Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. As is well known, aggregation and accumulation of amyloid β42 (Aβ42) causes AD, and Aβ42 oligomers exhibit strong neurotoxicity (Dahlgren et al., 2002, Doody et al., 2014, Kumar et al., 2015, Scheuner et al., 1996, Sevigny et al., 2016). Therefore, Aβ42 is a logical target for therapeutics. Accordingly, many natural compounds and synthetic molecules have been tested for their capacity to inhibit the aggregation of these proteins (Hatakawa et al., 2021) (Ai et al., 2021, Dhouafli et al., 2018, Kwon et al., 2011, Naldi et al., 2012, Pagano et al., 2020, Young et al., 2015). However, effective new drugs are urgently required to prevent and treat AD. The use of model mice is important for the development of therapeutic drugs for AD. Therefore, knock-in amyloid precursor protein (APP) and Tau genes have been developed for AD model mice; however, these model mice are considered unsuitable for drug development due to the long time to onset and the variation of symptoms (Drummond and Wisniewski, 2017, Myers and McGonigle, 2019, Oddo et al., 2003). Recent studies have been conducted using Alzheimer's disease model mice created by intraventricular (ICV) administration of amyloid β25–35 (Aβ25–35). ICV administration of Aβ25–35 causes cognitive functional decline and loss of nerve cells in the brain (D’ezio et al., 2021, Maurice et al., 2019, Nakamura et al., 2023, Ramírez-Hernández et al., 2023, Shen et al., 2002, Sun and Alkon, 2002, Wang et al., 2015). Aβ25–35 coheres strongly and becomes fibrous when incubated at 37 °C, as does Aβ42 (Blivet et al., 2018, Millucci et al., 2010, Naldi et al., 2012). Three-dimensional analyses of Aβ25–35 have shown that it aggregates by forming β sheets. When administered to neurons, Aβ25–35 aggregates induce apoptosis and morphological changes (Wang et al., 2015). For these reasons, Aβ25–35 is an attractive target for screening compounds as potential AD therapeutics (Blivet et al., 2018, D'Agostino et al., 2012).

We recently found a synthetic nonapeptide, JAL-TA9 (YKGSGFRMI), derived from the Box A region of Transducer of ErbB-2.1 (Tob1) protein, which possesses serine protease-like proteolytic activity (Nakamura et al., 2019a, Nakamura et al., 2019b). Generally, proteases are macromolecular proteins that act alone or in large complexes. Protease-like activity has not been reported in small synthetic peptides; thus, we named this hydrolytic peptide, “Catalytide” (Nakamura et al., 2019b). The Box A region of Tob1 protein is a highly conserved domain of the Tob/BTG family of proteins, comprising Tob1, Tob2, BTG1, BTG2, BTG3/ANA, and BTG4 (Horiuchi et al., 2009, Matsuda et al., 1996, Schulze-Topphof et al., 2013). These proteins possess antiproliferative activities and are involved in the regulation of tumorigenesis. However, there have been no reports on the function of the Box A region. JAL-TA9 cleaves Aβ1–20, Aβ11–29 and Aβ28–42, a fragment of Aβ42. Although the substrate specificity of JAL-TA9 is not clear, it has been reported that JAL-TA9 shows strong proteolytic activity against Aβ11–29 in the central region containing the aggregation core of Aβ42 (Nakamura et al., 2019a). In addition, JAL-TA9 has been shown to exhibit strong proteolytic activity after aggregate formation for human prion protein (hPrP) 180–192, a fragment peptide derived from prion protein. This reveals that JAL-TA9 has a tendency to degrade highly aggregating peptides as a substrate specificity. Furthermore, JAL-TA9 cleaves both soluble form Aβ42 and solid form Aβ42 at multiple sites, especially in the central region (Nakamura et al., 2019a) suggesting that JAL-TA9 may be applicable to AD treatment from early stage to late stage. This is an attractive advantage in comparison to antibody drugs, such as Lecanemab and Aducanumab. Moreover, JAL-TA9 is easy to handle due to its stability at room temperature. In addition, the enzyme activity is kept after treatment with an organic solvent. These natures are suitable for clinical use.

In the current study, we confirmed the utility of JAL-TA9 as an AD drug. Aβ42 is not easy to handle in vitro and in vivo experiments due to its solubility and multiple fibril formation. Thus, we investigated the effect of JAL-TA9 on soluble and aggregated forms of Aβ25–35, which is commonly used as a model peptide in Alzheimer’s disease (AD) research due to its aggregation and neurotoxicity properties, by using Thioflavin-T assays and scanning electron microscopy. The proteolytic activity of JAL-TA9 against Aβ25–35 was evaluated using HPLC and mass spectrometry. In addition, we evaluated the effect of JAL-TA9 against Aβ25–35-induced cytotoxicity.