Sortilins in the blood-brain barrier: impact on barrier integrity

Sortilin was discovered in 1997 by Petersen and his colleagues. The single transmembrane receptor was isolated as a 95 kDa membrane glycoprotein by receptor-associated protein affinity chromatography and therefore initially named gp95. The cDNA library screening revealed sequence homology between the luminal segment of gp95/sortilin and the yeast vacuolar sorting protein 10 protein (Vps10p) domain (Petersen et al., 1997). The Vps10p domain was first identified in the Saccharomyces cerevisiae protein Vps10p that directs lysosomal enzymes from the Golgi to the vacuole (Marcusson et al., 1994). The trafficking path is even more complex for the mammalian Vps10p domain receptors that can shuttle between vesicles in the endo-lysosomal system and the cell (Nielsen et al., 2001). The Vps10p domain is evolutionary conserved and has a unique 10-bladed β-propeller structure (Quistgaard and Thirup, 2009). In mammalians, five receptors with Vps10p domains have been reported: sortilin (also known as neurotensin receptor 3), sortilin-related receptor central nervous system expressed 1, 2, and 3 (SORCS1, SORCS2, SORCS3), and sorting protein-related receptor with A-type repeats (SORLA; also known as LR11 and SORL1). The members of this gene family are collectively known as Vps10p-domain receptors or simply sortilins. They are all type-1 transmembrane receptors with a short cytoplasmatic tail and a large extracellular domain. The cytoplasmatic domain contains sorting motifs for specific adaptor proteins that direct intracellular trafficking. The Vps10p domain is located on the extracellular part that is the place for protein binding. Sortilin has this basic structure, while the other receptors have additional motifs (Figure 1A), indicating a more comprehensive target-recognition profile (Willnow et al., 2008). The sortilins are involved in several physiological functions of the cells and are therefore characterized as multifunctional receptors. Sortilin acts as a receptor or co-receptor in endocytosis, cell survival, signal transduction and directs proteins such as neurotrophins. Similarly, SORLA sorts different kinases, phosphatases, and signaling receptors to their correct intracellular location. SORLA is also a receptor for apolipoprotein E in the brain and is involved in processing amyloid precursor protein. SORCS1 and SORCS3 regulate energy homeostasis as intracellular trafficking receptors for tropomyosin-related kinase B. Furthermore, SORCS1 participate also in amyloid precursor protein processing and insulin secretion in the pancreas. SORCS2 is a co-receptor for proneurothrophins and contributes to producing the oxygen species scavenger glutathione under oxidative stress in neurons. A complete list of sortilins’ binding partners can be found in a more recent review (Malik and Willnow, 2020). Sortilins are expressed throughout the central and the peripheral nervous system in defined cell populations (Willnow et al., 2008). Accordingly, they have mainly been investigated in neurons and glial cells, but their role in the cells of the blood-brain barrier (BBB) has been completely neglected until now.

Figure 1.

Structure of sortilins and the blood-brain barrier (BBB).

(A) Structural organization of sortilins or vacuolar protein sorting 10 protein (Vps10p) domain receptors from yeast the Vps10p receptor and vertebrates sortilin, sorting protein-related receptor with A-type repeats (SORLA), sortilin-related receptor central nervous system expressed 1, 2, and 3 (SORCS1, SORCS2, SORCS3). These receptors have a short cytoplasmatic tail, transmembrane, and a large extracellular domain. The cytoplasmatic domain contains sorting motifs for specific adaptor proteins that direct intracellular trafficking. The Vps10p domain is located on the extracellular part that is the place for protein binding. (B) The BBB is a highly specialized structure of the microvessels in the brain. The anatomical basis of BBB is formed by the endothelial cells closely attached with interendothelial junctions. These endothelial cells constantly communicate with their neighborhood cells, astrocytes, pericytes, microglias, and neurons. (C) Double immunocytochemistry shows sortilin (green) in rat brain microvessels. Brain endothelial cells are identified by claudin-5 staining (red). Nuclei are labelled with blue. Scale bars: 20 μm. Cartoons made in BioRender. Figure 1A and B were created with BioRender.com. Methods and materials for Figure 1C: All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Following antibodies were used: anti-sortilin antibody (AF2934, 1:300; R&D Systems, Minneapolis, MN, USA) and anti-claudin-5 antibody (SAB4502981, 1:300), Alexa Fluor 488-conjugated anti-goat, Alexa Fluor 568-conjugated anti-mouse or anti-rabbit antibodies (all from Invitrogen, Denmark, 1:500). Capiliraies, staining and microscopy were done as described in a study by Toth et al. (2021). Unpublished data.

The BBB is a highly specialized structure of the microvessels in the brain. The anatomical basis of BBB is formed by the endothelial cells closely attached with the mixture of tight and adherent junctions. These endothelial cells are in constant communication with their neighborhood cells, astrocytes, pericytes, microglias, and neurons, enabling them to develop and maintain a unique barrier structure (Figure 1B). The barrier ensures the regulated exchange of gases and nutritions between the blood and the brain. At the same time, it protects the brain parenchyma from toxic insults by restringing the entry of harmful agents. Therefore, the brain´s homeostasis heavily depends on the proper function of the BBB. Dysfunction of the BBB has been observed in several pathological conditions and has led to the development of diseases or further neuronal damage (Sweeney et al., 2019). Despite decades of intensive research, the factors and signaling pathways for maintaining and modulating the integrity of the BBB are not fully elucidated. We recently reported that sortilin can be found in the brain microvessels (Figure 1C) and is expressed in the cells of the BBB, such as endothelial cells, astrocytes, and pericytes. It plays an essential role in regulating the BBB’s integrity. Rats lacking sortilin have a leaky BBB due to disturbed localization of tight and adherent junctional proteins in brain endothelial cells. We revealed that sortilin influences the interendothelial junctions via the fine-tuning of the Akt/MAPK/ERK signaling pathway (Toth et al., 2021). Disturbance of junctional proteins is a common hallmark in BBB pathologies (Sweeney et al., 2019). Thus, our observations impact the understanding of sortilin’s role in the healthy and diseased BBB. We are currently in the process of analyzing the involvement of other Vps10p domain receptors in the integrity of the BBB. SORLA is not expressed in brain endothelial cells and pericytes but affects the BBB via its expression in astrocytes (unpublished results).

We hope that our research will open the door for future studies to explore further the function and importance of the sortilins receptor family in the BBB. Significantly, sortilins bind and transport several growth factors and influence signaling pathways (Malik and Willnow, 2020), which can be necessary for the communication between brain endothelial cells and their neighborhood cells. The role of sortilins in astrocytes, pericytes, and microglias would also be important to study under healthy and pathological conditions. A recent paper serves as a perfect example to support this statement. After a stroke, astrocytes release endostatin to help angiogenesis in the damaged tissue. Malik et al. (2020) proved that SORCS2 is vital for the correct intracellular protein sorting and release of endostatin in astrocytes during post-stroke recovery. Sortilins are primarily sorting receptors that mediate the intracellular trafficking of various proteins, lipids, and receptors involved in essential physiological as well as pathological processes (Malik and Willnow, 2020). In many of these diseases, the damage of the BBB has been reported (Sweeney et al., 2019). For example, sortilin is downregulated in Alzheimer’s disease, hypercholesterolemia, and diabetes. In peripheral endothelial cells, sortilin is involved in vascular impairment (Di Pietro et al., 2022) and myocardial hypoxia/reoxygenation injury (Yu et al., 2018). Therefore, circulating sortilin has been suggested as a biomarker for hypertension (Varzideh et al., 2022). SORLA, SORCS1, and SORCS3 have also been linked to Alzheimer’s disease and other age-related dementias (Willnow et al., 2008; Malik and Willnow, 2020). Taken together, revealing sortilins’ role in the diseased BBB has an enormous potential to identify new drug targets and find novel solutions for the unmet medical needs of our society.

AET and MSN received funding from the Research Initiative on Brain Barriers and Drug Delivery funded by the Lundbeck Foundation (grant No. 2013-14113).