there was a major debate on how to classify Alzheimer's disease (AD). Should it be operationally defined as a distinct entity or should it be considered an open-ended disorder until more data were collected? In 1984 a working group led by Guy M. McKhann ultimately outlined a set of criteria to diagnose probable, possible, and definite AD. These criteria generally focused on evidence of neurodegeneration, thereby moving the field away from considering vascular risk factors.

However, a number of clinical studies over the years in patients with both marked cognitive impairments and vascular problems including unilateral infarctions, elevated body mass index, and chronic conditions like hypertension, heart disease, and diabetes, lent strong support to the idea that vascular dysfunction contributes to cognitive impairment. Given these findings Gorelick and his colleagues, on behalf of the American Heart Association and the American Stroke Association, developed an overall guide "for practitioners to gain a better understanding of vascular cognitive impairment (VCI) and dementia, prevention, and treatment." They defined VCI as a syndrome where there is evidence of clinical stroke or subclinical vascular brain injury and cognitive impairment affecting at least one cognitive domain. Gorelick noted that it can be difficult to diagnose VCI because of the presence of other cerebral or systemic disorders. Specifically, both Alzheimer’s disease and stroke are common as we age, and these disorders co-exist in many elderly persons causing a “mixed”-type of dementia or cognitive impairment.

The guidelines outlined by the group include choices that at a minimum could reduce the probability of stroke and other cardiovascular diseases such as: smoking cessation, controlling hypertension, lowering cholesterol and hyperglycemia, adopting a Mediterranean diet, and increasing exercise. Evaluation of research on potential pharmacological interventions suggest that off-label Donepezil might be useful for treating some cases, while the benefits of antiplatelet therapy, the cholinesterase inhibitor rivastigmine, and the NMDA receptor antagonist memantine are less clear.

Oxidative stress and blood flow homeostasis

Costantino Iadecola from Weill Cornell Medical College began his presentation by reminding the audience that the brain is a unique organ with respect to blood. Unlike other organs, the brain has no energy reserves and is therefore dependent on blood flow to deliver oxygen and remove waste. To maintain homeostasis, the brain is subject to several neurovascular regulatory processes that maintain blood flow to sufficient levels. Autoregulation buffers cerebral blood flow from the deleterious effects of changes in blood pressure. Functional hyperemia links neural activity to cerebral blood flow, assuring that active neurons receive sufficient blood flow to support their increased energy needs. One critical factor in this regulation is the signaling from endothelial cells to the myocytes in the cerebrovasculature. For example, endothelial cells release nitric oxide, a potent vasodilator when increased blood flow is required. These neurovascular regulatory components are compromised in hypertension (high blood pressure), in aging, and in AD. In addition, some patients with AD tend to have atherosclerosis, microvessel alterations, and altered patterns of cerebral blood flow.

Iadecola's group studies neurovascular regulation in mice overexpressing the amyloid precursor protein (APP), a condition that produces an AD-like condition in these mice. They found that all major neuroregulatory processes that maintain blood flow (autoregulation, functional hyperemia, and endothelial regulation) were disrupted in APP mice at very early timepoints—even before the onset of plaques and cognitive decline.

Furthermore, the group found that oxidative stress contributes to this dysfunction of cerebral blood vessels via amyloid-beta (Aβ) activation of NADPH oxidase. A scavenger receptor, CD36, mediates this activation. Deletion of NADPH oxidase genes or the CD36 gene blocks the development of neurovascular dysfunction in the mouse model. In aged APP mice, the neurovascular rescue is independent of effects on amyloid plaques and results in improved cognitive performance of the mice.

The interaction of Aβ with CD36 leads to the activation of NADPH oxidase, and ultimately oxidative stress. (Image courtesy of Costantino Iadecola)

Additionally, findings from a model of angiotensin-induced hypertension shows that some of the hallmark changes reported in other AD models are also observed in hypertension. For example, there are clear alterations in cerebral blood flow that are related to failure of endothelial and neural mechanisms.

Sources of damage to blood vessels and the sequelae

Vincent Marchesi of Yale University took a genetic approach to investigate neurovascular contributions to AD. In studies of older adults with and without AD, the angioarchitecture of both these groups shows a global reduction in capillaries and blood flow when compared to younger subjects. Marchesi's work aims to determine the mechanisms that underlie the diffuse small blood vessel damage seen in aged individuals. He posited that oxidative damage of nucleic acids causes somatic mutations and yields mutant proteins that contribute to blood vessel damage. Mutations in certain proteins can enhance acute and chronic inflammation or can affect endothelial cell function. Mutations may also affect known players in AD pathology, such as APP, presenilins, and tau. Hence somatic mutations may be the precursor of a cascade that eventually signals inflammatory responses and poor vascular outcomes such as ischemia, and subsequently amyloid dysregulation.

Steven Greenberg of Harvard Medical School addressed the issue of how small vessel disease eventually results in cognitive decline. Stroke is a huge risk factor for dementia, and small vessel pathology is the fundamental basis for many kinds of stroke. Greenberg is interested in examining the relationship between bleeding or brain injury from small blood vessels in the brain and cerebral amyloid angiopathy (CAA), a condition in which amyloid builds up in these vessels. Greenberg's group used T2*-weighted magnetic resonance imaging (MRI) and noninvasive amyloid imaging with Pittsburgh Compound B (PiB) to analyze the spatial relationship between CAA and microbleeds. They found that amyloid deposition gives rise to local islands of increased vascular amyloid with impaired physiology that have increased predilection for microbleeds. fMRI studies showed that people with CAA have smaller and slower responses to visual stimulation, indicating that there are behavioral consequences to the damage caused by the condition.

Classic neuropathology studies that used dyes injected into the brain have contributed to the understanding of how amyloid gains access to the outer walls of small vessels and smooth muscle. In addition, Greenberg said, work by Roy Wellers's group at the University of Southampton School of Medicine, UK, showed that after Aβ is degraded by proteases it crosses the blood brain barrier and drains from the brain with interstitial fluid along basement membranes of capillaries and arteries, resulting in deposition of Aβ in vessels as CAA.

Because of the pervasiveness and multiple sequelae associated with amyloid angiopathy in AD, Greenberg posited that any study of AD is in fact a study in amyloid angiopathy, and that moderate to severe CAA may be an independent contributor to cognitive dysfunction.

Cortical atrophy in vascular cognitive impairment

Bruce Reed of the UC Davis Alzheimer's Research Center focused on the next part of the puzzle—the relationship between vascular brain injury and vascular cognitive impairment (VCI), which ranges from the mild impairment that comes with normal aging through dementia. Epidemiological data show that cerebrovascular disease is the result of chain of pathological events that starts off with risk factors like hypertension, diabetes and smoking, moves through atherosclerosis and vascular brain injury and stroke, and ends with enormous damage to the brain.

Generally, the progression of damage due to these risk factors is mostly linear over time. However, there is a marked shift to later onset and more rapid progression when all-cause dementia is considered, which parallels the increasing presence of Aβ. While these data seem to cast doubt on the contribution of cerebrovascular damage to dementia a study by Bennett and Schneider—the community-based Memory and Aging Project—showed that postmortem samples from demented subjects had a higher prevalence of mixed pathology, a combination of vascular and AD lesions compared to those with Aβ deposition or infarction alone.

Reed proposed that the cortical atrophy often observed in the AD brain is in fact a type of vascular brain injury, and that this atrophy is a big driver of cognitive dysfunction. In MRI studies that looked at brain volumes prior to death, patients with either vascular or AD lesions had less gray matter than normal controls, but otherwise were not strikingly different from each other. Patients with mixed lesions had the most neurodegeneration—so the mixture of vascular and AD lesions in the brain may have doubled the impact on the cognitive processing areas.

Interestingly, Reed's group used tests of executive dysfunction and memory in combination with or PET imaging or post mortem neuropathology to assess the correlation of cognitive dysfunction with VCI and AD. They found that in VCI there are direct and detectable cognitive changes in normal elderly, even without the presence of Aβ. Furthermore, the spectrum of cognitive consequences from VCI was highest in normal aging, intermediate in those with mild cognitive impairment (MCI), and indistinct in those with dementia.

Speakers:
Kejal Kantarci, Mayo Clinic
Gustavo C. Román, Methodist Neurological Institute

Highlights

• Immunotherapy to clear Aβ can lead to microhemorrhages in the brain.
• Obstructive sleep apnea is a significant risk factor in the early cognitive decline in the elderly.

Complications of Aβ immunotherapy

To date, there are no effective treatments for Alzheimer's disease, and clinical trials have revealed potential problems with some approaches. Kejal Kantarci of the Mayo Clinic discussed one of these: the effects of Aβ immunotherapy on the vasculature. Efforts to combat AD by using antibodies against Aβ led the disturbing finding that this treatment apparently caused microhemorrhages in the brain in some individuals. The Alzheimer's Association Research Roundtable Workgroup was tasked with addressing the FDA's concerns on the abnormalities that stemmed from immunotherapy.

Two major classes of abnormalities were observed—amyloid-related imaging abnormalities with edema/effusions (ARIA-E), and amyloid-related imaging abnormalities with hemosiderin deposition (ARIA-H). ARIA-E is characterized by extravasation of protein-rich fluid, vasogenic edema, and sulcal effusion/exudate. The abnormality, which may be accompanied by transient symptoms of headache, confusion, and visual disturbances, is reversible. ARIA-E resembles CAA-related inflammation, and the risk factors associated with the condition are high-dose immunotherapy, increased clearance of Aβ, and the presence of the APOE ε4 allele.

Immunotherapy to clear Aβ is complicated by the finding that increased clearance is associated with ARIA-E. (Ostrowitzki et al. Mechanism of amyloid removal in patients with Alzheimer disease treated with Gantenerumab. Archives of Neurology 2012)

ARIA-H is more prevalent in AD and increases with age. It may be influenced by CAA and appears as a microhemorrhage with superficial siderosis, a condition of iron deposition in neuronal tissues thought to represent residual leakage of blood. ARIA-H risk factors include the prevalence of microhemorrhage, CAA, and vascular events. Interestingly, studies have shown that 35% of subjects with microhemorrhages developed ARIA-E, making ARIA-H a risk factor for ARIA-E. Close to 50% of subjects with ARIA-E were found to have microhemorrhages.

What pathophysiological processes underlie ARIA? One hypothesis is that ARIA is related to the processes associated with the clearance and movement of amyloid into vessel walls. For example, rapid clearance of amyloid from parenchyma to the perivascular space may cause a drainage backup and excess fluid shifts. Another explanation is that movement of amyloid into cerebral vessel walls may damage the vessels leading to microhemorrhages, a process described by other speakers at the symposium.

Unfortunately, there are no well-established models for ARIA in animals. The AD roundtable recommended that further clinical trials for Aβ immunotherapy should be accompanied by MRI to detect ARIA. The roundtable offered recommendations about ways to increase the sensitivity of MRI and to determine the frequency of scanning. Kantarci noted that future studies to investigate the pathophysiology, natural history, risk factors, and clinical course of ARIA are needed.

Obstructive sleep apnea

The second session was wrapped up with a talk by Gustavo C. Román of Methodist Neurological Institute on the role of obstructive sleep apnea in hypertension, cognitive decline, and dementia. Approximately 20% of middle-aged adults suffer from obstructive sleep apnea (OSA). OSA is a form of periodic breathing that can induce a variety of physiological responses including: altered oxygen states, muscle contractions, pressure dysfunction, arterial receptor activation, and cardiovascular arrhythmias. Several studies have shown that sleep is critical for cognitive function and OSA is a significant risk factor in the early cognitive decline in the elderly. OSA can also exacerbate the effects from vascular risk factors, such as obesity and hypertension, on small vessel brain disease.

A study that evaluated sleep in 106 older adults in an AD and dementia clinic revealed that the majority of patients had moderate to severe OSA, suggesting elderly patients may have a predisposition to airway obstruction. Possibilities for this disparity in the elderly might include relaxed musculature, less sleep requirements, as well as greater sensitivities to medicines and to noise disturbances. In addition, while the population in this study was not overwhelmingly obese, a significant percentage of subjects suffered from hypertension, hypercholesterolemia, cardiovascular disease, and diabetes. The cognitive status of subjects ranged from those with mild cognitive impairment (MCI) to various forms of dementia, AD, and normal pressure hydrocephalus. The findings also revealed that the worse the sleep apnea a person suffered, the more severe their cognitive deficits. In addition, MRI results revealed that 86% of subjects also suffered from small vessel disease, and subjects with multi-infarct dementia were shown to have a higher prevalence of OSA than AD subjects or controls. Though treatment for OSA can be challenging, the considerable improvements on cognitive tests and cortical volume measurements in those who have been treated are promising.

Speakers:
Barry W. Festoff, University of Kansas Medical Center and pHLOGISTIX LLC
Chris B. Schaffer, Cornell University
Sidney Strickland, The Rockefeller University
Cheryl L. Wellington, University of British Columbia

Highlights

• Thrombomodulin is a potent anti-inflammatory with potential as a therapeutic target for traumatic brain injury.
• The decreased brain blood flow seen in AD patients may be due to dysfunction at the capillary level and may exacerbate AD pathology by decreasing Aβ clearance.
• Aβ binding to fibrinogen affects clot structure and clot lysis.

Vascular coagulation-inflammatory pathways and therapeutic targets

Traumatic brain injury (TBI) and chronic traumatic encephalopathy (CTE) in particular are significant risk factors for Alzheimer's disease (AD). Barry W. Festoff of University of Kansas Medical Center and pHLOGISTIX LLC presented his group's work on determining the ways in which these injuries can lead to AD. The human body has evolved innate immune responses to detect and combat invading pathogens and physical damage and an importance component of these responses is the activation of coagulation and inflammatory cascades via the protein thrombin and its receptors—proteinase-activated receptors (PARs). Although some level of inflammation and clotting is useful as a protective response, thrombin can also be damaging because it disrupts vascular barriers. Thus, through a negative feedback loop the proteins thrombomodulin and activated Protein C (APC) ultimately dampen the inflammatory and anticoagulatory response initiated by thrombin by inhibiting the activity of that protein.

Mechanical injury leads to the activation of thrombin, which cleaves fibrinogen to release fibrin. Thrombomodulin inhibits the cascade. (Image courtesy of Barry Festoff)

Festoff decided to investigate the role of thrombin in blood brain barrier (BBB) disruption because it had been shown that the protein disrupts vascular barriers in the gut, lung, and other organs. In a cell culture BBB model, thrombin and PAR1-agonist peptides were shown by a variety of criteria to affect the BBB. Festoff's group went on to demonstrate that thrombomodulin can counter this effect.

Festoff's group designed a controlled cortical impact model that could recapitulate some of the conditions associated with CTE and TBI, and allowed them to record the speed of an impact as well as the depth of that impact on the brain. They found that in response to cortical impact, there is an increase in the levels of hgmb1 protein, a protein that binds to TLRs and RAGE to activate a pro-inflammatory response. Furthermore thrombomodulin binds to hgmb1 to counter this response. Hence thrombomodulin might prove useful for treating conditions that stem from TBI or CTE, and could eventually prove useful as a target therapy for AD.

Leukocyte plugging of capillary segments in AD mice

Several mouse models for AD have been developed that successfully recapitulate many aspects of AD pathology and symptoms. Because small vessel diseases play a significant role in AD patient populations, Chris Schaffer of Cornell University is using mouse models of AD to investigate microcirculatory deficits and small vessel dysfunction. Two-photon excited fluorescence microscopy through glass-covered craniotomies allows Schaffer and his group to observe the flow of red blood cells through individual capillaries in wild-type mice and mouse models of AD.

Schaffer's team observed stalled blood flow in only a very small percentage of capillaries in wild-type mice (0.3%). In AD mice, this number increased to approximately 2%. Furthermore, in wild-type mice, these stalls in blood flow resolved in a few minutes but in the mutant mice, only half of stalls resolved while the remaining half continued for multiple hours. AD mice also had increased variability in stall rate, stalls tended to reappear in the same capillaries, and the fraction of capillaries stalled decreased as age advanced.

What could be the reason for these stalls? In vivo imaging revealed that more than 60% of the stalls in the AD mice were caused by leukocytes, specifically monocytes, that were plugging a capillary segment. This suggests vascular inflammation, likely due to the inflammatory effects of Aβ, may underlie the capillary stalling phenomena. But why is it that stalling in only 2% of capillaries makes such a significant difference? A single stalled capillary causes reduced blood flow in multiple downstream vessel branches, so that increases in the number of stalled capillaries not only decreases the number of flowing vessels, but causes decreased flow in the remaining vessels as well. A simple model revealed that the 2% differential in stalled capillaries between wild-type and AD mice predicts a 30% decrease in blood flow, which recapitulates findings from clinical studies. As a result of this decreased blood flow Aβ clearance would decrease, producing a positive feedback loop where microvascular problems and AD pathology mutually exacerbate each other.

Thrombosis and fibrinolysis in AD

Sidney Strickland from the Rockefeller University continued the discussion on potential therapeutic targets for cerebrovascular dysfunction in AD. In addition to the cardiovascular lifestyle risk factors for AD outlined by Gorelick, other factors include high fibrinogen levels, atrial fibrillation, and the genetic disease associated with coagulation—Factor 5 Leiden, Strickland said. The molecular clotting cascade, described earlier by Festoff, suggests that upon injury a protease cascade that leads to thrombin, which cleaves fibrinogen to yield fibrin. Strickland's work using mouse models has shown that the inability to clear fibrin from the blood can have serious consequences. By 6 months of age, mice with a genetic condition that results in fibrin tissue deposits waste and die. They also show delayed wound healing, greater incidences of arthritis, glomerulonephritis, venous thrombosis, and conjunctivitis.

Strickland's working model is that in AD, Aβ binds to fibrinogen and affects clot structure and lysis. Because abnormal clots are resistant to degradation, the result is marked blood vessel damage and inflammation. In addition, when clotting is induced in vivo using ferric chloride, AD mutant mice were more prone than wild-type mice to clotting and the clotting persisted even after application of a clot-dissolving compound. Reducing fibrin levels was shown to boost memory performance in AD mouse models.

Aβ and fibrinogen show colocalization in vessels of AD patients. (Image courtesy of Sidney Strickland)

Studies that examined Aβ-fibrinogen co-localization in human brain revealed that the ApoE ε4/ε4 genotype was associated with increased fibrinogen deposition in CAA-positive vessels in AD patients. In terms of possible therapies, a logical question is could inhibiting the Aβ-fibrinogen interaction be beneficial in AD? And if the issue is a clotting irregularity, could clotting inhibition work? While interesting mechanistically, the possibility of hemorrhage makes this a less attractive option; a targeted inhibition of Aβ's action on fibrinogen is more likely. High-throughput screening identified the RU505 compound, which has shown some promise in controlling clotting and altering cognitive performance in a contextual fear conditioning experiment on AD mutant mice.

Cheryl Wellington of the University of British Columbia closed with a discussion of high-density lipoproteins (HDLs) and the HDL-like ApoA1. While much research has examined the role of HDL, also known as "the good cholesterol," as a fundamental component of cardiovascular health, less is known about the role of lipoproteins in the brain. HDLs are heterogeneous particles that differ in size, density, shape, surface charge, and composition. These particles act through several mechanisms to mediate reverse cholesterol transport, suppress inflammation, enhance immunity, and promote endothelial health. ApoE the brain's major HDL-like lipoprotein—accessing the brain through the cerebral spinal fluid—is one of the major genetic risk factors for AD.

ABCA1 is a cholesterol transporter that is the physiological transporter of lipids onto brain apoE. Wellington's group showed that in AD mouse models ABCA1 levels and corresponding lipidation levels of apo E affects amyloid load. ABCA1 knockout mice showed increased amyloid while transgenic mice overexpressing ABCA1 showed negligible amounts of amyloid deposits.

Coronal mouse sections through the hippocampus show that when ABCA1 reduced, mice have increased amyloid staining (left panel). Coronal sections from a transgenic AD mouse overexpressing ABCA1 showed increased amyloid staining (right panel). (Image courtesy of Cheryl Wellington)

Furthermore additional CNS effects were demonstrated in the ApoA-I knockout mice: CAA and behavioral deficits increased, while in overexpressing ApoA-I transgenic mice CAA decreased. These findings support the idea that ApoA1 has a beneficial effect on the CNS.

Novel probes developed by the company Accent Assays can be used to measure the exchange of lipid free ApoA1, on and off HDL particles. The assays can also help distinguish healthy patients from those with metabolic syndromes or diabetes. Future studies on cohorts with AD, an MCI with and without diabetes can help further explore their effectiveness. Because HDL dysfunction underlies many diseases including those known to be comorbid with AD, like type-2 diabetes, a method by which to measure HDL functionality has many potential uses.