Alzheimer's disease remains one of the most formidable neurodegenerative challenges globally, placing an immense burden on healthcare systems and caregivers. Pathologically, the condition involves amyloid accumulation, neurofibrillary tangle deposition, neuroinflammation, and extensive oxidative injury. Consequently, therapeutic strategies that restore proteostasis and cellular clearance have garnered immense scientific interest. In particular, investigating the utility of trehalose in Alzheimer's disease has gained momentum because this natural non-reducing disaccharide promotes cellular autophagy and stabilizes native protein conformations. Preclinical investigations have consistently shown that trehalose reduces lipid peroxidation, suppresses pro-inflammatory cascades, and supports neuronal viability. However, human clinical validation and objective molecular pharmacodynamic biomarkers have historically remained scarce. A phase two clinical trial has now evaluated intravenous trehalose administration in patients with Alzheimer's disease, tracking systemic epigenetic changes via circulating microRNAs.

Clinical Trial Protocol and Patient Randomization

To evaluate biological responses systematically, investigators launched a double-blind, randomized, placebo-controlled phase two study. The trial recruited twenty patients diagnosed with probable Alzheimer's disease based on standardized cognitive criteria. Researchers randomly allocated participants into two equal cohorts of ten individuals each. The intervention arm received intravenous trehalose infusions at a dosage of 15 grams per week over a total duration of 12 weeks. Meanwhile, the control group received matching weekly infusions of sterile normal saline solution. Clinicians collected peripheral venous blood samples at baseline before the initial infusion and immediately following the completion of the 12-week regimen. Importantly, the researchers sought to identify circulating epigenetic alterations rather than relying solely on subjective cognitive scores. By maintaining rigorous blinding and standardized administration protocols, the trial minimized confounding variables and ensured reliable comparative data across both cohorts.

Direct Hybridization Profiling of Circulating MicroRNAs

Evaluating circulating microRNAs requires highly sensitive analytical tools because these small non-coding RNA molecules circulate in minute quantities. Therefore, the investigative team utilized a direct hybridization approach rather than conventional amplification-dependent assays. This methodology quantified expression levels without the enzymatic biases frequently introduced during reverse transcription or polymerase chain amplification. Microarray profiling successfully tracked differential expression across thousands of microRNA transcripts in serum samples taken before and after the 12-week intervention. By comparing the trehalose-treated cohort directly against the saline placebo arm, the analysts isolated transcript alterations that occurred specifically due to trehalose exposure. Ultimately, this rigorous molecular profiling confirmed that parenteral disaccharide therapy provokes measurable, reproducible changes in circulating regulatory transcripts, opening new avenues for monitoring neurodegenerative drug responses.

Differentially Expressed MicroRNAs Identified Under Trehalose

The comparative analysis revealed significant expression changes in several distinct microRNAs exclusively within the trehalose intervention group. Most notably, four circulating microRNAs underwent substantial downregulation: hsa-miR-1268a, hsa-miR-3605-3p, hsa-miR-555, and hsa-miR-6511a-3p. In contrast, two distinct microRNAs showed marked upregulation following the 12-week treatment course, specifically hsa-miR-324-3p and hsa-miR-539-5p. Furthermore, previous clinical literature has implicated several of these altered sequences in oncogenic, inflammatory, or neurodegenerative pathways. Because microRNAs act as post-transcriptional regulators, their selective repression or elevation alters downstream translational networks. Thus, identifying this distinct expression signature demonstrates that trehalose influences peripheral epigenetic regulation in a consistent and quantifiable manner, providing a foundational baseline for future validation studies.

Bioinformatics Insights into Downstream Cellular Mechanisms

To decipher the biological consequences of these shifts, investigators performed extensive bioinformatics target prediction and pathway enrichment analyses. The algorithms identified 147 validated overlapping target genes regulated by the differentially expressed microRNAs. Subsequently, Kyoto Encyclopedia of Genes and Genomes pathway enrichment demonstrated that these targets heavily cluster within critical neurobiological domains. These pathways include actin cytoskeleton regulation, axon guidance networks, and neurotrophin signaling cascades. Furthermore, gene ontology mapping revealed that several targeted genes regulate macroautophagy, protein aggregate clearance, and reactive oxygen species defense. Because neurodegenerative pathology involves impaired lysosomal clearance and synaptic deterioration, the modulation of these gene networks suggests biologically meaningful activity. Therefore, trehalose appears to influence pathways central to neuronal structural preservation and resilience against toxic proteopathic insults.

Therapeutic Relevance of Trehalose in Alzheimer's Disease

The findings from this trial carry important translational implications for the future development of trehalose in Alzheimer's disease. Clinicians have long sought minimally invasive peripheral biomarkers to monitor drug engagement and molecular efficacy in dementia trials. Because lumbar punctures and advanced positron emission tomography scans remain costly and invasive, circulating microRNAs offer an appealing liquid biopsy alternative. Tracking these specific regulatory RNAs could allow researchers to verify target engagement during early-phase studies. Additionally, the study confirms that systemic disaccharide administration produces measurable molecular effects in clinical subjects without severe adverse reactions. However, future larger clinical trials must establish whether these transcriptomic modulations translate directly into delayed cognitive decline, structural brain preservation, or reduced clinical dementia rating progression.

Frequently Asked Questions

What was the primary objective of this clinical trial?

The primary objective was evaluating alterations in circulating microRNA profiles before and after 12 weeks of intravenous trehalose therapy in patients with Alzheimer's disease. Researchers aimed to determine whether this treatment induces specific epigenetic changes that could serve as minimally invasive pharmacodynamic biomarkers.

Which microRNAs showed significant modulation following trehalose treatment?

The study demonstrated significant downregulation of hsa-miR-1268a, hsa-miR-3605-3p, hsa-miR-555, and hsa-miR-6511a-3p. Conversely, two microRNAs, hsa-miR-324-3p and hsa-miR-539-5p, showed significant upregulation after 12 weeks of trehalose infusions compared with placebo.

Which cellular pathways were enriched by the identified gene targets?

Bioinformatics analyses revealed that target genes significantly participate in macroautophagy, neuroinflammation modulation, and oxidative stress defense. Pathway enrichment highlighted prominent involvement in actin cytoskeleton dynamics, axon guidance, and neurotrophin signaling, which are critical pathways for neuronal integrity.

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References

Radbakhsh S et al. Direct hybridization and bioinformatics analysis of circulating microRNAs in patients with Alzheimer's disease under intravenous trehalose treatment. Brain Res. 2025 Jun 15. doi: 10.1016/j.brainres.2025.149607. PMID: 40187517.

Pupyshev AB et al. Therapeutic potential of trehalose in neurodegenerative diseases: the knowns and unknowns. Neural Regen Res. 2022;17(4):729-738. doi: 10.4103/1673-5374.322444.

Manna I et al. Insights into the Role of microRNAs as Clinical Tools for Diagnosis, Prognosis, and as Therapeutic Targets in Alzheimer's Disease. Int J Mol Sci. 2024;25(18):9936. doi: 10.3390/ijms25189936.