ORIGINAL RESEARCH published: 31 January 2022 doi: 10.3389/fnagi.2021.815361 Aged Cattle Brain Displays Alzheimer’s Disease-Like Pathology and Promotes Brain Amyloidosis in a Transgenic Animal Model Ines Moreno-Gonzalez 1,2,3,4*, George Edwards III 1 , Rodrigo Morales 1,4 , Claudia Duran-Aniotz 1,5,6 , Gabriel Escobedo Jr. 1 , Mercedes Marquez 7 , Marti Pumarola 7,8 and Claudio Soto 1* 1 Department of Neurology, Mitchell Center for Alzheimer’s Disease and Related Brain Disorders, University of Texas Health Science Center at Houston, Houston, TX, United States, 2 Departamento Biología Celular, Genética y Fisiología, Instituto de Investigacion Biomedica de Malaga-IBIMA, Universidad de Malaga, Malaga, Spain, 3 Center for Biomedical Research on Neurodegenerative Diseases (CIBERNED), Madrid, Spain, 4 Centro Integrativo de Biologia y Quimica Aplicada (CIBQA), Universidad Bernardo O’Higgins, Santiago, Chile, 5 Center for Social and Cognitive Neuroscience (CSCN), School of Psychology, Universidad Adolfo Ibáñez, Santiago, Chile, 6 Latin American Institute for Brain Health (BrainLat), Universidad Adolfo Ibanez, Santiago, Chile, 7 Department of Animal Medicine and Surgery, Veterinary Faculty, Animal Tissue Bank of Catalunya (BTAC), Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Valles), Barcelona, Spain, 8 Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Universitat Autonoma de Barcelona, Edited by: Bellaterra (Cerdanyola del Valles), Barcelona, Spain Nilton Custodio, Peruvian Institute of Neurosciences (IPN), Peru Alzheimer’s disease (AD) is one of the leading causes of dementia in late life. Although Reviewed by: the cause of AD neurodegenerative changes is not fully understood, extensive evidence Diana Laura Castillo-Carranza, University of Monterrey, Mexico suggests that the misfolding, aggregation and cerebral accumulation of amyloid beta (Aβ) M. Heather West Greenlee, and tau proteins are hallmark events. Recent reports have shown that protein misfolding Iowa State University, United States and aggregation can be induced by administration of small quantities of preformed *Correspondence: Ines Moreno-Gonzalez aggregates, following a similar principle by which prion diseases can be transmitted inesmoreno@uma.es by infection. In the past few years, many of the typical properties that characterize Claudio Soto prions as infectious agents were also shown in Aβ aggregates. Interestingly, prion Claudio.Soto@uth.tmc.edu diseases affect not only humans, but also various species of mammals, and it has been Specialty section: demonstrated that infectious prions present in animal tissues, particularly cattle affected This article was submitted to by bovine spongiform encephalopathy (BSE), can infect humans. It has been reported Alzheimer’s Disease and Related Dementias, that protein deposits resembling Aβ amyloid plaques are present in the brain of several a section of the journal aged non-human mammals, including monkeys, bears, dogs, and cheetahs. In this study, Frontiers in Aging Neuroscience we investigated the presence of Aβ aggregates in the brain of aged cattle, their similarities Received: 15 November 2021 Accepted: 17 December 2021 with the protein deposits observed in AD patients, and their capability to promote AD Published: 31 January 2022 pathological features when intracerebrally inoculated into transgenic animal models of Citation: AD. Our data show that aged cattle can develop AD-like neuropathological abnormalities, Moreno-Gonzalez I, Edwards G III, including amyloid plaques, as studied histologically. Importantly, cow-derived aggregates Morales R, Duran-Aniotz C, Escobedo G Jr, Marquez M, Pumarola M and accelerate Aβ amyloid deposition in the brain of AD transgenic animals. Surprisingly, Soto C (2022) Aged Cattle Brain the rate of induction produced by administration of the cattle material was substantially Displays Alzheimer’s Disease-Like Pathology and Promotes Brain higher than induction produced by injection of similar amounts of human AD material. Our Amyloidosis in a Transgenic Animal findings demonstrate that cows develop seeding-competent Aβ aggregates, similarly as Model. observed in AD patients. Front. Aging Neurosci. 13:815361. doi: 10.3389/fnagi.2021.815361 Keywords: amyloid, prions, Alzheimer’s disease, spreading, protein misfolding, seeding, cattle Frontiers in Aging Neuroscience | www.frontiersin.org 1 January 2022 | Volume 13 | Article 815361 Moreno-Gonzalez et al. Alzheimer’s Disease Pathology in Aged Cows INTRODUCTION induction has been observed by the addition of misfolded protein aggregates prepared in vitro using synthetic Aβ (Stöhr et al., Alzheimer’s disease (AD) is the most common form of dementia 2012). Accumulation of Aβ aggregates can be promoted by among elderly people and one of leading public health problems inoculation of very small amounts of aggregated seeds (Fritschi in developed countries. AD involves progressive brain atrophy, et al., 2014; Morales et al., 2015a) and titration experiments have neuronal death, synaptic dysfunction, astrogliosis and the shown that the rate of induction is proportional to the amount of accumulation of protein aggregates in the form of amyloid- seeds inoculated. Finally, disease transmission has been observed beta (Aβ) deposits, and tau neurofibrillary tangles. Although the even when seeds were administered systemically (Eisele et al., etiology of AD is not yet clear, extensive evidence suggests that 2010). the central pathological event is the misfolding, aggregation and The findings described above suggest that Aβ and other brain deposition of Aβ and tau (Soto, 2003; Huang and Mucke, misfolded protein aggregates can indeed behave as prions. Still, 2012; Masters and Selkoe, 2012). Amyloid accumulates as senile the main controversy is whether other misfolded proteins can plaques and diffuse deposits in the brain parenchyma and around act as infectious agents to transmit the disease among individuals cerebral blood vessels walls termed cerebral amyloid angiopathy under natural conditions (Fernández-Borges et al., 2013; Irwin (CAA) (Gomez-Isla et al., 2008). et al., 2013; Beekes et al., 2014; Collinge, 2016). Aside from this In addition to AD, various other protein misfolding diseases important point, another aspect that has not been explored is (PMDs) are thought to be caused by accumulation of misfolded the possibility that protein aggregates accumulating in animals aggregated proteins in various tissues, including highly prevalent may initiate the disease in humans. Indeed, prion diseases affect illnesses such as Parkinson’s disease (PD), type 2 diabetes and not only humans, but also various species of mammals. The more than 20 other diseases (Chiti et al., 2006; Soto, 2012). accumulation of Aβ aggregates has not been extensively analyzed Among the latter, prion diseases are quite intriguing because in animals, however, it has been reported that AD aggregates they are transmissible by infection through a proteinaceous are present in the brain of several aged non-human mammals, infectious agent known as prion (Prusiner, 1998). The molecular including monkeys, bears, dogs, and cheetahs (Moreno-Gonzalez mechanism responsible for prion infectivity depends on the and Soto, 2018). In bovine brains, Aβ has been as previously ability of the misfolded prion protein aggregates to act as observed as granular aggregates, but not depositing in plaques seeds, inducing the templated conversion of natively folded (Costassa et al., 2016). In this study, we investigated the presence prion protein into the developing aggregates (Soto, 2012). of AD-like pathology in aged cow brains and whether, in analogy In this manner, the pathological protein progressively grows to prion diseases, Aβ aggregates derived from cattle brain can by recruiting more and more of the normal protein. This induce Aβ misfolding and aggregation in a transgenic mice model process is often referred as seeding/nucleation polymerization. of AD amyloidosis. Importantly, protein aggregation of all proteins involved in PMDs follows the seeding-nucleation mechanism (Caughey and Lansbury, 2003; Soto, 2012). The similarities between prion MATERIALS AND METHODS replication and amyloid formation, and the intrinsic ability of Cattle Samples aggregated seeds to self-propagate the polymerization process led Cattle samples were obtained from the Animal Tissue Bank us and others to hypothesize over 10 years ago that misfolded of Catalunya (BTAC), Department of Animal Medicine and aggregates associated to other PMDs can spread by the prion Surgery, Veterinary Faculty, Universitat Autonoma de Barcelona, principle (Soto et al., 2006; Walker et al., 2006). Remarkably, Bellaterra (Cerdanyola del Valles), Barcelona, Spain. Brain a series of exciting recent reports have demonstrated that samples were obtained from slaughterhouses after the animals several PMDs can be experimentally transmitted by a prion- were sacrificed. Samples with a post-mortem time lower than like mechanism in various cellular and animal models of diverse 10 h were immersed in formol, processed and embedded in diseases (Prusiner, 2012; Walker and Jucker, 2015). Over the past paraffin. As shown in Supplementary Figure 1, we received 63 decade, many of the hallmark properties of prions as infectious samples from female cattle that were 13 to 23 years old. These agents have been shown to be shared by several of the prion- animals were from 10 different breeds, including Charolais, like misfolded proteins. For the specific case of Aβ, studies from Bruna of Pirineus, Pirinenca, Limousin, Montbeliard, Simmental, us and other groups have shown that inoculation of transgenic and crossbreeds. We also obtained ten samples of 10 month-old mouse models of amyloidosis with tissue homogenates from young crossbreed calves that were used as controls. Samples were patients affected by AD results in induction or acceleration of obtained from the temporal area and contained the hippocampal amyloid pathology in the recipient animals (Kane et al., 2000; area, entorhinal cortex, and part of the thalamus. Meyer-Luehmann et al., 2006; Morales et al., 2011; Watts et al., 2011). Moreover, in animals not genetically programmed to Human Samples develop the disease spontaneously, inoculation with AD brain AD brain hippocampal samples were acquired from the National homogenates leads to a completely de novo disease, more akin Disease Research Interchange (USA). Informed consent was to infectious prions (Morales et al., 2011; Rosen et al., 2011). obtained for experimentation with human subjects. The Code Importantly, pathological induction can be reduced by depleting of Ethics of the World Medical Association (Declaration of the inoculum of Aβ aggregates (Meyer-Luehmann et al., 2006; Helsinki) was followed to perform research on human samples Duran-Aniotz et al., 2014). Even more strikingly, efficient and they were manipulated following the universal precautions Frontiers in Aging Neuroscience | www.frontiersin.org 2 January 2022 | Volume 13 | Article 815361 Moreno-Gonzalez et al. Alzheimer’s Disease Pathology in Aged Cows for working with human samples and as directed by the age (Jankowsky et al., 2004). Animals were housed in groups of up Institutional Review Board of McGovern Medical School at The to five in individually ventilated cages under standard conditions University of Texas Health Science Center at Houston. (22◦ C, 12 h light–dark cycle) receiving food and water ad libitum. All animal experiments were carried out in accordance with the Brain Homogenate NIH regulations and approved by the committee of animal use Cattle and human brain tissue were homogenized at 10% w/v for research at The University of Texas Health Science Center at in PBS containing a cocktail of protease inhibitors for western Houston. Mice were sacrificed by CO2 inhalation and perfused blot and ELISA quantifications. For intracerebral inoculations, transcardially with PBS. Brains were removed, post-fixed into samples were homogenized at 40% w/v in the same solution and fixative solution (10% neutral buffered formalin) and embedded sterilized by the addition of 1% of antibiotic/antimycotic solution in paraffin. (Gibco) and γ-irradiated for 1 h. Animal Inoculation Western Blot For intracerebral inoculation, 30–40 days-old APP/PS1 mice 10% cattle brain homogenates were mixed with denaturing were injected with 10 µL of 40% (w/v) cattle or human brain loading buffer (Invitrogen), heated for 10 min at 95 ◦ C and homogenate in each hemisphere (n = 8 to 11 per group), without fractionated in 4–12% NuPAGE gels (Invitrogen). Proteins any purification or isolation. Briefly, mice were anesthetized were transferred to a nitrocellulose membrane (GE Healthcare), using isoflurane. Skin was incised and a small hole was drilled blocked with 10% milk, and incubated with rabbit anti-Aβ42 in the skull and samples were injected into both hippocampi. polyclonal antibody (Covance). After incubation with secondary At the end of the treatment, skin was closed using surgical antibody, Aβ42 was visualized by chemoluminescence using ECL suture. Animals were placed on a thermal pad until recovery and plus (GE Healthcare) in a dark chamber (BioRad). monitored daily for several days. ELISA Histological Quantification Mouse brain hemispheres were homogenized at 10% w/v in PBS Burden quantification was done through the lateromedial extent containing a cocktail of protease inhibitors. Brain extracts were of the cortical and hippocampal areas in the sagittal plane, centrifuged at 32,600 rpm for 1 h at 4◦ C in an ultracentrifuge being the first section of each animal randomly collected. (Beckman-Coulter). The pellets were resuspended in 200 µL Quantification was assessed in every tenth section (with a of 70% formic acid followed by sonication. Samples were distance of 100 µm among them), and four to six 10 µm centrifuged for 30 min in the same conditions and the sections were measured for each animal (n = 8–11 per group). supernatant was collected. This insoluble fraction was neutralized Photomicrographs were taken by using a DFC310 FX Leica in 1M Tris buffer, pH 11. Brain levels of Aβ42 were measured digital camera, imported into ImageJ 1.45s software (NIH) and using a human Aβ ELISA kit (Invitrogen) on an ELISA plate converted to black and white images. Threshold intensity was reader (EL800 BIO-TEK). manually set and kept constant, and the number of pixels Histology was determined for 4G8 immunostained sections to quantify Serial 10-µm-thick sections from all cattle, human, and mouse amyloid load in the hippocampal formation (CA1, CA2, CA3, groups were processed for immunostaining. After blocking and dentate gyrus) and several cortical areas (including motor, the endogenous peroxidase activity with 3% H2 O2 −10% somatosensory, visual, frontal, parietal and retrosplenial cortex). methanol for 20 min, sections were incubated overnight at Analysis for each was done by a single examiner blinded to room temperature in mouse anti-Aβ 4G8 (1:1,000 Covance). sample identities. Sections stained for Aβ were pretreated with 85% formic acid. Primary antibody was detected by incubating 1 h with goat Statistical Analysis anti-mouse HRP-linked secondary antibody and the peroxidase Graphs are expressed as means ± standard error (s.e.m.). After reaction was visualized using a DAB Kit (Vector) following confirming normal distribution with Skewness and Kurtosis the manufacturer’s instructions. For counterstaining, sections statistic test, T-test or one way analysis of variance (ANOVA) were incubated in Harris hematoxylin for 1 min (Fisher). For followed by a post-hoc Tukey’s multiple comparisons test were Thioflavin-S (ThS) staining, tissue slices were incubated in ThS used to analyze differences among groups. Statistical analyses solution (0.025% in 50% ethanol) for 8 min. Finally, all sections were performed using GraphPad Prism 5.0 software (GraphPad were dehydrated in graded ethanol, cleared in xylene, cover- Software Inc). Statistical differences for all tests were considered slipped with DPX mounting medium, and examined under a significant at the p < 0.05 level. bright field/epifluorescent microscope (DMI6000B, Leica). RESULTS Animals Hemizygous APP/PS1 (B6C3-Tg APPswe, PSEN1dE9 85Dbo/J, To investigate whether cattle develops AD-related abnormalities, The Jackson Laboratory) mice express human amyloid precursor we performed a detailed histological study for the presence protein (APP695swe) and a mutant human presenilin 1 (PS1- of amyloid-like deposits in the brain of cows of different dE9) in a B6C3 background. These animals develop amyloid ages. The majority of the samples were from female plaques and other AD-like features starting around 6 months of cattle of the Bruna of Pirineus and various crossbreed Frontiers in Aging Neuroscience | www.frontiersin.org 3 January 2022 | Volume 13 | Article 815361 Moreno-Gonzalez et al. Alzheimer’s Disease Pathology in Aged Cows (Supplementary Figure 1A). Old animal ages ranged from 13 to 23 years old (Supplementary Figure 1B). For this study, we analyzed the hippocampus, temporal cortex, and thalamus of more than 60 cows (Supplementary Figure 1C). For most of the specimens analyzed, we had access to both snap-frozen and formalin-fixed material. Positive and negative controls included human AD brains and 10 month-old calf brains, respectively. Paraffin embedded fixed tissue from all the specimens (cattle and human samples) were processed and stained for Aβ. The amino acid sequence of Aβ in both humans and cattle is exactly the same (Johnstone et al., 1991); therefore, we were able to use the same antibodies. After a complete histopathological analysis, we observed a scattered appearance of Aβ deposits in many slices of some aged cow brains (Figure 1A). Aβ deposits were detected in the cortex and hippocampus of various cows >13 years old, but not in any young animals. Cow amyloid deposits were reactive against human anti-Aβ antibody (4G8), depositing as mature plaques similar to those seen in human AD patients’ brains (Figure 1A, left panels). We also observed perivascular deposits, which are highly reminiscent of cerebral amyloid angiopathy lesions commonly seen in human AD brains (Figure 1A, center FIGURE 1 | Accumulation of Aβ deposits in aged cattle brain. (A) Histological panels). In addition, intracellular Aβ staining was observed in analysis of the brain of old cattle (top panels) showed the presence of diverse both cattle and human brains (Figure 1A, right panels). Most types of Aβ−immunoreactive lesions, including parenchymal amyloid plaques of the deposits observed in cattle brain corresponded to fibrillar (left), perivascular deposits (center) and intracellular aggregates (right). These deposits were similar to those observed in patients affected by AD (bottom amyloid aggregates, as confirmed by the positive staining of panels). Staining was done using the 4G8 anti-Aβ antibody in sections of the the plaques using thioflavin-S (ThS) in the consecutive slice temporal area. (B) The parenchymal deposits were also ThS-positive when (Figure 1B). In total, 14 out of 63 brains from cows older than 13 stained in the consecutive slide, showing that cattle aggregates also acquire years of age displayed Aβ aggregates in the brain areas studied, fibrillar structure. (C) Histology data was analyzed to quantify the Aβ burden, although just nine of them presented Aβ plaques similar to those defined as the percentage of the brain area occupied by Aβ−reactive deposits. Data represent the mean ± standard error of the mean and differences were observed in humans (14.3%). To examine the relative amount analyzed by an unpaired student T-test (p < 0.001, N = 5–14). Scale bar in (A) of amyloid deposits observed in cattle brain and compare it and (B) corresponds to 50 µm. ***p < 0.001. with that present in patients affected by AD, we performed image analysis of the temporal area from 14 old cows exhibiting amyloid pathology and five patients with AD (Figure 1C). The results show that the brains of old cows have a substantial same amount of old cow brain homogenate in which amyloid amount of amyloid deposits. Indeed, in the hippocampal region, deposition was not detected by histological or biochemical the animals analyzed showed an amyloid burden of 0.37 ± analysis (old cow/Aβ−); (iii) a control group injected with 0.07%, which means that 0.37% of the brain area analyzed was 40% brain extract from a young cow; (iv) a negative control occupied by amyloid. Nevertheless, this burden is significantly consisting of untreated animals; and (v) a positive control in smaller (>10-fold lower) than the one estimated on the AD animals injected with human AD brain homogenate. Before patients (3.82 ± 1.0%) analyzed (Figure 1C). We could not treatment, the inocula for the different groups were thoroughly detect any Aβ aggregates in young cattle brain samples. analyzed by histological and biochemical techniques to measure We and others have shown that inoculation of Aβ−rich the presence and quantity of Aβ aggregates. Figure 2A shows the brain homogenate from AD patients or transgenic mice is able comparative histological staining of representative brain sections to induce an early AD-like pathology in recipient transgenic from the different subjects used for inoculation. No amyloid animals (Meyer-Luehmann et al., 2006; Eisele et al., 2010; staining was seen in any of the slices analyzed from the young Morales et al., 2011). Knowing that the sequence of cattle cow or the old cow/Aβ−. In contrast, the old cow/Aβ+ exhibited Aβ peptide is identical to the human one, we wanted to test scattered appearance of 4G8 positive deposits reminiscent of whether cattle brain harboring amyloid aggregates exacerbate AD amyloid plaques, similar in morphology to human AD brains pathology in susceptible mice. To that end, we intracerebrally (Figure 2A). Biochemical analysis of cattle brain homogenate by (i.c.) injected amyloid-containing cow brain tissue extracts into a western blot demonstrated that the specimen selected for the double transgenic (APP/PS1) mouse model of brain amyloidosis experimental group (old cow1/Aβ+) presented a wide range of (Jankowsky et al., 2004) and analyzed the possible acceleration of high molecular weight species reactive with an antibody that the pathology. For the experiment, the following five groups (n = recognizes specifically human Aβ42 (Figure 2B). Only some of 8–11 per group) of animals were used: (i) the experimental group these bands were detectable and at a much lesser intensity in 10- injected with 40% brain homogenate from an old cow containing month-old young cow or the old cows/Aβ−, used as age-matched amyloid deposits (Old cow/Aβ+); (ii) a group injected with the controls. The samples indicated with an asterisk in Figure 2B Frontiers in Aging Neuroscience | www.frontiersin.org 4 January 2022 | Volume 13 | Article 815361 Moreno-Gonzalez et al. Alzheimer’s Disease Pathology in Aged Cows were significantly higher than in young animals, they represent only around 1% of the levels detectable in human AD brain homogenate (2,788.78 pg/mg) (Figure 2C). To evaluate the ability of Aβ aggregates deposited in cattle brain to seed amyloid formation in vivo, double transgenic animals were injected i.c. at 30–40 days old with 10 µL of 40% amyloid-containing old cow brain homogenate into both hippocampal hemispheres through stereotactic surgery (Tg + Old cow/Aβ+). In addition, various groups of control animals were treated with the diverse inocula described above. Animals were sacrificed at 6 months-old, when they normally start developing plaques due to the transgenic expression of the mutant genes. 4G8 immunostaining showed that animals injected with amyloid-containing cattle brain homogenate displayed more amyloid plaques in the cortical area than the control groups (Figure 3A, top panels). When Aβ load was measured by image analysis, we could observe that there was a statistically significant increase in Aβ burden (stained area/total area analyzed) in the group injected with old cow/Aβ+. The increase was as much as 2-fold (0.24 ± 0.04 vs. 0.11 ± 0.03 %) compared to non-treated animals (Figure 3B). Analysis of Aβ deposition in the hippocampus, where the inoculation occurred, showed similar results (Figure 3A, bottom panels). Quantification of the hippocampal amyloid burden in experimental and control groups also showed that animals injected with Aβ−containing cattle brain homogenate doubled the regular amount of Aβ (0.11 ± 0.02 vs. 0.05 ± 0.01%) found in the hippocampus of APP/PS1 at this age (Figure 3C). Moreover, staining of fibrillar aggregates by ThS also showed that animals injected with Aβ−containing cow brain tissue displayed more ThS-positive plaques in both cortex and hippocampus FIGURE 2 | Histological and biochemical characterization of materials used for than control groups (Figure 4). These results indicate that in vivo transmission experiments. (A) Representative pictures of cattle brains amyloid deposits present in cattle brain homogenate contain stained with the 4G8 anti-Aβ antibody that were utilized for in vivo inoculation, seeding-competent Aβ aggregates that are able to accelerate including young animals and old cows with (Aβ+) and without Aβ (Aβ−). The Aβ deposition when inoculated into a transgenic animal model figure also shows the staining for the AD human brain utilized for these of AD. experiments as a positive control. (B) Aliquots of 10 µL from the total brain extracts from old and young cows were analyzed by western blot using a To compare the seeding capability of cattle and human Aβ rabbit-Aβ42 polyclonal antibody. For comparison, the same amount of the AD aggregates to induce amyloid deposition, APP/PS1 transgenic brain extract was also included. Samples labeled with an asterisk (*) were used animals were also inoculated with human brain extracts from for the in vivo transmission studies described in Figure 3. A black line a patient affected by AD. The amyloid deposition pattern and separating the lanes indicates gel splicing. (C) Quantification of insoluble Aβ42 burden was analyzed by immunohistochemistry in both groups. aggregates by serial extraction, centrifugation and ELISA (see Methodology) expressed in pg per mg of brain tissue. Data represent the average ± standard As shown in Figure 5A, the human inoculum was able to induce error of the mean for three replicates. Differences on the levels between human amyloid deposition to a substantially higher extent than the AD and old cow (Aβ+) brains were evaluated by unpaired student T-test (***p cattle inoculum in both cortex and hippocampus. In the cortical < 0.001). Insoluble Aβ was not detected (N/D) in either the old cow (Aβ−) or area, the human brain injection increases amyloid burden 4-fold the young cow brain extracts. more than the cattle inoculum (0.92 ± 0.22 vs. 0.24 ± 0.04%) (Figure 5B, p = 0.003 T-test), whereas in the hippocampus the increase was ∼10-fold (1.59 ± 0.30 vs. 0.15 ± 0.2%) (Figure 5C, p < 0.0001 T-test). However, when the induction ratio (amyloid were used for the transmissibility experiments described below. burden/concentration of insoluble Aβ42 injected) was calculated, Finally, we measured the amount of Aβ42 in the insoluble the data indicates that cattle material promotes amyloid fraction after extraction with formic acid in each of the inocula deposition >10-fold better than an equivalent quantity of human (Figure 2C). The results showed a concentration of insoluble Aβ aggregates (Figures 5D,E). This surprising data suggests Aβ42 of 31.72 pg/mg of tissue in the old cow/Aβ+. In contrast, that although the cattle sample contains less concentration of no detectable insoluble Aβ42 was observed in the young cow aggregated Aβ, these structures appear to be more competent or the old cow/Aβ−. It is important to highlight that even to seed amyloid deposition than human samples. In addition, though the levels of insoluble Aβ42 in the old cow/Aβ+ brain the human aggregates produced a higher proportion of smaller Frontiers in Aging Neuroscience | www.frontiersin.org 5 January 2022 | Volume 13 | Article 815361 Moreno-Gonzalez et al. Alzheimer’s Disease Pathology in Aged Cows FIGURE 4 | Aβ−rich cattle tissue induced high amounts of fibrillar plaques. Representative pictures of ThS staining in APP/PS1 transgenic mice inoculated with brain extracts from Aβ−containing old cattle, old cow with no detectable Aβ aggregates, and young cattle. For a negative control, we used transgenic mice left untreated. Scale bar: 250 µm. these diseases and the development of novel strategies for therapeutic intervention (Prusiner, 2012; Soto, 2012; Walker and Jucker, 2015). The prion principle of disease transmission posits that a misfolded protein aggregate is able to transfer biological information by converting the normal form of the FIGURE 3 | Acceleration of amyloid pathology in transgenic mice by injecting protein into more of the abnormal, disease-associated form. This Aβ−rich cow brain extract. APP/PS1 double transgenic mice (30–40 days old) process initiates the progressive generation of misfolded protein were injected intra-cerebrally (both hemispheres at the level of the aggregates that spread to other areas of the brain and accumulate hippocampus) with 10 µL of 40% (w/v) brain extracts of a young cow or old over time to produce brain damage and disease. The prion- cows with (Aβ+) or without (Aβ−) Aβ. Control animals were left untreated. Animals were sacrificed at 6 months of age and their brains were fixed for like transmission of the pathological folding—operating at the histological analysis. (A) Representative pictures of 4G8 immunostaining of the molecular and cellular levels—is probably at the root of the cortex and hippocampal areas. Scale bar: 250 µm. Aβ burden was quantified spreading of damage throughout the brain that is characteristic by image analysis in the cortex (B) and the hippocampus (C). Data of neurodegenerative diseases (Moreno-Gonzalez and Soto, 2011; corresponds to the average ± standard error of the mean of all animals analyzed in each group (n = 7–9). Differences were analyzed by one-way Thal et al., 2014; Walker and Jucker, 2015). However, in prion ANOVA followed by the Tukey’s multiple comparison post-test. *p < 0.05, **p diseases transmission also occurs among different individuals in < 0.01, ***p < 0.001. which prions act as bona fide infectious agents to spread disease in animal or human populations. Moreover, in the case of prions the disease can be transmitted across animals from different species (Béringue et al., 2008), with the landmark case being the parenchymal and perivascular amyloid aggregates and in areas generation of a new human disease, termed variant Creutzfeldt- where plaques are not generally found in the non-treated animal, Jakob disease, produced by exposure to cattle affected by bovine such as in the corpus callosum (Figure 5A), whereas cow seeds spongiform encephalopathy (Collinge et al., 1996; Bruce et al., seem to trigger the formation of larger plaques. The differences 1997; Scott et al., 1999). Evidence gathered over the past 10 years in the seeding competency and the profile of aggregates induced has demonstrated that both protein aggregates implicated in AD suggest that Aβ aggregates present in human and cattle brain may (Aβ and tau) can propagate under experimental conditions as represent different arrangements or “conformational strains” prions (Prusiner, 2012; Soto, 2012; Morales et al., 2015a,b; Walker of Aβ seeds. and Jucker, 2015). The main goal of our study was to investigate whether cattle DISCUSSION present AD-like lesions in the brain, and if these pathological features display prion-like seeding activity. For this purpose, we The prion-like induction and spreading of misfolded protein characterized the brains from many cows at different ages for aggregates implicated in several protein misfolding disorders the presence of amyloid deposits and subsequently the ability is a recently recognized process with potentially important of those aggregates to induce amyloid pathology in a mouse implications to understand the etiology and progression of model of amyloid deposition. The results of this study show Frontiers in Aging Neuroscience | www.frontiersin.org 6 January 2022 | Volume 13 | Article 815361 Moreno-Gonzalez et al. Alzheimer’s Disease Pathology in Aged Cows FIGURE 5 | Comparison of amyloid induction by injecting Aβ− rich old cow and human AD brain extracts. (A) Representative pictures of 4G8 immunostaining in APP/PS1 transgenic mice inoculated with Aβ− containing old cattle brain extract or AD human brain homogenate. Pictures of the cortex and hippocampus regions are shown. Aβ burden in the cortex (B) and the hippocampus (C) was quantified by image analysis and expressed as the percentage of the Aβ immunoreactive area in relation to the total area analyzed. Data represents the average ± standard error of the animals analyzed (n = 7–9 per group). Since the amount of insoluble Aβ aggregates in the cow and human inoculum was substantially different, we estimated the induction ratio by dividing the Aβ burden (%) obtained in transgenic mice by the quantity of insoluble Aβ injected (pg/mg of brain) x 1,000 (arbitrary units, AU). Induction ratio was determined in the cortex (D) and the hippocampus (E) which was expressed as the average ± standard error of the mean. Data in panels B-E was analyzed by student T-test, **p < 0.01, ***p < 0.001. cc, corpus collosum; CA1, Cornu Ammonis area 1; DG, dentate gyrus. that some old cows spontaneously develop amyloid deposits in were not used as inoculum for the intracerebral inoculation study their brain. Interestingly, these deposits were similar to those to specifically evaluate the effect of Aβ aggregates. We cannot present in human cases of AD. Out of the 63 cattle brain samples exclude that brains could also contain other types of amyloid analyzed, nine of them (14.3%) displayed typical Aβ aggregates in seeds including tau oligomers that we were unable to detect. the analyzed areas. Therefore, appearance of amyloid deposits in Although tau aggregates can modulate Aβ toxicity, it has been cattle brain appears at a frequency comparable to the prevalence reported that they are not able to induce Aβ aggregation (Bloom, of AD pathological abnormalities in humans, since it is estimated 2014; Nisbet et al., 2015). This, together with the use of APP mice, that AD affects about 6% of people 65 years and older, and allows evaluating the seeding capability of Aβ using complete the incidence doubles every 5 years. Of course, many more brain homogenates, excluding the effect of potential other seeds elderly non-demented people display amyloid pathology in present in the inoculum. their brains, a condition that is usually considered as pre- Although behavioral or cognitive abnormalities were not clinical or prodromal AD (Tagliavini et al., 1988; Price et al., analyzed in cows, the amount of Aβ deposits observed parallels 2009). pre-clinical AD. Importantly, brain extracts from old cows Cattle brains were also analyzed for hyperphosphorylated tau containing Aβ aggregates were able to significantly increase (ptau) and tangle formation. We found that only three specimens amyloid deposition when injected intra-cerebrally into a double had elevated levels of ptau using AT8 antibody. These brains transgenic mouse model of AD amyloidosis. Though the total Frontiers in Aging Neuroscience | www.frontiersin.org 7 January 2022 | Volume 13 | Article 815361 Moreno-Gonzalez et al. Alzheimer’s Disease Pathology in Aged Cows level of pathological induction was lower than the one produced cattle are susceptible to develop pathological features similar by inoculation of AD brain homogenates, the cow material to AD, and that misfolded Aβ present in their brain is showed a higher promotion activity than human tissue when seeding competent. the rate of induction was corrected by the amount of Aβ aggregates injected. This surprising result suggests that cattle DATA AVAILABILITY STATEMENT aggregates are better seeds than their human counterparts. The most likely explanation for this result is that cattle deposits The raw data supporting the conclusions of this article will be are smaller, less compact and contain smaller amounts of other made available by the authors on reasonable request. components compared to human aggregates, which presumably remain deposited in the brain for much longer periods of time. ETHICS STATEMENT This interpretation is supported by our previous observations showing that brain tissue from people affected by mild cognitive The studies involving human participants were reviewed and impairment (MCI), which is considered a pre-clinical form of approved by Institutional Review Board, The University of AD, produced significantly higher Aβ induction than tissue Texas Health Science Center at Houston. Informed consent was from established AD patients (Duran-Aniotz et al., 2013). obtained by the National Disease Research Interchange (NDRI). Moreover, Jucker and colleagues have shown that small, soluble The animal study was reviewed and approved by Committee of Aβ oligomers are better seeds in vivo than large fibrillar Animal Use for Research, The University of Texas Health Science aggregates (Langer et al., 2011). We have recently published Center at Houston. data demonstrating that AD patients displaying different amyloid pathology induce different pathological features in the same AUTHOR CONTRIBUTIONS transgenic models used in this study (Duran-Aniotz et al., 2021). In that line, the distinctive pathology observed between cattle IM-G designed the experiments, participated in animal and human AD patients may be responsible of their dissimilar manipulation, performed the histological, biochemical, image, seeding activity. It could well be that these differences are due and statistical analyses, prepared the figures, and wrote the to the fact that cattle develop different strain(s) of Aβ, which manuscript. GEIII participated in animal manipulation and are able to accelerate aggregation with smaller amounts of the performed the histological analysis. RM participated in animal original seeds or more efficiently. A more in-depth study would manipulation and participated in the experimental design. CD-A be needed to determine the presence of a different Aβ strain performed histological, image, and statistical analysis. GEJr in cattle. participated in histological analysis. MM and MP provided cattle The prion-like transmission of Aβ aggregates has been samples and performed their initial neuropathological analysis. extensively reported in animal models and likely plays an CS supervised the entire project, designed the research plan, important role in the progressive spreading of pathological and wrote the manuscript. All authors read and approved the abnormalities throughout the brain (Moreno-Gonzalez and Soto, final manuscript. 2011; Thal et al., 2014; Walker and Jucker, 2015). Nevertheless, whether this phenomenon ever operates in the inter-individual FUNDING transmission of disease pathology in humans remains highly debatable. Recent studies have provided evidence for the This work was partially funded by the Alzheimer’s Association induction of Aβ aggregation in people receiving human pituitary- (NIRP-12-257323 to IM-G, AARGD-18-566576 to RM), derived growth hormone (Jaunmuktane et al., 2015; Ritchie the Spanish Ministry of Science and Innovation PID2019- et al., 2017). However, when the risk of AD development, 107090RA-I00 and Ramon y Cajal Program RYC-2017-21879 and not only amyloid pathology, was studied no evidence (IM-G), the National Institutes of Health RF1AG072491 (RM) was found for disease transmission (Irwin et al., 2013). The and 3P01AI077774-09S1 to CS, and an award from the Mitchell findings of our current study suggest that Aβ aggregates present Foundation (CS) 2018-AARG-591107, ANID/FONDEF in the brains of old cattle are competent to seed amyloid ID20I10152, ANID/FONDECYT 1210622, and Anillo deposition in vivo. This induction has also been observed ACT210096 to CD-A. with other protein aggregates such as AA amyloid (Rising et al., 2021). However, the potential transmission of Aβ cattle- SUPPLEMENTARY MATERIAL derived seeds to humans is unlikely, considering that repeated oral administration of AD brain extracts to susceptible mice The Supplementary Material for this article can be found failed to accelerate pathological features (Morales et al., 2021). online at: https://www.frontiersin.org/articles/10.3389/fnagi. The results presented in this manuscript suggest that aged 2021.815361/full#supplementary-material REFERENCES Parkinson-associated protein particles? Acta Neuropathol. 128, 463–476. doi: 10.1007/s00401-014-1324-9 Beekes, M., Thomzig, A., Schulz-Schaeffer, W. J., and Burger, R. (2014). Béringue, V., Vilotte, J. L., and Laude, H. (2008). Prion agent diversity and species Is there a risk of prion-like disease transmission by Alzheimer- or barrier. Vet. 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U S A. 108, 2528–2533. is cited, in accordance with accepted academic practice. No use, distribution or doi: 10.1073/pnas.1019034108 reproduction is permitted which does not comply with these terms. Frontiers in Aging Neuroscience | www.frontiersin.org 10 January 2022 | Volume 13 | Article 815361