Estudio neuropatológico del hipocampo y giro parahipocampal en la enfermedad de Alzheimer: de modelos transgénicos a humanos

La actual ausencia de tratamientos efectivos para la enfermedad de Alzheimer (AD) pone de manifiesto la necesidad urgente de caracterizar la evolución de la patología en muestras de pacientes para así entender mejor los mecanismos de la enfermedad y desarrollar buenos modelos animales con valor predictivo para la experimentación preclínica. En esta Tesis Doctoral, se han analizado mediante técnicas celulares y moleculares las principales características histopatológicas (placas amiloides, patología fosfo-tau, respuesta inflammatoria y pérdida neuronal) en el hipocampo y giro parahipocampal de muestras humanas post mortem desde estadios Braak II (asintomáticos) a Braak V-VI (dementes). En paralelo, se han analizado características patológicas similares en el modelo PS1M146L/APP751SL con objeto de comparar los resultados con los obtenidos he pacientes. Los resultados más relevantes son: 1) la patología fosfo-tau es mucho más prominente que la patología amiloide en los pacientes; además, la progresión de la patología amiloide muestra una gran variabilidad regional e inter-individual en pacientes con afectación del hipocampo sólo en etapas avanzadas. Los altos niveles de fosfo-tau en contraste con el bajo contenido de Aβ en la fracción soluble sugiere que son las formas solubles de fosfo-tau las responsables del proceso neurodegenerativo en humanos. 2) hay una limitada activación microglial junto con un proceso significativo de disfunción/degeneración de esta población celular en los pacientes en etapas avanzadas de la enfermedad, mientras que en modelos animales la microglía se activa y prolifera gradualmente durante la progresión de la enfermedad. Por tanto, el concepto clásico de la respuesta microglial citotóxica asociada con esta enfermedad en los pacientes debe ser reevaluado, un hecho que se ve reforzado por el fracaso de las estrategias terapéuticas anti-inflamatorias probadas hasta la fecha en pacientes. En este sentido, proponemos que la prevención de la disfunción/degeneración microglial podría ser una novedosa y potencial estrategia terapéutica. Por otra parte, y en contraste a la respuesta microglial atenuada, hay una patente hiperactivación de los astrocitos, células gliales además altamente resistentes a el proceso degenerativo; 3) existe una pérdida sustancial de interneuronas que expresan somatostatina y parvalbúmina en los pacientes de AD. Una pérdida similar ha sido documentada previamente por nuestro grupo en el hipocampo y corteza entorrinal del modelo APP/PS1, y ahora aquí hemos extendido estos resultados a la corteza perirrinal; 4) el humo del tabaco produjo un aumento significativo de las lesiones neuropatológicas asociadas con la enfermedad (carga amiloide, la fosforilación de tau y el proceso de neuroinflammatorio) en un modelo animal APP/PS1. Estos resultados indican que el humo del cigarrillo podría acelerar la progresión de la patología siendo un potencial factor de riesgo ambiental para esta enfermedad. En general, estos resultados muestran que los modelos que sobreexpresan APP y desarrollan placas amiloides no mimetizan los principales aspectos de la patología humana, especialmente la respuesta inflamatoria asociada con las células microgliales. Por tanto, sería necesario desarrollar nuevos modelos, o mejorar los ya existentes, mediante la disminución de la respuesta inmune innata para imitar así mejor lo observado en los pacientes

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

Plaque-Associated Oligomeric Amyloid-Beta Drives Early Synaptotoxicity in APP/PS1 Mice Hippocampus: Ultrastructural Pathology Analysis

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder characterized by initial memory impairments that progress to dementia. In this sense, synaptic dysfunction and loss have been established as the pathological features that best correlate with the typical early cognitive decline in this disease. At the histopathological level, post mortem AD brains typically exhibit intraneuronal neurofibrillary tangles (NFTs) along with the accumulation of amyloid-beta (Abeta) peptides in the form of extracellular deposits. Specifically, the oligomeric soluble forms of Abeta are considered the most synaptotoxic species. In addition, neuritic plaques are Abeta deposits surrounded by activated microglia and astroglia cells together with abnormal swellings of neuronal processes named dystrophic neurites. These periplaque aberrant neurites are mostly presynaptic elements and represent the first pathological indicator of synaptic dysfunction. In terms of losing synaptic proteins, the hippocampus is one of the brain regions most affected in AD patients. In this work, we report an early decline in spatial memory, along with hippocampal synaptic changes, in an amyloidogenic APP/PS1 transgenic model. Quantitative electron microscopy revealed a spatial synaptotoxic pattern around neuritic plaques with significant loss of periplaque synaptic terminals, showing rising synapse loss close to the border, especially in larger plaques. Moreover, dystrophic presynapses were filled with autophagic vesicles in detriment of the presynaptic vesicular density, probably interfering with synaptic function at very early synaptopathological disease stages. Electron immunogold labeling showed that the periphery of amyloid plaques, and the associated dystrophic neurites, was enriched in Abeta oligomers supporting an extracellular location of the synaptotoxins. Finally, the incubation of primary neurons with soluble fractions derived from 6-month-old APP/PS1 hippocampus induced significant loss of synaptic proteins, but not neuronal death. Indeed, this preclinical transgenic model could serve to investigate therapies targeted at initial stages of synaptic dysfunction relevant to the prodromal and early AD.Instituto de Salud Carlos III (ISCiii) FEDER funds PI18/01557 and PI18/01556Junta de Andalucia UMA18-FEDERJA-211, P18-RT-2233 and US-126273Spanish Minister of Science and Innovation PID2019-108911RA-100, PID2019-107090RA-I00 and RYC-2017-21879Malaga University B1-2019_07 and B1-2019_0

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

Transgenic Mouse Models of Alzheimer’s Disease: An Integrative Analysis

Alzheimer’s disease (AD) constitutes the most prominent form of dementia among elderly individuals worldwide. Disease modeling using murine transgenic mice was first initiated thanks to the discovery of heritable mutations in amyloid precursor protein (APP) and presenilins (PS) genes. However, due to the repeated failure of translational applications from animal models to human patients, along with the recent advances in genetic susceptibility and our current understanding on disease biology, these models have evolved over time in an attempt to better reproduce the complexity of this devastating disease and improve their applicability. In this review, we provide a comprehensive overview about the major pathological elements of human AD (plaques, tauopathy, synaptic damage, neuronal death, neuroinflammation and glial dysfunction), discussing the knowledge that available mouse models have provided about the mechanisms underlying human disease. Moreover, we highlight the pros and cons of current models, and the revolution offered by the concomitant use of transgenic mice and omics technologies that may lead to a more rapid improvement of the present modeling batterThis research was funded by INSTITUTO DE SALUD CARLOS III (ISCiii) of Spain, cofinanced by FEDER funds from European Union, through grants PI21/00915 (to AG) and PI21/00914 (to JV); by JUNTA DE ANDALUCIA CONSEJERÍA DE ECONOMÍA Y CONOCIMIENTO through grants UMA18-FEDERJA-211 (to AG), UMA20-FEDERJA-104 (to IMG), P18-RT-2233 (to AG) and US-1262734 (to JV) co-financed by Programa Operativo FEDER 2014–2020 and CONSEJERIA DE SALUD grant PI-0276-2018 (to JAGL); by SPANISH MINISTER OF SCIENCE AND INNOVATION grant PID2019-108911RA-100 (to DBV), BEATRIZ GALINDO PROGRAM BAGAL18/00052 (to DBV), Alzheimer Association AARG-22-928219 (to DBV), grant PID2019-107090RA-100 (to IMG) and RAMON Y CAJAL PROGRAM RYC-2017-21879 (to IMG); and by MALAGA UNIVERSITY grant B1-2019_07 (to ESM), grant B1-2020_04 (to JAGL), grant B1-2019_06 (to IMG) and NASARD grant 27565 2018 (to IMG). M.M.-O. held a predoctoral contract from Malaga University, J.J.F.-V. held a postdoctoral contract from Malaga University, and E.S.-M. a postdoctoral contract (DOC_00251) from Junta de Andalucia. Partial funding for open access charge: Universidad de Málaga

Distinct disease-sensitive GABAergic neurons in the perirhinal cortex of Alzheimer's mice and patients

Neuronal loss is the best neuropathological substrate that correlates with cortical atrophy and dementia in Alzheimer’s disease (AD). Defective GABAergic neuronal functions may lead to cortical network hyperactivity and aberrant neuronal oscilla-tions and in consequence, generate a detrimental alteration in memory processes. In this study, using immunohistochemical and stereological approaches, we report that the two major and non-overlapping groups of inhibitory interneurons (SOM-cells and PV-cells) displayed distinct vulnerability in the perirhinal cortex of APP/PS1 mice and AD patients. SOM-positive neurons were notably sensitive and exhibited a dramatic decrease in the perirhinal cortex of 6-month-old transgenic mice (57% and 61% in areas 36 and 35, respectively) and, most importantly, in AD patients (91% in Braak V–VI cases). In addition, this interneuron degenerative process seems to occur in parallel, and closely related, with the progression of the amyloid pathol-ogy. However, the population expressing PV was unaffected in APP/PS1 mice while in AD brains suffered a pronounced and significant loss (69%). As a key component of cortico-hippocampal networks, the perirhinal cortex plays an important role in memory processes, especially in familiarity-based memory recognition. Therefore, disrupted functional connectivity of this cortical region, as a result of the early SOM and PV neurodegeneration, might contribute to the altered brain rhythms and cognitive failures observed in the initial clinical phase of AD patients. Finally, these findings highlight the failure of amyloidogenic AD models to fully recapitulate the selective neuronal degeneration occurring in humans.Instituto de Salud Carlos III (ISCiii) de España y fondos FEDER de la Unión Europea. PI18/01557 y PI18/01556Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía. Proyecto de Excelencia CTS-2035Universidad de Málaga. PPIT.UMA.B1.2017/2

Microglia in Alzheimer’s Disease: Activated, Dysfunctional or Degenerative

Microglial activation has been considered a crucial player in the pathological process of multiple human neurodegenerative diseases. In some of these pathologies, such as Amyotrophic Lateral Sclerosis or Multiple Sclerosis, the immune system and microglial cells (as part of the cerebral immunity) play a central role. In other degenerative processes, such as Alzheimer’s disease (AD), the role of microglia is far to be elucidated. In this “mini-review” article, we briefly highlight our recent data comparing the microglial response between amyloidogenic transgenic models, such as APP/PS1 and AD patients. Since the AD pathology could display regional heterogeneity, we focus our work at the hippocampal formation. In APP based models a prominent microglial response is triggered around amyloid-beta (Aβ) plaques. These strongly activated microglial cells could drive the AD pathology and, in consequence, could be implicated in the neurodegenerative process observed in models. On the contrary, the microglial response in human samples is, at least, partial or attenuated. This patent difference could simply reflect the lower and probably slower Aβ production observed in human hippocampal samples, in comparison with models, or could reflect the consequence of a chronic long-standing microglial activation. Beside this differential response, we also observed microglial degeneration in Braak V–VI individuals that, indeed, could compromise their normal role of surveying the brain environment and respond to the damage. This microglial degeneration, particularly relevant at the dentate gyrus, might be mediated by the accumulation of toxic soluble phospho-tau species. The consequences of this probably deficient immunological protection, observed in AD patients, are unknown.España, Instituto de Salud Carlos III PI15/00957, PI15/00796España Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucia Proyecto de Excelencia CTS-203

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)

Phagocytic clearance of presynaptic dystrophies by reactive astrocytes in Alzheimer's disease

Reactive astrogliosis, a complex process characterized by cell hypertrophy and upregulation ofcomponents of intermediate filaments, is a common feature in brains of Alzheimer’s patients. Reac-tive astrocytes are found in close association with neuritic plaques; however, the precise role ofthese glial cells in disease pathogenesis is unknown. In this study, using immunohistochemical tech-niques and light and electron microscopy, we report that plaque-associated reactive astrocytesenwrap, engulf and may digest presynaptic dystrophies in the hippocampus of amyloid precursorprotein/presenilin-1 (APP/PS1) mice. Microglia, the brain phagocytic population, was apparentlynot engaged in this clearance. Phagocytic reactive astrocytes were present in 35% and 67% ofamyloid plaques at 6 and 12 months of age, respectively. The proportion of engulfed dystrophicneurites was low, around 7% of total dystrophies around plaques at both ages. This fact, alongwith the accumulation of dystrophic neurites during disease course, suggests that the efficiency ofthe astrocyte phagocytic process might be limited or impaired. Reactive astrocytes surroundingand engulfing dystrophic neurites were also detected in the hippocampus of Alzheimer’spatientsby confocal and ultrastructural analysis. We posit that the phagocytic activity of reactive astrocytesmight contribute to clear dysfunctional synapses or synaptic debris, thereby restoring impairedneural circuits and reducing the inflammatory impact of damaged neuronal parts and/or limitingthe amyloid pathology. Therefore, potentiation of the phagocytic properties of reactive astrocytesmay represent a potential therapy in Alzheimer s disease.Fondo de Investigación Sanitaria (FIS). Instituto de Salud Carlos III (ISCiii) de España y fondos FEDER de la Unión Europea. PI15/00796 y PI15/00957Fundación La Marató-TV3 de Cataluña, España. 20141432, 20141431, 20141433, y 20141430Centro de investigación en red de enfermedades neurodegenerativas (CIBERNED) de España. PI2015-2/02Junta de Andalucía. Proyecto de Excelencia CTS-203