Increasing evidence suggests that intestinal dysfunctions may represent early events in Alzheimer’s disease and contribute to brain pathology. This study examined the relationship between onset of cognitive impairment and colonic dysfunctions in a spontaneous AD model before the full development of brain pathology. SAMP8 mice underwent Morris water maze and assessment of faecal output at four, six and eight months of age. In vitro colonic motility was examined. Faecal and colonic Aβ, tau proteins, α-synuclein and IL-1β were assessed by ELISA. Colonic citrate synthase activity was assessed by spectrophotometry. Colonic NLRP3, caspase-1 and ASC expression were evaluated by Western blotting. Colonic eosinophil density and claudin-1 expression were evaluated by immunohistochemistry. The effect of Aβ on NLRP3 signalling and mitochondrial function was tested in cultured cells. Cognitive impairment and decreased faecal output occurred in SAMP8 mice from six months. When compared with SAMR1, SAMP8 animals displayed: (1) impaired in vitro colonic contractions; (2) increased enteric AD-related proteins, IL-1β, active-caspase-1 expression and eosinophil density; and (3) decreased citrate synthase activity and claudin-1 expression. In THP-1 cells, Aβ promoted IL-1β release, which was abrogated upon incubation with caspase-1 inhibitor or in ASC^-/- cells. Aβ decreased mitochondrial function in THP-1 cells. In SAMP8, enteric AD-related proteins deposition, inflammation and impaired colonic excitatory neurotransmission, occurring before the full brain pathology development, could contribute to bowel dysmotility and represent prodromal events in AD.

Alzheimer’s Disease (AD) is the most common Neurodegenerative Disease (ND), primarily characterised by neuroinflammation, neuronal plaques of β-amyloid (Aβ), and neurofibrillary tangles of hyperphosphorylated tau. α-Synuclein (α-syn) and its heteroaggregates with Aβ and tau have been recently included among the neuropathological elements of NDs. These pathological traits are not restricted to the brain, but they reach peripheral fluids as well. In this sense, Red Blood Cells (RBCs) are emerging as a good model to investigate the biochemical alterations of aging and NDs. Herein, the levels of homo- and heteroaggregates of ND-related proteins were analysed at different stages of disease progression. In particular, a validated animal model of AD, the SAMP8 (Senescence-Accelerated Mouse-Prone) and its control strain SAMR1 (Senescence-Accelerated Mouse-Resistant) were used in parallel experiments. The levels of the aforementioned proteins and of the inflammatory marker interleukin-1β (IL-1β) were examined in both brain and RBCs of SAMP8 and SAMR1 at 6 and 8 months. Brain Aβ, tau, and phospho-tau (p-tau) were higher in SAMP8 mice than in control mice and increased with AD progression. Similar accumulation kinetics were found in RBCs, even if slower. By contrast, α-syn and its heterocomplexes (α-syn-Aβ and α-syn-tau) displayed different accumulation kinetics between brain tissue and RBCs. Both brain and peripheral IL-1β levels were higher in SAMP8 mice, but increased sooner in RBCs, suggesting that inflammation might initiate at a peripheral level before affecting the brain. In conclusion, these results confirm RBCs as a valuable model for monitoring neurodegeneration, suggesting peripheral Aβ, tau, and p-tau as potential early biomarkers of AD.

The allele epsilon 4 (ε4) of apolipoprotein E (ApoE) is the strongest genetic risk factor for Alzheimer’s disease (AD). ApoE protein plays a pivotal role in the synthesis and metabolism of amyloid beta (Aβ), the major component of the extracellular plaques that constitute AD pathological hallmarks. Regular exercise is an important preventive/therapeutic tool in aging and AD. Nevertheless, the impact of physical exercise on the well-being of erythrocytes, a good model of oxidative stress and neurodegenerative processes, remains to be investigated, particularly depending on ApoE polymorphism. Herein, we evaluate the oxidative status, Aβ levels, and the membrane’s composition of erythrocytes in a cohort of human subjects. In our hands, the plasma antioxidant capability (AOC), erythrocytes membrane fluidity, and the amount of phosphatidylcholine (PC) were demonstrated to be significantly decreased in the ApoE ε4 genotype and non-active subjects. In contrast, erythrocyte Aβ content and lipid peroxidation increased in ε4 carriers. Regular physical exercise was associated with an increased plasma AOC and membrane fluidity, as well as to a reduced amount of erythrocytes Aβ. Altogether, these data highlight the influence of the ApoE genotype on erythrocytes’ well-being and confirm the positive impact of regular physical exercise.

Aim: Extracellular vesicles (EVs) have emerged as promising vehicles for the delivery of small non-coding RNAs (sncRNAs); however, their clinical translation is hindered by the lack of standardized manufacturing methods, RNA loading protocols, and dosing strategies in both preclinical and clinical settings. This review aims to analyze the current landscape of EV-based RNA therapeutics to identify key trends and discrepancies, providing insight for the clinical development of future sncRNA-loaded EVs. Methods: PubMed and Google Scholar were used to identify 74 published articles using cell-derived EVs loaded with sncRNA. EV source, EV surface modifications, type of loaded RNA, loading methods, and dosages used in preclinical studies were quantitatively analyzed to identify trends and discrepancies. Results: Most studies utilize naïve EVs derived from stem or immortalized cells, with electroporation and donor cell transfection being the predominant RNA loading strategies. EV loading and dosage schemes in preclinical studies are mainly based on protein content, while only a minority of studies use particle number. More generally, the variability in measurement units reflects the absence of standardized guidelines for both RNA loading and treatment dosing, generating variability and challenges in comparing results across studies. Conclusion: Reliable dosing strategies are extremely important for determining the therapeutic potential of EVs in preclinical settings and ensuring clinical translatability. However, a standardized framework for EVs as robust platforms for RNA delivery remains to be established. We underscore the critical need for universal quantification methods, standardized measurement units, and reproducible protocols for EV production and application.