Introduction

Our brain is the central organ of the human nervous system. It controls all necessary body functions whilst constantly receiving, processing and reflecting stimuli from the outside environment. About 20 to 25 % of the body’s blood is constantly pumped into the brain which used about 20 % of all oxygen and glucose even when our body is in a state of rest. And yet, our brain makes out only two percent of our total body weight (Mergenthaler, Lindauer et al. 2013). By a process called neurovascular coupling the brain can rapidly increase blood blow and oxygen supply to the activated regions of the brain (Zlokovic 2005). Energy supply in different brain regions is facilitated by a dense vascular tree of large arteries, smaller arteries, arterioles and tiny capillaries that branch into every inch of the brain. Capillaries make up 85 % of the vasculature and are the smallest blood vessels in our brain. Altogether, approximately 644 kilometers of brain vessels run through the human brain (Pardridge 2003). It has been determined that any cerebral cell is situated no more that approximately 10 μm away from the next blood vessel (Bovetti, Hsieh et al. 2007). Diffusion across these distances, even for large molecules, is instantaneous. Thus, in principle, every neuron has its own capillary for oxygen supply and the elimination of metabolic waste products (Pardridge 2002, Pardridge 2003, Meyer, Ulmann-Schuler et al. 2008).

As the central controlling element of the organism, the human brain is well-protected from exogenous and endogenous hazards. The thick bones of our scull and the suspension of cerebrospinal fluid (CSF) that surrounds our brain tissue mechanically protect our brain from external damages by alleviating potential impacts on our head (Oldendorf 1972, Pardridge 2002, Pardridge 2003). However, our brain is also safeguarded from endogenous, blood-derived hazards by a composition of different cells types that form the neurovascular unit that forms the so called blood-brain barrier, a term used to describe these unique characteristics of the cerebrovasculature (Daneman and Prat 2015). The functional interplay of the different cells types (Figure 1A) around the blood vessels in the brain dictates the capillaries to maintain a highly controlled microenvironment by tightly regulating the exchange of molecules between the blood and the brain. Thereby, in the healthy brain, cerebral capillaries prevent the entry of unwanted blood-derived products, xenobiotics, pathogens, and immune cells that are associated with inflammatory and immune responses that initiate neurodegeneration and by that, control internal hazards (Pardridge 2003). However, the barrier characteristics of the vessels in the central nervous system (CNS) are not homogenous throughout the brain. Small capillaries seem to show higher biochemical and physiological properties related to the barrier functions than larger vessels. Moreover, sensory organs or elements of the neuroendocrine system involved in secretion such as area postrema, the subfornical organ and the organum vasculosum laminae terminalis, the pineal gland, the posterior pituitary, the intermediate lobe of the pituitary gland, the median eminence and the subcommissural organ allow the passive diffusion of molecules and are therefore leaky (Wilhelm, Nyul-Toth et al. 2016, Noumbissi, Galasso et al. 2018). Consequently, the different areas of the brain allow their blood vessels molecule permeability depended of the regional requirements.

Figure 1:

(A) Capillaries of the brain show distinct characteristics because of the interplay of the different cells of the neurovascular unit. Endothelial cells built up tube-like structures for blood flow. Pericytes sit on top of endothelial cells and control barrier characteristics. Astrocytes ensheet the capillary and contribute to the barrier phenotype.

(B) Endothelial cells of the central nervous system exhibit low rates of pinocytosis, low leukocyte adhesion. As polarized cells they show distinct profiles of receptor and transporter expression on the luminal and abluminal side. Brain entry of blood-derived substances is restricted by the presence of tight junctions that connect two endothelial cells. Efflux transporters at the luminal side prevent lipophilic molecules from passing through the cell membrane.

The unique composition and physiology of brain capillaries along with the functional interplay of different cell types maintain a milieu of proper brain function. It has become evident that in many neurodegenerative diseases or neurological disorders such as Alzheimer’s disease (AD), multiple sclerosis, Parkinson’s disease, amyotrophic lateral sclerosis, or huntingtin’s disease, alterations at the brain vasculature lead to insufficiencies in maintaining proper brain homeostasis (Iadecola 2004, Zlokovic 2011). In recent years, more and more reports illuminate that the impact of cerebrovascular function on progression and onset of many diseases that affect the brain has been underestimated for the longest time (Zlokovic 2005, Bell and Zlokovic 2009, Campos-Bedolla, Walter et al. 2014, Daneman and Prat 2015). In order to understand cerebrovascular impact on health and disease, one has to look closely at the underlying physiological mechanisms and unique characteristics of the brain-blood interfaces that separate the CNS from the periphery.

Composition of the blood brain-barrier

The specialized function and composition of the cerebrovasculature is essential for the regulation of the brain homeostasis and thus, synaptic function and neuronal connectivity. Unlike vasculature from the periphery, the vasculature of the CNS possesses unique characteristics and an entirely different physiology (Figure 1B). As such, the vasculature in neuronal tissues is mostly impermeable for passive diffusion whereas peripheral vasculature mostly allows the passive exchange of molecules from the blood stream and adjacent tissues). The barrier function of the cerebral vasculature restricts the uncontrolled entry of blood-derived toxins, xenobiotics or cells from entering the brain and has a crucial protective function for the CNS.

Endothelial cells form the tube-like structure for blood flow and are the central element of the vessels and the neurovascular unit (Figure 1A). They are in direct contact with the blood and many restrictive functions of the BBB are due to their unique characteristics. Endothelial cells are polarized cells have luminal side facing the blood stream as well as the abluminal side facing the brain parenchyma show distinct receptor and transporter expression. These different membrane compartments facilitate the regulated transport of molecules from and into the brain. Through physical interaction and communication with neighboring pericytes and astrocytes, endothelial cells in CNS tissue become very tight as the adjacent cells induce barrier function in the endothelium. In the CNS, adjacent endothelial cells restrict the diffusion of molecules through the paracellular space by the presence of endothelial tight junctions (Figure 1B), interweaving protein networks consisting of different claudins, occludins, zona occludens and junctional adhesion molecules (Huber, Egleton et al. 2001). These interconnected protein strands seal the paracellular space between two endothelial cells (Reese and Karnovsky 1967, Brightman and Reese 1969, Westergaard and Brightman 1973) to restrict passive diffusion. Besides tight junctions additional junctional complexes are present in the paraendothelial space: With their major component vascular endothelial-cadherin adherens junctions are essential for endothelial survival and angiogenesis (Carmeliet and Collen 1999).

The barrier phenotype in CNS endothelial cells is further enhanced by extremely low rates of pinocytosis (Figure 1B). Therefore, transcellular movement of molecules through the cell is limited. It has been shown that this feature is mainly induced by pericytes that are in direct contact with cerebrovascular endothelium and sit on top of them (Daneman and Prat 2015). However, also astrocytes contribute to this phenotype (Abbott 2002). Compared to peripheral endothelial cells, CNS microvascular endothelial cells are extremely thin (110–300 nm) and enable short transport routes for molecules (e. g. glucose, amino acids, etc.) that need to be transported into and out of the brain (Coomber and Stewart 1985, Pardridge 2003, Wilhelm, Nyul-Toth et al. 2016). Thus, brain endothelial cells show a high amount of mitochondria which are believed to be critical for providing energy for ATP-dependent transport functions. Large essential molecules and hormones such as insulin, leptin or transferrin are transported via rapid receptor-mediated transcytosis processes in both directions across the endothelium (Pardridge 2005).

The entry of molecules from blood into the brain is further restricted by the expression of efflux transporters at the luminal side of endothelial cells (Daneman and Prat 2015). These transmembrane proteins shuffle lipophilic molecules that pass the cell membrane back into the bloodstream (Figure 1B). The function of these efflux carriers are the main reason why many potentially CNS-active drugs do not reach their site of action.

Also, endothelial cells control immune surveillance. Due to their low expression of leukocyte adhesion molecules (Figure 3D) in CNS endothelial cells, compared to endothelial cells of the periphery, the binding and brain entry of leukocytes is limited (Henninger, Panes et al. 1997). During aging and in neurodegenerative diseases like AD, amyotrophic lateral sclerosis or Parkinson’s disease, we see an alteration of the brain vasculature. Transporter, receptor and tight junction protein expression changes, transcellular transport processes increase, thus making the BBB more permeable and allowing the brain extravasation of molecules and immune cells from the periphery (Silverberg, Miller et al. 2010, Garbuzova-Davis, Hernandez-Ontiveros et al. 2012, Elahy, Jackaman et al. 2015, Gray and Woulfe 2015, Montagne, Barnes et al. 2015, Osgood, Miller et al. 2017).

Alzheimer’s disease as neurovascular disorder

AD is a neurodegenerative disorder and the most common form of dementia (Prince, Bryce et al. 2013). Although much research has been done since its initial description in 1906 little is known about the pathophysiological mechanisms underlying the disease. Therefore, there is no available treatment that cures or even slows down the progression of the disease (Iadecola 2016). In the brains of AD patients, intracellular hyper-phosphorylated tau protein and extracellular aggregates of the potential neurotoxic amyloid-beta (Aβ) protein can be detected (Selkoe 2001, Bloom 2014). Consequently, much data suggests both proteins play a central role in AD pathogenesis. Aβ seems to act upstream of tau in AD (Bloom 2014) pathogenesis and therefore the role of Aβ in the pathophysiological processes is extensively studied. Besides, various epidemiological studies have shown that cardiovascular factors are closely associated with AD (Iadecola 2016). Moreover, it seems vascular alterations are one of the earliest events in AD pathology even earlier than Aβ pathology (Iturria-Medina, Sotero et al. 2016). There is no general accepted hypothesis how AD develops or progresses. However, since vascular pathology often coincides with Aβ and tau pathology, a two-hit vascular hypothesis of AD was developed that states that Aβ accumulation in the brain is a second insult (hit 2) that is initiated by vascular damage (hit 1) (Zlokovic 2011) which seems to be consistent with recent clinical findings (Iturria-Medina, Sotero et al. 2016). Studies in humans have shown that in most AD cases Aβ accumulation is not the result of an overproduction but due to insufficient clearance (Bateman, Munsell et al. 2006, Mawuenyega, Sigurdson et al. 2010).

Over the years, it has been shown that the BBB plays a major role in AD pathology by clearance of brain Aβ, infiltration of macrophages into the brain and mediating inflammation (Zlokovic 2005).

Blood brain-barrier transport of amyloid-beta

With a surface area of approximately 20 square meters, the BBB proves a large surface area for the rapid removal of neurotoxic Aβ from brain. Studies in mice have shown that the BBB clears up to 75 % of all brain Aβ (Tarasoff-Conway, Carare et al. 2015). In endothelial cells, several receptors and transporters have been described to be involved in the translocation of Aβ from brain into the periphery (Figure 2).

Figure 2:

Various carriers for amyloid-beta (Aβ) have been described in brain endothelial cells. Organic-anion-transporting polypeptide transporters (OATPs) have been reported to transport Aβ from the periphery into the brain as well as the receptor for advanced glycosylation end products (RAGE). Low-density lipoprotein receptors like LRP1 and ABC transporters like P-glycoprotein or breast cancer-resistant protein (BCRP) have been shown to be involved in Aβ efflux from brain.

Many of them show altered protein expression during aging or in mouse models of AD (Hartz, Miller et al. 2010, Silverberg, Miller et al. 2010, Osgood, Miller et al. 2017). Once in the blood stream, Aβ seems to be rapidly degraded by liver, spleen and kidneys (Shibata, Yamada et al. 2000). Receptors from the low density receptor family (LDLR) have been implicated to play a crucial role in the clearance of Aβ from brain (Shibata, Yamada et al. 2000, Storck, Meister et al. 2016). The low-density receptor-related protein 1 (LRP1) rapidly effluxes soluble Aβ from brain interstitial fluid and thereby tightly regulates the levels of soluble Aβ in brain (Storck, Meister et al. 2016). It has been shown that brain endothelial-specific inactivation of LRP1 in mice significantly reduced the brain efflux of injected Aβ. Moreover, in a mouse model of AD brain endothelial inactivation of LRP1 did not influence plaque pathology but elevated soluble Aβ in the interstitial fluid (ISF) and aggravated learning and memory deficits (Storck, Meister et al. 2016). Studies in humans have shown that soluble oligomeric Aβ levels correlate with AD (McLean, Cherny et al. 1999, Bao, Wicklund et al. 2012, Ferreira, Lourenco et al. 2015) however plaque pathology does not. As LRP1 expression decreases with aging and in AD (Kang, Pietrzik et al. 2000, Shibata, Yamada et al. 2000, Silverberg, Messier et al. 2010), brain endothelial LRP1 might be potential candidate to target Aβ accumulation in AD (Storck and Pietrzik 2017). Aβ bound to Apolipoprotein J is transported via LRP2 out of the brain (Bell, Sagare et al. 2007). The neonatal Fc receptor (FcRn) has also been described to assist in Aβ clearance by transcytosis of Aβ bound to immunoglobulins (Deane, Sagare et al. 2005). Besides receptor-mediated transcytosis, Aβ can be transported by several transmembrane channels. For example, it has been shown that many ABC transporters are involved in the efflux of Aβ (Hartz, Miller et al. 2010, Elali and Rivest 2013, Dodacki, Wortman et al. 2017). The most prominent member of this family is probably Abcb1 also known as P-glycoprotein (P-gp). As P-gp is located at the luminal side of endothelial cells is has long been discussed how P-gp gets access to a protein that is mainly produced in the brain. A potential LRP1-dependend mechanism has been suggested, however, conclusive studies are still lacking (Hartz, Miller et al. 2010).

In addition to receptors and transporters involved in Aβ efflux from brain, other receptors have been described that are involved in an uptake of Aβ from the periphery. For example, it has been shown in mice that the receptor for advanced glycosylation end products (RAGE), expressed in endothelial cells can take up Aβ from the blood and specific RAGE inhibitors were able to block that transport and reduce amyloid pathology in an mouse model of AD (Deane, Du Yan et al. 2003, Deane, Singh et al. 2012). Also, some organic-anion-transporting polypeptide transporters (OATPs) have been reported to transport Aβ from the periphery into the brain (Do, Bedussi et al. 2013). Aging and disease models show a dysregulation of many of these receptors and carrier. This suggests that BBB-mediated Aβ clearance is affected in AD. But how does the accumulation of Aβ affect the vasculature of the brain? Aβ induces neurovascular stress (Deane, Singh et al. 2012) and has a direct effect on the downregulation of tight junction proteins (Park, Kim et al. 2014, Keaney, Walsh et al. 2015) that seal the space between adjacent endothelial cells. The opening of junctions, however, allows the entry of blood-derived molecules in the brain and disturbs the tightly controlled environment of the CNS. Moreover, in mouse models it has been shown that the deposition of Aβ causes the vessels to degenerate resulting in blood flow alterations and structural modifications of vessels throughout the brain (Meyer, Ulmann-Schuler et al. 2008). In principle, the accumulation of Aβ due to a reduction of clearance has a huge impact on vessel function and distribution.

Figure 3:

The blood brain barrier in Alzheimer’s disease shows alterations to normal conditions. Decreased expression of tight junction proteins and efflux transporters, as well as a loss of function, promote the passive diffusion of blood-borne molecules into brain triggering neurodegeneration and the accumulation of Aβ. The loss and degeneration of pericytes furthermore promotes brain extravasation of plasma substances through the upregulation of transcellular transport processes.

The Choroid plexus in AD

Another interface that separates the periphery from the brain and shows alterations during aging and in disease is the choroid plexus (CP). The CP is vascular tissue found in all cerebral ventricles and the major site of CSF production. There is a high of turnover CSF in the healthy human brain. The 140 mL of CSF fill up four ventricles (20 mL), the spinal sub-arachnoid space (30 mL), and the cranial sub-arachnoid space (90 mL) and is produced at a rate of approximately 20 mL per hour (Oldendorf 1972, Pardridge 2011). Compared to the BBB, the CP seems relatively small: with a surface area of about 210 square centimeters the CP is roughly 0.1 % of the brain capillaries’ surface (Dohrmann and Bucy 1970, Damkier, Brown et al. 2013). Different from the BBB, the blood-CSF barrier at the CP is formed by epithelial cells and their epithelial tight junctions. To increase the surface area of the apical membrane, microvilli are present on the CSF-facing surface of CP epithelial cells. These microvilli regulate CSF composition and are believed to be involved in fluid secretion. The capillaries of the CP do not form a diffusion barrier; they lack endothelial tight junctions and are highly fenestrated. The brain endothelium and the CP epithelium are anatomically distinct membrane barriers with different permeability profiles. The CP is leaky with respect of electrical resistance across the barrier and entry of plasma protein into the CNS compared to brain vasculature (Pardridge 2016). However, the CP plays an important role in brain homeostasis by regulating CSF composition, immune responses, its signaling function and clearance of metabolites (Oldendorf 1972, Deane, Zheng et al. 2004, Crossgrove, Li et al. 2005, Fujiyoshi, Tachikawa et al. 2011, Damkier, Brown et al. 2013, Balusu, Van Wonterghem et al. 2016, Gorle, Blaecher et al. 2018). It has been suggested that Aβ is cleared across the blood-CSF barrier via receptor- and transporter mediated processes, similar to the BBB (Fujiyoshi, Tachikawa et al. 2011). However, exact clearance mechanisms or the capacity of Aβ clearance across the blood-CSF-barrier of the CP is still poorly understood. One reason for this is that genetic knockout studies addressing blood-CSF-barrier-mediated Aβ clearance across are still lacking. Similar to the vasculature of the brain, the CP shows alterations during ageing and in disease (Pascale, Miller et al. 2011). These changes affect proper CP function, for example the flux of molecules, metabolic activities or CSF production. Although physiological and ultrastructural differences at blood facing barriers in the CNS exist, they both are important for brain homeostasis and may have an impact on disease progression or onset in pathophysiological conditions like AD.

Pathological blood brain-barrier aberrations in Alzheimer’s disease

However, diminished clearance of Aβ is not the only pathological abnormality that is found in patients and AD models (Figure 3). Clinical studies in AD patients have also shown an impaired function of P-gp (Deo, Borson et al. 2014) not only leading to a reduction in efflux but also allowing the passage of xenobiotics from plasma into brain. In humans it has been shown that the BBB becomes leaky even before hippocampal atrophy (Montagne, Barnes et al. 2015) which is usually seen in early AD. This suggests that vascular abnormality precede neurodegeneration. Also, microbleeds or microhaemorrhages often coincide with AD leading to an extravasation of blood-derived substances into the brain causing inflammation and immune responses (Cullen, Kocsi et al. 2005). In addition, impaired transport of glucose, the most important energy source of the brain, across the BBB has been reported in AD due to a lower endothelial expression of glucose transporters and reduced blood flow (Kalaria and Harik 1989). Moreover, a degeneration of pericytes has been found in AD (Sengillo, Winkler et al. 2013, Halliday, Rege et al. 2016). As these cells are critical for the barrier characteristics of brain endothelial cells, the BBB is compromised under these conditions as well.

Therapeutic approaches to AD

Despite extensive research on the pathological features of AD in the last decades, little has been achieved in term of developing effective treatment strategies. The major reason for that is that the research we do is based on hypotheses (Hardy 2006). Up to now, we do not know the causes of AD. In order to develop efficient treatment strategies, our goal should be to better understand the pathophysiological pathways and connection during disease progression. AD is a multifaceted disease and, to date, the complexity and the various pathways that go wrong during disease progression are beyond our current understanding. As AD-causing mutations in the Aβ production pathways have been discovered some decades ago, industry has tried to develop strategies to lower Aβ production with little success (Abbott and Dolgin 2016). In recent years, it has not only been shown that in most AD cases clearance pathways are affected. Also, many novel AD risk factor genes have been discovered that affect Aβ clearance mechanisms including transport across the BBB (Jun, Naj et al. 2010, Kamboh, Minster et al. 2012, Apostolova, Risacher et al. 2018).

It remains uncertain whether targeting the BBB will help to treat, prevent or slow down AD pathology. However, the barriers of the CNS are crucial mediators for proper brain function. Therefore, neutralizing the alterations during aging and disease progression would probably be beneficial for our health and cognition.

Conclusion

In conclusion, on can say that the BBB is the gatekeeper for brain homeostasis and important for information processing, neuronal connectivity and synaptic functioning. Although, to date, we do not know if BBB dysfunction is cause or consequence of AD, many lines of evidence suggest that vascular impairment precedes neurodegeneration. Therefore, therapeutic strategies should target BBB breakdown in combination with other approaches in order to prevent, treat and reveres neurodegeneration in AD.

Acknowledgements: We thank Michael Plenikowski and Dr. Sabrina Meister for contributing to the illustrations. This article was funded in part by DFG (PI 379/8-1), Bundesministerium für Bildung und Forschung (01ED1605) to C.U.P., S.S. was supported by the intramural funding program of the University Medical Center of the Johannes Gutenberg University Mainz.