Homogentisic Acid Disrupts the Blood–Brain Barrier and Promotes Aβ Aggregation in Alzheimer’s Disease

Abstract

Objective

Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with metabolic dysregulation. This study aimed to investigate the role of homogentisic acid (HGA), a tyrosine metabolite, in AD pathogenesis and explore its potential as a noninvasive diagnostic biomarker.

Methods

Human saliva samples from AD patients and controls were analyzed. In vivo experiments were conducted using APP/PS1 (Aβ-driven) and P301S (tauopathy-focused) mouse models, which received exogenous HGA via gavage. Key techniques included behavioral tests (Morris water maze, novel object recognition, fear conditioning), Western blot, immunofluorescence, real-time PCR, and mass spectrometry to assess cognitive function, blood–brain barrier (BBB) integrity, Aβ aggregation, synaptic protein expression, and HGA metabolism. In vitro experiments were performed on HT22, SY5Y cells, and primary brain microvascular endothelial cells (BMECs) to verify HGA’s direct effects.

Results

Salivary HGA levels were higher in AD patients than in controls, correlating with BBB impairment. Exogenous HGA significantly exacerbated cognitive deficits, BBB leakage, Aβ deposition, and loss of synaptic proteins (PSD93, synaptophysin) in mice, with effects more pronounced in the APP/PS1 than in the P301S model. In vitro, HGA exerted dose-dependent neurotoxicity, promoted Aβ aggregation, and downregulated tight junction proteins (claudin-5, occludin, ZO-1) in BMECs. Mechanistically, AD patients showed reduced expression of HGA-metabolizing enzymes (homogentisate 1,2-dioxygenase, maleylacetoacetate isomerase) and downstream metabolites, indicating impaired HGA catabolism. These findings confirm HGA promotes AD progression via two mutually reinforcing pathways: (1) accelerating Aβ aggregation and synaptic dysfunction; (2) disrupting BBB integrity through downregulating tight junction proteins.

Conclusion

This study identifies salivary HGA as a potential noninvasive biomarker and highlights targeting HGA metabolism or BBB protection as promising strategies for early AD intervention.

Keywords

Homogentisic acid / Alzheimer’s disease / Blood–brain barrier / Aβ aggregation / Salivary biomarker / Metabolic dysregulation

References

[1] Alzheimer’s disease facts and figures. Alzheimer’s Dement, 2023, 19(4): 1598-1695

[2] Sortino M, Amato A, Musumeci G. Advanced technologies applied to physical exercise for dementia and Alzheimer’s disease management: a narrative review. Adv Technol Neurosci, 2024, 1(1): 72-85

[3] Dong Y, Song X, Wang Xet al.. The early diagnosis of Alzheimer’s disease: blood-based panel biomarker discovery by proteomics and metabolomics. CNS Neurosci Ther, 2024, 30(11): e70060

[4] Liu Y, Guo Y, Yu J. Blood-based biomarkers of Alzheimer’s disease: standardization and comprehensiveness. Neuroprotection, 2024, 24246-254

[5] Marksteiner J, Humpel C. Glial fibrillary acidic protein as a biomarker for diagnosis of Alzheimer’s disease in cerebrospinal fluid, plasma, and saliva measured with Lumipulse technology: a narrative review. NeuroMarkers, 2025, 2(1): 100038

[6] Lehtonen Š, Jolkkonen J. Novel therapeutic strategies and common mechanisms of neurodegenerative diseases. Neuroprotection, 2024, 2265-66

[7] Yassine HN, Self W, Kerman BEet al.. Nutritional metabolism and cerebral bioenergetics in Alzheimer’s disease and related dementias. Alzheimer’s Dement, 2023, 19(3): 1041-1066

[8] Ahmed H, Wang Y, Griffiths WJet al.. Brain cholesterol and Alzheimer’s disease: challenges and opportunities in probe and drug development. Brain, 2024, 147(5): 1622-1635

[9] Andersen JV, Schousboe A, Verkhratsky A. Astrocyte energy and neurotransmitter metabolism in Alzheimer’s disease: integration of the glutamate/GABA-glutamine cycle. Prog Neurobiol, 2022, 217: 102331

[10] Trillo L, Das D, Hsieh Wet al.. Ascending monoaminergic systems alterations in Alzheimer’s disease. Translating basic science into clinical care. Neurosci Biobehav Rev, 2013, 37(8): 1363-1379

[11] Saggu S, Bai A, Aida Met al.. Monoamine alterations in Alzheimer’s disease and their implications in comorbid neuropsychiatric symptoms. Geroscience, 2025, 47(1): 457-482

[12] González-Domínguez R, García-Barrera T, Gómez-Ariza JL. Metabolite profiling for the identification of altered metabolic pathways in Alzheimer’s disease. J Pharm Biomed Anal, 2015, 107: 75-81

[13] Bloemendaal M, Froböse MI, Wegman J, et al. Neuro-cognitive effects of acute tyrosine administration on reactive and proactive response inhibition in healthy older adults. eNeuro. 2018;5(2):ENEURO.0035–ENEURO.0017.2018.

[14] Motta C, Assogna M, Bonomi CGet al.. Interplay between the catecholaminergic enzymatic axis and neurodegeneration/neuroinflammation processes in the Alzheimer’s disease continuum. Eur J Neurol, 2023, 30(4): 839-848

[15] Mathar D, ErfanianAbdoust M, Marrenbach Tet al.. The catecholamine precursor Tyrosine reduces autonomic arousal and decreases decision thresholds in reinforcement learning and temporal discounting. PLoS Comput Biol, 2022, 18(12): e1010785

[16] Hughes JH, Liu K, Plagge Aet al.. Conditional targeting in mice reveals that hepatic homogentisate 1, 2-dioxygenase activity is essential in reducing circulating homogentisic acid and for effective therapy in the genetic disease alkaptonuria. Hum Mol Genet, 2019, 28233928-3939

[17] Introne WJ, Perry MB, Troendle Jet al.. A 3-year randomized therapeutic trial of nitisinone in alkaptonuria. Mol Genet Metab, 2011, 103(4): 307-314

[18] Mavra M, Chadha G, Henein Met al.. Alcaptonuria with myelopathy and neuropathy. Med Princ Pract, 1999, 8(1): 73-76

[19] Hegedus ZL. The probable involvement of soluble and deposited melanins, their intermediates and the reactive oxygen side-products in human diseases and aging. Toxicology, 2000, 145(2–3): 85-101

[20] Foret MK, Orciani C, Welikovitch LAet al.. Early oxidative stress and DNA damage in Aβ-burdened hippocampal neurons in an Alzheimer’s-like transgenic rat model. Commun Biol, 2024, 7(1): 861

[21] Millucci L, Spreafico A, Tinti Let al.. Alkaptonuria is a novel human secondary amyloidogenic disease. Biochim Biophys Acta, 2012, 1822111682-1691

[22] Braconi D, Millucci L, Bernini Aet al.. Homogentisic acid induces aggregation and fibrillation of amyloidogenic proteins. Biochim Biophys Acta Gen Subj, 2017, 1861(2): 135-146

[23] Braconi D, Millucci L, Bernardini Get al.. Oxidative stress and mechanisms of ochronosis in alkaptonuria. Free Radic Biol Med, 2015, 88: 70-80

[24] Xie Y, Lv X, Ni Det al.. HPD degradation regulated by the TTC36-STK33-PELI1 signaling axis induces tyrosinemia and neurological damage. Nat Commun, 2019, 10(1): 4266

[25] Wang X, Sun G, Feng Tet al.. Sodium oligomannate therapeutically remodels gut microbiota and suppresses gut bacterial amino acids-shaped neuroinflammation to inhibit Alzheimer’s disease progression. Cell Res, 2019, 2910787-803

[26] Langen UH, Ayloo S, Gu C. Development and cell biology of the blood–brain barrier. Annu Rev Cell Dev Biol, 2019, 35: 591-613

[27] Shi J, Xiao Y, Zhang Net al.. HMGB1 from astrocytes promotes EAE by influencing the immune cell infiltration-associated functions of BMECs in mice. Neurosci Bull, 2022, 38(11): 1303-1314

[28] Zhan F, Dong Y, Zhou Let al.. Minocycline alleviates LPS-induced cognitive dysfunction in mice by inhibiting the NLRP3/caspase-1 pathway. Aging, 2024, 16(3): 2989-3006

[29] Peng S, Yang B, Duan MYet al.. The disparity of impairment of neurogenesis and cognition after acute or fractionated radiation exposure in adolescent BALB/c mice. Dose Response, 2019, 17: 1559325818822574

[30] Rehman IU, Park JS, Choe Ket al.. Overview of a novel osmotin abolishes abnormal metabolic-associated adiponectin mechanism in Alzheimer’s disease: peripheral and CNS insights. Ageing Res Rev, 2024, 100: 102447

[31] Al-Sarayreh SA, Al-Tarawneh IN, Al-Sbou MSet al.. Measurements of homogentisic acid levels in alkaptonuria patients using an optimized and validated gas chromatography method/mass spectrometry. Jordan J Biol Sci, 2014, 7(3): 195-198

[32] Wiesehan K, Stöhr J, Nagel-Steger Let al.. Inhibition of cytotoxicity and amyloid fibril formation by a D-amino acid peptide that specifically binds to Alzheimer’s disease amyloid peptide. Protein Eng Des Sel, 2008, 21(4): 241-246

[33] Mirdha L. Aggregation behavior of amyloid beta peptide depends upon the membrane lipid composition. J Membr Biol, 2024, 257(3–4): 151-164

[34] Li Y, Zhao T, Li Jet al.. Oxidative stress and 4-hydroxy-2-nonenal (4-HNE): implications in the pathogenesis and treatment of aging-related diseases. J Immunol Res, 2022, 2022: 2233906

[35] Škovierová H, Vidomanová E, Mahmood Set al.. The molecular and cellular effect of homocysteine metabolism imbalance on human health. Int J Mol Sci, 2016, 17(10): 1733

[36] Wu J, Yin X, Ye Het al.. Structure relationship of metalloporphyrins in inhibiting the aggregation of hIAPP. Int J Biol Macromol, 2021, 167: 141-150

[37] Wang Y, Du W, Hu Xet al.. Targeting the blood–brain barrier to delay aging-accompanied neurological diseases by modulating gut microbiota, circadian rhythms, and their interplays. Acta Pharm Sin B, 2023, 13(12): 4667-4687

[38] Merlini M, Rafalski VA, Rios Coronado PEet al.. Fibrinogen induces microglia-mediated spine elimination and cognitive impairment in an Alzheimer’s disease model. Neuron, 2019, 101(6): 1099-1108.e6

[39] Cortes-Canteli M, Strickland S. Fibrinogen, a possible key player in Alzheimer’s disease. J Thromb Haemost, 2009, 7(Suppl 1): 146-150

[40] Csiha S, Hernyák M, Molnár Áet al.. Alpha-lipoic acid treatment reduces the levels of advanced end glycation products in type 2 diabetes patients with neuropathy. Biomedicines, 2025, 13(2): 438

[41] Mastroeni P, Trezza A, Geminiani Met al.. HGA triggers SAA aggregation and accelerates fibril formation in the C20/A4 alkaptonuria cell model. Cells, 2024, 13171501

[42] Ghemrawi R, Khair M. Endoplasmic reticulum stress and unfolded protein response in neurodegenerative diseases. Int J Mol Sci, 2020, 21176127

[43] Wang S, Li B, Cai Z, et al. Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD. Res Sq. 2024:rs.3.rs–rs.4373201.

[44] Jia D, Wang F, Yu H. Systemic alterations of tricarboxylic acid cycle enzymes in Alzheimer’s disease. Front Neurosci, 2023, 17: 1206688

[45] Zha BS, Zhou H. ER stress and lipid metabolism in adipocytes. Biochem Res Int, 2012, 2012: 312943

[46] Yarbro JM, Han X, Dasgupta Aet al.. Human and mouse proteomics reveals the shared pathways in Alzheimer’s disease and delayed protein turnover in the amyloidome. Nat Commun, 2025, 1611533

[47] Phornphutkul C, Introne WJ, Perry MBet al.. Natural history of alkaptonuria. N Engl J Med, 2002, 347262111-2121

[48] Hughes JH, Wilson PJM, Sutherland Het al.. Dietary restriction of tyrosine and phenylalanine lowers tyrosinemia associated with nitisinone therapy of alkaptonuria. J Inherit Metab Dis, 2020, 43(2): 259-268

[49] Mayrink FD, Dorneles G, da Silva IMet al.. Efficacy and safety of nitisinone for patients with alkaptonuria: a systematic review with metanalysis. Mol Genet Metab, 2025, 1451109099

Funding

Hubei Provincial Natural Science Foundation of China for Distinguished Young Scholars(Grant No. 2022CFA104)

Key Research and Development Program of Wuhan(Grant No. 2024020802030159)

The National Natural Science Foundation of China(Grant No. 82371195)

Research Fund of Jianghan University(Grant  No. 2022XKZX26)

RIGHTS & PERMISSIONS

The Author(s), under exclusive licence to the Huazhong University of Science and Technology