Distinct Microglial Responses in Two Transgenic Murine Models of TAU Pathology

Microglial cells are crucial players in the pathological process of neurodegenerative diseases, such as Alzheimer’s disease (AD). Microglial response in AD has been principally studied in relation to amyloid-beta pathology but, comparatively, little is known about inflammatory processes associated to tau pathology. In the hippocampus of AD patients, where tau pathology is more prominent than amyloid-beta pathology, a microglial degenerative process has been reported. In this work, we have directly compared the microglial response in two different transgenic tau mouse models: ThyTau22 and P301S. Surprisingly, these two models showed important differences in the microglial profile and tau pathology. Where ThyTau22 hippocampus manifested mild microglial activation, P301S mice exhibited a strong microglial response in parallel with high phospho-tau accumulation. This differential phospho-tau expression could account for the different microglial response in these two tau strains. However, soluble (S1) fractions from ThyTau22 hippocampus presented relatively high content of soluble phospho-tau (AT8-positive) and were highly toxic for microglial cells in vitro, whereas the correspondent S1 fractions from P301S mice displayed low soluble phosphotau levels and were not toxic for microglial cells. Therefore, not only the expression levels but the aggregation of phospho-tau should differ between both models. In fact, most of tau forms in the P301S mice were aggregated and, in consequence, forming insoluble tau species.We conclude that different factors as tau mutations, accumulation, phosphorylation, and/or aggregation could account for the distinct microglial responses observed in these two tau models. For this reason, deciphering the molecular nature of toxic tau species for microglial cells might be a promising therapeutic approach in order to restore the deficient immunological protection observed in AD hippocampus.CIBERNEDJunta de Andalucía. Consejería de Economía, Innovación, Ciencia y Empleo CTS-2035Fundación Tatiana Pérez de Guzmán el BuenoMinisterio de Ciencia, Innovación y UniversidadesInstituto de Salud Carlos III. Fondo de Investigación Sanitaria. PI15/00957 PI15/00796Fondo Europeo de Desarrollo Regional PI15/00957 PI15/0079

Should we open fire on microglia? Depletion models as tools to elucidate microglial role in health and alzheimer’s disease

Microglia play a critical role in both homeostasis and disease, displaying a wide variety in terms of density, functional markers and transcriptomic profiles along the different brain regions as well as under injury or pathological conditions, such as Alzheimer’s disease (AD). The generation of reliable models to study into a dysfunctional microglia context could provide new knowledge towards the contribution of these cells in AD. In this work, we included an overview of different microglial depletion approaches. We also reported unpublished data from our genetic microglial depletion model, Cx3cr1CreER /Csf1rflx/flx, in which we temporally controlled microglia depletion by either intraperitoneal (acute model) or oral (chronic model) tamoxifen administration. Our results reported a clear microglial repopulation, then pointing out that our model would mimic a context of microglial replacement instead of microglial dysfunction. Next, we evaluated the origin and pattern of microglial repopulation. Additionally, we also reviewed previous works assessing the effects of microglial depletion in the progression of Aβ and Tau pathologies, where controversial data are found, probably due to the heterogeneous and time-varying microglial phenotypes observed in AD. Despite that, microglial depletion represents a promising tool to assess microglial role in AD and design therapeutic strategies.La Marato-TV3 Foundation 20141432, 20141431Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas CB06/05/0094, CB06/05/1116Junta de Andalucía US-1262734, UMA18-FEDERJA-211, P18-RT-223

Disruption of Amyloid Plaques Integrity Affects the Soluble Oligomers Content from Alzheimer Disease Brains

The implication of soluble Abeta in the Alzheimer's disease (AD) pathology is currently accepted. In fact, the content of soluble extracellular Abeta species, such as monomeric and/or oligomeric Abeta, seems to correlate with the clinicopathological dysfunction observed in AD patients. However, the nature (monomeric, dimeric or other oligomers), the relative abundance, and the origin (extra-/intraneuronal or plaque-associated), of these soluble species are actually under debate. In this work we have characterized the soluble (defined as soluble in Trisbuffered saline after ultracentrifugation) Abeta, obtained from hippocampal samples of Braak II, Braak III-IV and Braak V-VI patients. Although the content of both Abeta40 and Abeta42 peptides displayed significant increase with pathology progression, our results demonstrated the presence of low, pg/mg protein, amount of both peptides. This low content could explain the absence (or below detection limits) of soluble Abeta peptides detected by western blots or by immunoprecipitation-western blot analysis. These data were in clear contrast to those published recently by different groups. Aiming to explain the reasons that determine these substantial differences, we also investigated whether the initial homogenization could mobilize Abeta from plaques, using 12-month-old PS1xAPP cortical samples. Our data demonstrated that manual homogenization (using Dounce) preserved the integrity of Abeta plaques whereas strong homogenization procedures (such as sonication) produced a vast redistribution of the Abeta species in all soluble and insoluble fractions. This artifact could explain the dissimilar and somehow controversial data between different groups analyzing human AD sample

Amyloid-β impairs the phagocytosis of dystrophic synapses by astrocytes in Alzheimer's disease

Reactive astrocytes and dystrophic neurites, most aberrant presynaptic elements, are found surrounding amyloid-β plaques in Alzheimer's disease (AD). We have previously shown that reactive astrocytes enwrap, phagocytose, and degrade dystrophic synapses in the hippocampus of APP mice and AD patients, but affecting less than 7% of dystrophic neurites, suggesting reduced phagocytic capacity of astrocytes in AD. Here, we aimed to gain insight into the underlying mechanisms by analyzing the capacity of primary astrocyte cultures to phagocytose and degrade isolated synapses (synaptoneurosomes, SNs) from APP (containing dystrophic synapses and amyloid-β peptides), Tau (containing AT8- and AT100-positive phosphorylated Tau) and WT (controls) mice. We found highly reduced phagocytic and degradative capacity of SNs-APP, but not AT8/AT100-positive SNs-Tau, as compared with SNs-WT. The reduced astrocyte phagocytic capacity was verified in hippocampus from 12-month-old APP mice, since only 1.60 ± 3.81% of peri-plaque astrocytes presented phagocytic structures. This low phagocytic capacity did not depend on microglia-mediated astrocyte reactivity, because removal of microglia from the primary astrocyte cultures abrogated the expression of microglia-dependent genes in astrocytes, but did not affect the phagocytic impairment induced by oligomeric amyloid-β alone. Taken together, our data suggest that amyloid-β, but not hyperphosphorylated Tau, directly impairs the capacity of astrocytes to clear the pathological accumulation of oligomeric amyloid-β, as well as of peri-plaque dystrophic synapses containing amyloid-β, perhaps by reducing the expression of phagocytosis receptors such as Mertk and Megf10, thus increasing neuronal damage in AD. Therefore, the potentiation or recovery of astrocytic phagocytosis may be a novel therapeutic avenue in AD.Centro de Invesitgacion Biomedica en Red Enfermedades Neurodegenetativas (CIBERNED). CB06/05/0094 y CB06/05/1116Instituto de Salud Carlos III y fondos FEDER de la Unión Europea. PI18/01556 y PI18/01557Consejería de Economía y Conocimiento de la Junta de Andalucía y el Programa Operativo FEDER 2014-2020. PY18-RT-2233, UMA18-FEDERJA-211 y US-1262734Fundación La Marató-TV3. 20141430, 20141431, 2014143

Regional difference in inflammatory response to LPS-injection in the brain: Role of microglia cell density

To elucidate whether density of cells could contribute to the extent of microglial activation, we performed in vitro assays using three different densities of N13 microglia stimulated with LPS. Our results showed that induction of pro-inflammatory factors as TNF-α and iNOS was directly related to cell density, meanwhile the induction of the anti-inflammatory IL-10 was inversely related to cell density. Accordingly, in vivo assays showed that after LPS-injection, iNOS expression was more intense in substantia nigra, a brain area showing specific susceptibility to neurodegeneration after microglia activation, whereas IL-10 expression was more sustained in striatum, an area resistant to damage. These results support that microglia density is pivotal to control the balance between pro- and anti-inflammatory factors release.Instituto de Salud Carlos III PI060781, PI060567Junta de Andalucía CVI-90

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

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

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

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)

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