Introduction
Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases and the leading cause of late-onset dementia. Patients with AD typically exhibit progressive deficits in learning and memory. They are also affected by a range of neuropsychiatric disorders, such as anxiety and depression [1]. As of 2023, China's aging population poses a pressing healthcare challenge, with 21.1% of its citizens aged 60 or older and 15.4% exceeding 65 years. The newly released China Alzheimer's Disease Report 2024 reveals a staggering 16.99 million diagnosed cases of AD and related dementias nationwide. This neurodegenerative condition not only severely impairs patients' quality of life but also imposes multidimensional burdens. The progressive nature of AD necessitates long-term care, thereby placing a substantial burden on both family support systems and institutional care facilities [2]. AD is characterized by a constellation of neuropathological hallmarks, including extracellular Aβ plaque deposition, intraneuronal neurofibrillary tangles formed by aberrant tau hyperphosphorylation, and chronic microglial activation with associated neuroinflammatory responses. These interconnected mechanisms drive progressive neurite atrophy, synaptic loss, and neuronal death, ultimately culminating in cognitive decline [[3], [4], [5]]. Aβ accumulation in the cerebral cortex and hippocampus initiates a cascade of glial hyperactivation, in which microglia and astrocytes become chronically activated, creating a self-perpetuating cycle of inflammatory cytokine release and neuronal damage [6]. While the Aβ cascade hypothesis has dominated AD pathogenesis models for decades, emerging evidence highlights the dual role of neuroinflammation: initially serving as a protective response to amyloid toxicity, but ultimately evolving into a maladaptive process that exacerbates neurodegeneration. This paradigm shift underscores the necessity for therapeutic strategies targeting both amyloidogenic and neuroinflammatory pathways [7].
Tβ4, a 43-amino-acid peptide (molecular weight [MW] 4963 Da) originally purified from bovine thymus, represents the most abundant member of the β-thymosin family. It is widely expressed across mammalian tissues, with particularly high levels in the nervous system [[8], [9], [10]]. This evolutionarily conserved molecule exhibits multifunctional bioactivities, ranging from anti-inflammatory effects to the augmentation of cellular motility. Emerging evidence further underscores its neuroprotective roles in modulating synaptic plasticity, promoting axonal growth, and facilitating dendritic remodeling [[11], [12], [13]]. Notably, Tβ4 has demonstrated an excellent safety profile in both animal and human studies, with no indications of dose- dependent toxicity or serious adverse events [14,15]. These findings suggest that Tβ4 holds considerable promise as a potential therapeutic candidate for AD. Tβ4 encompasses several key functional domains, among which the N-terminal tetrapeptide Ac-SDKP and the actin-binding motif TB500 (LKKTETQ) are of particular interest. The N-terminal region also includes a longer fragment (ac-SDKPDMAEIEKFDKS, residues 1–15) associated with cell survival and anti-apoptotic activity. Ac-SDKP is well established for its anti-inflammatory and anti-fibrotic properties. In parallel, TB500, a synthetic peptide derived from Tβ4, has been shown to promote angiogenesis, wound healing, and cell migration. It facilitates muscle regeneration and enhances strength by modulating muscle cell proliferation and differentiation [16], and further contributes to tissue repair and endothelial protection in vascular injury models [17,18]. Similarly, Ac-SDKP, a 487.5 Da fragment of Tβ4, exhibits broad anti-inflammatory, pro-angiogenic, and cardioprotective effects across multiple pathological settings [19]. It also regulates cellular processes such as proliferation and differentiation, as evidenced by its ability to inhibit HL-60 cell growth while inducing their differentiation and apoptosis [20]. Given that TB500 and Ac-SDKP constitute the principal active motifs of Tβ4 and that the full-length Tβ4 protein exhibits limited blood-brain barrier (BBB) permeability owing to its high molecular weight, we focused our investigations on these smaller bioactive fragments.
In this study, the 5 × FAD murine model was utilized for systematic in vivo assessments, complemented by in vitro experiments using an established Aβ-induced AD cell model to investigate the neuroprotective mechanisms of Ac-SDKP and TB500. Through a comprehensive analysis of glial activation patterns, neuronal apoptosis dynamics, serum inflammatory cytokine profiles, and associated gene expression signatures, we identified the key molecular pathways that underlie the therapeutic effects of these compounds. These findings not only expand the existing evidence based on Ac-SDKP and TB500 in AD research but also provide a solid theoretical foundation for future translational studies.
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Section snippets
Neuronal culture, cell viability assay, and neurite outgrowth assessment
HT22 cell were maintained in high-glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco, 11995065) supplemented with 10% fetal bovine serum (FBS, ZETA, Z7186FBS-500) and 1% penicillin-streptomycin (Gibco, 15140122) under 5% CO2 at 37 °C. Cells were seeded at 8 × 103 cells/cm2 in 96-well or 4 × 105 cells/cm2 in 6-well plates for 24 h, following differentiated with N-2 (Gibco,17,502–408) supplemented medium for 24 h. Oligomeric Aβ25–35 (10 μM, HY-P0128, MCE; pre-aggregated in sterile ddH2O at
TB500 and Ac-SDKP protected against Aβ25–35-induced neurite atrophy and neuronal death
To investigate the direct neuroprotective effects of Tβ4-derived TB500 and Ac-SDKP on Aβ-induced neuronal damage, we utilized differentiated HT22 cells to establish an in vitro AD model (Fig. 1A). As expected, treatment of HT22 cells with various doses of Aβ resulted in a dose-dependent reduction in cell viability (Supplementary Fig. 1). Specifically, compared to the control group, cell viability decreased to 86.7% after 48 h treatment with 10 μM Aβ25–35. In contrast, TB500 treatment
Discussion
AD pathogenesis is multifaceted, involving neuroinflammation, Aβ deposition, tau pathology, and genetic factors such as apolipoprotein E4 (APOE4), yet current therapeutic strategies remain inadequate due to the complexity of these intertwined mechanisms [[29], [30], [31]]. Tβ4 exhibits extensive expression throughout the central nervous system. Experimental evidence demonstrates its therapeutic potential in facilitating neural remodeling post injury, which correlates with significant
Conclusion
This study highlights TB500 and Ac-SDKP as multi-modal therapeutics capable of counteracting AD-related cognitive decline through neuroprotection, anti-neuroinflammation, and neurite regeneration. Their efficacy in improving memory despite persistent Aβ plaques challenges the Aβ-centric therapeutic paradigm, emphasizing the need for complementary strategies targeting neuronal resilience. Further investigations into blood-brain barrier penetration, direct neuronal targets, and sex-dependent
CRediT authorship contribution statement
Haiyan Ou: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Ruiye Chen: Writing – review & editing, Methodology, Investigation, Formal analysis. Longjian Zhou: Writing – review & editing, Data curation. Yi Zhang: Writing – review & editing, Data curation. Shuai Zhao: Writing – review & editing, Funding acquisition, Data curation. Zhiyou Yang: Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization.
Declaration of generative AI and AI-assisted technologies in the writing process
During the preparation of this work the authors didn't use AI and AI-assisted technologies.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This research was funded by Guangdong University Featured Innovation Program Project (2024KTSCX199), the Sustainable Development Program of Shenzhen Science and Technology Major Program (KCXFZ20240903093925033), Special Funds for Science and Technology Development of Zhanjiang City (2021E05027), the Key Project in Higher Education of Guangdong, China (2022ZDZX2029), and the program for scientific research start-up funds of Guangdong Ocean University (R19039 and R19035). The graphical abstract
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