Alzheimer's disease (AD) accounts for up to 80% of dementia cases with 44 million people currently affected worldwide (GBD 2019 Dementia Forecasting Collaborators, 2022). AD is a neurodegenerative disease associated with memory deficits, cognitive decline and executive dysfunctions in late stages. Genetic studies revealed that familial AD forms are caused by missense mutations in the genes coding for amyloid precursor protein (APP) and presenilin 1 and 2 (Weggen and Beher, 2012). The two neuropathological hallmarks of AD are extracellular amyloid plaques formed by aggregated β-amyloid peptides (Aβ) and intracellular neurofibrillary tangles formed primarily by hyperphosphorylated tau filaments. Oligomeric Aβ is a marked driver of neurotoxicity (Broersen et al., 2010; Tu et al., 2014; Viola and Klein, 2015). The accumulation of misfolded proteins in the central nervous system (CNS) is paralleled by the activation of microglia, the resident immune cells of the CNS (Krstic et al., 2012). Their function and phenotypes are progressively altered throughout the disease. The implication of the immune system in the pathogenesis of AD is further corroborated by the association of the disease with mutations in immune-related genes such as TREM2 and CD33, and variants of TREM2 correlating with elevated risk of developing AD (Ulland et al., 2017).

The accumulation of Aβ aggregates and tau proteins in the brains of AD patients suggests a deficit in the general protein degradation machineries, the ubiquitin proteasome system (UPS) and autophagy. Whereas the UPS is central to intracellular protein quality control, autophagy is responsible for the removal of large aggregates or damaged organelles by their isolation in double membrane vesicles (autophagosomes), their fusion to lysosomes and the subsequent degradation by lysosomal enzymes. Both cellular protein degradation systems are necessary for the regulation of metabolic activity and the maintenance of homeostasis to ensure cell survival and growth.

The mammalian target of rapamycin (mTOR) controls autophagy by nutrient-consuming anabolic processes and is an inhibitor of autophagy initiation (Zhang et al., 2014). It has been implicated in the metabolism of Aβ and tau with its signalling increased by Aβ (Li et al., 2017). A role of mTOR in inflammation has also been reported (Srivastava et al., 2016; Thomson et al., 2009). Importantly, mTOR activation is necessary for microglia viability and proliferation in response to bacterial lipopolysaccharide or proinflammatory cytokines (Dello Russo et al., 2009). mTOR is composed of two functionally different complexes: mTOR complex 1 (mTORC1) that is selectively inhibited by rapamycin, and mTOR complex 2 (mTORC2) that is insensitive to short-term rapamycin treatment (Sarbassov et al., 2006).

An accumulation of aberrant ubiquitinated proteins has also been observed in the brains of AD patients, suggesting the disruption of the UPS. The UPS is responsible for the clearance of abnormal proteins, protein quality control, but also for the control of many signalling molecules (Zhao and Goldberg, 2016). The proteasome, the degradation machinery of the UPS, harbors a barrel-shaped 20S core composed of two outer α-rings and two inner β-rings with 7 subunits each with 19S regulatory complexes attached. In response to (neuro)inflammation, an alternative proteasome isoform, the immunoproteasome, is activated, via incorporation of the three immuno-subunits β1i/LMP2/PSMB9, β2i/MECL-1/PSMB10, and β5i/LMP7/PSMB8 to improve the proteolytic capacity (Krüger and Kloetzel, 2012; Orre et al., 2013). Immunoproteasomes were also shown to control activation of innate immune signalling and microglial function (Çetin et al., 2022). In AD, induction of proteasome isoforms has been reported in human patients and in the APP/PS1 transgenic mouse model of AD (Mishto et al., 2006; Orre et al., 2013; Wagner et al., 2017). Notably, mTOR inhibition not only increases the activity of autophagy, but also increases UPS activity and UPS-mediated protein degradation (Zhao et al., 2015).

Rapamycin was discovered in 1972 and is clinically used as an immunosuppressant to prevent organ transplant rejection (Guertin and Sabatini, 2009). Rapamycin can cross the blood-brain barrier and increases autophagy by inhibition of mTORC1 phosphorylation through specific binding of the protein FKBP12 (FK 506-binding protein of 12 kDa). As a result, protein synthesis and cell growth are decreased (Stanfel et al., 2009). Inhibition of mTOR has been correlated with a significant increase of lifespan (Harrison et al., 2009; Vellai et al., 2003), and numerous studies confirmed not only a life-prolonging effect of rapamycin in wild-type mice but also a role in delaying ageing phenotypes (Richardson et al., 2015). Moreover, rapamycin reduces inflammation in brain injury models (Erlich et al., 2007), suggesting neuroprotective effects of mTOR inhibition. Currently two small phase 2 clinical trials are underway/have been completed to assess the safety (REACH) and efficacy (ERAP) of rapamycin in AD (Svensson et al., 2024; Svensson et al., 2025; Kaeberlein and Galvan, 2019). However, the benefit of rapamycin treatment in AD patients versus potential side effects are still debated. Along these lines, a recent paper demonstrated that rapamycin treatment downregulated Trem2 expression in microglia, reduced Aβ clearance, and exacerbated Aβ plaque load (Shi et al., 2022).

In this study, we wanted to test the effects of long-term treatment with rapamycin in 5xFAD mice, a transgenic mouse model of amyloid pathology that develops Aβ plaques and gliosis from 2 months of age and impaired spatial-working memory from the age of 4–5 months (Oakley et al., 2006).

In our study, we first validated the effect of Aβ deposition on mTOR signalling in organotypic brain slice cultures (OBSCs) from wild-type mice, and then assessed the effects of long-term rapamycin treatment on microglia and peripheral immune cells in 5xFAD mice. We also determined Aβ load and immunoproteasome content and activity in the brains of 5xFAD mice. Finally, we assessed spontaneous exploratory activity in the open field and place recognition after 30 min in the Y-maze in rapamycin-treated 5xFAD mice at 5 months of age.