FIGURE 1 HERE
Figure 1. Altered proteins with an important role in Alzheimer’s disease identified in mouse tissues. This figure illustrates the heatmap of identified altered proteins (p < 0.05 and at least 1.5-fold change) in the (A) brain, (B) hippocampus, and (C) prefrontal cortex of mice treated daily with 1 mg kg-1 MTK for one week. The LFQ intensity scale represents the normalised relative quantification across all samples. Adapted from Marques et al. (2022d), with permission from Elsevier.

Hippocampus proteomics of MTK-treated mice

Regarding the hippocampus (Figure 1B), some identified proteins could be interesting from a repurposing perspective, particularly presenilin 1 (Psen1). This protein, one of the components of the γ‑secretase complex that is involved in the final cleavage of the β-C-terminal fragment (amyloidogenic pathway), originating the Aβ1-40 and Aβ1-42 peptides (Chen et al. , 2017; De Strooperet al. , 2010), was found to be up-regulated in the hippocampus (2.28-fold higher) of MTK treated mice. High levels of protrudin, a membrane protein that regulates polarized vesicular trafficking in neurons (3.93-fold higher in MTK-treated mice), were also found in hippocampus tissue. This protein has been associated with beneficial effects on the development of axon growth, particularly in regeneration (Petrova et al. , 2020). Finally, polyunsaturated fatty acid 5-lipoxygenase (5‑LOX), a protein involved in the production of leukotrienes, was found to be down-regulated in the hippocampus of treated animals (1.81-fold lower in MTK treated mice).

Prefrontal cortex proteomics of MTK-treated mice

Prefrontal cortex (PFC) proteomics (Figure 1C) also provided interesting data regarding relevant proteins in Alzheimer’s disease. The 14-3-3 protein family is among the most abundant proteins expressed in the brain, binding specific phosphoserine- and phosphothreonine-containing motifs from kinases, phosphatases, and transcription factors. These protein-protein interactions regulate cellular processes such as cell cycle, transcription, intracellular trafficking, apoptosis, and autophagy (Gu et al. , 2020); in neurons, these proteins are involved in differentiation, migration, survival, neurite growth, and ion channel regulation (Gu et al. , 2020). From the seven 14-3-3 isoforms identified in the human frontal cortex, five [η (4.72-fold), γ (5.68-fold), ε (6.43-fold), ζ/δ (7.38-fold), and β/α (4.45-fold)] were found to be up-regulated in the PFC of treated mice, indicating an up-regulation of this family of proteins upon MTK treatment.
Another significantly altered protein is hexokinase 1 (HK1, 7.17-fold higher in MTK-treated mice). The brain is the organ that consumes the greatest amount of energy, and neurons require large amounts of energy to maintain their normal activity (Yan et al. , 2020). Thus, HK1 is fundamental for glucose conversion to glucose-6-phosphate, the first step in glycolysis. Moreover, the tricarboxylic acid cycle enzymes malate dehydrogenase 1 (5.41-fold higher), and isocitrate dehydrogenase subunit α (3.92-fold higher) were also found to be up-regulated, likely due to the feed-forward glycolytic stimulation.
Regarding proteins involved in Aβ processing, the reticulon-3 protein, a negative regulator of BACE1 (Deng et al. , 2013; Kume et al. , 2009), was found to be 5.62-fold up-regulated in the PFC of MTK-treated mice, similarly to the insulin-degrading enzyme (3.78-fold higher in MTK-treated mice) and neprilysin (3.43-fold higher in MTK-treated mice). Conversely, the subunit of the γ‑secretase, nicastrin, was found to be decreased in MTK-treated mice (1.60-fold lower).
The reinterpretation of proteomics results from brain, hippocampus and PFC of MTK-treated mice shows that energy generating pathways and amyloid clearance processes are the mechanisms whose modulation in the brain would most likely be affected by MTK, providing a rationale for a possible impact of MTK on the management of neurodegenerative disorders.

Altered proteins in chicken embryo neurons exposed to MTK

To further explore MTK’s effect on neuronal viability, mature isolated embryonic chicken neurons treated with MTK were collected and their proteome was analysed as before.
The data from mature neurons treated with 1 μM MTK for 48 h suggested that MTK interferes with the α‑adrenergic signalling pathway (Marqueset al. , 2022d). This pathway involves various G‑protein coupled receptors that are targeted by catecholamines such as adrenaline and noradrenaline, modulating the synaptic transmission, as well as learning and memory (Perez, 2020); however, no biological processes specifically associated to the etiology or progression of AD were identified as being dysregulated.
By contrast, when mature neurons were exposed to 5 μM MTK for 48 h (Marques et al. , 2022d), we were able to follow a pathway-enrichment strategy, using the altered proteins (Figure 2 ). The data indicate that MTK interferes with microtubule polymerization and depolymerisation, as well as neurogenesis and the development of the nervous system (not shown). Furthermore, pathway enrichment also points to alterations in the AD presenilin-dependent pathway. Five altered proteins were found in this enriched pathway: amyloid-β A4 (APP, 2.04-fold higher), Wnt-11 (1.84-fold higher), Wnt5a (1.66-fold higher), histone acetyltransferase (KAT7) (1.56-fold higher), and tumour necrosis factor α converting enzyme (a subunit of the α‑secretase complex known as ADAM17, 1.72-fold lower).
The peroxisome proliferator-activated receptor-γ coactivator 1α (PGC‑1α), a regulator of liver gluconeogenesis, was also altered in treated neurons (1.90-fold higher in mature neurons exposed to 5 μM MTK). The glucagon-like peptide 1 receptor, involved in energy supply (Athauda and Foltynie, 2016; Li et al. , 2010), was also found to be up-regulated in treated neurons (2.86-fold times higher).