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

Microtubule stabilization protects cognitive function and slows down the course of Alzheimer's like pathology in an amyloidogenic mouse model

Cognitive decline in Alzheimer's disease (AD) is highly related to synaptic dysfunction and neuronal loss. In AD, the hyperphosphorylation of tau compromises axonal transport and leads to the generation of dystrophic neurites, contributing to synaptic impairment. In addition to phospho-tau, AD brains accumulate amyloid-beta. This study evaluated the effect of the brain-penetrant microtubule-stabilizing agent, Epothilone D (EpoD) in the progression of the disease in a double transgenic mouse model of amyloidosis. Young APP/PS1 mice were weekly treated with intraperitoneal injections of EpoD (2 mg/kg) or vehicle solution for 3 months. Memory performance was tested using object-recognition tasks, Y-maze and Morris water maze. EpoD-treated mice improved their performance of cognitive tests, while hippocampal phospho-tau and Aβ levels, especially soluble oligomers, decreased significantly. β/γ-secretase activities were not affected by EpoD in vitro. A significant amelioration of synaptic/neuritic pathology was found. Remarkably, EpoD exerted a neuroprotective effect on SOM-interneurons, a highly AD-vulnerable GABAergic subpopulation. In conclusion, EpoD improved microtubule dynamics and axonal transport in an AD-like context, reducing tau and Abeta accumulation, and promoting neuronal and cognitive protection. These results underline the crosstalk between cytoskeleton pathology and proteinopathy. Therefore, microtubule-stabilizing drugs could be candidates for slowing AD progression at both tau and Aβ pathologies.Supported by PI18/01557 (to AG) and PI18/01556 (to JV) grants from ISCiii of Spain, co-financed by FEDER funds from European Union, CIBERNED collaborative grant (to AG and JV), and by PPIT.UMA.B1.2017/26 grant (to RSV). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

Systemic and Local Hypoxia Synergize Through HIF1 to Compromise the Mitochondrial Metabolism of Alzheimer's Disease Microglia

Microglial cells are key contributors to Alzheimer’s disease (AD), constituting the first cellular line against Aß plaques. Local hypoxia and hypoperfusion, which are typically present in peripheral inflammatory foci, are also common in the AD brain. We describe here that Aß deposits are hypoxic and hypoperfused and that Aß plaque-associated microglia (AßAM) are characterized by the expression of hypoxia-inducible factor 1 (HIF1)-regulated genes. Notably, AßAM simultaneously upregulate the expression of genes involved in anaerobic glycolysis and oxidative mitochondrial metabolism, show elongated mitochondria surrounded by rough endoplasmic reticulum, and blunt the HIF1-mediated exclusion of pyruvate from the mitochondria through the pyruvate dehydrogenase kinase 1 (PDK1). Overstabilization of HIF1 –by genetic (von Hippel-Lindau deficient microglia) or systemic hypoxia (an AD risk factor)– induces PDK1 in microglia and reduces microglial clustering in AD mouse models. The human AD brain exhibits increased HIF1 activity and a hypoxic brain area shows reduced microglial clustering. The loss of the microglial barrier associates with augmented Aß neuropathology both in the chronic hypoxia AD mouse model and the human AD brain. Thus, the synergy between local and systemic AD risk factors converges with genetic susceptibility to cause microglial dysfunction.Peer reviewe

Hypoxia compromises the mitochondrial metabolism of Alzheimer’s disease microglia via HIF1

Genetic Alzheimer’s disease (AD) risk factors associate with reduced defensive amyloid β plaque-associated microglia (AβAM), but the contribution of modifiable AD risk factors to microglial dysfunction is unknown. In AD mouse models, we observe concomitant activation of the hypoxia-inducible factor 1 (HIF1) pathway and transcription of mitochondrial-related genes in AβAM, and elongation of mitochondria, a cellular response to maintain aerobic respiration under low nutrient and oxygen conditions. Overactivation of HIF1 induces microglial quiescence in cellulo, with lower mitochondrial respiration and proliferation. In vivo, overstabilization of HIF1, either genetically or by exposure to systemic hypoxia, reduces AβAM clustering and proliferation and increases Aβ neuropathology. In the human AD hippocampus, upregulation of HIF1α and HIF1 target genes correlates with reduced Aβ plaque microglial coverage and an increase of Aβ plaque-associated neuropathology. Thus, hypoxia (a modifiable AD risk factor) hijacks microglial mitochondrial metabolism and converges with genetic susceptibility to cause AD microglial dysfunction.R.M.-D. was the recipient of a Sara Borrell fellowship from Instituto de Salud Carlos III (ISCIII) (CD09/0007). N.L.-U., C.O.-d.S.L., C.R.-M. and M.I.A.-V. were the recipients of FPU fellowships from Spanish Ministry of Education, Culture and Sport (FPU14/02115, AP2010‐1598, FPU16/02050 and FPU15/02898, respectively). A.H.-G. was the recipient of an FPI fellowship from the Spanish Ministry of Education, Culture and Sport (BES-2010-033886). This work was supported by grants from the Spanish MINEICO, ISCIII and FEDER (European Union) (SAF2012‐33816, SAF2015‐64111‐R, SAF2017-90794-REDT and PIE13/0004 to A.P.); by the Regional Government of Andalusia co-funded by CEC and FEDER funds (European Union) (‘Proyectos de Excelencia’; P12‐CTS‐2138 and P12‐CTS‐2232 to A.P.); by the ‘Ayuda de Biomedicina 2018’, Fundación Domingo Martínez (to A.P.) ; by the ISCIII of Spain, co-financed by FEDER funds (European Union) through grants PI18/01556 (to J.V.) and PI18/01557 (to A. Gutierrez); by Junta de Andalucía, co-financed by FEDER funds (grants UMA18-FEDERJA-211 (to A. Gutierrez) and US‐1262734 (to J.V.)); and by Spanish MINEICO (BFU2016-76872-R and BES-2011-047721 to E.B.).Peer reviewe

Astroglial reactivity in response to b-amyloidosis is associated with mithocondrial pathology in the hippocampus of Alzheimer's transgenic mice

Aims: Alzheimer’s diseaase (A)D is associated with early energy hypometabolism, synaptic and mitochondrial dysfunction, oxidative stress, inflammation, abnormal proteostasis and progressive neurodegeneration. During the pathogenic process, amyloid-beta and phospho-tau pathologies have a detrimental effect on neurons and glial cells, affecting neuronal stability, and compromising ATP production and energy metabolism. Though mitochondrial dysfunction is thought to be an early event in the pathogenesis of this AD, the vast majority of studies have focused on neurons, and little is known about the functional characteristics and dynamics of mitochondria in astrocytes. Here, we aim to analyze mitochondrial subcellular features of reactive astrocytes in APP/PS1 mice hippocampus in order to a better understanding of this pathology. Methods: Mitochondrial features were observed by transmission electron microscopy and immunogold labeling. Image analysis was performed to assess morphological changes. Results: Our results show mitochondrial structural alterations including mitochondrial cristae loss, broken double membrane structure and fragmentation. In addition, an increase in both number and size of mitochondria in this transgenic model compared to age-matched WT mice, was found. Conclusions: Since mitochondrial morphology is directly related to mitochondrial fusion/fission, the ultrastructural pathology of astrocytic mitochondria in this amyloidogenic model suggests dynamics abnormalities in these organelles that might lead to astroglial functional deficits compromising neuronal survival. Supported by ISCiii grants PI21-0915 (AG) and PI21-00914 (JV) co-financed by FEDER funds from European Union, by Junta de Andalucia grants P18-RT-2233 (AG) and US-1262734 (JV) co-financed by Programa Operativo FEDER 2014-2020, and by grant PPIT.UMA.B1-2021_32 (LTE).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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.Research funding: Centro de Invesitgacion Biomedica en Red Enfermedades Neurodegenetativas (CIBERNED). Grant Numbers: CB06/05/0094, CB06/05/1116; Instituto de Salud Carlos III co‐financed by FEDER funds from European Union. Grant Numbers: PI18/01556, PI18/01557; Junta de Andalucia Consejería de Economía y Conocimiento co‐financed by Programa Operativo FEDER 2014‐2020. Grant Numbers: PY18‐RT‐2233, UMA18‐FEDERJA‐211, US‐1262734; La Marató‐TV3 Foundation. Grant Numbers: 20141430, 20141431, 20141432

Enhancing microtubule stabilization rescues cognitive deficits and ameliorates pathological phenotype in an amyloidogenic Alzheimer’s disease model

In Alzheimer’s disease (AD), and other tauopathies, microtubule destabilization compromises axonal and synaptic integrity contributing to neurodegeneration. These diseases are characterized by the intracellular accumulation of hyperphosphorylated tau leading to neurofibrillary pathology. AD brains also accumulate amyloid-beta (Aβ) deposits. However, the effect of microtubule stabilizing agents on Aβ pathology has not been assessed so far. Here we have evaluated the impact of the brain-penetrant microtubule-stabilizing agent Epothilone D (EpoD) in an amyloidogenic model of AD. Three-month-old APP/PS1 mice, before the pathology onset, were weekly injected with EpoD for 3 months. Treated mice showed significant decrease in the phospho-tau levels and, more interesting, in the intracellular and extracellular hippocampal Aβ accumulation, including the soluble oligomeric forms. Moreover, a significant cognitive improvement and amelioration of the synaptic and neuritic pathology was found. Remarkably, EpoD exerted a neuroprotective effect on SOM-interneurons, a highly AD-vulnerable GABAergic subpopulation. Therefore, our results suggested that EpoD improved microtubule dynamics and axonal transport in an AD-like context, reducing tau and Aβ levels and promoting neuronal and cognitive protection. These results underline the existence of a crosstalk between cytoskeleton pathology and the two major AD protein lesions. Therefore, microtubule stabilizers could be considered therapeutic agents to slow the progression of both tau and Aβ pathology.This study was supported by Instituto de Salud Carlos III (ISCiii) of Spain, co-financed by FEDER funds from European Union, through grants PI15/00796 and PI18/01557 (both to AG), PI15/00957 and PI18/01556 (both to JV), and CIBERNED (CB06/05/1116 to AG and CB06/05/0094 to JV); by Malaga University grant PPIT.UMA.B1.2017/26 (to RSV). JJFV and CMC were supported by FPU PhD fellowships (Spanish Ministry of Science, Innovation and Universities). RSV held a postdoctoral contract from Malaga University. IMG is recipient of a senior postdoctoral contract from Ramon y Cajal Program (Spain Government)