Forum Labo Paris March 2023

The blood–brain barrier and neurodegenerative disease

Enhanced leakiness of the blood–brain barrier has been associated with neurodegenerative disease, and assessing this leakiness has even been suggested as a possible method for early diagnosis of such disease. In light of the recent promising results from the lecanemab therapy for Alzheimer’s disease, CLI caught up with Dr Fiona McLean (University of Dundee, UK) to find out more about the blood–brain barrier – what it is, how it functions and what happens to it during neurodegeneration.

What is the blood–brain barrier and how does it normally function?
Delivery of oxygen, fuel and nutrients to organs is essential  for them to function properly. This is dependent on the network of blood vessels which span throughout our bodies. In our brains, these blood vessels contain a unique group of cells. Together, these cells form a tightly regulated barrier between the blood and the brain parenchyma, and is called the ‘blood–brain barrier’ (BBB). This barrier is vital for maintaining a stable microenvironment in which neuronal circuits can function optimally.

The BBB primarily consists of endothelial cells, which line the interior of the blood vessels, and function in partnership with other cells, including pericytes, astrocytes and neurons. Endothelial cells are essential for maintaining brain homeostasis by acting as (1) a physical barrier due to tight junctions found between adjacent endothelial cells which limits the paracellular movement of molecules, and (2) a selective barrier due to the presence of transporters, including solute carriers, active efflux transporters, carrier-mediated transporters, receptor-mediated transporters, absorptive-mediated transporter and ion transporters, which selectively regulate the transcellular movement of molecules across the barrier. Importantly, this selective barrier not only uptakes molecules into the brain, but also acts as a barricade to prevent neurotoxins and pathogens entering, while also removing waste products from the brain into the blood for disposal. These characteristics make the endothelial cells in the BBB uniquely distinct from other vascular endothelial cells.

What happens to the BBB during neurodegenerative disease?
Changes to the BBB occur during healthy aging, but in neurodegenerative diseases there is an exacerbation and acceleration of breakdown and dysfunction. Impairment of the BBB is known to occur in several neurodegenerative diseases including Parkinson’s disease, Huntington’s disease, multiple sclerosis, amyotrophic lateral sclerosis, vascular dementia and Alzheimer’s disease [1,2].

Breakdown of the BBB can result in barrier dysfunction and a loss of integrity. Leakiness can occur owing to degradation of tight-junctions between endothelial cells and shrinkage of the cells themselves [1,3]. Expression and function of endothelial transporters and receptors can be affected, including glucose transporter 1, low density lipoprotein receptor-related protein 1, and P-glycoprotein 1. Importantly, these changes result in altered paracellular and transcellular movement of molecules across the BBB. Further disruption to the BBB includes loss of basement membrane composition and structure, dysfunction and loss of pericytes and astrocytes, activation of microglia, neuronal damage, and increase infiltration of leukocytes. The underlying cellular mechanisms which can drive these problems include oxidative stress and inflammation, which can disrupt cellular signalling mechanisms for transportation, structural organization and energy production [3,4]. Inflammation has been found to drive increases in nitric oxide in endothelial cells, which is the main oxidative species that contributes to cellular damage in the BBB [5]. Whereas normal levels of nitric oxide are required for essential brain functions including vascular tone control, hemostasis control, immune functions and neurotransmission, elevated levels of nitric oxide are associated with increased permeability of the BBB. Furthermore, nitric oxide is associated with the build-up of pathological proteins amyloid and α-synuclein in Alzheimer’s and Parkinson’s diseases respectively [6,7], highlighting it as a key signalling pathway in BBB dysfunction in neurodegenerative diseases.

To help prevent and treat neurodegenerative diseases, it should be a priority to understand how and when the BBB is altered in neurodegenerative diseases. Notably, the BBB can be repaired endogenously by both astrocytes and microglia [1]. Further research to learn about the role these cells play in barrier maintenance can lead to the generation of knowledge that can be harnessed to repair the barrier. Moreover, there should be a drive to develop therapeutics that restore the integrity and functionality of the BBB and protect the microenvironment of the brain.

How can the BBB be studied?
The BBB is notoriously difficult to study. It comprises multiple cell types in a 3D, tubular formation, with cell polarity and shear stress influenced by blood flow on the blood-facing side of the vessel and complex neuronal networks spanning the parenchymal side of the vessel. The main approaches to studying the BBB are in vitro cell models, in vivo animal models, and human clinical models [8]. In vitro models are essential for high-throughput drug discovery but struggle to recapitulate the complexities of the BBB. However, new methodologies, such as microfluidic chambers to emulate blood flow and transwell models to enable multiple cell types to be grown together and make functional connections, are moving the field forward. Human induced pluripotent stem cells (iPSCs) from patients have allowed researchers to explore BBB properties in models which are more translational to humans, although there have been many challenges in accuracy of cell type and reproducibility. Nevertheless, this approach is important for learning about the BBB in human diseases. In vivo approaches are essential for BBB research as they provide whole-system models with complete vascular systems and can also include models of neurodegenerative diseases, metabolic manipulations, and targeted genetic models. Finally, studies in humans are imperative to understand the BBB in health and disease, to advance diagnostic tools to assess BBB status, and to develop therapeutics to target the BBB to preserve its integrity and function.

What are the challenges that BBB changes present in the diagnosis and treatment of neurodegenerative diseases?
BBB status is difficult to monitor in humans. Since the barrier is compromised early on in neurodegenerative diseases [2], developing tools to evaluate barrier integrity would be of great diagnostic value. Measuring cerebrospinal fluid levels of proteins associated with BBB leakiness, such as S100β, offers an accessible approach to assessing integrity [4]. However, the pitfall is that these markers may not be specific or sensitive enough for particular diseases. Advanced imaging techniques, such as contrast-enhanced magnetic resonance imaging (MRI) and dynamic contrast-enhanced computed tomography (CT) scanning, can also be used to investigate the BBB [9,10]. However, these techniques are expensive and not always easily accessed. Alternative approaches
to assessing the BBB should be developed. One such approach under investigation is using retinal vascular imaging to predict brain vascular health and vulnerability to developing neurodegenerative diseases. The retina can act as a window into the brain as they are connected via axons in the optic nerve and the retina contains the blood–retina barrier, which shares structural and function features with the BBB. The blood–retina barrier has been found
to breakdown in neurodegenerative diseases [11,12], such as Alzheimer’s disease, and therefore could be used as an indicator of BBB health.

Navigating the BBB has presented many challenges in developing therapeutics for neurodegenerative diseases. The main challenge is successfully delivering drugs across the BBB into the brain. This is because most drugs are too large to cross via the paracellular route and they cannot cross transcellularly as the transporters are selective [13]. Some strategies have tried to take advantage of the leaky BBB that can occur in neurodegenerative diseases. However, a compromised barrier does not necessarily mean that a drug can get into the brain more easily. This can be a result of change in functionality of the BBB in neurodegenerative diseases due to decreased expression or internalization of transporters and receptors [1,2].

There have been a number of investigations into delivery methods [13]. Some are invasive and act to purposely break down the tight junctions between endothelial cells to allow drugs to pass. These include the use of ultrasound, hyperosmotic solutions, or noxious agents. However, these approaches also have unwanted negative consequences. Direct injection or intraventricular infusion of therapeutics into the cerebrospinal fluid can bypass the barrier, but this requires intrathecal administration. There are some great strides in modifying the physiochemical properties of the drugs to enable passage into the brain including enhancing lipid solubility, mimicry of endogenous structures to allow use of existing transporters and carriers, using molecular ‘Trojan horses’, gene therapies, and nanoparticle-based technologies.

One of the biggest challenges in delivering therapeutics into the brain is that endothelial cells contain active efflux transporters which pump therapeutics back out of the brain and can cause drug resistance. The best characterized of these transporters are the ATP-binding cassette (ABC) transporters, which include breast cancer resistant protein, multidrug resistant proteins, and P-glycoprotein. Notably, upregulation of P-glycoprotein is also linked with drug-resistant epilepsy and tumours [14]. Additionally, endothelial cells contain drug-metabolizing enzymes, such as CYP450 enzymes, which can break down drugs before they can have a beneficial effect [4]. Drug discovery research to treat neurodegenerative diseases must consider efflux mechanisms.

Aging can lead to a decrease in the activity of drug-resistant functions in the BBB, which may be seen as useful for accumulating drugs in the brain; however, it also results in a reduction of efflux mechanisms for clearance of neurotoxins and waste products. This may be a factor in the development of neurodegenerative diseases as impaired efflux mechanisms may facilitate inappropriate protein accumulation and aggregation. For example, BBB endothelial cells express lipoprotein receptor-related protein 1 (LRP) and receptor for advanced glycation end products (RAGE) that facilitate amyloid efflux and influx respectively. In Alzheimer’s disease, expression of LRP decreases and RAGE increases, promoting accumulation and aggregation of amyloid, oxidative stress and neuroinflammation [1,15]. Preventing BBB breakdown in Alzheimer’s disease, as well as other neurodegenerative diseases, is a lucrative goal as it occurs early in pre-clinical stages and so may be involved in initiation and early disease progression making this a potential and attractive therapeutic target.

How does lifestyle affect neurodegenerative disease risk?
Altered characteristics of the BBB coincide with healthy aging, but the changes seen in neurodegenerative diseases are exacerbated and associate with changes in brain function, such as cognitive impairment. Further research into understanding why the BBB breaks down in neurodegenerative diseases is needed. It is still not known whether barrier breakdown is causative or a symptom of neurodegenerative diseases.

Environmental factors may trigger a cascade of problems in the BBB that lead to breakdown and dysfunction, leaving the brain vulnerable and predisposing it to neurodegenerative conditions. These factors can include unhealthy diets, lack of physical activity, high blood pressure, high cholesterol, diabetes, smoking, excessive alcohol consumption, air pollution and microplastics, traumatic brain injuries, and infections.

One of the key roles of the BBB is to prevent infectious agents (e.g. bacteria or viruses) from entering the brain. However, these agents can induce inflammatory responses by local or systemic infections which can lead to BBB breakdown. This can then facilitate entry into the brain. Neurological complications can then occur as a result of the infectious agent entering the brain and causing cellular damage. Owing to its rapid emergence and high occurrence, COVID-19 is one of the most studied infectious diseases. Evidence shows that the COVID-19 virus may not primarily pass into the brain paracellularly as the result of a leaky BBB, but that it harnesses transcellular transport routes across the BBB [16]. This is supported by evidence showing that tight-junction proteins remain intact during infection [17]. Further research into understanding the full breadth of mechanisms by which infectious agents can enter the brain is essential.

Smoking and excessive alcohol consumption have been well characterized as factors for poor health. Smoking is associated with an increased risk of atherogenic and thrombotic problems, but it has been understudied in terms of the effect on the BBB. It is known that it causes inflammation, oxidative stress and cell damage in endothelial cells [18]. The effect of alcohol on the BBB is better understood. Alcohol can pass through the BBB relatively easily as it is lipophilic and, as with smoking, induces inflammation and oxidative stress [19]. Moreover, alcohol is thought to be particularly detrimental to structural proteins, such as tight junction protein ZO-1, VE-cadherin and occludin, key for barrier integrity [20]. In human neuroimaging studies, it has been shown that not only do individuals with alcohol-use disorder have a leaky BBB, but also social drinkers [21,22]. Air pollutants, such as diesel exhaust particles, can trigger BBB breakdown. Evidence supports that these particles can trigger inflammatory responses in the brain, as well as oxidative stress,
and that upregulation of P-glycoprotein at the BBB is induced [23]. Concerningly, there is evidence in children and young adults that there is an association between long-term air pollution exposure and the accumulation of amyloid and α-synuclein, which are both pathological hallmarks of Alzheimer’s disease and Parkinson’s disease respectively [24]. Micro- and nano-plastic exposure is also a concern to brain health. Evidence supports that plastic particles can cause cellular damage through oxidative stress as well as alter neurotransmitter activity [25]. However, there is little information how plastic particles cross the BBB, the quantity and if they accumulate. Importantly, plastic particles may be able to cross the BBB more easily in individuals who have a leaky BBB and therefore be more vulnerable to the knock-on effects. Research into this is vital as the global abundance of plastics increases alongside increasing contamination into the environment and food chain.

There is evidence that metabolic disorders, which encompasses a myriad of non-communicable diseases (i.e. obesity, type 2 diabetes, high blood pressure, high cholesterol) can lead to BBB impairment. Metabolic disease is associated with chronic inflammation and oxidative stress and changes in blood glucose are associated with loss of barrier integrity and altered transport function [26,27]. Importantly, there is evidence that obesity and type 2 diabetes decrease LRP and increase RAGE in endothelial cells. This also occurs in Alzheimer’s disease, linking metabolic dysregulation to BBB dysfunction and Alzheimer’s disease causality [1,15]. Indeed, reduced expression of the glucose transporter, GLUT1, is reported in Alzheimer’s disease endothelial cells [28]. Metabolic dysregulation may be particularly detrimental to brain endothelial cells, as they have a high mito­chondria content and energy demand due to their transport functions relative to peripheral endothelial cells. Defective mitochondrial respiration is linked to Alzheimer’s disease as amyloid decreases endothelial cell oxidative phosphorylation, an effect exacerbated by hyperglycemia [29]. Notably, people with type 2 diabetes are twice
as likely to develop Alzheimer’s disease, and it is also a risk factor for vascular dementia and Parkinson’s diseases [30,31].

Eating healthily not only contributes to BBB health by negating metabolic disease, but nutrients can play a role as well through anti-inflammatory and antioxidant actions. One of the most well characterized is the omega-3 docosahexaenoic acid, which can freely diffuse across the barrier. Docosahexaenoic acid can only be provided through dietary intake, primarily from fish. It is essential in the brain as it is a key constituent of neuronal membranes. Notably, higher docosahexaenoic acid plasma levels correlate with better cognitive ability in older adults, with Alzheimer’s patients showing depleted levels of docosahexaenoic acid [32]. Practising a healthy diet high in docosahexaenoic acid is a viable strategy to reduce the risk of cognitive impairment associated with aging and neurodegenerative diseases.

In general, exercise restores and protects the BBB from permeability. The positive effects of exercise are attributed to reduced inflammation, reduced oxidative stress, strengthened tight junctions, improved clearance of neurotoxins and waste products from the brain [33]. However, the type of exercise should be considered as sports which can lead to head trauma can actually cause damage to the BBB. There is a growing body of evidence that players of football, American football and rugby have a higher rate of incidence of dementia than the general population [34]. Nevertheless, exercise should be recognized as an important factor in looking after brain vascular health and may be a viable method of treatment of neurodegenerative disease as there is evidence in multiple sclerosis and Alzheimer’s disease patients that exercise can improve symptoms [33].

It is now recognized that poor lifestyle choices increase the risk of developing neurodegenerative diseases, and there is evidence that they are a factor in BBB breakdown and dysfunction. However, lifestyle choices are modifiable and present an opportunity to slow-down or prevent the development of neurodegenerative diseases. Educating society about risk factors is essential to help people protect their brain health.

The expert

Dr Fiona McLean PhD
School of Medicine,
University of Dundee,
Dundee, UK

Email: f.mclean@dundee.ac.uk

References

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