1

NEURODEGENERATION AND THE BRAIN BARRIERS

Co-chairs: Paula Grammas1, John Nutt2

Contributors: Conrad Johanson3, Edward G. Stopa4, Joseph Quinn5, Leeza Maron6, Dinah Sah7, Jeffrey

Wu8, Nico Leenders9, Berislav Zlokovic10

1Texas Tech University Health Sciences Center, Garrison Institute on Aging, 6630 S Quaker Street, Suite

E, Lubbock, Texas, USA 97413, paula.grammas@ttuhsc.edu, 806-743-7821

2Oregon Health & Science University, Department of Neurology, 3181 SW Sam Jackson Park Road, OP-

32, Portland, Oregon, USA 97239, nuttj@ohsu.edu, 503-494-9054

3 Brown Medical School, Providence, Rhode Island, USA

4 Brown Medical School, Providence, Rhode Island, USA

5 Oregon Health & Science University, Portland, Oregon, USA

6 Oregon Health & Science Unversity, Portland, Oregon, USA

7 Alnylam Pharmaceuticals, Cambridge, Massachusets, USA,

8Oregon Health & Science University, Portland, Oregon, USA

9 Groningen University Hospital, Groningen, Netherlands

10 University of Rochester, Rochester, New York, USA

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Introduction

Intense research efforts in the past two decades to understand the pathogenesis of neurodegenerative

diseases and design effective therapeutics have been focused mainly on neurons. Although this so-called

“neuro-centric” view has contributed to our understanding of neuronal dysfunction, death pathways, and

accumulation of proteinaceous aggregates during chronic neurodegenerative processes, this approach

has not resulted in disease-modifying therapeutics. This suggests that the pathogenesis of

neurodegenerative disorders is more complex than previously thought, and that the lack of success of

neurocentric-based therapies may be due to the participation of non-neuronal cells in the disease

process.

Although Alzheimer’s’ disease (AD) has been traditionally classified as a neurodegenerative dementia

without cerebrovascular changes, current epidemiological, pathological, experimental, and imaging

studies suggest that such classification is no longer tenable. Many vascular risk factors such as

hypertension, diabetes mellitus, hypercholesterolemia, obesity, homocysteinemia, apoE4 genotype have

recently been shown to increase the risk of AD. Growing evidence suggests that silent brain infarcts and

cerebral hypoperfusion can increase risk for dementia and cognitive impairment in the elderly.

Considerable data suggest that there are important pathogenic mechanisms such as inflammation,

oxidative stress, and apoE4 expression common to both AD and cardiovascular disease. Endothelial cells

are widely recognized as key players in the development of cardiovascular disease and increasing

evidence implicates dysfunction of brain endothelial cells in the pathogenesis of neurodegenerative

diseases. Indeed, vascular dysfunction has been linked to a growing number of neurodegenerative

disorders including, AD, Parkinson’s disease (PD), ALS, spinal cord injury and Huntington’s disease.

In the clinical arena of neurodegenerative disorders, the blood-brain barrier (BBB) is traditionally

envisioned as irrelevant or as simply a barrier to treatment. However, recent studies have raised the

possibility that the BBB and blood-CSF barrier (BCSFB) may play important roles in pathology and

progression in a broad spectrum of CNS disorders including AD, PD, ALS, and multiple sclerosis. From

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both a clinical and basic science perspective, the barrier systems of the brain are the keys to brain

homeostasis and the preservation of neuronal integrity. It has become clearer since 2000 that there are

alterations of the cerebral microcirculation/BBB in neurodegenerative disease that need to be explored

and may provide possible new targets for therapeutic development.

State of science in brain barriers research

Key areas of emphasis from the basic science perspective:

BBB and A� transport in and out of the brain: Role of RAGE and LRP-1

Changes in BBB permeability: ischemia, age and apoE

Cerebral microcirculation as both a source and a target of inflammatory factors

Oxidative stress and the BBB: reactive oxygen species and nitric oxide

Aberrant angiogenesis in neurodegenerative diseases

BBB and A� transport in and out of the brain: Role of RAGE and LRP-1: The receptor for advanced

glycation end products (RAGE) is thought to be a primary transporter of A� from systemic circulation

across the BBB and into brain, while the low-density lipoprotein receptor-related protein-1 (LRP-1)

mediates transport of A� in the other direction, that is, out of the brain.1 AD is associated with changes in

relative distribution of RAGE and LRP-1 receptors in human hippocampus and cortex, suggesting AD

may develop as a clearance storage disorder with the resulting accumulation of A� and

neurodegeneration2 [Figure 1]. Based on the BBB transport-clearance hypothesis, various novel immune

and non-immune anti-A� clearance treatments for AD are currently under development. Among others

these include inhibitors of RAGE-A� interaction at the BBB which results in reduced A� transport into the

brain, reduced neuroinflammation and increased responses of cerebral blood flow to brain activation.3

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Changes in BBB permeability: ischemia, age and apoE: In the past, the absence of a gross disruption of

BBB function, as measured by current technologies, has led to the assumption that the BBB is unaltered

in neurodegenerative disease. However, more recent data have suggested that a focally dysfunctional

BBB is unable to regulate the influx/efflux of neurotoxic amyloid peptides and could participate in the

pathogenesis of AD lesions.4 Furthermore, there are regional (focal) differences in the presence of AD

lesions and there appears to be a pathogenic relationship among development of AD lesions, A� positive

vessels, and impaired BBB function.5 Indeed, both increased BBB permeability to A� and reduced

elimination of A� have been shown to precede senile plaque formation in an AD model.6 In the Tg2576

AD mice, vascular CBF dysfunction and dysfunction in BBB transport were evident in animals as young

as 4 months; implying that structural changes to the BBB caused by elevated A� could play a central role

in AD development and might define an early point of intervention for designing effective therapy against

the disease.

Another area with emerging interest is the role of apoE in regulating BBB integrity. It is well documented

that inheritance of the apoE allele ε4 increases the risk of developing cardiovascular disease and AD.7 It

has been recently shown that both blood- and tissue-derived apoE are important for BBB function and

integrity. Furthermore, there is an age-dependent defect in BBB function that is exacerbated in apoE-/-

mice.8 ApoE4 has also been associated with increased loss of agrin, a major basal lamina associated

heparin-sulfate proteoglycan, from the cerebral capillary basement membrane.9 Since vascular

dysfunction is found in patients with age-related neurodegenerative diseases, such as AD, age-related

BBB-dysfunction could underlie these defects and may thus be an important contributor to the cumulative

neuronal damage characteristic of these diseases. Finally, CNS hypoperfusion may be an underlying

defect/contributor in several neurodegenerative diseases. Cerebral ischemia induces an increase in BBB

permeability, upregulates expression of amyloid precursor protein, and aggravates ALS functional deficits

and pathology.10-12

Cerebral microcirculation as both a source and a target of inflammatory factors: A substantial literature

demonstrates activation of inflammatory processes in pathologically vulnerable regions of the AD brain

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and documents the presence of a large number of inflammatory molecules (13).13 Also, in both human

and animal studies anti-inflammatory drugs appear to reduce the risk of AD pathology and in some cases

enhance cognitive performance.14,15 Microvascular endothelial cells are a rich source of both cytokines

and chemokines and release inflammatory factors in response to a wide variety of stimuli.16 Isolated brain

microvessels from AD patients have high levels of both cell-associated and soluble cytokines and

chemokines including IL-1� , IL-6, IL-8, TNF� , TGF-� and monocyte chemoattractant protein-1 compared

to vessels from age-matched non-AD controls.17-19 Since 2000 a large body of work has demonstrated

that the cerebral microcirculation is in an “activated pro-inflammatory” state in neurodegenerative

diseases such as AD, MS, ALS and others.

Oxidative stress and the BBB: Reactive oxygen species and nitric oxide: Considerable evidence points to

oxidative stress as an important trigger in the complex chain of events leading to neurodegenerative

diseases such as AD and PD.20,21 Postmortem analysis of AD brains show elevated markers of oxidative

stress including protein nitrotyrosine, carbonyls in proteins, lipid oxidation products, and oxidized DNA

bases.22,23 Despite widespread consensus that oxidative stress is an important feature of the injured CNS,

the source of radical species as well as the factors that regulate their release/synthesis are not well

defined. Recently, a role for the microcirculation as a source of reactive oxygen species (ROS) in

neurodegenerative disease has been investigated. In this regard, both oxidized and nitrated proteins are

found in the vessel walls in AD.24,25 Also, it has been shown that brain microvessels when injured by

anoxia/reoxygenation (an in vitro model of ischemia/reperfusion injury) release oxygen free radicals

(specifically hydroxyl radicals).26 Homocysteine is thought to promote cardiovascular disease through

endothelial dysfunction and oxidative stress. Homocysteine has been implicated in the pathogenesis of

AD by data showing that serum homocysteine levels are significantly higher in AD patients than in

controls.27

In the past decade, nitric oxide has emerged as important neuromodulator in the brain as well as a

neurotoxin, at high concentrations.28 In the cerebral microcirculation a significant increase in nitric oxide

release has been demonstrated in the AD brain. In these AD vessels the levels of both endothelial nitric

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oxide synthase and inducible nitric oxide synthase are high.29 High levels of inducible nitric oxide

synthase are consistent with the pro-inflammatory state of the microcirculation in AD. Elevated vascular

production of nitric oxide, a potentially neurotoxic mediator in the CNS, may contribute to the susceptibility

of neurons to injury and cell death in neurodegenerative disease.

Aberrant angiogenesis in neurodegenerative diseases: Recent work has discovered the role of the

mesenchyme homeobox gene 2 (MEOX2) in neurovascular dysfunction in AD.30 MEOX2-gene is

vascularly restricted gene expressed exclusively in the vascular system in brain and particularly at the

BBB. Its downregulation in AD leads to aberrant brain capillary morphogenesis, abnormal responses to

angiogenic growth factors, improper barrierogenesis and downregulation of A� clearance receptors

associated with reduced brain capillary flow. Moreover, low levels of MEOX2 gene in AD endothelium

abort the angiogenesis at an early stage of brain capillary morphogenesis resulting in formation on non-

patent capillaries and reduced capillary network. Since in the presence of low MEOX2 and A� , VEGF

FGF and other pro-angiogenic factors fail to restore normal vascular remodeling, search for new

therapies to regulate angiogenesis in AD is warranted.

Factors and processes characteristic of vascular activation and angiogenic stimulation have been

documented in the AD brain. Genome-wide expression profiling in the AD brain has identified a marked

upregulation of genes that promote angiogenesis.31 Expression of angiogenic factors may result from

chronic hypoxia which is known to stimulate angiogenesis as well as contribute to the clinical and

pathological manifestations of AD. It has been shown that in AD brain microvessels express or release

inflammatory proteins, including thrombin, vascular endothelial growth factor, angiopoietin-2, tumor

necrosis factor-α, transforming growth factor-β, interleukin (IL) IL-1� , IL-6, IL-8, monocyte

chemoattractant protein-1, hypoxia inducible factor-1� , matrix metalloproteinases, and integrins (� V� 3

� V� 5), all of which have been implicated in endothelial activation and regulation of angiogenesis.17-19,32

Despite increases in several pro-angiogenic factors in the AD brain, evidence for increased vascularity in

AD is lacking. On the contrary, the present evidence suggests that the angiogenic process and vascular

remodeling are aberrant and/or impaired in aged tissues, with several studies showing decreased

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microvascular density and aberrant capillary tube formation in the AD brain.33-36 In this regard, it has been

also demonstrated that A� peptides have strong anti-angiogenic properties in several models of

angiogenesis in vivo and in vitro.37-39

A recently proposed model suggests that in response to a persistent stimulus, such as cerebral

hypoperfusion, one of the major clinical features in AD, brain endothelial cells become activated or

acquire a senescent, pro-inflammatory state. Senescent endothelial cells overexpress a number of

adhesion proteins and may produce a surge of inflammatory cytokines/chemokines depending on the

type. Despite the continued presence of the stimulus, an imbalance of pro- and anti-angiogenic factors

and aborted angiogenic signaling prevents new vessel growth. Therefore, in the absence of feedback

signals to shut off vascular activation, endothelial cells remain activated and elaborate a large number of

proteases, inflammatory proteins and other gene products with biologic activity that could injure or kill

neurons32 [Figure 2]. This phenotypic modulation of brain endothelial cells is important given an

increasing recognition for the role of the neurovascular unit and neurovascular dysregulation/dysfunction

in the development of cognitive decline in AD. Identification of “vascular activation” as a target in AD

would stimulate translational investigations in this newly defined area and may lead to novel therapeutic

approaches for the treatment of this devastating disease.

Key areas of emphasis from the clinical perspective:

BBB function and etiology of neurodegeneration

BBB and rate of progression of neurodegeneration

Circumvention of the BBB for treatment

BBB and etiology of neurodegenerative disorders: The etiology of the vast majority of PD is presumed to

be due to a combination of environmental and genetic factors. Environmental factors are most commonly

thought to be exposure to endogenous or exogenous toxins that gain access to the central nervous

system. Genetic factors are envisioned as multiple genes that positively or negatively influence the

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probability of developing PD by affecting the absorption or metabolism of these putative toxins. Variability

in transport of putative toxins into and out of the brain is another obvious potential mechanism for

sensitivity to toxins.

Evidence for variability of BBB transport processes contributing to PD comes from studies of

polymorphisms of the multiple drug resistance (MDR1) gene in Chinese populations that suggest that

some polymorphisms reduce the risk of developing PD.40 Further, a direct measure of the MDR1 function

in PD and controls by examining the retention of a positron labeled verapamil (a substrate for MDR1) by

PET scanning found greater retention in PD subjects than in normal controls.41 This observation suggests

that MDR1 activity was less in PD and could lead to increased retention of a putative toxin in the PD

brain. Interestingly, the increased retention of verapamil was primarily in the midbrain and not throughout

the brain, suggesting that there might be regional differences in transporters within the brain. Paraquat, a

widely used herbicide, can produce a parkinsonism model in rodents. The effect of paraquat in this

model can be blocked by competitive inhibition of the BBB large neutral amino acid transporter giving

further credence to the importance of access of putative toxins to the brain.42

BBB and progression of neurodegeneration: Alterations in the BBB and B-CSF-B could alter the rate of

deterioration with neurodegenerative disorders. Slowing the rate of progression could make a large

impact on disability in neurodegenerative disorders.

There is evidence of alteration of the BBB in AD as discussed above that may potentially exist before AD

becomes clinically apparent. Clinical evidence for increased BBB permeability in AD is an increased

concentration of the prothrombin in the brain parenchyma in advanced AD.43 Alterations of BBB or

BCSFB permeability are also implicated by an elevation of CSF albumin in the CSF of a subset of AD

which is associated with more rapid progression of AD.44 This does not appear to be a nonspecific

finding associated with neurodegeneration; it is not present in PD.45 These effects could be related to the

mild inflammatory response seen in neurodegenerative disorders although this explanation would not

explain why an elevated albumin index is not seen in PD where there are also indications of an

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inflammatory response. It will be important to define whether these changes are a response to

neurodegeneration or are independent contributors to disease progression.

In rats made hemiparkinsonian by unilateral injections of 6-hydroxydopamine, a catecholamine

neurotoxin, and treated with levodopa, there is evidence of endothelial proliferation and increased

permeability to levodopa.46 The endothelial proliferation and changes in levodopa entry were present only

in the basal ganglia, subventricular zone and hippocampal dentate gyrus and not elsewhere, suggesting

regional regulation of BBB transport. The changes in regional capillary endothelial cells have been

correlated with the development of dyskinesia, a troublesome adverse effect of long-term levodopa

treatment in PD. Thus, disease treatments may alter the BBB and thereby the course of the disease and

response to therapy.

A final example of change in BBB and BCSFB that may alter rate of progression of neurodegeneration is

CSF production, clearance and composition. CSF production is reduced in AD but not PD. The reduction

in CSF formation reduces CSF turnover which has important implications if the CSF acts as a sink for

toxic molecules such as A� .47 A clinical observation that bolsters the suspicion that CSF turnover may be

important is the fact that the clinical triad of dementia, gait disorder and urinary incontinence of normal

pressure hydrocephalus (NPH) is often associated with pathological evidence of AD, subcortical

microvascular disease and possibly other neurodegenerative disorders.48 Further, patients with NPH and

these other disorders may respond, at least for many months, to ventricular shunting.48,49

Circumvention of the BBB: One of the great successes in neurodegenerative disorders was the discovery

of depletion of dopamine in PD and the recognition that the precursor of dopamine, L-DOPA, was

transported across the BBB and provided symptomatic benefit to patients. This therapy was further

refined by preventing the catabolism of L-DOPA in the periphery by DOPA decarboxylase with carbidopa,

a compound that inhibited DOPA decarboxylase but did not cross the BBB to inhibit the central DOPA

decarboxylase which is essential to L-DOPA’s conversion to dopamine.

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However, other therapies for PD have been thwarted by the BBB. A good example is the saga with glial

derived neurotrophic factor (GDNF). GDNF is a potent neurotrophin that will revive damaged

dopaminergic nerve terminals in animal models of PD.50 This peptide will not cross the BBB and

alternative methods of administration have been attempted. In PD subjects, intracerebroventricular

administration by implanted reservoirs and ventricular catheters proved that GDNF was biologically active

but was without clinical efficacy.51 A postmortem on a patient dying of other causes found no evidence

that intraventricular GDNF affected dopaminergic nerve terminals in the putamen which lies centimeters

from the lateral ventricle. A subsequent study infused GDNF directly into the putamen with implanted

catheters producing PET evidence of enhanced dopaminergic function in the immediate vicinity of the

catheter tip but no clinical improvement.52 A clinical trial that is just beginning will use gene therapy with

an adenovirus carrying the gene for neurturin, another neurotrophin from the GDNF family. This

treatment will require four needle passes and eight injections into each putamen. Another strategy being

pursued includes programming stem cells to produce GDNF and implanting them into the putamen.

Another alternative under consideration is methods to enhance the synthesis and release of endogenous

GDNF with smaller molecules that will cross the BBB, as has been done with brain-derived neurotrophic

factor (BDNF) in animal models.53 Even activity may influence neurotrophin activity in brain.54 The

development of less invasive methods to deliver therapeutic molecules such as peptides, small RNAs and

genes to specific regions of the CNS is clearly very necessary.

We need to consider other routes into the CNS. One novel route under investigation is intranasal delivery

of peptides. Novel routes to the brain may also be linked to the etiology of PD. Braak has shaken up the

PD scientific community by presenting evidence that PD begins in the dorsal motor nucleus of the vagus

nerve (in the medulla) and not in the midbrain dopaminergic neurons as has been generally assumed for

the past 4 decades.55 Further, since this site is connected to the periphery by the vagus nerve, he has

proposed that some toxic factor enters the CNS via the vagus nerve and the pathological process then

progresses up the neuroaxis. Blood-nerve barriers may be critical to this hypothesis.

Barriers to progress in the field

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For transport studies: The normal clearance pathways for most metabolites that are not degraded in situ

are the barriers of the CNS: the capillary endothelium, the choroid plexus epithelium and the CSF

circulation. A better understanding of the continuum between normal aging and the age-related

dementias requires that we explore the changes in the barrier structure, receptors and their expression,

that underlie the transport of metabolites into and out of the CNS at all of the barrier sites.

Good standardized in vitro and in vivo models of the BBB and BCSFB are critical. Models to study

transport dynamics and equilibrium at the BBB are not widely available and can be found in a small

number of highly specialized laboratories. And how do we model the BBB and altered neurovascular

signaling that occurs in various diseases? Can we make the BBB and its broader neurovascular signals a

much larger part of the CNS disorders consciousness? Right now, we are still way too focused on the

“neurobiology" of disease rather than the integrative biology of the entire brain which should include an in-

depth understanding of the BBB functions. Comparative transport studies of the BBB and BCSFB within a

given model will furnish more insight on the complex interplay between the cerebral interstitial fluid and

the large-cavity CSF. Such interaction of barrier systems is a key factor in understanding the homeostatic

mechanisms that assure the proper extracellular environment of neurons.

For permeability studies: There has been a lack of emphasis on developing comorbidity models which

include vascular factor models (such as ischemia/reperfusion injury) and models of neurodegenerative

disorders, as well as lack of well-defined models of the age-related dementias. With the increased aging

of the population such research will become imperative. Also, improvement of neuroimaging techniques

that will allow identification/resolution of subtle, even transient, changes in BBB permeability will be

extremely important.

For inflammatory and neurovascular unit cross-talk studies: Challenges for the future include

understanding the cross-talk between non-neuronal cell types (glia, microglia) and cells of the vessel wall.

Identifying how these cells respond to, process, and/or synthesize inflammatory mediators is critical to

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understanding how they regulate the neuronal microenvironment. Despite a large body of data implicating

inflammation in the pathogenesis of neurodegenerative disease and epidemiological studies showing that

sustained use of anti-inflammatory drugs may prevent or slow down the progression of

neurodegenerative diseases the small number of clinical trials carried out so far using anti-inflammatory

drugs, were minimal and equivocal in their outcome. Potential reasons for these mixed results include

timing of drug administration, nonselective inhibition of cyclooxygenase (COX), inappropriate use of

particular anti-inflammatory drugs for a given disease or disease progression/ severity, sub-optimal dose

in target site, or limited penetration to the brain through the BBB. Therefore, design of anti-inflammatory

drugs for the treatment of neurodegenerative diseases based upon better BBB penetration, and with

minimal adverse events, would be appropriate. In addition, relevant genetic differences among patients

should be considered in planning new anti-inflammatory drugs, for improved efficacy. Furthermore, due to

the possible co-involvement of oxidative stress and excitotoxicity in the pathogenesis of these diseases,

combination therapy with antioxidants or glutamate antagonists or a multi-potent drug might be much

more effective in successfully treating neurodegenerative diseases.

For oxidative stress studies: New technologies and approaches to measuring short-lived ROS in vivo in

real time could significantly improve our understanding of how oxidants contribute to brain injury. It is also

important to develop new generation of antioxidant agents. Because ROS as well as nitric oxide have

important functions in cellular signaling and in the regulation of gene expression, separate from their toxic

effects, development of site or level specific antioxidants will be needed to effectively block the toxic

effects of these molecules.

For aberrant angiogenesis: Future work to test angiogenic capabilities should assess the temporal or

causal association between acquisition of the activated-angiogenic phenotype and the onset of disease.

A causal link between the angiogenic phenotype and disease progression could be evaluated using

antiangiogenic drugs. Administration of these drugs to animals prior to the onset of behavioral changes

and AD pathology will determine whether inhibiting the activated-angiogenic phenotype affects the course

of disease. Development of new treatments to curb aberrant angiogenesis caused by A� , MEOX2,

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senescent endothelial phenotype, and injured or activated endothelial cells deserve special attention in

future studies. On the other hand, VEGF2R anti-angiogenic therapy has been shown to aggravate the

disease process and enhance disease onset in models of ALS and cautions use of this approach for

other neurodegenerative disorders.

For etiology of neurodegeneration: A general lack of knowledge is the major barrier to developing this

area. There needs to be an exploration of transport mechanisms for substances of interest in

neurodegenerative disorders. Transport of proteins that accumulate in the disorders, analogous to beta

amyloid, are candidates. Alpha synuclein, a major component of the Lewy body in PD, TAU, a

component of neurofibrillary tangles, paraquat, other insecticides and other potential toxins are

candidates. Exploring polymorphisms in these transporters to look for genetic susceptibility to

neurodegenerative disorders is a priority. If transporters are contributors to susceptibility or progression

of neurodegeneration, manipulation of transport processes may offer neuroprotective and treatment

strategies.

For progression of neurodegeneration: The question of whether the heterogeneity of the BBB contributes

to the selective vulnerability in neurodegeneration is less important in PD since Braak emphasized that

PD began in the medulla and appeared to progress up the neuroaxis. However, the heterogeneity of

BBB could be very important in targeting therapies for neurodegeneration to particular brain areas. For

example the efforts to get genes, small RNAs and peptides into the putamen would be much more

effective if these agents could be targeted to the putamen or striatum selectively by utilizing transport

mechanisms unique to the striatum. Mapping the heterogeneity of the BBB should be a high priority.

For circumvention of BBB: The critical need for this area is a noninvasive method to evaluate CSF

turnover and preferentially be able to separate problems with production and problems with clearance.

Molecules that would selectively be secreted by the choroid plexus and could be imaged by some means

would perhaps allow a better assessment of CSF dynamics.

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Delivery of therapeutic agents across the BBB is critical but is being covered in another section of this

report. Systemic administration that could be targeted for specific areas of the brain, such as the striatum

for parkinsonism, would be ideal. However, research is also needed to explore methods to employ

delivery from the ventricular CSF to brain and the safe use of convection enhanced delivery of agents

directly into parenchyma.

Recommendations to advance the field and resources required

� Increase the number of scientists/clinicians interested in BBB/cerebrovasculature.

� Fund and promote inter-institutional, multidisciplinary teams.

� Develop interactive, web-based database of BBB scientists/clinicians.

� Develop studies that support cause and effect rather than just associations.

� Explore the genome of the neurovascular unit and choroid plexus to:

– Examine polymorphisms linked to various neurodegenerative disorders.

– Create conditional knockouts of genes important to microvasculature structure (tight

junctions, basement membrane, etc).

– Spin off new transport systems for drug delivery.

� Develop new vascular-based clearance strategies.

� Design strategies to control aberrant brain angiogenesis

� Develop in vivo imaging of brain microcirculation in models of human neurodegenerative

disorders to follow the kinetics of the disease process.

Summary and single most important issue to advance the field

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Because ageing is the most important risk factor for neurodegeneration, we hypothesize changes in

the normal neuronal milieu caused by alterations of the cerebral microvasculature, CSF circulation

and disruption of the neurovascular unit that occur with aging predisposes the CNS to

neurodegeneration. We propose examining the changes in the neurovascular unit and CSF turnover

that occur with aging. Specifically, we need studies of: 1. Secretion of endothelial toxins and

inflammatory factors, 2. Alterations in other structural components of the neurovascular unit (tight

junctions, basal lamina, pericytes, astrocytic foot processes, etc), 3. CSF production, clearance and

composition, 4/ Amyloid clearance and clearance of other proteinaceous aggregates which

accumulate in brain interstitial fluid or cells, 5. Role of non-neuronal cells in the pathogenesis of

neurodegenerative disorders and 6. Aberrant brain angiogenesis.

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References

1. Deane R, Wu Z, Zlokovic BV. RAGE (yin) versus LRP (yang) balance regulates alzheimer

amyloid beta-peptide clearance through transport across the blood-brain barrier. Stroke

2004;35(11 Suppl 1):2628-31.

2. Donahue JE, Flaherty SL, Johanson CE, Duncan JA, 3rd, Silverberg GD, Miller MC, Tavares R,

Yang W, Wu Q, Sabo E, Hovanesian V, Stopa EG. RAGE, LRP-1, and amyloid-beta protein in

Alzheimer's disease. Acta Neuropathol (Berl) 2006;112(4):405-15.

3. Deane R, Zlokovic BV. Role of the blood-brain barrier in the pathogenesis of Alzheimer's disease.

Curr Alzheimer Res 2007;4(2):191-7.

4. Pluta R. Is the ischemic blood-brain barrier insufficiency responsible for full-blown Alzheimer's

disease? Neurol Res 2006;28(6):665-71.

5. Jeynes B, Provias J. The possible role of capillary cerebral amyloid angiopathy in Alzheimer

lesion development: a regional comparison. Acta Neuropathol (Berl) 2006;112(4):417-27.

6. Ujiie M, Dickstein DL, Carlow DA, Jefferies WA. Blood-brain barrier permeability precedes senile

plaque formation in an Alzheimer disease model. Microcirculation 2003;10(6):463-70.

7. Strittmatter WJ, Saunders AM, Schmechel D, Pericak-Vance M, Enghild J, Salvesen GS, Roses

AD. Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4

allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci U S A 1993;90(5):1977-81.

8. Hafezi-Moghadam A, Thomas KL, Wagner DD. ApoE deficiency leads to a progressive age-

dependent blood-brain barrier leakage. Am J Physiol Cell Physiol 2007;292(4):C1256-62.

9. Salloway S, Gur T, Berzin T, Tavares R, Zipser B, Correia S, Hovanesian V, Fallon J, Kuo-

Leblanc V, Glass D, Hulette C, Rosenberg C, Vitek M, Stopa E. Effect of APOE genotype on

microvascular basement membrane in Alzheimer's disease. J Neurol Sci 2002;203-204:183-7.

17

10. Pluta R, Ulamek M, Januszewski S. Micro-blood-brain barrier openings and cytotoxic fragments

of amyloid precursor protein accumulation in white matter after ischemic brain injury in long-lived

rats. Acta Neurochir Suppl 2006;96:267-71.

11. Sadowski M, Pankiewicz J, Scholtzova H, Li YS, Quartermain D, Duff K, Wisniewski T. Links

between the pathology of Alzheimer's disease and vascular dementia. Neurochem Res

2004;29(6):1257-66.

12. Lambrechts D, Carmeliet P. VEGF at the neurovascular interface: therapeutic implications for

motor neuron disease. Biochim Biophys Acta 2006;1762(11-12):1109-21.

13. Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, Cooper NR, Eikelenboom P,

Emmerling M, Fiebich BL, Finch CE, Frautschy S, Griffin WS, Hampel H, Hull M, Landreth G, Lue

L, Mrak R, Mackenzie IR, McGeer PL, O'Banion MK, Pachter J, Pasinetti G, Plata-Salaman C,

Rogers J, Rydel R, Shen Y, Streit W, Strohmeyer R, Tooyoma I, Van Muiswinkel FL, Veerhuis R,

Walker D, Webster S, Wegrzyniak B, Wenk G, Wyss-Coray T. Inflammation and Alzheimer's

disease. Neurobiol Aging 2000;21(3):383-421.

14. McGeer PL, Schulzer M, McGeer EG. Arthritis and anti-inflammatory agents as possible

protective factors for Alzheimer's disease: a review of 17 epidemiologic studies. Neurology

1996;47(2):425-32.

15. Lim GP, Yang F, Chu T, Chen P, Beech W, Teter B, Tran T, Ubeda O, Ashe KH, Frautschy SA,

Cole GM. Ibuprofen suppresses plaque pathology and inflammation in a mouse model for

Alzheimer's disease. J Neurosci 2000;20(15):5709-14.

16. Gimbrone MA, Jr., Topper JN, Nagel T, Anderson KR, Garcia-Cardena G. Endothelial

dysfunction, hemodynamic forces, and atherogenesis. Ann N Y Acad Sci 2000;902:230-9;

discussion 239-40.

17. Grammas P, Ovase R. Inflammatory factors are elevated in brain microvessels in Alzheimer's

disease. Neurobiol Aging 2001;22(6):837-42.

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18. Grammas P, Ovase R. Cerebrovascular transforming growth factor-beta contributes to

inflammation in the Alzheimer's disease brain. Am J Pathol 2002;160(5):1583-7.

19. Grammas P, Samany PG, Thirumangalakudi L. Thrombin and inflammatory proteins are elevated

in Alzheimer's disease microvessels: implications for disease pathogenesis. J Alzheimers Dis

2006;9(1):51-8.

20. Mariani E, Polidori MC, Cherubini A, Mecocci P. Oxidative stress in brain aging,

neurodegenerative and vascular diseases: an overview. J Chromatogr B Analyt Technol Biomed

Life Sci 2005;827(1):65-75.

21. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in

normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39(1):44-84.

22. Aksenov MY, Aksenova MV, Butterfield DA, Geddes JW, Markesbery WR. Protein oxidation in

the brain in Alzheimer's disease. Neuroscience 2001;103(2):373-83.

23. Lovell MA, Markesbery WR. Ratio of 8-hydroxyguanine in intact DNA to free 8-hydroxyguanine is

increased in Alzheimer disease ventricular cerebrospinal fluid. Arch Neurol 2001;58(3):392-6.

24. Hensley K, Williamson KS, Maidt MK, Gabbita SP, Floyd RA. Determination of Biological

Oxidative Stress Using High Performance Liquid Chromatography with Electrochemical Detection

(HPLC-ECD). Journal of High Resolution Chromatography 1999;22(8):429-437.

25. Williamson KS, Gabbita SP, Mou S, West M, Pye QN, Markesbery WR, Cooney RV, Grammas

P, Reimann-Philipp U, Floyd RA, Hensley K. The nitration product 5-nitro-gamma-tocopherol is

increased in the Alzheimer brain. Nitric Oxide 2002;6(2):221-7.

26. Grammas P, Liu GJ, Wood K, Floyd RA. Anoxia/reoxygenation induces hydroxyl free radical

formation in brain microvessels. Free Radic Biol Med 1993;14(5):553-7.

27. Miller JW. Homocysteine and Alzheimer's disease. Nutr Rev 1999;57(4):126-9.

19

28. Guix FX, Uribesalgo I, Coma M, Munoz FJ. The physiology and pathophysiology of nitric oxide in

the brain. Prog Neurobiol 2005;76(2):126-52.

29. Dorheim MA, Tracey WR, Pollock JS, Grammas P. Nitric oxide synthase activity is elevated in

brain microvessels in Alzheimer's disease. Biochem Biophys Res Commun 1994;205(1):659-65.

30. Wu Z, Guo H, Chow N, Sallstrom J, Bell RD, Deane R, Brooks AI, Kanagala S, Rubio A, Sagare

A, Liu D, Li F, Armstrong D, Gasiewicz T, Zidovetzki R, Song X, Hofman F, Zlokovic BV. Role of

the MEOX2 homeobox gene in neurovascular dysfunction in Alzheimer disease. Nat Med

2005;11(9):959-65.

31. Pogue AI, Lukiw WJ. Angiogenic signaling in Alzheimer's disease. Neuroreport 2004;15(9):1507-

10.

32. Thirumangalakudi L, Samany PG, Owoso A, Wiskar B, Grammas P. Angiogenic proteins are

expressed by brain blood vessels in Alzheimer's disease. J Alzheimers Dis 2006;10(1):111-8.

33. Edelberg JM, Reed MJ. Aging and angiogenesis. Front Biosci 2003;8:s1199-209.

34. Buee L, Hof PR, Bouras C, Delacourte A, Perl DP, Morrison JH, Fillit HM. Pathological alterations

of the cerebral microvasculature in Alzheimer's disease and related dementing disorders. Acta

Neuropathol (Berl) 1994;87(5):469-80.

35. Buee L, Hof PR, Delacourte A. Brain microvascular changes in Alzheimer's disease and other

dementias. Ann N Y Acad Sci 1997;826:7-24.

36. Jellinger KA. Alzheimer disease and cerebrovascular pathology: an update. J Neural Transm

2002;109(5-6):813-36.

37. Paris D, Townsend K, Quadros A, Humphrey J, Sun J, Brem S, Wotoczek-Obadia M, DelleDonne

A, Patel N, Obregon DF, Crescentini R, Abdullah L, Coppola D, Rojiani AM, Crawford F, Sebti

SM, Mullan M. Inhibition of angiogenesis by Abeta peptides. Angiogenesis 2004;7(1):75-85.

20

38. Paris D, Patel N, DelleDonne A, Quadros A, Smeed R, Mullan M. Impaired angiogenesis in a

transgenic mouse model of cerebral amyloidosis. Neurosci Lett 2004;366(1):80-5.

39. Paris D, Ait-Ghezala G, Mathura VS, Patel N, Quadros A, Laporte V, Mullan M. Anti-angiogenic

activity of the mutant Dutch A(beta) peptide on human brain microvascular endothelial cells. Brain

Res Mol Brain Res 2005;136(1-2):212-30.

40. Lee CG, Tang K, Cheung YB, Wong LP, Tan C, Shen H, Zhao Y, Pavanni R, Lee EJ, Wong MC,

Chong SS, Tan EK. MDR1, the blood-brain barrier transporter, is associated with Parkinson's

disease in ethnic Chinese. J Med Genet 2004;41(5):e60.

41. Kortekaas R, Leenders KL, van Oostrom JC, Vaalburg W, Bart J, Willemsen AT, Hendrikse NH.

Blood-brain barrier dysfunction in parkinsonian midbrain in vivo. Ann Neurol 2005;57(2):176-9.

42. McCormack AL, Di Monte DA. Effects of L-dopa and other amino acids against paraquat-induced

nigrostriatal degeneration. J Neurochem 2003;85(1):82-6.

43. Zipser BD, Johanson CE, Gonzalez L, Berzin TM, Tavares R, Hulette CM, Vitek MP, Hovanesian

V, Stopa EG. Microvascular injury and blood-brain barrier leakage in Alzheimer's disease.

Neurobiol Aging 2007;28(7):977-86.

44. Bowman GL, Kaye JA, Moore M, Waichunas D, Carlson NE, Quinn JF. Blood-brain barrier

impairment in Alzheimer disease: stability and functional significance. Neurology

2007;68(21):1809-14.

45. Haussermann P, Kuhn W, Przuntek H, Muller T. Integrity of the blood-cerebrospinal fluid barrier

in early Parkinson's disease. Neuroscience Letters 2001;300(3):182-184.

46. Westin JE, Lindgren HS, Gardi J, Nyengaard JR, Brundin P, Mohapel P, Cenci MA. Endothelial

proliferation and increased blood-brain barrier permeability in the basal ganglia in a rat model of

3,4-dihydroxyphenyl-L-alanine-induced dyskinesia. J Neurosci 2006;26(37):9448-61.

21

47. Silverberg GD, Mayo M, Saul T, Rubenstein E, McGuire D. Alzheimer's disease, normal-pressure

hydrocephalus, and senescent changes in CSF circulatory physiology: a hypothesis. Lancet

Neurol 2003;2(8):506-11.

48. Bech-Azeddine R, Hogh P, Juhler M, Gjerris F, Waldemar G. Idiopathic normal-pressure

hydrocephalus: clinical comorbidity correlated with cerebral biopsy findings and outcome of

cerebrospinal fluid shunting. J Neurol Neurosurg Psychiatry 2007;78(2):157-61.

49. Silverberg GD, Levinthal E, Sullivan EV, Bloch DA, Chang SD, Leverenz J, Flitman S, Winn R,

Marciano F, Saul T, Huhn S, Mayo M, McGuire D. Assessment of low-flow CSF drainage as a

treatment for AD: results of a randomized pilot study. Neurology 2002;59(8):1139-45.

50. Brundin P. GDNF treatment in Parkinson's disease: time for controlled clinical trials? Brain

2002;125(Pt 10):2149-51.

51. Nutt JG, Burchiel KJ, Comella CL, Jankovic J, Lang AE, Laws ER, Jr., Lozano AM, Penn RD,

Simpson RK, Jr., Stacy M, Wooten GF. Randomized, double-blind trial of glial cell line-derived

neurotrophic factor (GDNF) in PD. Neurology 2003;60(1):69-73.

52. Lang AE, Gill S, Patel NK, Lozano A, Nutt JG, Penn R, Brooks DJ, Hotton G, Moro E, Heywood

P, Brodsky MA, Burchiel K, Kelly P, Dalvi A, Scott B, Stacy M, Turner D, Wooten VG, Elias WJ,

Laws ER, Dhawan V, Stoessl AJ, Matcham J, Coffey RJ, Traub M. Randomized controlled trial of

intraputamenal glial cell line-derived neurotrophic factor infusion in Parkinson disease. Ann

Neurol 2006;59(3):459-66.

53. Martinez-Turrillas R, Del Rio J, Frechilla D. Sequential changes in BDNF mRNA expression and

synaptic levels of AMPA receptor subunits in rat hippocampus after chronic antidepressant

treatment. Neuropharmacology 2005;49(8):1178-88.

54. Smith AD, Zigmond MJ. Can the brain be protected through exercise? Lessons from an animal

model of parkinsonism. Exp Neurol 2003;184(1):31-9.

22

55. Braak H, Bohl JR, Muller CM, Rub U, de Vos RA, Del Tredici K. Stanley Fahn Lecture 2005: The

staging procedure for the inclusion body pathology associated with sporadic Parkinson's disease

reconsidered. Mov Disord 2006;21(12):2042-51.

56. Zlokovic BV. Neurovascular mechanisms of Alzheimer's neurodegeneration. Trends in

Neurosciences 2005;28(4):202-208.

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Figure Legend

Figure. I. Schematic of blood–brain barrier and blood–CSF barrier (inset) transport routes for A� . A�

efflux across the BBB can predict brain amyloid burden in AD models and the development of plaques

shifts the A� transport equilibrium. Double deletion of the genes encoding apoJ and apoE accelerates A�

pathology in APP-overexpressing mice , raising a possibility that these apolipoproteins affect A�

clearance and/or metabolism. Gp330/megalin/LRP-2, an apoJ receptor at the BBB and the choroid

epithelium, could participate in clearance of A� –apoJ complexes from the brain across the BBB, and from

CSF across the choroid epithelium of the blood–CSF barrier (inset) to maintain the sink action of CSF. P-

glycoprotein at the luminal side of the BBB could reduce brain endothelial A� by promoting its efflux into

blood. In addition to transport, interaction of A� with RAGE amplifies neurovascular stress and

inflammation. LRP, an endocytotic and signaling receptor that has ligands including A� , apoE, a2M and

APP, is linked genetically to AD and influences APP processing and A� clearance . APP-overexpressing

mice overexpressing the LRP mini-receptor in neurons, however, accumulate soluble A� in brain . By

contrast, LRP on brain capillary endothelium clears A� to blood with affinity inversely related to the

content of � -sheets in A� , and lipoprotein receptors on astrocytes promote apoE-dependent degradation

of A� deposits. Whether LRP on brain endothelium can also clear oligomeric A� , and whether chaperone

proteins can assist clearance of aggregated A� across the BBB, is not known. (Reprinted with permission

from Elsevier)56.

Figure 2. Proposed scheme for endothelial activation in Alzheimer’s disease.

Under normal conditions, endothelial cells in response to a stimulus such as hypoxia or IL-1� ( )

elaborate a large number of gene products with biological activity including inflammatory cytokines and

proteases ( ) . Endothelial cells then migrate, proliferate and form new blood vessels, which through a

negative feedback loop ( ) turn off the process. In AD, endothelial cell activation in response to stimuli

occurs, but no migration, proliferation or new blood vessels develop. Thus, in AD endothelial cells

continue to release inflammatory cytokines and proteases, with deleterious consequences for neurons.

24

Figure 1

Reprinted from Trends in Neuroscience, vol. 28, Berislav Zlokovic, Neurovascular mechanisms of Alzheimer’s

neurodegeneration, pages 202-208, Copyright 2005, with permission from Elsevier.

25

Figure 2

EC migration and proliferation

No new blood vessel formation AD

EC

AD pathology

EC

New blood vessel formation EC migration and proliferation

Normal