The ageing BBB: process and highlights

The process of ageing is an inevitable, continuous and time-dependent decline in tissues and organs with progressive loss of function, decreasing fertility and increasing mortality (Balcombe and Sinclair 2001). The brain, as any other organ in the body is affected by ageing at both cellular and molecular level, with the hippocampus and the cerebral cortex amongst the most affected regions (Anderton 2002). Ageing in the CNS has been reported to include changes in gene and protein expression, abnormal protein accumulation or mitochondrial dysfunction (Brunk and Terman 2002; Liang et al. 2007; Trojanowski and Mattson 2003). These age-related changes have been implicated in promoting neurodegeneration, declined cognitive function, reduced cerebral blood flow and vasculopathies such as stroke (Cabeza et al. 2018; Desjardins et al. 2014; Gupta et al. 2012; Wyss-Coray 2016). Accordingly, the BBB has been described to undergo several alterations in function and structure during ageing (Krause, Fautsmann, and Dermietzel 2002; Pelegrí et al. 2007).

As previously discussed, the BBB plays a critical role in maintaining the homeostasis of the brain. Alterations and declined integrity of the BBB have been implicated in a variety of age-related neurodegenerative conditions, such as vascular cognitive impairment, Alzheimer’s disease (AD) and Parkinson’s disease (PD) (Marques et al. 2013; Zlokovic 2008). As the percentage of individuals over 60 years has increased from 9.2% in 1990 to 11.7% in 2013 and is projected to be 21.1% by 2050 (Sander et al. 2015), understanding the mechanisms that underlie age-related changes in the BBB in health and disease is of increasing importance (Aunan et al. 2016).

1.2.1 Breakdown of the BBB during ageing

During ageing, the cerebrovasculature undergoes several pathophysiological changes that lead to its dysfunction. For instance, cerebrovascular ageing is characterised by alterations in blood flow dynamics which induce changes in the expression of mechanosensitive genes that in turn promote vascular remodelling or pro-atherogenic modifications in the vessel wall

17 (Ungvari et al. 2010). Indeed, cerebral microvessels have been reported to become tortuous and show severe changes such as increased wall stiffness or decreased density, that contribute to a decline in cerebrovascular efficiency and cerebral blood flow (Riddle, Sonntag, and Lichtenwalner 2003). Interestingly, as cerebrovascular efficiency falls, the brain also suffers from impaired transport of essential elements like glucose or oxygen (Peters 2006; Yamazaki et al. 2016). In addition, aged cerebrovasculature might also undergo process of neuroinflammation and oxidative stress (Enciu, Gherghiceanu, and Popescu 2013). BBB alterations have also been observed to vary depending on the brain region in both aged humans and rodents, contributing to the complexity of the ageing process (Goodall et al. 2018; Hawkes et al. 2013).

BBB dysfunction during ageing has been related to numerous alterations in the endothelium, at both cellular and molecular levels (Figure 3). These age-related changes include:

▪ Increased BM thickness due to the accumulation of ECM components such as collagen IV, laminin and other proteoglycans, which in turn induces an increase in stiffness of the vessel wall (Candiello, Cole, and Halfter 2010). Higher BM thickness and stiffness may have detrimental effects on cerebral blood flow and clearance mechanisms across the BBB that may contribute to cognitive decline and neurodegeneration (Ito et al. 2013).

▪ Weakening of TJ protein structures, which may lead to higher BBB permeability (Montagne et al. 2015). For example, levels of TJ proteins such as occludin, Cldn-5 and ZO-1 have been reported to decrease in aged rodent brain compared to their young counterparts (Elahy et al. 2015; Mooradian, Haas, and Chehade 2003; Stamatovic et al. 2019). Also, previous studies have shown an age-induced increase in matrix metalloproteinase (MMP) expression, including MMP-9 and MMP-2, in the aged mice upon brain injury, which contributes to the cleavage of ZO-1 and claudin-5 and the increase in barrier permeability (Lee et al. 2012). Additionally, MMP-mediated disruption of TJ protein complexes was enhanced by increased levels of cytokines (e.g. Tumour necrosis factor α, TNF-α) in previous studies using an in vitro dynamic BBB model with rat BEC cells (Krizanac-Bengez et al. 2006). Similarly, TJ disruption may also be

18 worsened by exposure to reactive oxygen species (ROS) derived from oxidative stress, which may induce redistribution and loss of occludin or claudin-5 as reported in rat BECs in vitro (Schreibelt et al. 2007).

▪ BBB transport systems have been described to be altered with ageing, including enhanced pinocytosis or decreased expression of glucose transporter, insulin receptor or low density lipoprotein-related protein-1 (LRP-1), as observed in the ageing human and rodent brain (Cholerton, Baker, and Craft 2011; Mooradian et al. 1991; Ramanathan et al. 2015). Similarly, Pgp expression has been reported to decrease in the ageing BBB, although only in humans (Bartels et al. 2009). In any case, these changes lead to a severe imbalance in brain vasculature nutrient uptake/waste elimination function (Silverberg, Messier, et al. 2010; Silverberg, Miller, et al. 2010).

▪ Mitochondrial dysfunction is another prominent feature in the process of ageing in general and at the BBB in particular. Previous studies have described the age- related decrease in mitochondrial number and impairment in the BBB of several species including human, mouse and monkey, mostly related to oxidative stress and pro-inflammatory processes (Burns et al. 1979; Sure et al. 2018). As stated above, oxidative stress might contribute to vascular and BBB disruption. In fact, age-induced increase in production of ROS has been linked to endothelial cell dysfunction (Brandes, Fleming, and Busse 2005).

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Figure 3. BBB cellular and molecular alterations in ageing.

During ageing, several alterations can be observed at structural and functional levels as the breakdown of the BBB progresses. A prominent feature is the increase in BM thickness due to the accumulation of extracellular matrix components. TJ protein structure becomes loose and weaker, which may lead to increased permeability. Transport systems are modified in the ageing BBB, including increased pinocytotic vesicles trafficking through the endothelium, as well as altered expression of transporters and membrane proteins such as glucose and insulin transporters and Pgp. In addition, there is a decrease in mitochondrial density and accumulation of ROS that contribute to oxidative stress and neuroinflammation, worsening the features of the ageing BBB. (Diagram by

Eduardo Frías Anaya).

Several of these age-related changes at both cellular and molecular level appear to correlate with changes in gene expression to a certain degree (Osgood et al. 2017; Stamatovic et al. 2019). Therefore, connecting age-related changes in structure and function with concomitant alterations in gene expression profiles might shed light on the mechanisms that promote BBB dysfunction during ageing.