Discussion and conclusions

Montelukast has been associated with some neuropsychiatric side effects, especially among children. Our previous work suggests that these adverse effects may be associated with a dysregulation of the hypothalamic-pituitary-adrenal axis and an increase of oxidative stress in children’s brain (Marques et al. , 2022d). Since older people have a mature brain, they should be less susceptible to these effects, which could explain the decreased incidence of adverse drug reactions (ADRs) in adults. Moreover, specific montelukast effects in the older brain may underlie the proposed, yet mechanistically elusive, repurposing of the drug for the management of neurodegenerative disorders. In the current work, the same proteomics dataset generated in our previous work was interrogated from a repurposing perspective, to identify altered proteins known to be involved in the pathophysiology of Alzheimer’s disease.
The up-regulation of Nceh1 and HIVEP3 in the mouse brain suggests that MTK contributes to AD management. Indeed, while Nceh1 has been associated with a protective role against α‑synuclein toxicity (Zhanget al. , 2017) and has been found to have decreased levels in the blood of AD patients (Mandas et al. , 2012), HIVEP3 is underexpressed in a double transgenic APP/PS1 mouse model of early-onset AD (Islam et al. , 2021). Likewise, the hippocampus up-regulation of Psen1 mimics the effect observed upon γ‑secretase inhibition (Campanari et al. , 2014; Sogorb-Esteve et al. , 2018). This is in agreement with the observed up-regulation, in the PFC, of the reticulon-3 protein, a known inhibitor of the anterograde transport of the BACE1 subunit of β‑secretase, thus contributing to an improved clearance of amyloid deposits (Deng et al. , 2013; Kume et al. , 2009).
The insulin-degrading enzyme, up-regulated in mouse PFC, is involved in the degradation of α‑synuclein aggregates, and its dysregulation has been related to the occurrence of type 2 diabetes mellitus, whereby the patients have a high risk of developing AD or PD (Sousa et al. , 2021). Neprilysin was also found to be up-regulated in the PFC of MTK-treated mice. It has been proposed that the up-regulation of these two enzymes could be a promising therapeutic strategy for neurodegenerative disorders (Nalivaeva and Turner, 2019). It is noteworthy that the glucagon-like peptide 1 receptor, which has been associated with improved memory and learning in mice (During et al. , 2003), was also overexpressed in MTK-treated mature chicken embryo neurons supporting the hypothesis of an improvement of the amyloid clearance process caused by MTK.
The lower levels of nicastrin, coupled to the altered levels of Psen1 (another component of the γ‑secretase complex) in the PFC of MTK‑treated mice, also indicate that MTK may be involved in the inhibition of γ‑secretase, leading to a decrease of amyloid production. Factoring in the altered AD presenilin-dependent pathway, dominated by an up-regulation of the Wnt pathways, observed in treated neurons, MTK appears to have the potential to overcome the synaptic degeneration and cognitive deficits characteristic of AD. It is noteworthy that Wnt proteins are systematically found to be down-regulated in AD patients, and these lower levels are associated to activation of the amyloidogenic pathway (Palomer et al. , 2019). Thus, the proteomic shifts associated with MTK have the potential to counterbalance a number of pro-amyloidogenic mechanisms involved in AD’s etiology.
5-LOX is involved in the synthesis of cysteinyl leukotrienes, which have a pro-inflammatory effect antagonized by MTK (Marques et al. , 2022c). Interestingly, while 5-LOX levels are found to be higher in AD patients (Li et al. , 2017), the observed lower 5-LOX levels in the mouse hippocampus also suggest that MTK is able to, at least partially, restore the altered phenotype of AD. This is further corroborated by the observed up-regulation of the 14-3-3 protein family in the prefrontal cortex of treated mice. While decreased levels of the 14-3-3 proteins are associated with several AD and PD hallmarks (Guet al. , 2020), their MTK-driven overexpression is quite promising, since the modulation of these isoforms has been described as a potential target for neurodegenerative disorders (Pair and Yacoubian, 2021).
From a central metabolism standpoint, the ageing process is characterized by a lowering of cell basal metabolic rate, particularly in the case of neurons (Yan et al. , 2020). HK1 levels are consistently found to be decreased in AD patients (Yan et al. , 2020), whereby the observed increase of HK1 levels caused by MTK could play a neuroprotective role in the brain. This HK1 increase was observed in the PFC of treated mice, together with the up-regulation of the tricarboxylic acid (TCA) cycle enzymes malate dehydrogenase and isocitrate dehydrogenase. From a metabolic perspective, the observed overexpression of the glucagon-like peptide 1 receptor in chicken embryo neurons is a further link between the brain energy pathways and AD, as this protein is also involved in the clearance of amyloid and synuclein aggregates. Notably, the glucagon-like peptide 1 receptor has been proposed as a therapeutic target for neurodegenerative disorders (Athauda and Foltynie, 2016; Li et al. , 2010). The MTK-induced up-regulation of this receptor observed in our work, and its mechanistic implications, may thus provide, at least in part, the missing rationale for the repurposing of MTK in these diseases. It is also noteworthy that the proteomic alterations of TCA cycle enzymes reported herein are in agreement with the TCA changes we observed previously at a metabolomics level (Marques et al. , 2022d).
Taken together, the proteomics data reanalysed herein clearly indicate that montelukast is associated with a shift towards amyloid clearance, coupled to an up-regulation of specific enzymes involved in energy production (Figure 3 ). These are two fundamental biological processes that sustain MTK’s repurposing towards AD management, which is currently being assessed in various clinical trials (for example, ClinicalTrials.gov Identifiers NCT03991988 and NCT03402503). This wide anti-AD activity of montelukast is reminiscent of reports from studies on valproic acid, which has been shown to increase neprilysin expression and activity in hypoxic rats and in model mice, and is speculated to enhance amyloid clearance in AD patients (Nalivaeva et al. , 2012; Nalivaeva and Turner, 2019; Wang et al. , 2014). Overall, central nervous system-acting drugs of different classes offer new potential strategies for the management of AD and other neurological diseases. To be fulfilled, these promising approaches require further investigation of the biochemical mechanisms underlying such beneficial side effects.