Alzheimer’s disease (AD) is the most common form of dementia and one of the world’s leading causes of death. AD is a multifactorial neurodegenerative disease that involves a combination of the extracellular accumulation of beta-amyloid (Aβ) plaques, intracellular neurofibrillary tangles (NFTs), composed of hyperphosphorylated tau protein, chronic neuroinflammation, and reactive gliosis, ultimately leading to neuronal loss and declining cognition. However, effective treatments are still lacking (Authors, 2022; Karran and De Strooper, 2022). Fortunately, disease-modifying therapy (DMT) for AD is progressing through clinical trials; especially, Aβ-targeted monoclonal antibodies (mAb) (e.g., aducanumab, bapineuzumab, gantenerumab, solanezumab, and lecanemab), working on the amyloid hypothesis that an inability to clear Aβ contributes to the development and progression of AD, developed successfully and conducted in clinical trials (Cummings et al., 2022). These trials enrolled people with early AD, which includes people with mild cognitive impairment (MCI) or mild dementia due to AD. This implies that intervention at an early stage of AD is the key to promoting therapeutic success.

Burgeoning human data demonstrate that dysregulation of the immune system, including the innate and adaptive immune systems, is a cardinal feature of AD. Several studies indicate that dysregulation of inflammatory markers is evident in AD., which leads to an increased risk of the future development of all-cause dementia or AD (Swardfager et al., 2010; Koyama et al., 2013; Lai et al., 2017; Darweesh et al., 2018). Other evidence demonstrated that the peripheral homeostasis of T cells is partially altered in patients with AD, especially CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs). One study indicates that Tregs were more numerous in AD patients. Rosenkranz et al. (2007), while other studies found that the overall proportion of peripheral Tregs was lowered in MCI patients.in comparison to people with mild or severe AD dementia (Saresella et al., 2010; Le Page et al., 2017). Although strategies aimed at the depletion, amplification, or even transfer of Tregs are attracting increased attention as a potential treatment for Alzheimer’s disease, their precise contribution is complex and remains unclear (Yang et al., 2013; Baruch et al., 2015; Wang et al., 2015; Baek et al., 2016; Dansokho et al., 2016; Alves et al., 2017; Le Page et al., 2017).

Glatiramer acetate (GA), a drug approved by FDA for remitting–relapsing multiple sclerosis (RRMS), has been explored for its potential to reverse AD pathology and preserve memory and learning abilities in an AD mouse model (Butovsky et al., 2006, 2007; Bakalash et al., 2011; Koronyo et al., 2015; Rentsendorj et al., 2018; Doustar et al., 2020; Dionisio-Santos et al., 2021). As a mixture of four amino acid-based synthesized polypeptides, the method for GA administration in previous clinical and experimental research was subcutaneous injection due to the fact that most of a dose is slowly and sustainably degraded into smaller fragments in the subcutaneous department. In fact, several studies found that the therapeutic mechanisms of GA subcutaneous administration were attributed to reducing Tregs levels, enhancing recruitment of neuroprotective monocyte-derived macrophages in the brain, and rebalancing the levels of pro- and anti-inflammatory cytokines. While these encouraging therapeutic effects have been shown in early-symptomatic and in late-stage transgenic models of AD (7–20 months old), they have never previously been studied in an earlier stage prior to Aβ deposition. Given that a growing number of studies suggest that treatment intervention early in the process of the disease would provide better clinical benefits, we evaluated the impacts of GA immunomodulation in various stages of AD in APPswe/PSEN1dE9 (APP/PS1) transgenic mice, subcutaneously injected GA for 12 weeks at different time points (2- and 6-month-old), and, respectively, sacrificed the mice at 6 and 10 months of old (Figure 1). The results indicated that early subcutaneous injection of GA in APP/PS1 mice induced therapeutic benefits against AD, including expanding the frequency of peripheral Tregs, reducing the activation of microglia, and regulating the neuroinflammation by increasing the level of anti-inflammatory cytokines, which eventually delayed the impairment of cognitive function imposed by amyloid accumulation.

In this study, we showed that GA therapy may help the amplification of Tregs to reduce microglia recruitment and elevate protective anti-inflammatory cytokines at early stages of AD, thereby altering amyloid pathology and improving cognitive performance.

GA has been used for a commonly prescribed disease-modifying therapy for RRMS for more than two decades. Although the mechanism of action of GA is still not fully understood, recent preclinical research using GA found its potential neuroprotective effects in neurodegenerative disease models, such as AD, Parkinson’s disease (PD), and Huntington’s disease (HD) (Kasindi et al., 2022). Studies examining in symptomatic transgenic mouse models of AD, including 5xFAD, 3xFAD or APP_SWE/PS1_ΔE9, have found that weekly GA vaccination for 8 weeks reversed plaque pathology and prevented cognitive decline (Butovsky et al., 2006; Bakalash et al., 2011; Baruch et al., 2015; Koronyo et al., 2015; Dansokho et al., 2016; Doustar et al., 2020; Dionisio-Santos et al., 2021). Nevertheless, the potential of long-term GA treatment in young mice with no amyloid pathology development has yet to be investigated. Our study found that early GA treatment for 12 weeks in the young APP/PS1 mice without amyloid depositing attenuated amyloid pathology and improved spatial learning and memory in the MWM and Y-maze tests. Interestingly, in contrast to the positive impact of early GA treatment, our results showed that APP/PS1 mice following GA injections in a pre-symptomatic stage of amyloid-deposition did not exhibit any beneficial effect on either cognitive performance or plaque load. One possible reason for this discrepancy could be that the impact of GA in AD depends on multifactor influences, such as varying session duration, drug frequency, or disease states. For instance, some research evaluated the effects of administering GA to transgenic mice with AD on a daily versus weekly basis and found quite different results; weekly treatment showed an advantage, while daily GA injections even had a mildly negative effect on plaque load and cognitive function (Baruch et al., 2015). Additionally, certain studies examining the effects of daily versus biweekly GA administration in Amyotrophic Lateral Sclerosis (ALS) patients found that daily treatment improve Th2 cytokines, whereas twice-weekly treatment was linked to an increase in Th1 cytokines (Mosley et al., 2007). These results highlight the need for further investigation into the mechanism of action of GA at multiple dosages, session durations, and disease states.

It is important to note that growing evidence from genome-wide association studies (GWAS) has shown that a variety of AD risk genes are involved in inflammation signaling that influences the development and course of the disease (Marioni et al., 2018; Jansen et al., 2019), supporting the notion that sustained inflammation caused by abnormally folded Aβ and tau proteins accumulation plays a crucial role in the pathophysiology of Alzheimer’s disease. Considering the Tregs’ pivotal role in maintaining self-tolerance and in limiting excessive immunological reactions harmful to the host (Sakaguchi et al., 2008), emerging strategies attempt to examine the potential of Tregs regulation as a therapy in the progression of animal models of AD. In our study, APP/PS1 mice that received GA injections as young as 2 months old showed a significant amplification in the frequency of Tregs and an increase in the expression of anti-inflammatory cytokines compared to those in APP/PS1 mice treated with PBS. Consistent with our observation, (Dansokho et al., 2016) showed that using the 4–6-week-old AD mouse model (APP/PS1), the amplification of Tregs by peripheral chronic low-dose IL-2 administration restored cognitive functions, whereas the transient depletion of Tregs accelerated the onset of cognitive decline (Dansokho et al., 2016). Baek et al. (2016) showed that the adoptive transfer of Tregs into 4-month-old 3xTg-AD mice improved cognitive function and reduced Aβ burdens, whereas similarly, the depletion of Tregs in 4-week-old 3xTg-AD mice resulted in aggravation of the disease progression (Baek et al., 2016). Furthermore, our studies revealed that GA might have dichotomous effects on Tregs in APP/PS1 mice: the amplification of Tregs in the pre-depositing stage and the depletion of Tregs in the amyloid-depositing stage. Baruch et al. (2015) also found the similar result that weekly GA treatment in 6-month-old 5XFAD mice reduced splenocyte Tregs levels. However, differing from our finding that no effective impact of Tregs’ depletion on alleviating the development of AD was observed in amyloid-depositing APP/PS1 mice, they suggested that the depletion of Tregs induced the activation of the brain’s choroid plexus, followed by amyloid-β plaque clearance, mitigation of the neuroinflammatory response, as well as a reversal of cognitive deterioration (Baruch et al., 2015). Apparently, Tregs play an important role in the processing of AD, yet their specific contribution is complex and uncertain so far. We considered that Tregs might have had a beneficial role in the early stages of AD, while they become dysfunctional with the progression of the disease. This hypothesis is consistent with the previous studies that have demonstrated that anti-inflammatory and immunosuppressive Treg function was impaired in AD patients (Ciccocioppo et al., 2019; Faridar et al., 2020) and is supported by the finding that ex vivo expanded Tregs restored their immunomodulatory function and alleviated AD pathology in a preclinical model of AD (Faridar et al., 2022). Furthermore, a recent study of immune cells revealed that Tregs reside in the brain (Korin et al., 2017, 2018). Under neuroinflammatory circumstances, the central and peripheral immune responses result in Treg infiltration into the brain (Schetters et al., 2017; Machhi et al., 2020), where they exert a direct anti-inflammatory effect (Xie et al., 2015; Machhi et al., 2020). But as the disease progresses, the function of the meningeal lymphatics is impaired, resulting in the blockage of their infiltration (Da Mesquita et al., 2018). Perhaps it is the double hit of dysfunction of Tregs and meningeal lymphatics that leads to the complicating results of the study in the late period of AD. Of note, as more recent studies demonstrated that Aβ antigen-specific Tregs were more effective at alleviating neuroinflammation and recovering cognitive impairment in a model mouse of AD in contrast to polyclonal Tregs (Yang et al., 2022; Jung et al., 2023), the roles that different Treg subtypes play should not be ignored. Altogether, these results point to the involvement of Tregs in the pathophysiology of AD, but their actual contribution and phenotype in different disease stages need to be further studied.

Previous research has found that Aβ plaques and activated microglia colocalize in the brains of AD patients, suggesting an interaction between these two key pathogenic features of AD (McGeer et al., 1987; Tooyama et al., 1990). Likewise, our study found that GA-treated ahead of amyloid deposition not only significantly reduced microglia activation but also alleviated Aβ burden in APP/PS1 mice. However, the arguments about whether the disease is brought on by microglia surrounding the plaques or whether AD pathology triggers a subsequent reaction in the microglia are still ongoing. In a recent study, in order to investigate the role of microglia in the early phases of AD plaque formation, the researchers designed and synthesized a unique brain-penetrant CSF1R inhibitor (PLX5622) allowing for the sustained and specific elimination of microglia prior to and during the period of plaque formation in the 5xFAD mice. They found that, except in areas where microglia are still present, plaques do not develop in the parenchymal of the brain after microglial removal, suggesting that microglia play key roles in triggering plaque pathogenesis (Spangenberg et al., 2019). Intriguingly, microglia might not only be involved in the launch of Aβ formation but may also contribute to the seeding and spreading of Aβ pathology. For instance, microglia might excrete ASC specks, an adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), after the NACHT-,LRR- and pyrin (PYD)-domain-containing protein 3 (NLRP3) inflammasome activation induced by Aβ, causing them to bind rapidly to Aβ and promote the production of Aβ oligomers and aggregates (Venegas et al., 2017). Alternatively, excessive accumulation of Aβ within microglial lysosomes might cause cellular death, which could lead to the release of Aβ aggregates at the location of microglial death, resulting to Aβ expansion (Baik et al., 2016). Considering the above, the effectiveness of early GA intervention in AD may be attributed to preventing the formation of Aβ plaque by reducing microglia activation. Notably, transcriptomic studies in AD mouse models identified that a special microglial phenotype, termed disease associated microglia (DAM), which is close to Aβ plaques, participate in Aβ clearance, suggesting their protective role in AD (Keren-Shaul et al., 2017). Regrettably, considering the limitation of our study is the lack of identification of microglia subsets, the mechanism linking different microglia subsets to GA and AD pathology in the transgenic mouse model is required for further study.

This work substantiated that weekly GA-administration for 12 weeks during the early stage of AD prior to amyloid deposition improved cognitive performance and alleviated Aβ pathology in APP/PS1 mice through its effect on the amplification of peripheral Tregs, reduction of microglia activation, and increased the level of anti-inflammatory cytokines. But current results lacked proof of mutual causality about the relationship between Aβ deposition and central and peripheral immune or inflammatory changes. We hope further investigation will find a hypothesized mechanism, especially their actual contribution and phenotype in different disease stages. Taken together, GA administration in the early stages of AD may be a highly valuable therapeutic strategy for halting or delaying AD disease development.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was approved by the Institutional Animal Care and Use Committee of Guangdong General Hospital. The study was conducted in accordance with the local legislation and institutional requirements.

ZH: Formal analysis, Investigation, Methodology, Visualization, Writing – original draft. ZG: Formal analysis, Investigation, Writing – original draft. YL: Investigation, Writing – original draft. FY: Investigation, Writing – original draft. WC: Formal analysis, Writing – review & editing. ShaX: Formal analysis, Writing – original draft. YH: Methodology, Writing – review & editing. HX: Methodology, Writing – review & editing. ShuX: Supervision, Writing – review & editing. JD: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing.