Caracterización neuropatológica y evaluación preclínica de potenciales estrategias terapéuticas en modelos animales transgénicos de la enfermedad de Alzheimer

La enfermedad de Alzheimer (EA), principal causa de demencia en personas mayores de 65 años, constituye uno de los problemas socio-sanitarios más importantes de nuestra época. Su etiología no es conocida y, actualmente, no existen tratamientos farmacológicos efectivos para paliar o retrasar las deficiencias neurológicas que se producen durante su evolución. La ausencia de terapias que modifiquen el curso de la enfermedad es debida, principalmente, a la ausencia de buenos modelos animales. Por ello, la generación y caracterización de modelos que mimeticen la patología de los pacientes es clave para avanzar en la lucha contra esta devastadora enfermedad. Los modelos transgénicos, que portan una o varias mutaciones de las formas familiares del Alzheimer, son herramientas de gran valor para probar in vivo los posibles efectos terapéuticos/preventivos de fármacos potenciales, así como para investigar la evolución temporal de esta enfermedad desde estadios iniciales hasta fases más avanzadas. Por ello, y dentro de la línea de investigación de nuestro grupo, el objetivo principal de este trabajo de Tesis Doctoral ha consistido en profundizar en la caracterización neuropatológica de modelos transgénicos de la EA y su uso en la evaluación preclínica de potenciales estrategias terapéuticas para esta enfermedad. El primer objetivo específico se ha centrado en la caracterización de distintos aspectos patológicos claves de la EA en diversos modelos transgénicos de la enfermedad con objeto de aumentar su valor predictivo. Para ello, se ha estudiado la muerte neuronal de células principales e interneuronas, la patología axonal y sináptica, y la acumulación de Abeta en la región del subículo de la formación hipocampal de un modelo APP/PS1. Los resultados demuestran que este modelo presenta un fenotipo neurodegenerativo similar a lo observado en pacientes, y por lo tanto se trata de un modelo animal de gran valor predictivo para probar el efecto neuroprotector de nuevas terapias para el Alzheimer. Además, se ha determinado la existencia de un proceso degenerativo del sistema colinérgico del telencéfalo basal en modelos APP/PS1, PS1 y Tau, similar a lo ocurre en pacientes. En relación a esto, proponemos la implicación de la patología de Tau y, lo que es más novedoso, la alteración del complejo beta-secretasa en la degeneración de la población colinérgica. Como segundo objetivo específico, se han realizado dos ensayos preclínicos utilizando carbonato de litio (fármaco utilizado en el tratamiento de trastornos bipolares) y una nueva estatina. En ambos casos se ha determinado un potente efecto neuroprotector con reducción de la progresión de la patología en modelos APP/PS1, manifestando estos compuestos un gran potencial para futuros ensayos clínicos preventivos. Por último, y como tercer objetivo específico, hemos avanzado en el conocimiento del papel de la respuesta inflamatoria en la progresión de la patología mediante manipulación genética de uno de estos modelos. Para ello, se ha llevado a cabo la deleción de la interlequina antiinflamatoria IL-4 en un modelo APP. Los resultados proponen que una posible modulación farmacológica de la diferenciación microglial hacia el fenotipo de activación alternativo (anti-inflamatorio), mediada por IL-4, podría representar una nueva y potencial estrategia terapéutica

Neurons and astrocytes derived from human iPSCs to model Alzheimer´s disease

Alzheimer's disease (AD) is characterized by presenting a complex pathology, not fully resolved yet. This fact, together with the lack of reliable models, has impeded the development of effective therapies. Recently, several studies have shown that functional glial cell defects have a key role in the pathology of AD. However, this glial dysfunction, currently, cannot be correctly modeled using the available animal models, so we hypothesized that cells derived from Alzheimer's patients can serve as a better platform for studying the disease. In this sense, human pluripotent stem cells (hPSC) allow the generation of different types of neural cells, which can be used for disease modeling, identification of new targets and drugs development. Methods: We have a collection of hiPSCs derived from patients with sporadic forms of AD stratified based on APOE genotype. We have differentiated these cells towards neural cells and mature them to neurons or astrocytes using a serum-free approach, to assess intrinsic differences between those derived from AD patients or healthy controls. Results: We have implemented a serum-free approach and generated neural precursors and astrocytes from all the lines tested. We observe differences at the phenotypic level and a reduced capacity to differentiate towards neural lineage in those lines derived from APOE4 carriers. Conclusions: Our preliminary data suggest intrinsic differences in the neural differentiation capacity between cell lines derived from APOE4 or APOE3 carrier subjects. Further experiments would contribute to elucidate novel pathogenic pathways associated with neurodegeneration and susceptible of therapeutic modulation, likely contributing to the development of new effective drugs against AD.This study was supported by Instituto de Salud Carlos III (ISCiii) of Spain, co-financed by FEDER funds from European Union, through grants PI21/00915 (to AG) and PI21/00914 (to JV); by Junta de Andalucia through Consejería de Economía y Conocimiento grants PY18-RT-2233 (to AG) and US-1262734 (to JV) co-financed by Programa Operativo FEDER 2014-2020, Consejeria de Salud grant PI-0276-2018 (to JAGL) and Programa Operativo de Empleo Juvenil SNGJ4-11 to LCP. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Glia and neurons from human iPSCs to address the pathology of Alzheimer´s disease

Alzheimer's disease (AD) is characterized by presenting a complex pathology, not fully resolved yet. This fact, together with the lack of reliable models, has impeded the development of effective therapies. Recently, several studies have shown that functional glial cell defects have a key role in the pathology of AD. However, this glial dysfunction, currently, cannot be correctly modeled using the available animal models, so we hypothesized that cells derived from Alzheimer's patients can serve as a better platform for studying the disease. In this sense, human pluripotent stem cells (hPSC) allow the generation of different types of neural cells, which can be used for disease modeling, identification of new targets and drugs development. Methods: We have a collection of hiPSCs derived from patients with sporadic forms of AD stratified based on APOE genotype. We have differentiated these cells towards neural cells and mature them to neurons or astrocytes using a serum-free approach, to assess intrinsic differences between those derived from AD patients or healthy controls. Results: We have implemented a serum-free approach and generated neural precursors and astrocytes from all the lines tested. We observe differences at the phenotypic level and a reduced capacity to differentiate towards neural lineage in those lines derived from APOE4 carriers. Conclusions: Our preliminary data suggest intrinsic differences in the neural differentiation capacity between cell lines derived from APOE4 or APOE3 carrier subjects. Further experiments would contribute to elucidate novel pathogenic pathways associated with neurodegeneration and susceptible of therapeutic modulation, likely contributing to the development of new effective drugs against AD.This study was supported by Instituto de Salud Carlos III (ISCiii) of Spain, co-financed by FEDER funds from European Union, through grants PI21/00915 (to AG) and PI21/00914 (to JV); by Junta de Andalucia through Consejería de Economía y Conocimiento grants UMA20-FEDERJA-048 (to JAGL), PY18-RT-2233 (to AG) and US-1262734 (to JV) co-financed by Programa Operativo FEDER 2014-2020, and Programa Operativo de Empleo Juvenil SNGJ4-11 to LCP. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Co-cultures between neurons and astrocytes to address Alzheimer´s disease pathology.

Background: Alzheimer's disease (AD) is characterized by presenting a complex pathology, not fully resolved yet. This fact, together with the lack of reliable models, has impeded the development of effective therapies. Recently, several studies have shown that functional glial cell defects have a key role in the pathology of AD. However, this glial dysfunction, currently, cannot be correctly modeled using the available animal models, so we hypothesized that cells derived from Alzheimer's patients can serve as a better platform for studying the disease. In this sense, human pluripotent stem cells (hPSC) allow the generation of different types of neural cells, which can be used for disease modeling, identification of new targets and drugs development. Methods: We have a collection of hiPSCs derived from patients with sporadic forms of AD stratified based on APOE genotype. We have differentiated these cells towards neural cells and mature them to neurons or astrocytes using a serum-free approach, to assess intrinsic differences between those derived from AD patients or healthy controls. Results: We have implemented a serum-free approach and generated neural precursors and astrocytes from all the lines tested. We observe differences at the phenotypic level and a reduced capacity to differentiate towards neural lineage in those lines derived from APOE4 carriers. Conclusions: Our preliminary data suggest intrinsic differences in the neural differentiation capacity between cell lines derived from APOE4 or APOE3 carrier subjects. Further experiments would contribute to elucidate novel pathogenic pathways associated with neurodegeneration and susceptible of therapeutic modulation, likely contributing to the development of new effective drugs against AD.This study was supported by ISCiii (Spain), co-financed by FEDER funds, through grants PI21/00915 (AG) and PI21/00914 (JV); by Junta de Andalucia through Consejería de Economía and Conocimiento grants UMA20-FEDERJA-048 (JAGL), PY18-RT-2233 (to AG) and US-1262734 (JV), co-financed by Programa Operativo FEDER 2014-2020, and SNGJ4-11 (LCP). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Involvement of different aβ-associated myeloid populations in the human alzheimer’s brain

Parenchymal microglia, the brain-resident immune cells, have been postulated as a critical factor in Alzheimer´s disease (AD) since the identification of genetic risk factors related to their functions. Though the role of microglia in the AD progression/development is still unknown, a dysfunctional response has recently gained support. However, the different phenotypes and the implication of others myeloid cells in the human pathology have not been determined yet. In this work, we analyzed the phenotypic profile displayed by damage-associated myeloid cells in two AD vulnerable brain regions, the frontal cortex and hippocampus. For this purpose, immunohistochemistry and image analysis approaches have been carried out in postmortem brain samples from patients with AD (Braak VVI stage) and aged controls without neurological symptoms (Braak 0-II stage). Damage-associated microglial cells were clustered around amyloid plaques and expressed Iba1, CD32,TMEM119, CD68,Trem2 and CD45high. A subset of these cells also expressed ferritin and Gal-3. However, and even though some Braak II individuals accumulated reactive CD45 and CD68-positive plaques, only AD patients exhibited parenchymal infiltration of CD163-positive monocyte-derived cells that invaded plaque near blood vessels. While the frontal cortex showed strong microglial activation similar to that reported in amyloidogenic mice, the hippocampus of the same patients showed an attenuated microglial activation with a degenerative phenotype. These results reveal the co-existence of distinct myeloid populations associated with amyloid plaques during disease progression, as well their region-specific contribution to neuroimmune protection. These findings open the opportunity to design targeted therapies, not only to microglia, but also to the population of macrophages to modulate amyloid pathology and provide a better understanding of the immunological mechanisms underlying AD progression.Supported by ISCiii grants (PI21-0915 (AG), PI21-00914 (JV)); FEDER funds from European Union, by Junta de Andalucia grants (P18-RT-2233 (AG), US-1262734 (JV)); Programa Operativo FEDER 2014-2020, and by grant PPIT.UMA.B1-2019-07 (ESM). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Lithium, as a neuroprotective therapy for Alzheimer’s disease pathology, modifies abeta plaque toxicity

BACKGROUND: Despite the relatively large information about the Alzheimer’s disease (AD) pathology, no effective disease-modifying treatment has been yet developed. Lithium, a primary drug to treat bipolar disorder, has been suggested as a potential treatment against AD. In this work we have evaluated whether lithium treatment could ameliorate the neuropathology progression of the transgenic PS1M146L/APPSwe-London mice. Unlike most transgenic animal models, which do not exhibit the neurodegenerative spectrum of disease observed in the patient population, this AD model exhibits a prominent amyloid pathology along with a selective and significant neuronal loss in the hippocampus and entorhinal cortex. Therefore, this model is highly valuable for evaluating the effectiveness of potential neuroprotective therapies for AD. METHODS: For lithium treatment, PS1/APP mice (3 month old at the beginning of treatment) were fed, ad libitum, with diet supplemented with lithium carbonate (1.2g/kg, Harlan, Spain). The treatment lasts 6 months. After behavioural studies, mice were anesthetized and brains dissected out (hippocampus and cortex). Hemibrains were processed for immunohistochemistry, stereological and image analysis quantification, and the other hemibrains for RT-PCR and Western blot studies. RESULTS: Our data demonstrate that chronic oral administration of lithium, before the pathology onset, resulted in less toxic plaque formation that significantly ameliorated the degenerative processes and behavioural/memory deficits occurring during disease progression in our PS1/APP model. Specifically, and of great relevance for AD prevention, early lithium intervention was able to arrest neuronal loss in hippocampus and entorhinal cortex of highly vulnerable populations. Besides, lithium reduced the axonal dystrophic pathology, associated to amyloid plaques, by increasing the Abeta compaction. Moreover, a significant lower accumulation of phospho-tau, LC3-II and ubiquitinated proteins was detected. Our study highlights that the switch of plaque quality by lithium could be mediated by astrocyte activation and the release of heat shock proteins, which concentrated in the core of the plaques.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Diversity of plaque-associated myeloid cells subtypes in human alzheimer’s disease brain

Aims: Parenchymal microglia, as well other myeloid cells, have been postulated as a critical factor in Alzheimer´s disease (AD) pathogenesis since the identification of genetic risk factors related to their functions. However, the different phenotypes and the implication of the diverse immune cells in the human pathology have not been determined yet. In this work, we have further analyzed the phenotypic profile of the damage-associated myeloid cells in two AD vulnerable brain regions, the frontal cortex and hippocampus. Methods: Immunohistochemistry and image analysis approaches have been carried out in postmortem brain samples from patients with AD (Braak V-VI) and aged controls without neurological symptoms (Braak II). Results: Damage-associated microglial cells were clustered around amyloid plaques and expressed Iba1, TMEM119, CD68, Trem2 and CD45high. Moreover, AD brains exhibited parenchymal infiltration of CD163-positive monocyte-derived cells that invaded plaque near blood vessels. While the frontal cortex showed strong microglial activation similarly to that reported in amyloidogenic mice, the hippocampus of the same patients showed an attenuated microglial activation with a degenerative phenotype. Conclusions: These findings suggest the existence of different myeloid populations associated with Aβ plaques that correlates with disease severity. These results open the opportunity to design targeted therapies, not only to microglia, but also to the population of macrophages to modulate amyloid pathology and provide a better understanding of the immunological mechanisms underlying AD progression.Supported by ISCiii of Spain grants PI18/01557 (AG), PI18/01556 (JV) co-financed by FEDER funds from EU, by Junta de Andalucia grants UMA18-FEDERJA-211(AG), P18-RT-2233(AG) and US-1262734(JV) co-financed by Programa Operativo FEDER 2014-2020, and by B1-2019_07 Universidad de Malaga. Campus de Excelencia Internacional Andalucia Tech (ESM)

Peripheral myeloid cells infiltrate the hippocampus of Alzheimer's disease patients.

Microglia, the brain-resident myeloid cells, play a major role in the immune responses of the nervous system and in the pathogenesis of Alzheimer's disease (AD). However, the presence of peripheral myeloid cells in the AD brains remain to be demonstrated. Cellular and molecular approaches have been carried out in post-mortem hippocampal samples from patients with AD and age-matched controls without neurological symptoms. Our study provides evidence that circulating monocytes infiltrate the AD brains. Our findings showed that a high proportion of demented cases was associated with up-regulation of genes rarely expressed by microglial cells and abundant in monocytes-derived cells (MDC), among which stands the scavenger receptor Cd163. These Cd163-positive MDC invaded the brain parenchyma, acquired a microglial-like morphology, and were located in close proximity to blood vessels. These cells infiltrated the nearby amyloid plaques contributing to plaque-associated myeloid cell heterogeneity. Besides, control individuals with high amyloid pathology, showed no signs of MDC brain infiltration or plaque invasion. The MDC infiltration was associated with the progression and severity of AD pathology.These results reveal the co-existence of distinct myeloid populations associated with amyloid plaques during disease progression, as well their region-specific contribution to neuroimmune protection. The recruitment of monocytes could be a consequence rather than the cause of the severity of the disease. Whether monocyte infiltration is beneficial or detrimental to AD pathology remains to be fully elucidated. These findings open the opportunity to design targeted therapies, not only to microglia, but also to peripheral immune cell population to modulate amyloid pathology and provide a better understanding of the immunological mechanisms underlying AD progression.Supported by ISCiii grants(PI21-0915(AG),PI21-00914(JV)co-financed by FEDER funds from European Union;Junta de Andalucia grants P18-RT-2233(AG) and US-1262734(JV)co-financed by Programa Operativo FEDER 2014-2020;PPIT.UMA.B1-2019-07(ESM). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

WAT ALTERATIONS IN DIABETIC MICE: ITS CONNECTION AND IMPLICATION IN AD PATHOGENESIS

Alzheimer’s disease (AD) is a complex disorder and multiple cellular and molecular mechanisms are involved in AD onset and progression. Recent evidences have suggested that metabolic alterations are an important pathological feature in disease progression in AD. Likewise, diabetes and obesity, two mayor metabolic illnesses associated with white adipose tissue expansion, are risk factors for AD. Here, we hypothesize that the white adipose tissue may serve as a key communicator organ between the brain and peripheral metabolic illnesses. We used histological stains, immunohistochemistry and biochemical means to determine changes in the white adipose tissue from WT and db/db mice. Moreover, similar techniques were used in the brain of 3xTg-AD mice that received white fat pads from WT and db/db donors to determine any changes in amyloid and tau pathology. Our study shows that recipient 3xTg-AD mice from db/db fat pads mice develop profound changes in tau pathology due to increased CDK5/p25 expression compared to 3xTg-AD mice that received fad pads from WT mice. This increment in tau level was associated with elevated levels in IL-1β and microglial activation. However, we found that Aβ levels were reduced in recipient 3xTg-AD mice from db/db fat pads compared to 3xTg- AD mice that received fad pads from WT mice. These reduction in Aβ levels were correlated with an increment in microglia phagocytic capacity. Overall, our study demonstrates a novel important crosstalk between AD and diabetes type II through white adipose cells and a differential effect on tau and Aβ pathology

Mitochondrial ultrastructural defects in reactive astrocytes of Alzheimer's mice hippocampus.

Alzheimer's disease (AD) is a complex neurodegenerative condition that causes progressive memory loss and dementia. In AD brains astrocyte become reactive potentially contributing to cognitive decline. Astrocyte reactivity is a highly complex phenomenon with remarkable morphologic and molecular phenotype changes, and the role of astrocytes in the development of AD is still unknown. Astrocytes are the prevalent glial cells in the brain and have a large number of functions aimed at maintaining brain homeostasis including regulation of brain energy metabolism, maintenance of the blood-brain barrier, ion homeostasis, synaptic activity and plasticity, among many other functions. Any disruption regarding the normal roles of astrocytes can result in morphological and functional changes that ensue in pathological consequences. Mitochondrial dysfunction is an early event in the pathogenesis of AD, although most studies have focused on neurons and little is known about the functional characteristics and the dynamics of astrocyte mitochondria. We had performed an ultrastructural analysis using transmission electron microscopy in the hippocampus of amyloidogenic (APP/PS1) and tauopathy (P301S) mice. Our results show structural alterations in mitochondria that include double membrane rupture, cristae loss, and fragmentation together with a loss of their circularity. Since mitochondrial morphology is directly related to mitochondrial fusion/fission processes, the ultrastructural changes observed in astrocyte mitochondria in these amyloidogenic and tauopathy models suggest dynamic abnormalities in these organelles that may lead to deficits in astroglial function compromising their capability to maintain brain homeostasis and support neuronal energy metabolism and survival. A better understanding of cell type-specific mitochondrial dysfunction as a pathological feature of AD might hold great potential for the exploration of novel molecular targets for therapeutic development.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech