Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases worldwide. It is currently estimated to affect more than 44 million people, which is expected to double by 2050 (Dumurgier and Sabia, 2020). This condition is characterized by the presence of two histopathological hallmarks in the central nervous system (CNS), senile plaques, protein aggregates of amyloid beta (Aβ) peptide and neurofibrillary tangles (NFT), aggregates of hyperphosphorylated tau protein, leading to cognitive impairment and dementia (Zhao and Huai, 2023). In the last years, these two features of the disease have been the main therapeutic targets for AD drug development. However, there is still no cure for the disease, so further studies of the different mechanisms involved in the disease are needed.

The dorsal subiculum is one of the earliest regions affected in AD (Ye et al., 2024). It is crucial for learning and memory processes in rodents, as it serves as the main pathway for information leaving the CA1 region of hippocampus (O'Mara and Aggleton, 2019; Frost et al., 2021) and is a key structure within the hippocampal formation, playing an important role in spatial representation and navigation information processing (de Melo et al., 2023). This early involvement of the dorsal subiculum in AD suggests that it may be particularly vulnerable to the harmful effects of chronic neuroinflammation, which plays a significant role in disease progression.

Chronic neuroinflammation is a common feature of numerous neurodegenerative diseases, such as AD, Parkinson´s disease (PD) or multiple sclerosis (Glass et al., 2010). However, despite existing evidence of the importance of neuroinflammation, there is still a lot of controversy regarding its role and relevance in AD (Calsolaro and Edison, 2016; Heneka et al., 2015). The CNS immune response detects the abnormal protein aggregation as harmful, leading to astrogliosis around senile plaques and morphological microglial changes leading to the secretion of pro-inflammatory cytokines (Boche and Nicoll, 2008). Normally, once the damage has ceased, glial cells would stop the inflammatory response and return to the basal state. Nevertheless, in the event of persistent damage, glial cells are activated long-term, leading to uncontrolled neuroinflammation that can lead to neuronal dysfunction and cell death, promoting the progression of the disease (Carriba and Comella, 2015). All in all, compounds that can modulate neuroinflammation and neuroimmune responses in the CNS, could be a potential therapeutic target for AD (Yu et al., 2021).

Pleiotrophin (PTN) is an important cytokine for CNS repair, neuronal survival, and differentiation (Herradón and Pérez-García, 2014). It is widely expressed during development, while its pattern of expression in adults is restricted to a few cell types in different organs, including the brain, where it is mainly expressed in neurons in healthy murine models (Deuel et al., 2002; Herradon et al., 2019; Silos-Santiago et al., 1996; Vanderwinden et al., 1992) being its highest expression in the CNS during embryonic and neonatal periods (Wang, 2020). However, after an injury or noxious stimulus, its expression increases in different cells including microglia and macrophages (Martin et al., 2011; Jin et al., 2009; Muramatsu, 2011; González-Castillo et al., 2014). PTN is overexpressed in different brain areas in situations with inflammatory component, as in brain damage due to ischemia, in neuropathic pain, after administration of different drugs of abuse such as amphetamine, alcohol and opioids, even in neurodegenerative processes, in senile plaques in the brain of patients with AD and in the substantia nigra of patients with PD (Herradón and Pérez-García, 2014; Alguacil and Herradón, 2015), suggesting a modulatory role of PTN in these processes. PTN is a potent modulator of neuroinflammation in different contexts (Herradon et al., 2019; Fernández-Calle et al., 2017; Vicente-Rodríguez et al., 2016; Rodríguez-Zapata et al., 2024; Rodríguez-Zapata et al., 2023) and binds to different receptors in many organs, being Receptor Protein Tyrosine Phosphatase β/ζ (RPTPβ/ζ) mainly expressed in the adult CNS in neurons and glial cells (Canoll et al., 1996; Shintani et al., 1998; Lafont et al., 2009). RPTPβ/ζ is important for neuronal and microglial viability (Del Campo et al., 2021), and it is the most relevant in modulating neuroinflammation (Herradon et al., 2019). Pleiotrophin binds to the extracellular domain of RPTPβ/ζ (Maeda et al., 1996; Maeda et al., 1999), inactivating its phosphatase activity and therefore increasing the phosphorylation levels of its substrates, such as TrkA (Shintani and Noda, 2008) and Fyn kinase (Pariser et al., 2005; Panicker et al., 2015), both with known roles in neuroinflammation.

To further characterize the functions of RPTPβ/ζ, we designed and synthesized MY10, a selective inhibitor of RPTPβ/ζ permeable to the blood-brain barrier (BBB). MY10 interacts with the intracellular domain PD1 of RPTPβ/ζ, and it inactivates its tyrosine phosphatase activity, simulating the inhibitory action of PTN on this receptor (Pastor et al., 2018).

Based on these considerations, we aim to demonstrate that RPTPβ/ζ modulates neuroinflammation and neurodegeneration, using the selective inhibitor of RPTPβ/ζ, MY10, in a mouse model of AD.

Alzheimer’s disease is a major public health concern worldwide. Every year, there are almost 10 million new cases, and there is still no cure (Passeri et al., 2022). There is an urgent need to find new therapeutic targets for this disease. Recent studies point out that modulating neuroinflammation seems to represent a valuable therapeutic approach. Therefore, compounds that modulate the immune response in the CNS may have therapeutic potential in AD. In this context, we previously demonstrated that the PTN/RPTPβ/ζ axis regulates glial responses and neuroinflammation induced by different stimuli (Herradon et al., 2019; Fernández-Calle et al., 2017; Vicente-Rodríguez et al., 2016). In the present work, we aimed to test the impact of RPTPβ/ζ inhibition with MY10 on neuroinflammation and neurodegeneration in AD, using the APP/PS1 animal model. Our findings reveal, for the first time, that treatment with MY10 significantly reduced the number and size of Aβ plaques in the dorsal subiculum of the hippocampus of APP/PS1 mice, suggesting that RPTPβ/ζ plays an important role modulating Aβ plaques formation in the hippocampus. The CNS immune response detects the abnormal protein aggregation as harmful, leading to astrogliosis and morphological microglial changes around senile plaques, resulting in pro-inflammatory cytokines secretion (Boche and Nicoll, 2008). Prolonged glial cells activation, causes uncontrolled neuroinflammation, neuronal dysfunction, and cell death, which promote disease progression (Carriba and Comella, 2015). On the other hand, the clearance of Aβ from the brain involves the active participation of glial cells amongst others (Yoon and Jo, 2012). They surround compacted Aβ plaques, forming a barrier to prevent the generation of new Aβ monomers and protect neurons (Ullah and Lee, 2023). Insufficient clearance of Aβ has been identified as the major pathological mechanism of AD (Cai et al., 2023). Given this context, our study aimed to test the possibility that RPTPβ/ζ inhibition with MY10 modulates glial responses in APP/PS1 mice. Our results indicate that treatment with MY10 decreased the number and size of astrocytes, as well as the number of microglial cells in male and female APP/PS1 mice. It is known that activated glial cells like microglia and astrocytes are key in promoting a neuroinflammatory response that can be the neuropathological event leading to neurodegeneration in AD (Fakhoury, 2018). Many reactive glial cells are found near senile plaques in AD patients, which suggests the role of these cells in the pathogenesis of the disease (Zotova et al., 2011). In this study, the decreased numbers of glial cells caused by MY10 treatment are associated with a reduction of Aβ, suggesting that a diminished persistent glial reactivity against this pathologic protein aggregation may contribute to mitigate the progression of neuroinflammation and neurodegeneration in the context of the disease. However, not only the reduction of gliosis is important but the proximity of glial cells to Aβ plaques and the type of molecules they express.

PTN is primarily expressed in the CNS but is significantly upregulated in various cells, including microglia and inflammatory macrophages, following injury (Martin et al., 2011; Jin et al., 2009; Muramatsu, 2011; González-Castillo et al., 2014). In APP/PS1 mice, we observed that PTN was predominantly expressed in astrocytes and, to a lesser extent, in microglia. Treatment with MY10 reduced the overall PTN marked area. Recent studies demonstrate that PTN is accumulated in senile plaques, and it has an impact on amyloid deposition by accelerating senile plaques aggregation (Levites et al., 2024). This suggests that MY10 treatment could reduce senile plaque aggregation by decreasing PTN expression. Specifically, MY10 tended to decrease PTN expression in microglial cells, but not in astrocytes, suggesting different regulatory mechanisms and cellular responses in astrocytes and microglia in AD. As expected, both astrocytes and microglia were spatially associated with Aβ plaques in APP/PS1 mice. Here, we show that MY10 treatment significantly reduced the number of astrocytes and microglial cells surrounding Aβ plaques in APP/PS1 mice. These results strongly support the modulation of neuroinflammation through RPTPβ/ζ inhibition and its potential association with the clearance of Aβ plaques in the hippocampus.

In response to Aβ plaque accumulation, microglia produces proinflammatory cytokines, leading to chronic neuroinflammation (Hansen et al., 2018). Interestingly, in the hippocampus of APP/PS1 mice, Tnfa and Hmgb1 mRNA expression levels were significantly reduced by MY10. On the other hand, Il6, Il1b, Ptgs2 and Cd68 expression levels did not seem to be significantly modulated by MY10, suggesting that RPTPβ/ζ inhibition with MY10 modulates neuroinflammation by regulating Tnfa and Hmgb1 mRNA expression levels in APP/PS1 mice. TNFα and HMGB1 are key pro-inflammatory molecules implicated in neuroinflammation, which plays a crucial role in AD progression. Several anti-inflammatory treatments targeting microglial activation have been shown to significantly decrease TNFα expression, reducing synaptic dysfunction and cognitive impairment in AD (Decourt et al., 2017). Similarly, extracellular HMGB1 is thought to contribute to AD pathology by inhibiting microglial phagocytosis and stabilizing Aβ42 oligomers. It has been demonstrated that inhibition of HMGB1 reduces neuroinflammation and enhances Aβ clearance (Fang et al., 2012). These findings support the idea that pharmacological treatments that modulate these inflammatory pathways, can suppress the activation of glial cells and promote Aβ clearance.

Microglia and astrocytes also promote the breakdown of Aβ fibrils and oligomers by secreting multiple Aβ-degrading enzymes (Abud et al., 2017) such as BACE1 (Ulku et al., 2023; Hampel et al., 2021), MMP9 (Hernandes-Alejandro et al., 2020) and IDE (Tian et al., 2023). Interestingly, we found that MY10 differentially modulates the hippocampal mRNA expression levels of these enzymes. Treatment with MY10 significantly increased Bace1 mRNA expression in the hippocampus compared to vehicle-treated APP/PS1 mice. The beta-site amyloid precursor protein (APP) cleaving enzyme BACE1 has been known for years for its amyloidogenic activity, contributing to the production of Aβ peptides (Hampel et al., 2021). However, recent studies have established an amyloidolytic activity of BACE1, degrading longer Aβ peptides into a non-toxic Aβ34 intermediate (Ulku et al., 2023). This dual functionality of BACE1 suggests a complex regulatory role in amyloid metabolism. Specifically, while an excess of APP promotes the amyloidogenic Aβ peptide production, an excess of BACE1 facilitates increased Aβ peptides degradation (Liebsch et al., 2019). In this context, MY10 treatment may modulate Aβ plaques formation by enhancing the amyloidolytic activity of BACE1, thereby promoting the degradation of potentially toxic Aβ peptides.

Recent studies have elucidated that MMP9 contributes to the clearance of Aβ by degrading amyloid plaques and facilitating their removal from the brain (Fragkouli et al., 2014). However, our findings demonstrate that treatment with MY10 reduces the levels of Mmp9 in the hippocampus of APP/PS1 mice. MMP9 is also involved in various physiological and pathological processes beyond Aβ degradation, such as inflammation. A decrease in Mmp9 could also reflect changes in the neuroinflammatory environment or alterations in tissue homeostasis, which might indirectly affect Aβ plaque dynamics (Fujimoto et al., 2008). Inhibition of MMP9 facilitates Aβ clearance across the BBB and it also decreases tissue damage, neutrophil infiltration, oxidative stress and neuronal degeneration (Ringland et al., 2021; Wang and Tsirka, 2005; Romanic et al., 1998; Rosenberg et al., 1998). MMP9 binds and proteolyzes lipoprotein receptors inducing ectodomain shedding and reducing the ability to transport Aβ out of the brain (Shackleton et al., 2019). Further studies are needed to unravel if and how MY10-induced reduction of Mmp9 levels is involved in the beneficial effects of MY10 treatment in the APP/PS1 mouse model.

In addition, hippocampal Ide mRNA expression was upregulated in male mice treated with MY10, whereas no significant differences were observed in females. IDE cleaves several peptides, such as insulin and Aβ (Qiu et al., 1998). However, its relevance lies in the fact that it is the primary soluble Aβ degrading enzyme at neutral pH in the human brain (Baranello et al., 2015; Dorfman et al., 2010). Accordingly, reduced IDE activity has been linked to increased Aβ accumulation and AD pathology (Tian et al., 2023). It is known that sex hormones modulate Aβ via induction of IDE among others (George et al., 2013). Nevertheless, the gender-specific increase in Ide activity in MY10-treated male mice needs further investigation and underscores the importance of considering biological sex in AD research and treatment development.

While this study provides important insights, certain limitations should be acknowledged. The temporal scope of our observations does not clarify whether the reduction in Aβ deposition reflects a lasting effect or a delay in progression, highlighting the need for further longitudinal studies. In addition, the precise mechanisms underlying these effects of MY10 remain unclear. Our findings are consistent with previous work in different cell lines and in vivo models; However, we did not directly examine the effects of MY10 on RPTPβ/ζ activity in primary neural cell types, which could render a deeper understanding of the mechanisms underlying these effects. Future studies are needed to address these limitations and build upon the current findings.

This study provides for the first time convincing evidence that RPTPβ/ζ inhibition with MY10 significantly reduces Aβ plaque formation, which seems related to the capacity of MY10 to regulate glial responses and the proinflammatory signal characteristic of AD, and to modulate the expression of Aβ aggregate-degrading enzymes. The data support that the PTN/RPTPβ/ζ signalling pathway could be a novel therapeutic target in AD.