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

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

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

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

Litio como terapia neuroprotectora en el modelo appsl/ps1m146l de la enfermedad de Alzheimer

El litio se utiliza desde hace varias décadas en el tratamiento de trastornos bipolares y la depresión, y recientemente se debate su uso potencial en patologías neurodegenerativas como la enfermedad de Alzheimer (AD)

Adipose tissue as a therapeutic target for vascular damage in Alzheimer's disease

Adipose tissue has recently been recognized as an important endocrine organ that plays a crucial role in energy metabolism and in the immune response in many metabolic tissues. With this regard, emerging evidence indicates that an important crosstalk exists between the adipose tissue and the brain. However, the contribution of adipose tissue to the development of age-related diseases, including Alzheimer's disease, remains poorly defined. New studies suggest that the adipose tissue modulates brain function through a range of endogenous biologically active factors known as adipokines, which can cross the blood–brain barrier to reach the target areas in the brain or to regulate the function of the blood–brain barrier. In this review, we discuss the effects of several adipokines on the physiology of the blood–brain barrier, their contribution to the development of Alzheimer's disease and their therapeutic potential.Funding for open access charge; Universidad de Málaga / CBU

Defective lysosomal proteolysis and axonal transport are early pathogenic events that worsen with age leading to increased APP metabolism and synaptic Abeta in transgenic APP/PS1 hippocampus

Background: Axonal pathology might constitute one of the earliest manifestations of Alzheimer disease. Axonal dystrophies were observed in Alzheimer’s patients and transgenic models at early ages. These axonal dystrophies could reflect the disruption of axonal transport and the accumulation of multiple vesicles at local points. It has been also proposed that dystrophies might interfere with normal intracellular proteolysis. In this work, we have investigated the progression of the hippocampal pathology and the possible implication in Abeta production in young (6 months) and aged (18 months) PS1(M146L)/APP(751sl) transgenic mice. Results: Our data demonstrated the existence of a progressive, age-dependent, formation of axonal dystrophies, mainly located in contact with congophilic Abeta deposition, which exhibited tau and neurofilament hyperphosphorylation. This progressive pathology was paralleled with decreased expression of the motor proteins kinesin and dynein. Furthermore, we also observed an early decrease in the activity of cathepsins B and D, progressing to a deep inhibition of these lysosomal proteases at late ages. This lysosomal impairment could be responsible for the accumulation of LC3-II and ubiquitinated proteins within axonal dystrophies. We have also investigated the repercussion of these deficiencies on the APP metabolism. Our data demonstrated the existence of an increase in the amyloidogenic pathway, which was reflected by the accumulation of hAPPfl, C99 fragment, intracellular Abeta in parallel with an increase in BACE and gamma-secretase activities. In vitro experiments, using APPswe transfected N2a cells, demonstrated that any imbalance on the proteolytic systems reproduced the in vivo alterations in APP metabolism. Finally, our data also demonstrated that Abeta peptides were preferentially accumulated in isolated synaptosomes. Conclusion: A progressive age-dependent cytoskeletal pathology along with a reduction of lysosomal and, in minor extent, proteasomal activity could be directly implicated in the progressive accumulation of APP derived fragments (and Abeta peptides) in parallel with the increase of BACE-1 and gamma-secretase activities. This retard in the APP metabolism seemed to be directly implicated in the synaptic Abeta accumulation and, in consequence, in the pathology progression between synaptically connected regions

Abnormal accumulation of autophagic vesicles correlates with axonal and synaptic pathology in young Alzheimer’s mice hippocampus

Dystrophic neurites associated with amyloid plaques precede neuronal death and manifest early in Alzheimer’s disease (AD). In this work we have characterized the plaque-associated neuritic pathology in the hippocampus of young (4- to 6-month-old) PS1M146L/ APP751SL mice model, as the initial degenerative process underlying functional disturbance prior to neuronal loss. Neuritic plaques accounted for almost all fibrillar deposits and an axonal origin of the dystrophies was demonstrated. The early induction of autophagy pathology was evidenced by increased protein levels of the autophagosome marker LC3 that was localized in the axonal dystrophies, and by electron microscopic identification of numerous autophagic vesicles filling and causing the axonal swellings. Early neuritic cytoskeletal defects determined by the presence of phosphorylated tau (AT8-positive) and actin–cofilin rods along with decreased levels of kinesin-1 and dynein motor proteins could be responsible for this extensive vesicle accumulation within dystrophic neurites. Although microsomal Ab oligomers were identified, the presence of A11-immunopositive Ab plaques also suggested a direct role of plaque-associated Ab oligomers in defective axonal transport and disease progression. Most importantly, presynaptic terminals morphologically disrupted by abnormal autophagic vesicle buildup were identified ultrastructurally and further supported by synaptosome isolation. Finally, these early abnormalities in axonal and presynaptic structures might represent the morphological substrate of hippocampal dysfunction preceding synaptic and neuronal loss and could significantly contribute to AD pathology in the preclinical stages.Fondo de Investigación Sanitaria (FIS). Instituto de Salud Carlos III, España. PS09/00099, PS09/00151, PS09/00848 y PS09/00376Junta de Andalucía. SAS P09/496 y CTS-479

Visceral adipose tissue triggers tau pathogenesis in transgenic mice

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, are risk factors for AD. These two overwhelming diseases are associated with a significant expansion of visceral adipose tissue. Here, we hypothesize that the visceral adipose tissue may serve as a key communicator organ between the brain and peripheral metabolic illnesses and affecting both types of disorders. Method: We used histological stains, immunohistochemistry and biochemical means to determine changes in the visceral adipose tissue from WT and db/db mice. Moreover, similar techniques were used in 3xTg-AD mice that received white fat pads from WT and db/db donors to determine any changes in amyloid and tau pathology. Result: Our study shows that recipient 3xTg-AD mice from db/db fat pads mice develop profound changes in tau pathology due to increased CDK5 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 profound microglia activation. Moreover, we found the opposite effect on amyloid pathology, in which insoluble Aβ levels and Thioflavin positive plaques 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. Conclusion: Overall, our study demonstrate a novel important crosstalk between Alzheimer´s disease and obesity/diabetes type II through visceral adipose cells and a differential effect on tau and Aβ pathology mediated by an activated immune response.This study was supported by Minister of Science and Innovation grant PID2019-108911RA-100 (D.B.V.), Alzheimer's Association grant AARG-22-9282/9, Beatriz Galindo program BAGAL18/00052 (D.B.V.), University of Malaga grant PPIT.UMA.B1- 2021_32(LTE) and Institute of Health Carlos III (ISCiii) grant PI18/01557 (A.G.) cofinanced by FEDER funds from European Union

Visceral adipose tissue triggers tau pathogenesis in transgenic mice through CDK5/P25 pathway.

Alzheimer’s disease (AD) is a complex disorder and multiple molecular mechanisms are involved in AD onset and progression. Recent evidences have suggested that metabolic alterations are an important pathological feature in this disease progression. Likewise, diabetes and obesity, two mayor metabolic illnesses, are risk factors for AD. These two overwhelming diseases are associated with a significant expansion of visceral adipose tissue (VAT). Here, we hypothesize that the VAT may serve as a key communicator organ between the brain and peripheral metabolic illnesses and affecting both types of disorders. We used histological stains, immunohistochemistry and biochemical means to determine changes in the visceral adipose tissue from WT and db/db mice. Moreover, similar techniques were used in 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 mice fat pads develop profound changes in tau pathology due to increased CDK5 expression compared to 3xTg-AD mice that received fat pads from WT mice. This increment in tau level was associated with elevated levels in IL-1β and profound microglia activation. Moreover, we found the opposite effect on amyloid pathology, in which insoluble Aβ levels and Thioflavin positive plaques 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 demonstrate a novel important crosstalk between Alzheimer´s disease and obesity/diabetes type II through visceral adipose cells and a differential effect on tau and Aβ pathology mediated by an activated immune response.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech