Introduction

An increase in the lifespan due to advancement in the healthcare system has resulted in an increase in the incidence of age associated comorbidities including neurodegenerative, cardiovascular, and metabolic diseases. Cognitive impairment has emerged as a significant health issue particularly in the elderly as it is difficult to diagnose it at an early stage. A global meta analysis based on 85 research articles revealed that the prevalence of mild cognitive impairment (MCI) increases with age; 6.5% to 34% in 50–59 years old, 5.1% to 37.5% in 60–69 years old, and 11.6% to 41% in individual ≥ 70 years old [1]. A previous study reported that around 20–40% of MCI cases can progress to dementia [2]. In addition, the majority of available therapeutic interventions provide symptomatic relief without targeting the underlying pathological mechanisms. The condition further poses an immense economic and emotional burden on the family members as it requires prolonged nursing care. Therefore, it is imperative to understand the key mechanisms involved during the early stage and arrest the progression to the advanced stage [3].

Blood-brain barrier

There are several vascular anomalies that may play important roles in the pathophysiology of cognitive impairment, including endothelial dysfunction [4] and blood-brain barrier (BBB) leakage [5, 6]. BBB refers to the unique property of brain vasculature by which it tightly regulates the movement of molecules and cells between the brain and blood [7]. The major component of the BBB is endothelial cells, smooth muscle cells, and pericytes. A high resistance paracellular barrier is established in the cerebral vascular with the help of a tight junction that holds together the endothelial cells. The structure of tight junctions is maintained with the help of homotypic interactions between proteins such as claudins, occludins, and zona occludins [8]. Mice deficient in these proteins display significant impairment in the BBB integrity and thus promote leakage across the barrier [9].

Assessment of BBB

In postmortem brain tissues, BBB leakage can be determined by measuring the extravasation of fibrinogen into brain parenchyma from the blood [10]. In addition, the ratio of albumin in cerebrospinal fluid (CSF) to the serum albumin called as albumin quotient (Qalb), can also be used to evaluate the integrity of BBB [11]. The Qalb ratio offers an advantage as it does not require postmortem tissue and can be performed in the patients by collecting blood and CSF. However, these methods are not completely reliable as inappropriate perfusion and tissue degradation during sample processing may affect the results. However, changes in the Qalb does not specifically indicates the specific region of the brain where BBB disruption took place. Montagne et al. [12] devised a high resolution magnetic resonance imaging (MRI) method to determine the BBB leakage by calculating blood-to-brain transfer constant of gadolinium (K_trans).

Changes in the BBB with age and cognitive impairment

In post-mortem brain tissues from patients suffering from Alzheimer’s disease (AD), Hultman et al. [10] reported an increased BBB leakage. Previous reports suggest that Qalb increases in individuals having a higher risk of developing AD [13, 14]. Montagne et al. [12] reported a linear increase with age in K_trans in the CA1 and dentate gyrus region of hippocampus. The same report further suggested that K_trans was about 60% higher in individuals suffering from MCI when compared to healthy controls with normal cognitive scores.

BBB impairment can lead to cognitive impairment and neurodegeneration as per several in vivo and clinical studies and is recognized as a major pathological event occurring during the early stages of cognitive impairment [10, 12, 13, 14]. It has been reported that BBB function can be compromised with AD [15–17]. In addition, neurodegeneration and cognitive impairment associated with high fat and fructose diet (HFF) were shown to be proceeded by impairment in BBB [18]. Various alterations at the cellular and molecular are known to take place in the endothelial cells with age that can contribute towards age-associated neurodegeneration, neuroinflammation, and cognitive impairment. P Glycoprotein 1 (PgP1) along with LDL receptor-related protein-1 (LRP1) is responsible for the clearance of drug molecules and aggregation-prone amyloid-beta (Aβ) species, a known hallmark of AD, from the brain parenchyma [15, 17]. Interestingly, the expression of PgP1 is known to decline with murine [19] as well as human aging [16]. Collectively, these reports suggest that BBB breakdown is an early event during the aging process which precede cognitive impairment. Therefore, identification of pathways and interventions that can control the progression of BBB leakage will help reduce the burden of cognitive impairment in aged individuals by allowing us to arrest it in early stage. The molecular signaling behind this BBB impairment is still a mystery and understanding of involved pathway(s) will enable us to limit age-related cognitive impairment.

Neutrophils extracellular traps and their role in aging and cognitive impairment

Release of neutrophil extracellular traps (NETs) which was first discovered by Takei et al. [20] refers to the release of NETs and involves chromatin decondensation that occurs through peptidyl arginine deiminase 4 (PAD4)-dependent deimination of nuclear histone proteins [21]. NETs consists of various components such as myeloperoxidase (MPO), citrullinated histone H3 (H3Cit), elastase, and histones. Previous literature suggests an increase in the NETosis and thus establishes its potential involvement in age related cognitive impairment. For instance, previously Kumar et al. [3] and Jylhävä et al. [22] have reported an increase in the circulating markers of NETosis such as cell free DNA and H3Cit with age. In addition, severe infiltration of H3Cit‐positive neutrophils was found in elderly patients suffering from severe vasculitis [23]. Moreover, cardiac and lung fibrosis occurring in aged mice was found to be associated with increased NETosis [24]. Specifically, aging is associated with an increase in the levels of reactive oxygen species (ROS) including hydrogen peroxide [25–30]. Furthermore, in vitro, the treatment of cultured neutrophils with hydrogen peroxide can induce NETosis [31]. In our previous study, we had shown that the mice overexpressing glutathione peroxidase 1 (Gpx1, enzyme responsible for converting hydrogen peroxide to water) exhibit lower levels of circulating markers of NEtosis such as cell free DNA [3].

Taken together, this evidence suggests that aging is associated with increased NETosis, however, the cause-effect relationship still needs to be confirmed.

Suttorp et al. [32] have shown neutrophil elastase (a component of NETs) can increase the permeability of cultured pulmonary endothelial cell monolayers. Furthermore, neutrophil depletion reduces BBB breakdown and increases neovascularization following stroke [33]. In addition, depletion of neutrophils using anti-Lymphocyte antigen 6 complex locus G6D (Ly6G) can attenuate the severity of AD in transgenic models 5xFAD and 3xTg-AD murine models [34]. Together, these points suggest that the release of NETs contributes to BBB leakage and cognitive impairment in mice. The effects could be mediated through alteration in the expression of PgP1, which also changes with age. However, these claims still need to be ascertained with experimental studies. Therefore, it is highly expected that overactivation of NETosis can promote age associated BBB impairment and cognitive impairment.

Inhibitors of NETs formation

Inhibition of PAD4 either by genetic manipulation [24] or pharmacological methods results in the suppression of NETs formation [35]. Similarly, administration of deoxyribonuclease 1 (DNase 1) can reduce the burden of NETs and protect against diseases like deep vein thrombosis [3]. Owing to the expected role of NETs in age-related BBB leakage and cognitive impairment, there is a critical need for the development of novel inhibitors of NET. Although several inhibitory molecules are available, they are limited by factors such as suboptimal inhibition, limited bioavailability, and non-specificity. Although DNase 1 can degrade the NETs and formulations are available for the human usage, it doesn’t treat the underlying cause of the condition [3]. GSK-484 was identified as a highly potent reversible selective inhibitor of PAD4 [35] and inhibits NETosis in several models such as ischemia-reperfusion injury, atherosclerosis, and cancer [36–38]. Although GSK-484 provided a proof-of-concept to inhibit NETs formation, this compound does not exhibit a strong potency as it requires micromolar concentrations to be effective, and therefore, it is not suitable for clinical development [39]. Recently, Martinez et al. [40] reported that tetrahydroisoquinolines (THIQs) inhibits NETosis via inhibition of neutrophil elastase at micromolar concentration. Thus, THIQs holds the potential to inhibits NETs formation, but need further derivatization to enhance the efficiency. However, till date, no systematic study has been conducted to decipher the therapeutic role of NETosis inhibition in age related cognitive impairment.

Conclusions

Taken together, all these studies indicates that NETosis activation with age can increase the BBB leakage and promotes cognitive impairment. Inhibition of NETosis using different pharmacological approaches can prevent the onset of cognitive impairment in aged individuals via attenuation of BBB leakage and thus pave the path for the development of novel therapeutic approaches. It is noteworthy that age-related cognitive impairment places an immense financial burden on the healthcare system and therefore it is imperative to examine the underlying mechanism and develop novel therapeutic interventions.