Impact of Alpha-Synuclein Pathology on the Blood-Brain Barrier
The blood-brain barrier protects the delicate neuronal environment from peripheral immune cells, ion fluctuations in the blood and toxic substances, ultimately making it one of the most important structures in our central nervous system.
A majority of drugs given to Parkinson`s disease (PD) patients and compounds currently in preclinical and clinical trials, such as small molecules, antibodies and enzymes, have to cross the blood-brain barrier. However, the blood-brain barrier changes with aging and more importantly it is also altered in neurodegenerative diseases like PD. Since there is growing evidence that blood-brain barrier alterations in PD occur early in disease progression, it seems unlikely that they occur merely in response to advanced-stage hallmarks of PD, such as nigrostriatal neurodegeneration and dopamine loss, or physiological aging. It is rather possible that they constitute a relevant pathophysiological aspect to the disease. Moreover, it is reasonable that pathology of alpha-synuclein (aSyn), the major protein component of Lewy bodies, another hallmark in the brains of PD patients, could be a contributing factor to blood-brain barrier impairments. Therefore, there is urgent need to resolve the underlying pathomechanisms of blood-brain barrier dysfunction in PD.
Endothelial cells, linked by tight junction proteins, form a semipermeable layer and thereby contribute a major part to the blood-brain barrier. This inner layer is enclosed by a basal lamina and surrounded by pericytes, astrocytic end-feet, microglia and neurons. Altogether these cells are often called the neurovascular unit. Each cell-type could individually be affected by disease progression, but deficiency in one part would influence the other components, thereby likely exacerbating impairments of the barrier.
We aimed to investigate how alpha-synuclein pathology effects distinct blood-brain barrier components as well as possible impact on the barriers`overall integrity.
To replicate aSyn pathology and dopamine loss, our research group uses a progressive mouse model of PD with overexpression of human aSyn (Thy1-aSyn, line 61, Chesselet, Richter et al. 2012 Neurotherapeutics). Male Thy1-aSyn and wild-type age-matched controls were used at different ages reflecting different stages of disease progression, ranging from prodromal to clinical disease stage. The aim was to detect whether the blood-brain barrier in our model is already altered at an early time point, reflecting when the PD patients get diagnosed or if it`s a rather late manifestation. We were able to isolate brain capillaries from cortex and striatum, only consisting of the endothelial cell, pericytes and basal lamina. Thus, we could continue our work with a pure form of the blood-brain barrier. Protein extracts from these capillaries were subjected to Western blotting for analysis of barrier function-associated proteins. We performed immunocytochemistry with isolated capillaries and histological staining of blood vessels using whole brain Our first hypothesis was that aSyn is located at the blood-brain barrier. This is important because its presence could directly induce modifications in the barrier, for example by promoting inflammation or activating endothelial cells. By histological staining and protein expression analysis, alpha-synuclein was detected in brain capillaries of Thy1-aSyn mice.
This led to our second hypothesis if the present alpha-synuclein affects transporter molecules at the blood-brain barrier. For example, expression of transporter proteins possibly involved in alpha-synuclein transport could be altered as a compensatory mechanism. Indeed, we could show low-density lipoprotein receptor related protein-1 (LRP-1) upregulation and P-glycoprotein downregulation in the isolated capillaries. This indicates that endothelial cells are actively trying to change the transportation pathways of specific molecules in alpha-synuclein overexpressing mice.
Presumably, alpha-synuclein and the formation of aggregates trigger inflammation and stress response in the brain. To test this third hypothesis in isolated capillaries, we measured the protein expression levels of two inflammatory factors, vascular cell adhesion molecule-1 and matrix metalloproteinase-3. As expected, we saw an upregulation of both proteins.
Increased inflammation markers raise the fourth hypothesis, whether the blood-brain barrier is still intact. There is a broad discussion about the permeability of blood vessels in brains affected by PD, but the majority agrees that especially in early disease stages there is no overt leakage of blood molecules into the brain parenchyma. By staining the blood molecule immunoglobulin G (IgG) we were not able to detect leakage into brain parenchyma as well. It is of note that leakage could be so subtle that we did not recognize it using IgG staining.
PD-affected brains show not only alterations at the blood-brain barrier, but also a modification of the whole microvasculature pervading the brain parenchyma. In a histological staining of the blood vessels in brain sections of our Thy1-aSyn mice, we could detect a decrease in vessel density after the brain was exposed to alpha-synuclein pathology. Additionally, aquaporine-4 (AQP4), a water channel located directly where the end-feet of astrocytes connect to a blood vessel, was reduced. This indicates that either the number of astrocytes is reduced or the water channel AQP4 is dispersed in Thy1-aSyn mice.
All of these results indicate that alpha-synuclein pathology leads to regional specific and disease stage-dependent alterations of the blood-brain barrier and the microvasculature in our model, and potentially in PD patient brains. Targeting alpha-synuclein pathology in cells beyond neurons (including endothelial and glia cells) may be beneficial for the patients and could improve efficacy of other treatments. Especially, when one takes into account that systemically applied compounds are required to cross the blood-brain barrier and thereby potentially encounter alpha-synuclein decompositions in endothelial cells.
Kristina Lau, PhD student at the Department of Pharmacology, Toxicology, and Pharmacy; University of Veterinary Medicine Hannover, Germany. She presented her work as part of a guided poster tour at the WPC 2023 in Barcelona.
Ideas and opinions expressed in this post reflect that of the author solely. They do not reflect the opinions or positions of the World Parkinson Coalition®