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The 5xFAD transgenic mouse model widely used in Alzheimer’s disease (AD) research recapitulates many AD-related phenotypes with a relatively early onset and aggressive age-dependent progression. Besides developing amyloid peptide deposits alongside neuroinflammation by the age of 2 months, as well as exhibiting neuronal decline by the age of 4 months that intensifies by the age of 9 months, these mice manifest a broad spectrum of behavioural impairments. In this review, we present the extensive repertoire of behavioural dysfunctions in 5xFAD mice, organised into four categories: motor skills, sensory function, learning and memory abilities, and neuropsychiatric-like symptoms. The motor problems, associated with agility and reflex movements, as well as balance and coordination, and skeletal muscle function, typically arise by the time mice reach 9 months of age. The sensory function (such as taste, smell, hearing, and vision) starts to deteriorate when amyloid peptide buildups and neuroinflammation spread into related anatomical structures. The cognitive functions, encompassing learning and memory abilities, such as visual recognition, associative, spatial working, reference learning, and memory show signs of decline from 4 to 6 months of age. Concerning neuropsychiatric-like symptoms, comprising apathy, anxiety and depression, and the willingness for exploratory behaviour, it is believed that motivational changes emerge by approximately 6 months of age. Unfortunately, numerous studies from different laboratories are often contradictory on the conclusions drawn and the identification of onset age, making preclinical studies in rodent models not easily translatable to humans. This variability is likely due to a range of factors associated with animals themselves, housing and husbandry conditions, and experimental settings. In the forthcoming studies, greater clarity in experimental details when conducting behavioural testing in 5xFAD transgenic mice could minimise the inconsistencies and could ensure the reliability and the reproducibility of the results.

Alzheimer’s disease (AD), the leading cause of dementia, presents a significant global health challenge with no known cure to date. Central to our understanding of AD pathogenesis is the β-amyloid cascade hypothesis, which underlies drug research and discovery efforts. Despite extensive studies, no animal models of AD have completely validated this hypothesis. Effective AD models are essential for accurately replicating key pathological features of the disease, notably the formation of β-amyloid plaques and neurofibrillary tangles. These pathological markers are primarily driven by mutations in the amyloid precursor protein (APP) and presenilin 1 (PS1) genes in familial AD (FAD) and by tau protein mutations for the tangle pathology. Transgenic mice models have been instrumental in AD research, heavily relying on the overexpression of mutated APP genes to simulate disease conditions. However, these models do not entirely replicate the human condition of AD. This review aims to provide a comprehensive evaluation of the historical and ongoing research efforts in AD, particularly through the use of transgenic mice models. It is focused on the benefits gathered from these transgenic mice models in understanding β-amyloid toxicity and the broader biological underpinnings of AD. Additionally, the review critically assesses the application of these models in the preclinical testing of new therapeutic interventions, highlighting the gap between animal models and human clinical realities. This analysis underscores the need for refinement in AD research methodologies to bridge this gap and enhance the translational value of preclinical studies.

Alzheimer’s disease (AD) is the most common form of dementia, affecting over 50 million people globally. Since 1906, efforts to understand this neurodegenerative disease and to develop effective treatments have continued to this day. Recognizing docosahexaenoic acid (DHA, 22:6n-3) as a safe, inexpensive and vital nutrient for brain health and cognitive protection due to its key role in brain development and function, this study explores novel, sustainable non-fish sources as potential dietary supplements to prevent or mitigate AD, within a blue biotechnology framework. Forty 5×FAD male mice, five weeks old, were allocated to five body weight-matched dietary groups (n = 8) and fed isocaloric diets based on AIN-93M standard chow for 6 months. Each diet, except the control feed (non-supplemented group), enclosed a modified lipid fraction supplemented with 2% of the following: (1) linseed oil (LSO, rich in alpha-linolenic acid (ALA,18:3n-3)); (2) cod liver oil (fish oil, FO, rich in both DHA and eicosapentaenoic acid (EPA, 20:5n-3)); (3) Schizochytrium sp. microalga oil (Schizo) with 40% of DHA; and (4) commercial DHASCO oil (DHASCO) with 70% of DHA. The different diets did not affect (p > 0.05) growth performance criteria (e.g., final body weight, daily feed intake, and body weight gain) suggesting no effect on the overall caloric balance or mice growth, but n-3 long-chain polyunsaturated-fatty acid (n-3 LCPUFA) supplementation significantly reduced total cholesterol (p < 0.001) and total lipids (p < 0.001). No systemic inflammation was detected in 5×FAD mice. In parallel, a beneficial modulation of lipid metabolism by DHA-enriched diets was observed, with polyunsaturated fatty acid incorporation, particularly DHA, across key metabolic tissues, such as the liver (p < 0.001) and the brain (p < 0.001). No behavioural variations were detected using an open-field test after 6 months of diet (p > 0.05). While mice fed a standard diet or LSO diet showed cognitive deficit, the incorporation of FO, Schizo or DHASCO oils into dietary routine showed promising protective effects on the working memory (p < 0.05) and the last two diets also on the recognition memory (p < 0.05) Increased neuronal count (p < 0.05), reflecting neuronal survival, was clearly observed with the fish oil diet. In turn, the number of TAU-positive cells (p < 0.05) was reduced in the Schizo diet, while β-amyloid deposition (p < 0.01) and the neuroinflammatory marker, IBA1 (p < 0.05), were decreased across all DHA-enriched diets. These promising findings open new avenues for further studies focused on the protective effects of DHA derived from sustainable and underexploited Schizochytrium sp. microalga in the prevention of AD.

Funding Information: This work was financially supported by FCT (Fundação para a Ciência e a Tecnologia, Lisboa, Portugal) through the FCT.2022.08133.PTDC project, UIDB/00276/2020 grant to CIISA, the LA/P/0059/2020 grant to AL4AnimalS, and the UIDP/04138/2020 grant to iMed.ULisboa. Publisher Copyright: © 2025 by the authors. Alzheimer’s disease (AD) is the most common form of dementia, affecting over 50 million people globally. Since 1906, efforts to understand this neurodegenerative disease and to develop effective treatments have continued to this day. Recognizing docosahexaenoic acid (DHA, 22:6n-3) as a safe, inexpensive and vital nutrient for brain health and cognitive protection due to its key role in brain development and function, this study explores novel, sustainable non-fish sources as potential dietary supplements to prevent or mitigate AD, within a blue biotechnology framework. Forty 5×FAD male mice, five weeks old, were allocated to five body weight-matched dietary groups (n = 8) and fed isocaloric diets based on AIN-93M standard chow for 6 months. Each diet, except the control feed (non-supplemented group), enclosed a modified lipid fraction supplemented with 2% of the following: (1) linseed oil (LSO, rich in alpha-linolenic acid (ALA,18:3n-3)); (2) cod liver oil (fish oil, FO, rich in both DHA and eicosapentaenoic acid (EPA, 20:5n-3)); (3) Schizochytrium sp. microalga oil (Schizo) with 40% of DHA; and (4) commercial DHASCO oil (DHASCO) with 70% of DHA. The different diets did not affect (p > 0.05) growth performance criteria (e.g., final body weight, daily feed intake, and body weight gain) suggesting no effect on the overall caloric balance or mice growth, but n-3 long-chain polyunsaturated-fatty acid (n-3 LCPUFA) supplementation significantly reduced total cholesterol (p < 0.001) and total lipids (p < 0.001). No systemic inflammation was detected in 5×FAD mice. In parallel, a beneficial modulation of lipid metabolism by DHA-enriched diets was observed, with polyunsaturated fatty acid incorporation, particularly DHA, across key metabolic tissues, such as the liver (p < 0.001) and the brain (p < 0.001). No behavioural variations were detected using an open-field test after 6 months of diet (p > 0.05). While mice fed a standard diet or LSO diet showed cognitive deficit, the incorporation of FO, Schizo or DHASCO oils into dietary routine showed promising protective effects on the working memory (p < 0.05) and the last two diets also on the recognition memory (p < 0.05) Increased neuronal count (p < 0.05), reflecting neuronal survival, was clearly observed with the fish oil diet. In turn, the number of TAU-positive cells (p < 0.05) was reduced in the Schizo diet, while β-amyloid deposition (p < 0.01) and the neuroinflammatory marker, IBA1 (p < 0.05), were decreased across all DHA-enriched diets. These promising findings open new avenues for further studies focused on the protective effects of DHA derived from sustainable and underexploited Schizochytrium sp. microalga in the prevention of AD.

A indústria farmacêutica e a indústria cosmética estão entre as que mais investem em pesquisa e desenvolvimento, o que leva ao constante aparecimento de novos fármacos e cosméticos. Todos estes produtos, antes de serem introduzidos no mercado, necessitam de passar por uma avaliação criteriosa relativamente à sua eficácia e toxicidade, pois podem provocar efeitos nocivos no organismo humano. Anteriormente, estas avaliações eram realizadas recorrendo à experimentação animal, no entanto com o passar dos anos desenvolveu-se uma consciencialização relativamente ao uso de animais para este fim, o que levou a que restrições legais fossem criadas (em diversos países de todo o mundo) e por conseguinte a uma utilização muito limitada desta prática. Em substituição deste método começaram a desenvolver-se métodos alternativos que permitissem a reprodutibilidade, quantidade e predictibilidade das avaliações. No que diz respeito aos produtos com aplicação tópica, não só a sua eficácia e toxicidade têm de ser testadas mas também a sua eficiência de penetração. A pele sendo a interface entre o organismo e o seu ambiente externo, constitui uma barreira de proteção contra todas as interferências exteriores e é dividida fundamentalmente em 3 camadas: a hipoderme, a derme e a epiderme, esta última a principal responsável pela função de barreira apresentada por este órgão. A epiderme é a camada mais exterior da pele e é constituída por queratinócitos os quais sofrem um processo continuo de diferenciação originando um tecido estratificado. Para que o produto aplicado topicamente penetre a pele e tenha o efeito desejado, esta camada tem de ser transposta. Hoje em dia já existem diversos modelos in vitro de pele humana para testar estes produtos, alguns contendo apenas a epiderme, outros apenas a derme e outros ainda contendo ambas as camadas (epiderme e derme). Todos estes modelos se assemelham morfologicamente e bioquimicamente à pele in vivo, no entanto ainda não exibem uma adequada propriedade de barreira, evidenciando maiores permeabilidades que a pele nativa. Esta disparidade parece estar relacionada com a camada apical da epiderme - stratum corneum - que integra células mortas e achatadas envoltas numa matriz lipídica, onde se observam variações tanto no perfil lipídico como na sua estruturação. Dado verificar-se uma maior permeabilidade nos tecidos in vitro do que nos tecidos in vivo, sempre que um produto é submetido a avaliações toxicológicas e de eficiência de penetração, extrapolações têm de ser realizadas para fases clínicas o que torna o processo pouco preciso. Surge assim a necessidade de descobrir quais os parâmetros que influenciam a formação da barreira da pele durante a cultura de equivalentes epidérmicos humanos in vitro, de forma a gerar um modelo que mais se assemelhe com a epiderme humana nativa, tanto em termos de morfologia como de propriedade barreira. Assim, este projeto pretendeu produzir equivalentes epidérmicos in vitro sob quatro condições distintas, resultantes da alteração independente de quatro fatores relativamente às condições standard de cultura, de forma a avaliar o efeito dessas alterações nos tecidos produzidos e nas suas propriedades de barreira.