Komagataella pastoris KM71H attenuates cognitive deficits and depressive-like behavior by the TLR4/NF-κB signaling pathway in an Aβ 1-40 -induced AD-like mouse model

Komagataella pastoris KM71H attenuates cognitive deficits and depressive-like behavior by the TLR4/NF-κB signaling pathway in an Aβ 1-40 -induced AD-like mouse model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Komagataella pastoris KM71H attenuates cognitive deficits and depressive-like behavior by the TLR4/NF-κB signaling pathway in an Aβ 1-40 -induced AD-like mouse model Paloma T. Birmann, Giuliana Petiz Zugno, Airton Sinott, Rafael R. Rodrigues, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6431395/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Aug, 2025 Read the published version in Molecular Neurobiology → Version 1 posted 13 You are reading this latest preprint version Abstract Komagataella pastoris is a promising probiotic for modulating the microbiota-gut-brain axis, and growing evidence has demonstrated the relevance of this axis in the pathophysiology of neurological and psychiatric disorders, such as Alzheimer disease (AD) and major depressive disorder. This study investigated the ameliorated cognitive impairment and antidepressive-like effect of K. pastoris treatment on an AD-like mouse model induced by amyloid β 1−40 peptide (Aβ 1−40 ). Behavioral tests revealed that Aβ 1−40 administration (400 pmol/mouse, icv) increased immobility time in the tail suspension test, decreased spatial and working memory, and increased the time animals took to reach the chocolate cookies, and these behavioral changes were attenuated by the K. pastoris KM71H treatment (8 log CFU − 1 /mouse, ig). Biochemical changes also occurred as the applied protocol decreased reactive species and lipid peroxidation in the hippocampi and small intestine and reduced NRLP3, TLR4, Nfҡb, and IL-1β expression in the small intestine of mice. We believe that the ameliorated cognitive impairment and antidepressant effects of K. pastoris KM71H are due to its antioxidant activity and the modulation of the TLR4/NF-κB pathway, and our findings led us to conclude that K. pastoris KM71H is a promising therapeutic strategy for treating Alzheimer disease and major depressive disorder. Yeast probiotic depression cognitive microbiota-gut-brain axis Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Probiotics are live microorganisms that provide health benefits when ingested in adequate amounts [ 1 ]. However, preclinical and clinical research has focused primarily on bacterial strains (e.g., Lactobacillus ), and only a handful of studies have covered yeast [ 2 ]. This scenario is changing as more studies have sought to investigate the probiotic activities of fungi. One positive aspect of their use is that they are not affected by antibacterial agents and can be combined to prevent intestinal dysbiosis [ 3 ]. Yeasts are well-known unicellular eukaryotic fungi utilized in industrial settings to produce fermented and biotechnological food products [ 4 ]. Komagataella pastoris stands out among such products as this methylotrophic yeast is widely employed as a protein expression system [ 5 ], although little has been known about its probiotic effects until recently. Evidence has shown that K. pastoris has probiotic properties with remarkable antibacterial activity against Salmonella Typhimurium [ 6 ], which was recently corroborated by reports demonstrating its psychobiotic activity and antidepressant-like effects in rodent depression models [ 7 , 8 ]. These studies revealed that K. pastoris is resistant in the gastrointestinal tract, has antibacterial, antioxidant, and immunomodulatory properties, does not damage intestinal epithelial tissues, modulates gut microbiota, maintains the integrity of the intestinal and blood-brain barrier, and does not alter blood levels of aspartate transferase, alanine transaminase, urea, and creatinine. Hence, these data collectively imply that all these effects improve depressive-like behavior in rodents. Given this context, we are eager to continue investigating the potential benefits of K. pastoris KM71H supplementation. Therefore, this study aimed to evaluate whether this yeast attenuates cognitive deficits and depressive-like behavior in an Alzheimer disease (AD) animal model and its potential mechanisms. Roughly 55 million people worldwide have dementia, and this number is expected to reach 139 million by 2050; AD is a progressive disease with debilitating consequences and the most common form of dementia, accounting for up to 70% of cases. Despite cognitive and functional decline being the main symptoms, major depressive disorder (MDD) affects nearly half of all AD patients [ 9 ]. Recent evidence has demonstrated the crucial role of the gut microbiota in sustaining host health, indicating that the gut microbiota communicates with the central nervous system (CNS) via the microbiota-gut-brain axis [ 10 , 11 ]. Dysbiosis, an imbalance in the gut microbiota, has been suggested as a mechanism of neuroinflammation and a contributing factor in promoting amyloidosis, which leads to AD and, eventually, MDD [ 12 ]. Nevertheless, probiotic supplementation may mitigate gut dysbiosis [ 13 ], and a growing body of research has shown that probiotic supplementation improves overall cognitive function and counteracts AD progression in humans and animals [ 14 – 17 ]. 2. Materials and methods 2.1. Animals and experimental protocol This study was approved by the Animal Care and Use Committee of the Federal University of Pelotas (no. 026745/2021-90). Swiss male mice were provided by the Central Animal Facility of the Federal University of Pelotas and housed under constant temperature conditions (22 ± 1°C), 12/12-h light-dark cycle (from 7:00 am to 7:00 pm), and water and food ad libitum . All tests were performed between 8:00 am and 5:00 pm, and all efforts were made to minimize the number of animals and their suffering. The animals were randomLy divided into four groups (n = 8): the control (phosphate-buffered saline [PBS] + PBS), K. pastoris KM71H + PBS, amyloid β 1−40 peptide (Aβ 1−40 ) + PBS, and K. pastoris KM71H + Aβ 1−40 . Mice were intragastrically treated with 8 log CFU of K. pastoris KM71H or PBS ( K. pastoris KM71H vehicle) for 14 days. On the fourth day, Aβ 1−40 or PBS (Aβ 1−40 vehicle) were intracerebroventricularly (icv) injected. On the fifteenth day, the mice were subjected to a series of behavioral tests, including the open field test (OFT), tail suspension test (TST), Y-maze test, and buried food-finding test (BFFT). Afterward, the mice were euthanized by isoflurane inhalation and their hippocampi and small intestine were removed for ex vivo analyses and stored in ice (Fig. 1 ). 2.2. Aβ administration Aβ 1−40 was purchased from Sigma (St. Louis, MO, USA), dissolved in PBS (pH 7.4), and incubated at 37°C for four days to induce aggregation; Aβ 1−40 (4 µL/400 pmol) was administered icv, as this dose was reported to effectively induce depressive-like behavior in rodents. The icv injection was performed freehand under isoflurane inhalation anesthesia as described elsewhere, with the bregma fissure as a reference [ 18 – 20 ]. Bregma was found by lightly rubbing the tip of the needle over the skull until the suture was felt through the skin. Aβ 1−40 or PBS (4 µL) was injected over 30 s, and the needle remained in place for another 30 s to avoid the reflux of the substances injected. In summary, a 25-µL microsyringe (Hamilton) was inserted unilaterally 1 mm to the midline point equidistant from each eye, at an equal distance between the eyes and ears and perpendicular to the plane of the skull and no more than 2 mm into the brain and following the coordinates: 0.8 mm posterior to bregma, 1.0 mm lateral to the sagittal suture, and 3.0 mm beneath the surface of the brain [ 21 ]. 2.3. Behavioral tests 2.3.1. Open field test Each animal was placed in a box (30 × 30 × 15 cm) with the floor divided into nine equal quadrants and observed for 5 min. The number of quadrants crossed with all four paws (crossings) indicated locomotor activity, and the number of rearings indicated exploratory activity [ 22 ]. 2.3.2. Tail suspension test Each mouse was suspended by the tail at 50 cm above the floor and was observed for 6 min; the immobility time was measured in the last 4 min (the first 2 min were for habituation) [ 23 ]. Increased immobility time was considered indicative of depressive-like behavior. 2.3.3. Y-maze test The Y-maze tests spatial working memory in rodents and consists of a three-arm horizontal maze (40 cm long and 3 cm wide with 12 cm high walls). The percentage of spontaneous alternations was employed; the mice were initially placed in one arm (A), and the arm entry sequence and the number of arm entries were recorded for 6 min. Alternation indicates sequential entries into all three arms and by the formula: % alternation = [(number of alternations × 3)/(total arm entries − 2)] × 100 [ 24 ]. Decreased spontaneous alternation was considered indicative of cognitive deficits. 2.3.4. Buried food finding test All mice were familiarized with chocolate cookies two days before the test, and the animals received various 1.6-g pieces with water ad libitum . Twelve hours before testing, mice received 30% less food. For testing, each mouse was placed into clear cages (30 x 19 x 13 cm), in which a piece of a chocolate cookie was hidden under 1.5 cm of standard bedding at the end of the cage. The animals were placed in the right corner at the other end of the cage to test their olfactory memory, and the food-finding time was measured. If a mouse failed to locate the food within 300 s, the testing session was terminated, and the latency duration was defined as 300 s [ 25 ]. This test assesses olfactory memory, and increased latency time to find the cookie was considered indicative of cognitive deficits. 2.4. Biochemical analyses 2.4.1. Tissue collection The small intestine (removed ± 15 cm from the stomach exit, corresponding to the duodenum) was washed with 2 mL of saline solution (0.9%), sliced open, and the mucosa was collected by gently scraping with a scalpel. The hippocampi and small intestine were homogenized in 50 mM Tris-HCl pH 7.4 (1:4 w/v), followed by centrifugation at 2500 × g for 10 min at 4°C. The supernatant fraction was employed to determine reactive species and lipid peroxidation. For gene expression analysis, the hippocampi and small intestine were kept at -80°C until sample processing. 2.4.2. Quantification of reactive species Reactive species formation was quantified using 2’-7’-dichlorofluorescein diacetate (DCHF-DA), as described by Loetchutinat et al. [ 26 ]. An aliquot of the supernatant (10 µL) was incubated with 1 mM DCHF-DA (10 µL) and 10 mM Tris-HCl pH 7.4 (2980 µL). The oxidation of DCFH-DA to fluorescent dichlorofluorescein (DCF) is measured to detect intracellular reactive species, and DCF fluorescence intensity emission was recorded at 520 nm with 480 nm excitation. 2.4.3. Lipid peroxidation assay An aliquot of the supernatant (10 µL) was incubated with 8.1% sodium dodecyl sulfate (20 µL), 0.8% thiobarbituric acid (150 µL), and acetic acid/HCl (pH 3.4, 150 µL) at 95°C for 60 min, as described by Ohkawa et al. [ 27 ]. Thiobarbituric acid reactive species levels were measured spectrophotometrically at 532 nm. 2.4.4. Protein determination Protein concentrations were measured according to the method of Bradford [ 28 ] using serum bovine albumin as standard. 2.4.5. RNA extraction and gene expression analysis by qRT-PCR The total mRNA was extracted from the prefrontal cortex, hippocampus and small intestine using TRIzol reagent (Invitrogen, Carlsbad, USA), followed by DNase treatment with DNA-free kit (Ambion, USA) and mRNA quantification. The cDNA synthesis was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, UK) according to the manufacturer’s instructions. Amplification was made with UltraSYBR Mix (CoWin Bioscience Co., Pequim, China) using the Stratagene Mx3005P. Gene expressions were normalized using GAPDH as a reference gene, and the conditions for the reaction included 95°C for 15 s, 60°C for 60 s, and 72°C for 30 s. The delta-delta comparative threshold method was used to normalize the fold change in gene expressions. The following primers were used: nuclear factor kappa B (Nf-κb; fwd 5′-GCT TTC GCA GGA GCA TTA AC-3′, rev 5′-CCG AAG CAG GAG CTA TCA AC-3′), interleukin-1β (Il-1β; fwd 5´GCT GAA AGC TCT CCA CCT CAA TG-3´ rev- 5´-TGT CGT TGC TTG GTT CTC CTT GC-3´), toll-like receptor 4 (Tlr4, F: 5´-GCC TTT CAG GGA ATT AAG CTC C-3´, R: 5´-AGA TCA ACC GAT GGA CGT GTA A-3´), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh; fwd 5′-AGG TCG GTG TGA ACG GAT TTG-3′, rev 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′). 2.5. Statistical analyses Data from the behavioral and neurochemical analyses are expressed as mean ± standard error of the mean (SEM). The experimental results were analyzed by two-way analysis of variance (ANOVA), followed by Tukey’s test for post-hoc comparisons. Results were considered significant when p < 0.05. The main effects are only presented when the first-order interaction is non-significant. The statistical analysis was performed using the Graph Pad Prism software (version 9.0; San Diego, CA, USA). 3. Results 3.1. K. pastoris KM71H attenuated depressive-like behavior of Aβ 1−40 -induced AD-like mice The number of crossings and rearings were counted in the OFT before the other behavioral tests to assess whether the Aβ 1−40 + KM71H and K. pastoris KM71H treatments affected the mice’s locomotor and exploratory activities; no statistical difference was found in the number of crossings (Fig. 2 A) and rearings (Fig. 2 B) compared to the control (i.e., unaltered psychomotor and exploratory activities). The TST was performed to evaluate the antidepressant-like effect of K. pastoris KM71H, and Aβ 1−40 exposure significantly increased immobility time compared to the control (Fig. 2 C). Notably, the K. pastoris KM71H treatment reduced the immobility time compared to the Aβ group (interaction, F (1,32) = 84.8, p < 0.001). In addition, the K. pastoris KM71H treatment reduced immobility time in the TST compared to the control. 3.2. K. pastoris KM71H attenuated cognitive deficits of Aβ 1−40 -induced AD-like mice The animals were submitted to the Y-maze test to evaluate their spatial working memory, and Aβ 1−40 exposure was shown to significantly decrease spatial and working memory (Fig. 2 D). The K. pastoris KM71H treatment increased the percentage of spontaneous alternations, demonstrating its ability to ameliorate memory impairment (main effect, F (1,32) = 67, p < 0.001). The animals were submitted to the BFFT to evaluate their olfactory memory. As predicted, Aβ 1−40 administration increased the time the animals took to retrieve the chocolate cookies compared to the control, whereas the K. pastoris KM71H treatment decreased the necessary time to find the chocolate cookies compared to the Aβ group, indicating its ability to improve olfactory memory (interaction, F (1,32) = 17.2, p < 0.001) (Fig. 2 E). 3.3. Effect of oxidative stress of K. pastoris KM71H in the hippocampus and small intestine In neurodegenerative diseases such as AD, oxidative stress is often implicated in their etiologies (Cheignon et al. 2018). Due to high oxygen consumption and poor antioxidant defense, the brain is particularly vulnerable to oxidative damage [ 29 ]. Therefore, we investigated the antioxidant effect of K. pastoris KM71H treatment in Aβ 1−40 -induced AD-like mice, and our findings showed the Aβ 1−40 exposure significantly increased reactive species in the hippocampi (interaction, F (1,32) = 10.5, p < 0.001; Fig. 3 A) and small intestine (interaction, F (1,32) = 17.6, p < 0.001; Fig. 3 B), which was restored by K. pastoris KM71H treatment. The increased reactive species may damage biomolecules, including lipids and proteins. Aβ 1−40 administration increased the lipid peroxidation in the hippocampi (interaction, F (1,32) = 4.58, p < 0.001; Fig. 3 C) and small intestine (interaction, F (1,32) = 20.8, p < 0.001; Fig. 3 D), and the K. pastoris KM71H restored this alteration. Together, these results suggest that K. pastoris KM71H presents antioxidant activity, possibly related to improved cognition and depressive-like behavior. 3.4. Effects of K. pastoris KM71H in the TLR4/NF-κB signaling pathway in Aβ 1−40 -induced AD-like mice Evidence suggests that neuroinflammation is also involved in AD pathogenesis (Heneka et al. 2015), and TLR4 plays a key role in inflammation by inducing proinflammatory cytokine production, including IL-1β (Sun et al. 2020). Here, we demonstrated that Aβ 1−40 administration increased NRLP3 (interaction, F (1,16) = 13.2, p = 0.002; Fig. 4 A), TLR4 (interaction, F (1,16) = 8.15, p = 0.01; Fig. 4 B), Nfҡb (interaction, F (1,16) = 6.11, p = 0.03; Fig. 4 C), and IL-1β (main effect, F (1,16) = 12.3, p = 0.003; Fig. 4 D) expression in the small intestine compared to control group, and these alterations were restored by K. pastoris KM71H. In addition, Aβ 1−40 administration showed a trend of increasing IL-1β expression (main effect, F (1,16) = 1.49, p = 0.2; Fig. 4 E) in the hippocampi compared to the control group, and the K. pastoris KM71H treatment demonstrated a trend of decreasing IL-1β expression compared to the Aβ group. These results suggest that K. pastoris KM71H treatment improves cognitive deficits and depressive-like behavior via the TLR4/NF-κB signaling pathway. 4. Discussion This study revealed for the first time that K. pastoris KM71H ameliorated cognitive deficits and depressive-like behavior in Aβ 1−40 -induced AD-like mice. These effects are corroborated by the fact that K. pastoris KM71H increased the percentage of spontaneous alternations in the Y-maze test, decreased the time necessary for the animal to retrieve the chocolate cookies in the BFFT, and decreased the immobility time of Aβ 1−40 -exposed mice. Furthermore, improved cognitive impairment and depressive-like behavior in the animals treated with the yeast may be due to the antioxidant effects elicited by K. pastoris KM71H. This betterment was accompanied by lower levels of reactive species and lipid peroxidation in the hippocampi and small intestine, followed by modulation of the TLR4/NF-κB pathway as NRLP3, TLR4, Nfҡb, and IL-1β expression decreased in the small intestine. Alzheimer disease is a multifactorial disease involving the periphery and CNS and is mainly characterized by the accumulation of aggregated Aβ, neurofibrillary tangles, and neurodegeneration in the brain, as well as clinical manifestations, including cognitive deficits and neuropsychiatric symptoms [ 30 , 31 ]. A growing body of evidence has demonstrated that AD is a neurodegenerative disease with substantial comorbidity with MDD [ 32 ]; in fact, a meta-analysis conducted by Zhao et al. [ 9 ] showed that MDD is present in up to 50% of AD cases. Additionally, changes in the composition of the intestinal microbiota have been hypothesized to contribute to neuroinflammation and neurodegeneration in the CNS since these changes toward gut dysbiosis are a risk factor for both clinical conditions[ 33 – 36 ]. Indeed, numerous researchers have shown that the gut microbiota of depressed and AD patients exhibit different bacterial compositions compared to healthy controls and activation patterns of peripheral and central inflammatory cells [ 35 ]. Thus, probiotics are a promising therapeutic alternative for both conditions, as corroborated by the evidence of probiotic supplementation attenuating depressive-like behaviors and cognitive impairments [ 16 , 37 – 39 ]. Recent research with K. pastoris KM71H showed that it also decreased TST immobility time in different animal models of depression [ 7 , 8 ], and we believe the mechanism by which K. pastoris KM71H confers these benefits is the modulation of the intestinal microbiota, antioxidant activity, and immunomodulation. Here, we showed that K. pastoris KM71H attenuated depressive-like behavior and cognitive deficits in mice that received Aβ 1−40 icv injection, as supported by the decreased immobility time in the TST, increased percentage of spontaneous alternations in the Y-maze test, and decreased time for the animal to retrieve the chocolate cookies in the BFFT compared to the Aβ group. The intestinal microbiota can influence neurodegeneration by promoting Aβ aggregation and neuroinflammation. Within the given frame of reference, therapeutic strategies that modulate the intestine microbial composition toward increasing the population of beneficial bacteria, such as Bacteroidetes and Lactobacillus , are of the utmost importance [ 40 ]. In a previous study, we showed that K. pastoris increased the concentration of these genera of bacteria and, through interactions with the intestinal epithelial barrier and immune system, contributed to improving depressive-like behavior [ 8 ]. These findings may also be related to the improved cognitive deficit, although more robust experimental evidence is required to prove this hypothesis. Likewise, stimuli associated with inflammatory and neurodegenerative processes, including Aβ aggregates and reactive species, promote the activation of immune system components that, in turn, can alter the composition of the intestinal microbiota, leading to pathologies such as AD and MDD [ 35 , 41 ]. In the present study, we demonstrated that Aβ 1−40 administration elicits oxidative stress, parameters observed by the higher levels of reactive species and lipid peroxidation in the hippocampi and small intestines of mice. Our data corroborate previous studies demonstrating that Aβ aggregates promote oxidative stress, contributing to the pathogenesis and disease progression of AD [ 42 ]. Oxidative stress is caused by an imbalance between reactive species levels and the action of the antioxidant defense systems and is involved in the pathophysiology of several neuropsychiatric and neurodegenerative disorders [ 42 – 44 ]. Therefore, there is great interest in developing new antioxidant therapeutic strategies for treating MDD and AD. Supplementation with K. pastoris KM71H attenuated the increased levels of reactive species and lipid peroxidation in the hippocampi and small intestine of Aβ 1−40 -exposed mice, showing a promising antioxidant effect; nevertheless, these were unsurprising data as K. pastoris KM71H showed antioxidant activity in previous studies [ 7 , 8 ]. Oxidative stress and increased reactive species and lipid peroxidation in the intestinal environment may impair the integrity of the intestinal barrier and contribute to changes in intestinal microbiota composition, which may lead to inflammatory responses [ 45 ]. Moreover, the intestinal microbiota modulates the inflammatory responses mainly by stimulating toll-like receptors (TLRs) and/or NOD-like receptors (NLRs) [ 46 ]. The TLRs are the first line of defense, recognizing molecules present in a pathogen, known as pathogen-associated molecular patterns (PAMPs), triggering proinflammatory cytokine expression [ 47 ]. Notably, TLR4 is stimulated by numerous PAMPs, including lipopolysaccharide from Gram-negative bacteria inducing the release of proinflammatory cytokines necessary to activate potent immune responses [ 47 ]. Furthermore, Aβ is also a ligand for TLRs, which can initiate the inflammatory process in the gut and CNS, leading to the development of neurodegenerative diseases [ 46 ]. Indeed, animals that received Aβ 1−40 showed increased TLR4 expression in the small intestine, and the K. pastoris KM71H restored this alteration. We believe that the decreased TLR4 expression in the small intestine was due to yeast supplementation improving the integrity of the intestinal barrier, thereby preventing the translocation of enteric bacteria and their products into the lamina propria and preventing the inflammatory response. Although more studies are needed to confirm this premise, previous research has shown that K. pastoris KM71H helps maintain the integrity of the intestinal barrier and decreases TLR4 expression in the small intestine in a model of antibiotic-induced intestinal dysbiosis in mice [ 8 ]. There is also evidence that Aβ aggregates and reactive species promote nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. As a result, it can lead to a shift in intestinal microbiota towards a proinflammatory phenotype, contributing to inflammatory responses in brain disorders [ 35 ]. Evidence has also shown that patients with AD and MDD present enteric and brain activation of the NLRP3 inflammasome [ 48 – 50 ]. Here, Aβ 1−40 increased NLRP3 expression in the intestine, while the K. pastoris KM71H treatment restored this alteration. This effect may have contributed to improving cognitive deficit and depressive-like behavior in the animals since the NLRP3 is involved in regulating the microbiota-gut-brain axis, and their modulation counteracts CNS disease progression in animal models [ 51 , 35 , 49 ]. Different stimuli can promote canonical and/or non-canonical NLRP3 inflammasome activations; for example, Gram-negative bacteria activate the TLR4-MyD88 pathway, with consequent transcription of IL-1β and NLRP3 genes through NF-κB activation [ 35 ]. Indeed, we observed increased TLR4, NFKB, and IL-1β expression in the small intestine and a trend of increased IL-1β expression in the hippocampi of Aβ 1−40 -exposed mice. Supplementation with K. pastoris KM71H decreased NFKB and IL-1β expression in the small intestine and presented a trend to decrease IL-1β expression in the hippocampi. We believe that these effects are attributed to the ability of K. pastoris KM71H to modulate the gut-brain axis through its antioxidant and immunomodulatory activity. Clinical data have shown that patients with AD and MDD are characterized by changes in gut microbiota composition, oxidative stress, enteric, peripheral, and brain activation of the NLRP3 inflammasome, and impairment of intestinal and brain barriers, facilitating the activation of immune/inflammatory pathways [ 48 – 50 ]. In order to minimize these effects, therapeutic strategies are needed. Therefore, we highlight K. pastoris KM71H, which is a potent probiotic with antioxidant and immunomodulatory activity, restoring the integrity of the brain and intestinal barrier and promoting the modulation of the microbiota composition. In conclusion, our findings are the first to indicate that K. pastoris KM71H ameliorated cognitive deficits and depressive-like behavior in Aβ 1−40 -induced AD-like mice. Therefore, we hypothesize that K. pastoris KM71H can be a new target for developing new probiotic treatments for comorbidities between AD and MDD. Abbreviations AD, Alzheimer disease; BFFT, buried food-finding test; DCF, dichlorofluorescein; DCHF-DA, 2’-7’-dichlorofluorescein diacetate; ICV, intracerebroventricularly; MDD, major depressive disorder; OFT, open field test; TST, tail suspension test; Declarations Funding Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS). Data availability The datasets generated during and/or analyzed in the current study are available from the corresponding author upon reasonable request. Code availability Not applicable. CRediT authorship contribution statement P.T. Birmann performed the experimental design, behavioral tests, biochemical assessments, and data analysis and wrote the manuscript. L. Savegnago performed the experimental design, data analysis, writing, reviewing, and editing of the manuscript, supervised the experiments, acquired funding, and is the corresponding author*. G.P. Zugno and A. Sinott performed the behavioral tests and the biochemical assessments and wrote the manuscript. F.S.S. Sousa, F.K. Seixas, and T. Collares performed the qRT-PCR analyses. Rochedo and R.R. Rodrigues performed the cultivation and preparation of K. pastoris. L. Savegnago and F.C. Rochedo were responsible for funding acquisition and reviewing and editing the manuscript. Ethics approval The experiments with animals were carried out in accordance with the Institutional Animal Care and Use Committee of the Federal University of Pelotas (approval number of the ethics committee: 026745/2021-90), affiliated with the National Council for Animal Experimentation Control (CONCEA). Consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Acknowledgments The authors are grateful to UFPel, especially Biotechnology Graduate Program (UFPel), for supporting this work and thank CNPq, CAPES, and FAPERGS (PRONEM 16/2551-0000240-1, PqG 17/2551-00011046-9) for the financial support. L.S., T.C., F.K.S., and F.R.C. are recipients of CNPq fellowships. This study was partly financed by the CAPES - Finance Code 001. We would also like to thank Atlas Assessoria Linguística for language editing. 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Mol Psychiatry 21 (6):797-805. doi:10.1038/mp.2016.46 Amin FU, Shah SA, Kim MO (2017) Vanillic acid attenuates Abeta1-42-induced oxidative stress and cognitive impairment in mice. Sci Rep 7:40753. doi:10.1038/srep40753 Lushchak VI (2014) Free radicals, reactive oxygen species, oxidative stress and its classification. Chem Biol Interact 224:164-175. doi:10.1016/j.cbi.2014.10.016 Sies HJRb (2015) Oxidative stress: a concept in redox biology and medicine. 4:180-183 Wang H, Yang F, Xin R, Cui D, He J, Zhang S, Sun Y (2020) The gut microbiota attenuate neuroinflammation in manganese exposure by inhibiting cerebral NLRP3 inflammasome. Biomed Pharmacother 129:110449. doi:10.1016/j.biopha.2020.110449 Lin C, Zhao S, Zhu Y, Fan Z, Wang J, Zhang B, Chen Y (2019) Microbiota-gut-brain axis and toll-like receptors in Alzheimer's disease. Comput Struct Biotechnol J 17:1309-1317. doi:10.1016/j.csbj.2019.09.008 Lu YC, Yeh WC, Ohashi PS (2008) LPS/TLR4 signal transduction pathway. Cytokine 42 (2):145-151. doi:10.1016/j.cyto.2008.01.006 Cheung SG, Goldenthal AR, Uhlemann A-C, Mann JJ, Miller JM, Sublette MEJFip (2019) Systematic review of gut microbiota and major depression. 10:34 Saresella M, La Rosa F, Piancone F, Zoppis M, Marventano I, Calabrese E, Rainone V, Nemni R, Mancuso R, Clerici MJMn (2016) The NLRP3 and NLRP1 inflammasomes are activated in Alzheimer’s disease. 11 (1):1-14 Vogt NM, Kerby RL, Dill-McFarland KA, Harding SJ, Merluzzi AP, Johnson SC, Carlsson CM, Asthana S, Zetterberg H, Blennow KJSr (2017) Gut microbiome alterations in Alzheimer’s disease. 7 (1):1-11 Heneka MT, Carson MJ, El Khoury J, Landreth GE, Brosseron F, Feinstein DL, Jacobs AH, Wyss-Coray T, Vitorica J, Ransohoff RM, Herrup K, Frautschy SA, Finsen B, Brown GC, Verkhratsky A, Yamanaka K, Koistinaho J, Latz E, Halle A, Petzold GC, Town T, Morgan D, Shinohara ML, Perry VH, Holmes C, Bazan NG, Brooks DJ, Hunot S, Joseph B, Deigendesch N, Garaschuk O, Boddeke E, Dinarello CA, Breitner JC, Cole GM, Golenbock DT, Kummer MP (2015) Neuroinflammation in Alzheimer's disease. Lancet Neurol 14 (4):388-405. doi:10.1016/S1474-4422(15)70016-5 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 06 Aug, 2025 Read the published version in Molecular Neurobiology → Version 1 posted Editorial decision: Revision requested 03 Jun, 2025 Reviews received at journal 03 Jun, 2025 Reviews received at journal 23 May, 2025 Reviews received at journal 22 May, 2025 Reviewers agreed at journal 18 May, 2025 Reviewers agreed at journal 14 May, 2025 Reviewers agreed at journal 14 May, 2025 Reviewers agreed at journal 13 May, 2025 Reviewers agreed at journal 12 May, 2025 Reviewers invited by journal 12 May, 2025 Editor assigned by journal 30 Apr, 2025 Submission checks completed at journal 30 Apr, 2025 First submitted to journal 11 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6431395","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":456989394,"identity":"254bcb88-46e8-4346-9993-8e5e05715a03","order_by":0,"name":"Paloma T. Birmann","email":"","orcid":"","institution":"Federal University of Pelotas","correspondingAuthor":false,"prefix":"","firstName":"Paloma","middleName":"T.","lastName":"Birmann","suffix":""},{"id":456989395,"identity":"14c63659-8747-4f99-9479-204d78f3dea2","order_by":1,"name":"Giuliana Petiz Zugno","email":"","orcid":"","institution":"Federal University of Pelotas","correspondingAuthor":false,"prefix":"","firstName":"Giuliana","middleName":"Petiz","lastName":"Zugno","suffix":""},{"id":456989396,"identity":"b484a729-695c-4d9d-9dc7-44be16cf50fd","order_by":2,"name":"Airton Sinott","email":"","orcid":"","institution":"Federal University of Pelotas","correspondingAuthor":false,"prefix":"","firstName":"Airton","middleName":"","lastName":"Sinott","suffix":""},{"id":456989397,"identity":"48923cc3-478c-4a9c-b1c7-82767c07d1c3","order_by":3,"name":"Rafael R. Rodrigues","email":"","orcid":"","institution":"Federal University of Pelotas","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"R.","lastName":"Rodrigues","suffix":""},{"id":456989398,"identity":"f9a1aca9-79a2-46b5-85a8-9842720028ed","order_by":4,"name":"Fabricio R. Conceição","email":"","orcid":"","institution":"Federal University of Pelotas","correspondingAuthor":false,"prefix":"","firstName":"Fabricio","middleName":"R.","lastName":"Conceição","suffix":""},{"id":456989399,"identity":"c71f7cd3-eb04-496d-9704-d15ebc33e495","order_by":5,"name":"Fernanda Severo Sabedra Sousa","email":"","orcid":"","institution":"Federal University of Pelotas","correspondingAuthor":false,"prefix":"","firstName":"Fernanda","middleName":"Severo Sabedra","lastName":"Sousa","suffix":""},{"id":456989400,"identity":"bf86a7c9-6aea-4aea-bca5-3ae4c3194f63","order_by":6,"name":"Tiago Collares","email":"","orcid":"","institution":"Federal University of Pelotas","correspondingAuthor":false,"prefix":"","firstName":"Tiago","middleName":"","lastName":"Collares","suffix":""},{"id":456989401,"identity":"d20f5475-2151-47b4-9c72-e86754ae9cf3","order_by":7,"name":"Fabiana K. Seixas","email":"","orcid":"","institution":"Federal University of Pelotas","correspondingAuthor":false,"prefix":"","firstName":"Fabiana","middleName":"K.","lastName":"Seixas","suffix":""},{"id":456989402,"identity":"48943270-8d1a-4bc9-87a2-742e929c099b","order_by":8,"name":"Lucielli Savegnago","email":"data:image/png;base64,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","orcid":"","institution":"Federal University of Pelotas","correspondingAuthor":true,"prefix":"","firstName":"Lucielli","middleName":"","lastName":"Savegnago","suffix":""}],"badges":[],"createdAt":"2025-04-11 23:53:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6431395/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6431395/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12035-025-05267-9","type":"published","date":"2025-08-06T15:57:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82865314,"identity":"4902d077-0dc7-47d9-b331-78c3af74927e","added_by":"auto","created_at":"2025-05-16 07:45:52","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":78798,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentation of the experimental design. Mice were treated with \u003cem\u003eK. pastoris\u003c/em\u003e KM71H (8 log CFU\u003csup\u003e-1\u003c/sup\u003e ig) or PBS for 14 days. On the fourth day, the animals received an intracerebroventricular injection of Aβ\u003csub\u003e1-40\u003c/sub\u003e or PBS. On the fifteenth day, mice were submitted for behavioral tests, including an open field test (OFT), tail suspension test (TST), Y-maze test, and buried food-finding test (BFFT). After behavioral tests, the mice were euthanized and the hippocampi and small intestine were removed for \u003cem\u003eex vivo\u003c/em\u003e analyses.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6431395/v1/6f9cbce91b2f6e765d3fe744.jpeg"},{"id":82865315,"identity":"22130697-bfbd-493b-9253-c6c8916029f4","added_by":"auto","created_at":"2025-05-16 07:45:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":47910,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eK. pastoris\u003c/em\u003e KM71H on the number of (A) crossings and (B) rearings in the OFT, (C) immobility time in the TST, (D) spontaneous alternations (%) in the Y-maze test, and € latency to find the chocolate cookies in BFFT. Data are expressed as mean ± SEM. ### p \u0026lt; 0.001 compared to the control. \u003csup\u003e*\u003c/sup\u003e p \u0026lt;0.05 and \u003csup\u003e***\u003c/sup\u003e p \u0026lt;0.001 compared to the Aβ group (two-way ANOVA followed by Tukey’s test).\u003c/p\u003e","description":"","filename":"image2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6431395/v1/d142648ec76da13e76af8bbe.jpg"},{"id":82865316,"identity":"7f334d7c-c510-49f6-90a7-8872c5178eea","added_by":"auto","created_at":"2025-05-16 07:45:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46774,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eK. pastoris\u003c/em\u003e KM71H on levels of reactive species in (A) the hippocampi and (B) the small intestine on lipid peroxidation in (C) the hippocampi and (D) the small intestine. Data are expressed as mean ± SEM. ## p \u0026lt; 0.05 and ### p \u0026lt; 0.001 compared to the control.\u003csup\u003e *\u003c/sup\u003e p \u0026lt;0.05 and \u003csup\u003e***\u003c/sup\u003e p \u0026lt;0.001 compared to the Aβ group (two-way ANOVA followed by Tukey’s test).\u003c/p\u003e","description":"","filename":"image3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6431395/v1/4d2503877b8e94a9374b5cfd.jpg"},{"id":82865318,"identity":"a4ecac98-7117-429b-9ac4-c80da8cb73b0","added_by":"auto","created_at":"2025-05-16 07:45:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":35485,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eK. pastoris\u003c/em\u003e KM71H on (A) NRLP3 expression in the small intestine, (B) TLR4 expression in the small intestine, (C) NFҡB mRNA expression in the small intestine, and IL-1β mRNA expression in (D) small intestine and (E) the hippocampi. Data are expressed as mean ± SEM. # p \u0026lt; 0.05, ## p \u0026lt; 0.01 and ### p \u0026lt; 0.001 compared to the control. \u003csup\u003e* \u003c/sup\u003ep \u0026lt;0.05 and \u003csup\u003e** \u003c/sup\u003ep \u0026lt;0.01 compared to the Aβ group (two-way ANOVA followed by Tukey’s test).\u003c/p\u003e","description":"","filename":"image4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6431395/v1/d73b1fb95999aad8570a107c.jpg"},{"id":88814200,"identity":"30069115-818e-409c-b458-f59da65ff4a0","added_by":"auto","created_at":"2025-08-11 16:08:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1127663,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6431395/v1/c2e65cd0-15e5-41cf-ba1e-17256b38b6cd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Komagataella pastoris KM71H attenuates cognitive deficits and depressive-like behavior by the TLR4/NF-κB signaling pathway in an Aβ 1-40 -induced AD-like mouse model","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eProbiotics are live microorganisms that provide health benefits when ingested in adequate amounts [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, preclinical and clinical research has focused primarily on bacterial strains (e.g., \u003cem\u003eLactobacillus\u003c/em\u003e), and only a handful of studies have covered yeast [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This scenario is changing as more studies have sought to investigate the probiotic activities of fungi. One positive aspect of their use is that they are not affected by antibacterial agents and can be combined to prevent intestinal dysbiosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Yeasts are well-known unicellular eukaryotic fungi utilized in industrial settings to produce fermented and biotechnological food products [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. \u003cem\u003eKomagataella pastoris\u003c/em\u003e stands out among such products as this methylotrophic yeast is widely employed as a protein expression system [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], although little has been known about its probiotic effects until recently.\u003c/p\u003e \u003cp\u003eEvidence has shown that \u003cem\u003eK. pastoris\u003c/em\u003e has probiotic properties with remarkable antibacterial activity against \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], which was recently corroborated by reports demonstrating its psychobiotic activity and antidepressant-like effects in rodent depression models [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These studies revealed that \u003cem\u003eK. pastoris\u003c/em\u003e is resistant in the gastrointestinal tract, has antibacterial, antioxidant, and immunomodulatory properties, does not damage intestinal epithelial tissues, modulates gut microbiota, maintains the integrity of the intestinal and blood-brain barrier, and does not alter blood levels of aspartate transferase, alanine transaminase, urea, and creatinine. Hence, these data collectively imply that all these effects improve depressive-like behavior in rodents.\u003c/p\u003e \u003cp\u003eGiven this context, we are eager to continue investigating the potential benefits of \u003cem\u003eK. pastoris\u003c/em\u003e KM71H supplementation. Therefore, this study aimed to evaluate whether this yeast attenuates cognitive deficits and depressive-like behavior in an Alzheimer disease (AD) animal model and its potential mechanisms. Roughly 55\u0026nbsp;million people worldwide have dementia, and this number is expected to reach 139\u0026nbsp;million by 2050; AD is a progressive disease with debilitating consequences and the most common form of dementia, accounting for up to 70% of cases. Despite cognitive and functional decline being the main symptoms, major depressive disorder (MDD) affects nearly half of all AD patients [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent evidence has demonstrated the crucial role of the gut microbiota in sustaining host health, indicating that the gut microbiota communicates with the central nervous system (CNS) via the microbiota-gut-brain axis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Dysbiosis, an imbalance in the gut microbiota, has been suggested as a mechanism of neuroinflammation and a contributing factor in promoting amyloidosis, which leads to AD and, eventually, MDD [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Nevertheless, probiotic supplementation may mitigate gut dysbiosis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and a growing body of research has shown that probiotic supplementation improves overall cognitive function and counteracts AD progression in humans and animals [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals and experimental protocol\u003c/h2\u003e \u003cp\u003e This study was approved by the Animal Care and Use Committee of the Federal University of Pelotas (no. 026745/2021-90). Swiss male mice were provided by the Central Animal Facility of the Federal University of Pelotas and housed under constant temperature conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C), 12/12-h light-dark cycle (from 7:00 am to 7:00 pm), and water and food \u003cem\u003ead libitum\u003c/em\u003e. All tests were performed between 8:00 am and 5:00 pm, and all efforts were made to minimize the number of animals and their suffering. The animals were randomLy divided into four groups (n\u0026thinsp;=\u0026thinsp;8): the control (phosphate-buffered saline [PBS]\u0026thinsp;+\u0026thinsp;PBS), \u003cem\u003eK. pastoris\u003c/em\u003e KM71H\u0026thinsp;+\u0026thinsp;PBS, amyloid β\u003csub\u003e1\u0026minus;40\u003c/sub\u003e peptide (Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e)\u0026thinsp;+\u0026thinsp;PBS, and \u003cem\u003eK. pastoris\u003c/em\u003e KM71H\u0026thinsp;+\u0026thinsp;Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eMice were intragastrically treated with 8 log CFU of \u003cem\u003eK. pastoris\u003c/em\u003e KM71H or PBS (\u003cem\u003eK. pastoris\u003c/em\u003e KM71H vehicle) for 14 days. On the fourth day, Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e or PBS (Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e vehicle) were intracerebroventricularly (icv) injected. On the fifteenth day, the mice were subjected to a series of behavioral tests, including the open field test (OFT), tail suspension test (TST), Y-maze test, and buried food-finding test (BFFT). Afterward, the mice were euthanized by isoflurane inhalation and their hippocampi and small intestine were removed for \u003cem\u003eex vivo\u003c/em\u003e analyses and stored in ice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Aβ administration\u003c/h2\u003e \u003cp\u003eAβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e was purchased from Sigma (St. Louis, MO, USA), dissolved in PBS (pH 7.4), and incubated at 37\u0026deg;C for four days to induce aggregation; Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e (4 \u0026micro;L/400 pmol) was administered icv, as this dose was reported to effectively induce depressive-like behavior in rodents. The icv injection was performed freehand under isoflurane inhalation anesthesia as described elsewhere, with the bregma fissure as a reference [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Bregma was found by lightly rubbing the tip of the needle over the skull until the suture was felt through the skin. Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e or PBS (4 \u0026micro;L) was injected over 30 s, and the needle remained in place for another 30 s to avoid the reflux of the substances injected. In summary, a 25-\u0026micro;L microsyringe (Hamilton) was inserted unilaterally 1 mm to the midline point equidistant from each eye, at an equal distance between the eyes and ears and perpendicular to the plane of the skull and no more than 2 mm into the brain and following the coordinates: 0.8 mm posterior to bregma, 1.0 mm lateral to the sagittal suture, and 3.0 mm beneath the surface of the brain [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Behavioral tests\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Open field test\u003c/h2\u003e \u003cp\u003eEach animal was placed in a box (30 \u0026times; 30 \u0026times; 15 cm) with the floor divided into nine equal quadrants and observed for 5 min. The number of quadrants crossed with all four paws (crossings) indicated locomotor activity, and the number of rearings indicated exploratory activity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Tail suspension test\u003c/h2\u003e \u003cp\u003eEach mouse was suspended by the tail at 50 cm above the floor and was observed for 6 min; the immobility time was measured in the last 4 min (the first 2 min were for habituation) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Increased immobility time was considered indicative of depressive-like behavior.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Y-maze test\u003c/h2\u003e \u003cp\u003eThe Y-maze tests spatial working memory in rodents and consists of a three-arm horizontal maze (40 cm long and 3 cm wide with 12 cm high walls). The percentage of spontaneous alternations was employed; the mice were initially placed in one arm (A), and the arm entry sequence and the number of arm entries were recorded for 6 min. Alternation indicates sequential entries into all three arms and by the formula: % alternation = [(number of alternations \u0026times; 3)/(total arm entries\u0026thinsp;\u0026minus;\u0026thinsp;2)] \u0026times; 100 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Decreased spontaneous alternation was considered indicative of cognitive deficits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4. Buried food finding test\u003c/h2\u003e \u003cp\u003eAll mice were familiarized with chocolate cookies two days before the test, and the animals received various 1.6-g pieces with water \u003cem\u003ead libitum\u003c/em\u003e. Twelve hours before testing, mice received 30% less food. For testing, each mouse was placed into clear cages (30 x 19 x 13 cm), in which a piece of a chocolate cookie was hidden under 1.5 cm of standard bedding at the end of the cage. The animals were placed in the right corner at the other end of the cage to test their olfactory memory, and the food-finding time was measured. If a mouse failed to locate the food within 300 s, the testing session was terminated, and the latency duration was defined as 300 s [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This test assesses olfactory memory, and increased latency time to find the cookie was considered indicative of cognitive deficits.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Biochemical analyses\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Tissue collection\u003c/h2\u003e \u003cp\u003eThe small intestine (removed\u0026thinsp;\u0026plusmn;\u0026thinsp;15 cm from the stomach exit, corresponding to the duodenum) was washed with 2 mL of saline solution (0.9%), sliced open, and the mucosa was collected by gently scraping with a scalpel. The hippocampi and small intestine were homogenized in 50 mM Tris-HCl pH 7.4 (1:4 w/v), followed by centrifugation at 2500 \u0026times;\u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C. The supernatant fraction was employed to determine reactive species and lipid peroxidation. For gene expression analysis, the hippocampi and small intestine were kept at -80\u0026deg;C until sample processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Quantification of reactive species\u003c/h2\u003e \u003cp\u003eReactive species formation was quantified using 2\u0026rsquo;-7\u0026rsquo;-dichlorofluorescein diacetate (DCHF-DA), as described by Loetchutinat et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. An aliquot of the supernatant (10 \u0026micro;L) was incubated with 1 mM DCHF-DA (10 \u0026micro;L) and 10 mM Tris-HCl pH 7.4 (2980 \u0026micro;L). The oxidation of DCFH-DA to fluorescent dichlorofluorescein (DCF) is measured to detect intracellular reactive species, and DCF fluorescence intensity emission was recorded at 520 nm with 480 nm excitation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. Lipid peroxidation assay\u003c/h2\u003e \u003cp\u003eAn aliquot of the supernatant (10 \u0026micro;L) was incubated with 8.1% sodium dodecyl sulfate (20 \u0026micro;L), 0.8% thiobarbituric acid (150 \u0026micro;L), and acetic acid/HCl (pH 3.4, 150 \u0026micro;L) at 95\u0026deg;C for 60 min, as described by Ohkawa et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Thiobarbituric acid reactive species levels were measured spectrophotometrically at 532 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4. Protein determination\u003c/h2\u003e \u003cp\u003eProtein concentrations were measured according to the method of Bradford [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] using serum bovine albumin as standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.4.5. RNA extraction and gene expression analysis by qRT-PCR\u003c/h2\u003e \u003cp\u003eThe total mRNA was extracted from the prefrontal cortex, hippocampus and small intestine using TRIzol reagent (Invitrogen, Carlsbad, USA), followed by DNase treatment with DNA-free kit (Ambion, USA) and mRNA quantification. The cDNA synthesis was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, UK) according to the manufacturer\u0026rsquo;s instructions. Amplification was made with UltraSYBR Mix (CoWin Bioscience Co., Pequim, China) using the Stratagene Mx3005P. Gene expressions were normalized using GAPDH as a reference gene, and the conditions for the reaction included 95\u0026deg;C for 15 s, 60\u0026deg;C for 60 s, and 72\u0026deg;C for 30 s. The delta-delta comparative threshold method was used to normalize the fold change in gene expressions. The following primers were used: nuclear factor kappa B (Nf-κb; fwd 5\u0026prime;-GCT TTC GCA GGA GCA TTA AC-3\u0026prime;, rev 5\u0026prime;-CCG AAG CAG GAG CTA TCA AC-3\u0026prime;), interleukin-1β (Il-1β; fwd 5\u0026acute;GCT GAA AGC TCT CCA CCT CAA TG-3\u0026acute; rev- 5\u0026acute;-TGT CGT TGC TTG GTT CTC CTT GC-3\u0026acute;), toll-like receptor 4 (Tlr4, F: 5\u0026acute;-GCC TTT CAG GGA ATT AAG CTC C-3\u0026acute;, R: 5\u0026acute;-AGA TCA ACC GAT GGA CGT GTA A-3\u0026acute;), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh; fwd 5\u0026prime;-AGG TCG GTG TGA ACG GAT TTG-3\u0026prime;, rev 5\u0026prime;-TGT AGA CCA TGT AGT TGA GGT CA-3\u0026prime;).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Statistical analyses\u003c/h2\u003e \u003cp\u003eData from the behavioral and neurochemical analyses are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The experimental results were analyzed by two-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s test for \u003cem\u003epost-hoc\u003c/em\u003e comparisons. Results were considered significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The main effects are only presented when the first-order interaction is non-significant. The statistical analysis was performed using the Graph Pad Prism software (version 9.0; San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1. K. pastoris KM71H attenuated depressive-like behavior of Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e-induced AD-like mice\u003c/h2\u003e \u003cp\u003eThe number of crossings and rearings were counted in the OFT before the other behavioral tests to assess whether the Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;KM71H and \u003cem\u003eK. pastoris\u003c/em\u003e KM71H treatments affected the mice\u0026rsquo;s locomotor and exploratory activities; no statistical difference was found in the number of crossings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and rearings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) compared to the control (i.e., unaltered psychomotor and exploratory activities). The TST was performed to evaluate the antidepressant-like effect of \u003cem\u003eK. pastoris\u003c/em\u003e KM71H, and Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e exposure significantly increased immobility time compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Notably, the \u003cem\u003eK. pastoris\u003c/em\u003e KM71H treatment reduced the immobility time compared to the Aβ group (interaction, F\u003csub\u003e(1,32)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;84.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In addition, the \u003cem\u003eK. pastoris\u003c/em\u003e KM71H treatment reduced immobility time in the TST compared to the control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2. K. pastoris KM71H attenuated cognitive deficits of Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e-induced AD-like mice\u003c/h2\u003e \u003cp\u003eThe animals were submitted to the Y-maze test to evaluate their spatial working memory, and Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e exposure was shown to significantly decrease spatial and working memory (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The \u003cem\u003eK. pastoris\u003c/em\u003e KM71H treatment increased the percentage of spontaneous alternations, demonstrating its ability to ameliorate memory impairment (main effect, F\u003csub\u003e(1,32)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;67, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The animals were submitted to the BFFT to evaluate their olfactory memory. As predicted, Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e administration increased the time the animals took to retrieve the chocolate cookies compared to the control, whereas the \u003cem\u003eK. pastoris\u003c/em\u003e KM71H treatment decreased the necessary time to find the chocolate cookies compared to the Aβ group, indicating its ability to improve olfactory memory (interaction, F\u003csub\u003e(1,32)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;17.2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Effect of oxidative stress of K. pastoris KM71H in the hippocampus and small intestine\u003c/h2\u003e \u003cp\u003eIn neurodegenerative diseases such as AD, oxidative stress is often implicated in their etiologies (Cheignon et al. 2018). Due to high oxygen consumption and poor antioxidant defense, the brain is particularly vulnerable to oxidative damage [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, we investigated the antioxidant effect of \u003cem\u003eK. pastoris\u003c/em\u003e KM71H treatment in Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e-induced AD-like mice, and our findings showed the Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e exposure significantly increased reactive species in the hippocampi (interaction, F\u003csub\u003e(1,32)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;10.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and small intestine (interaction, F\u003csub\u003e(1,32)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;17.6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), which was restored by \u003cem\u003eK. pastoris\u003c/em\u003e KM71H treatment. The increased reactive species may damage biomolecules, including lipids and proteins. Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e administration increased the lipid peroxidation in the hippocampi (interaction, F\u003csub\u003e(1,32)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.58, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and small intestine (interaction, F\u003csub\u003e(1,32)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;20.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), and the \u003cem\u003eK. pastoris\u003c/em\u003e KM71H restored this alteration. Together, these results suggest that \u003cem\u003eK. pastoris\u003c/em\u003e KM71H presents antioxidant activity, possibly related to improved cognition and depressive-like behavior.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Effects of K. pastoris KM71H in the TLR4/NF-κB signaling pathway in Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e-induced AD-like mice\u003c/h2\u003e \u003cp\u003eEvidence suggests that neuroinflammation is also involved in AD pathogenesis (Heneka et al. 2015), and TLR4 plays a key role in inflammation by inducing proinflammatory cytokine production, including IL-1β (Sun et al. 2020). Here, we demonstrated that Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e administration increased NRLP3 (interaction, F\u003csub\u003e(1,16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;13.2, p\u0026thinsp;=\u0026thinsp;0.002; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), TLR4 (interaction, F\u003csub\u003e(1,16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8.15, p\u0026thinsp;=\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), Nfҡb (interaction, F\u003csub\u003e(1,16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.11, p\u0026thinsp;=\u0026thinsp;0.03; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), and IL-1β (main effect, F\u003csub\u003e(1,16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;12.3, p\u0026thinsp;=\u0026thinsp;0.003; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) expression in the small intestine compared to control group, and these alterations were restored by \u003cem\u003eK. pastoris\u003c/em\u003e KM71H. In addition, Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e administration showed a trend of increasing IL-1β expression (main effect, F\u003csub\u003e(1,16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.49, p\u0026thinsp;=\u0026thinsp;0.2; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) in the hippocampi compared to the control group, and the \u003cem\u003eK. pastoris\u003c/em\u003e KM71H treatment demonstrated a trend of decreasing IL-1β expression compared to the Aβ group. These results suggest that \u003cem\u003eK. pastoris\u003c/em\u003e KM71H treatment improves cognitive deficits and depressive-like behavior via the TLR4/NF-κB signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study revealed for the first time that \u003cem\u003eK. pastoris\u003c/em\u003e KM71H ameliorated cognitive deficits and depressive-like behavior in Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e-induced AD-like mice. These effects are corroborated by the fact that \u003cem\u003eK. pastoris\u003c/em\u003e KM71H increased the percentage of spontaneous alternations in the Y-maze test, decreased the time necessary for the animal to retrieve the chocolate cookies in the BFFT, and decreased the immobility time of Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e-exposed mice. Furthermore, improved cognitive impairment and depressive-like behavior in the animals treated with the yeast may be due to the antioxidant effects elicited by \u003cem\u003eK. pastoris\u003c/em\u003e KM71H. This betterment was accompanied by lower levels of reactive species and lipid peroxidation in the hippocampi and small intestine, followed by modulation of the TLR4/NF-κB pathway as NRLP3, TLR4, Nfҡb, and IL-1β expression decreased in the small intestine.\u003c/p\u003e \u003cp\u003eAlzheimer disease is a multifactorial disease involving the periphery and CNS and is mainly characterized by the accumulation of aggregated Aβ, neurofibrillary tangles, and neurodegeneration in the brain, as well as clinical manifestations, including cognitive deficits and neuropsychiatric symptoms [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. A growing body of evidence has demonstrated that AD is a neurodegenerative disease with substantial comorbidity with MDD [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]; in fact, a meta-analysis conducted by Zhao et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] showed that MDD is present in up to 50% of AD cases. Additionally, changes in the composition of the intestinal microbiota have been hypothesized to contribute to neuroinflammation and neurodegeneration in the CNS since these changes toward gut dysbiosis are a risk factor for both clinical conditions[\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Indeed, numerous researchers have shown that the gut microbiota of depressed and AD patients exhibit different bacterial compositions compared to healthy controls and activation patterns of peripheral and central inflammatory cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Thus, probiotics are a promising therapeutic alternative for both conditions, as corroborated by the evidence of probiotic supplementation attenuating depressive-like behaviors and cognitive impairments [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent research with \u003cem\u003eK. pastoris\u003c/em\u003e KM71H showed that it also decreased TST immobility time in different animal models of depression [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and we believe the mechanism by which \u003cem\u003eK. pastoris\u003c/em\u003e KM71H confers these benefits is the modulation of the intestinal microbiota, antioxidant activity, and immunomodulation. Here, we showed that \u003cem\u003eK. pastoris\u003c/em\u003e KM71H attenuated depressive-like behavior and cognitive deficits in mice that received Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e icv injection, as supported by the decreased immobility time in the TST, increased percentage of spontaneous alternations in the Y-maze test, and decreased time for the animal to retrieve the chocolate cookies in the BFFT compared to the Aβ group.\u003c/p\u003e \u003cp\u003eThe intestinal microbiota can influence neurodegeneration by promoting Aβ aggregation and neuroinflammation. Within the given frame of reference, therapeutic strategies that modulate the intestine microbial composition toward increasing the population of beneficial bacteria, such as \u003cem\u003eBacteroidetes\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e, are of the utmost importance [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In a previous study, we showed that \u003cem\u003eK. pastoris\u003c/em\u003e increased the concentration of these genera of bacteria and, through interactions with the intestinal epithelial barrier and immune system, contributed to improving depressive-like behavior [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These findings may also be related to the improved cognitive deficit, although more robust experimental evidence is required to prove this hypothesis. Likewise, stimuli associated with inflammatory and neurodegenerative processes, including Aβ aggregates and reactive species, promote the activation of immune system components that, in turn, can alter the composition of the intestinal microbiota, leading to pathologies such as AD and MDD [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the present study, we demonstrated that Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e administration elicits oxidative stress, parameters observed by the higher levels of reactive species and lipid peroxidation in the hippocampi and small intestines of mice.\u003c/p\u003e \u003cp\u003eOur data corroborate previous studies demonstrating that Aβ aggregates promote oxidative stress, contributing to the pathogenesis and disease progression of AD [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Oxidative stress is caused by an imbalance between reactive species levels and the action of the antioxidant defense systems and is involved in the pathophysiology of several neuropsychiatric and neurodegenerative disorders [\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Therefore, there is great interest in developing new antioxidant therapeutic strategies for treating MDD and AD. Supplementation with \u003cem\u003eK. pastoris\u003c/em\u003e KM71H attenuated the increased levels of reactive species and lipid peroxidation in the hippocampi and small intestine of Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e-exposed mice, showing a promising antioxidant effect; nevertheless, these were unsurprising data as \u003cem\u003eK. pastoris\u003c/em\u003e KM71H showed antioxidant activity in previous studies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOxidative stress and increased reactive species and lipid peroxidation in the intestinal environment may impair the integrity of the intestinal barrier and contribute to changes in intestinal microbiota composition, which may lead to inflammatory responses [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Moreover, the intestinal microbiota modulates the inflammatory responses mainly by stimulating toll-like receptors (TLRs) and/or NOD-like receptors (NLRs) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The TLRs are the first line of defense, recognizing molecules present in a pathogen, known as pathogen-associated molecular patterns (PAMPs), triggering proinflammatory cytokine expression [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Notably, TLR4 is stimulated by numerous PAMPs, including lipopolysaccharide from Gram-negative bacteria inducing the release of proinflammatory cytokines necessary to activate potent immune responses [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Furthermore, Aβ is also a ligand for TLRs, which can initiate the inflammatory process in the gut and CNS, leading to the development of neurodegenerative diseases [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Indeed, animals that received Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e showed increased TLR4 expression in the small intestine, and the \u003cem\u003eK. pastoris\u003c/em\u003e KM71H restored this alteration. We believe that the decreased TLR4 expression in the small intestine was due to yeast supplementation improving the integrity of the intestinal barrier, thereby preventing the translocation of enteric bacteria and their products into the lamina propria and preventing the inflammatory response. Although more studies are needed to confirm this premise, previous research has shown that \u003cem\u003eK. pastoris\u003c/em\u003e KM71H helps maintain the integrity of the intestinal barrier and decreases TLR4 expression in the small intestine in a model of antibiotic-induced intestinal dysbiosis in mice [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere is also evidence that Aβ aggregates and reactive species promote nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. As a result, it can lead to a shift in intestinal microbiota towards a proinflammatory phenotype, contributing to inflammatory responses in brain disorders [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Evidence has also shown that patients with AD and MDD present enteric and brain activation of the NLRP3 inflammasome [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Here, Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e increased NLRP3 expression in the intestine, while the \u003cem\u003eK. pastoris\u003c/em\u003e KM71H treatment restored this alteration. This effect may have contributed to improving cognitive deficit and depressive-like behavior in the animals since the NLRP3 is involved in regulating the microbiota-gut-brain axis, and their modulation counteracts CNS disease progression in animal models [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent stimuli can promote canonical and/or non-canonical NLRP3 inflammasome activations; for example, Gram-negative bacteria activate the TLR4-MyD88 pathway, with consequent transcription of IL-1β and NLRP3 genes through NF-κB activation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Indeed, we observed increased TLR4, NFKB, and IL-1β expression in the small intestine and a trend of increased IL-1β expression in the hippocampi of Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e-exposed mice. Supplementation with \u003cem\u003eK. pastoris\u003c/em\u003e KM71H decreased NFKB and IL-1β expression in the small intestine and presented a trend to decrease IL-1β expression in the hippocampi. We believe that these effects are attributed to the ability of \u003cem\u003eK. pastoris\u003c/em\u003e KM71H to modulate the gut-brain axis through its antioxidant and immunomodulatory activity. Clinical data have shown that patients with AD and MDD are characterized by changes in gut microbiota composition, oxidative stress, enteric, peripheral, and brain activation of the NLRP3 inflammasome, and impairment of intestinal and brain barriers, facilitating the activation of immune/inflammatory pathways [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In order to minimize these effects, therapeutic strategies are needed. Therefore, we highlight \u003cem\u003eK. pastoris\u003c/em\u003e KM71H, which is a potent probiotic with antioxidant and immunomodulatory activity, restoring the integrity of the brain and intestinal barrier and promoting the modulation of the microbiota composition.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings are the first to indicate that \u003cem\u003eK. pastoris\u003c/em\u003e KM71H ameliorated cognitive deficits and depressive-like behavior in Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e-induced AD-like mice. Therefore, we hypothesize that \u003cem\u003eK. pastoris\u003c/em\u003e KM71H can be a new target for developing new probiotic treatments for comorbidities between AD and MDD.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAD, Alzheimer disease; BFFT, buried food-finding test; DCF, dichlorofluorescein; DCHF-DA, 2\u0026rsquo;-7\u0026rsquo;-dichlorofluorescein diacetate; ICV, intracerebroventricularly; MDD, major depressive disorder; OFT, open field test; TST, tail suspension test;\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCoordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq), and Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado do Rio Grande do Sul (FAPERGS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed in the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.T. Birmann performed the experimental design, behavioral tests, biochemical assessments, and data analysis and wrote the manuscript. L. Savegnago performed the experimental design, data analysis, writing, reviewing, and editing of the manuscript, supervised the experiments, acquired funding, and is the corresponding author*. G.P. Zugno and A. Sinott performed the behavioral tests and the biochemical assessments and wrote the manuscript. F.S.S. Sousa, F.K. Seixas, and T. Collares performed the qRT-PCR analyses. Rochedo and R.R. Rodrigues performed the cultivation and preparation of K. pastoris. L. Savegnago and F.C. Rochedo were responsible for funding acquisition and reviewing and editing the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthics approval\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe experiments with animals were carried out in accordance with the Institutional Animal Care and Use Committee of the Federal University of Pelotas (approval number of the ethics committee: 026745/2021-90), affiliated with the National Council for Animal Experimentation Control (CONCEA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to UFPel, especially Biotechnology Graduate Program (UFPel), for supporting this work and thank CNPq, CAPES, and FAPERGS (PRONEM 16/2551-0000240-1, PqG 17/2551-00011046-9) for the financial support. L.S., T.C., F.K.S., and F.R.C. are recipients of CNPq fellowships. This study was partly financed by the CAPES - Finance Code 001. We would also like to thank Atlas Assessoria Lingu\u0026iacute;stica for language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eConsultation FWJRfFWE (2001) Evaluation of health and nutritional properties of powder milk and live lactic acid bacteria. \u003c/li\u003e\n\u003cli\u003eDidari T, Solki S, Mozaffari S, Nikfar S, Abdollahi MJEoods (2014) A systematic review of the safety of probiotics. 13 (2):227-239\u003c/li\u003e\n\u003cli\u003eShruthi B, Deepa N, Somashekaraiah R, Adithi G, Divyashree S, Sreenivasa MY (2022) Exploring biotechnological and functional characteristics of probiotic yeasts: A review. 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Lancet Neurol 14 (4):388-405. doi:10.1016/S1474-4422(15)70016-5\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Yeast, probiotic, depression, cognitive, microbiota-gut-brain axis","lastPublishedDoi":"10.21203/rs.3.rs-6431395/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6431395/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eKomagataella pastoris\u003c/em\u003e is a promising probiotic for modulating the microbiota-gut-brain axis, and growing evidence has demonstrated the relevance of this axis in the pathophysiology of neurological and psychiatric disorders, such as Alzheimer disease (AD) and major depressive disorder. This study investigated the ameliorated cognitive impairment and antidepressive-like effect of \u003cem\u003eK. pastoris\u003c/em\u003e treatment on an AD-like mouse model induced by amyloid β\u003csub\u003e1\u0026minus;40\u003c/sub\u003e peptide (Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e). Behavioral tests revealed that Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e administration (400 pmol/mouse, icv) increased immobility time in the tail suspension test, decreased spatial and working memory, and increased the time animals took to reach the chocolate cookies, and these behavioral changes were attenuated by the \u003cem\u003eK. pastoris\u003c/em\u003e KM71H treatment (8 log CFU\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e/mouse, ig). Biochemical changes also occurred as the applied protocol decreased reactive species and lipid peroxidation in the hippocampi and small intestine and reduced NRLP3, TLR4, Nfҡb, and IL-1β expression in the small intestine of mice. We believe that the ameliorated cognitive impairment and antidepressant effects of \u003cem\u003eK. pastoris\u003c/em\u003e KM71H are due to its antioxidant activity and the modulation of the TLR4/NF-κB pathway, and our findings led us to conclude that \u003cem\u003eK. pastoris\u003c/em\u003e KM71H is a promising therapeutic strategy for treating Alzheimer disease and major depressive disorder.\u003c/p\u003e","manuscriptTitle":"Komagataella pastoris KM71H attenuates cognitive deficits and depressive-like behavior by the TLR4/NF-κB signaling pathway in an Aβ 1-40 -induced AD-like mouse model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 07:45:48","doi":"10.21203/rs.3.rs-6431395/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-03T17:47:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-03T04:29:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-23T06:58:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-22T05:57:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176024847732694092041676453589615041964","date":"2025-05-19T03:07:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"190400062939285149091605702716282038653","date":"2025-05-15T02:07:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92047763012090099525142758719433918407","date":"2025-05-14T19:13:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221470110522016596378249992795181043951","date":"2025-05-13T05:02:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"184319696672487235265221052588982609101","date":"2025-05-13T01:50:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-12T19:09:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-01T00:13:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-01T00:10:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2025-04-11T23:37:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"99fa4d00-a23c-43a0-a343-d112c20e3659","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-11T16:03:45+00:00","versionOfRecord":{"articleIdentity":"rs-6431395","link":"https://doi.org/10.1007/s12035-025-05267-9","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2025-08-06 15:57:59","publishedOnDateReadable":"August 6th, 2025"},"versionCreatedAt":"2025-05-16 07:45:48","video":"","vorDoi":"10.1007/s12035-025-05267-9","vorDoiUrl":"https://doi.org/10.1007/s12035-025-05267-9","workflowStages":[]},"version":"v1","identity":"rs-6431395","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6431395","identity":"rs-6431395","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}