Epothilone-d rescues cognition and attenuates alzheimer’s disease-like pathology in APP/PS1 mice

AIMS: Cognitive decline in Alzheimer's disease (AD) patients has been linked to synaptic damage and neuronal loss. Hyperphosphorylation of tau protein destabilizes microtubules leading to the accumulation of autophagy/vesicular material and the generation of dystrophic neurites, thus contributing to axonal/synaptic dysfunction. In this study, we analyzed the effect of a microtubule-stabilizing compound in the progression of the disease in the hippocampus of APP751SL/PS1M146L transgenic model. METHODS: APP/PS1 mice (3 month-old) were treated with a weekly intraperitoneal injection of 2 mg/kg epothilone-D (Epo-D) for 3 months. Vehicle-injected animals were used as controls. Mice were tested on the Morris water maze, Y-maze and object-recognition tasks for memory performance. Abeta, AT8, ubiquitin and synaptic markers levels were analyzed by Western-blots. Hippocampal plaque, synaptic and dystrophic loadings were quantified by image analysis after immunohistochemical stainings. RESULTS: Epo-D treated mice exhibited a significant improvement in the memory tests compared to controls. The rescue of cognitive deficits was associated to a significant reduction in the AD-like hippocampal pathology. Levels of Abeta, APP and ubiquitin were significantly reduced in treated animals. This was paralleled by a decrease in the amyloid burden, and more importantly, in the plaque-associated axonal dystrophy pathology. Finally, synaptic levels were significantly restored in treated animals compared to controls. CONCLUSION: Epo-D treatment promotes synaptic and spatial memory recovery, reduces the accumulation of extracellular Abeta and the associated neuritic pathology in the hippocampus of APP/PS1 model. Therefore, microtubule stabilizing drugs could be considered therapeutical candidates to slow down AD progression. Supported by FIS-PI12/01431 and PI15/00796 (AG),FIS-PI12/01439 and PI15/00957(JV)Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

SYSTEMIC ADMINISTRATION OF EPOTHYLONE-D RECUES MEMORY AND AMELIORATES ALZHEIMER’S DISEASE-LIKE PATHOLOGY IN APP/PS1 MICE

Aims Cognitive and memory decline in Alzheimer's disease (AD) patients is highly related to synaptic dysfunction and neuronal loss. Tau hyperphosphorylation destabilizes microtubules leading to axonal transport failure, accumulation of autophagy/vesicular material and the generation of dystrophic neurites, thus contributing to axonal/synaptic dysfunction. In this study, we analyzed the effect of a microtubule-stabilizing drug in the progression of the disease in an APP751SL/PS1M146L transgenic model. Method APP/PS1 mice (3 month-old) were weekly treated with 2 mg/kg intraperitoneal injections of Epothilone-D (Epo-D) for 3 months. Vehicle-injected animals were used as controls. For memory performance, animals were tested on the object-recognition tasks, Y-maze and Morris water maze. Levels of Abeta, ubiquitin, AT8 and synaptic markers were analyzed by Western-blot. Hippocampal plaque burden, dystrophic and synaptic loadings were quantified after immunostaining by image analysis. Results Epo-D treated mice showed a significant improvement in the performance of hippocampus-associated cognitive tests compared to controls. This memory recovery correlated with a significant reduction in the AD-like hippocampal pathology. Abeta, APP and ubiquitin levels were significantly reduced in treated animals, and a decrease in both the plaque loading and the axonal pathology was also found. Finally, synaptic levels were significantly preserved in treated animals in comparison with controls. Conclusion Epo-D treatment promotes synaptic and cognitive improvement, reduces the accumulation of extracellular Abeta and the associated neuritic pathology in the hippocampus of APP/PS1 model. Therefore, microtubule stabilizing drugs could be considered therapeutical candidates to slow down AD progression.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Supported by FIS-PI15/00796 (AG), FIS-PI15/00957(JV) and co-financed by FEDER funds from European Union

Microglial responses in the human Alzheimer’s disease frontal cortex

The continuing failure to develop an effective treatment for Alzheimer’s disease (AD) reveals the complexity for this pathology. Increasing evidence indicates that neuroinflammation involving particularly microglial cells contributes to AD pathogenesis. The actual view, based on the findings in APP based models, gives a cytotoxic/proinflammatory role to activated microglia. However, we have previously reported a limited activation and microglial degeneration in the hippocampus of AD patients in contrast with that observed in amyloidogenic models. Here, we evaluated the microglial response in a different region of AD brains, the frontal cortex. Post mortem tissue from controls (Braak 0-II) and AD patients (Braak V-VI) including familial cases, were obtained from Spain Neurological Tissue Banks. Cellular (immunohistochemistry and image analysis) and molecular (qPCR and western blots) approaches were performed. Frontal cortex of AD patients (Braak V-VI) showed strong microglial activation similar to that observed in amyloidogenic mice. These strongly activated microglial cells, predominantly located surrounding amyloid plaques, could drive the AD pathology and, in consequence, could be implicated in the pathology progression. Furthermore, different microglial responses were observed between sporadic and familial AD cases. These findings in the frontal cortex were highly in contrast to the attenuated activation and degenerative morphology displayed by microglial cells in the hippocampus of AD patients. Regional differences in the microglial response suggest different functional states of microglial cells in a region-specific manner. All together, these data provide a better understanding of the immunological mechanisms underlying AD progression and uncover new potential therapeutic targets to fight this devastating neurodegenerative disease.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Supported by PI18/01557 (AG) and PI18/01556 (JV) grants from ISCiii of Spain co-financed by FEDER funds from European Unio

Microglial response differences between amyloidogenic transgenic models and Alzheimer’s disease patients

Aims: The continuing failure to develop an effective treatment for Alzheimer’s disease (AD) reveals the complexity for AD pathology. Increasing evidence indicates that neuroinflammation involving particularly microglial cells contributes to disease pathogenesis. Here we analyze the differences in the microglial response between APP/PS1 model and human brains. Methods: RT-PCR, western blots, and immunostaining were performed in the hippocampus of human post mortem samples (from Braak II to Braak V-VI) and APP751SL/PS1M146L mice. In vitro studies to check the effect of S1 fractions on microglial cells were assayed. Results: In APP based models the high Abeta accumulation triggers a prominent microglial response. On the contrary, the microglial response detected in human samples is, at least, partial or really mild. This patent difference could simple reflect the lower and probably slower Abeta production observed in human hippocampal samples, in comparison with models or could reflect the consequence of a chronic long-standing microglial activation. However, beside this differential response, we also observed a prominent microglial degenerative process in Braak V-VI samples 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 of the hippocampal formation, might be mediated by the accumulation of toxic soluble phospho-tau species. Conclusions: These differences need to be considered when delineating animal models that better integrate the complexity of AD pathology and, therefore, guarantee clinical translation. Correcting dysregulated brain inflammatory responses might be a promising avenue to restore cognitive function. Supported by grants FIS PI15/00796 and FIS PI15/00957 co-financed by FEDER funds from European Union, and by Junta de Andalucia Proyecto de Excelencia CTS385 2035.Financiado por FIS PI15/00796 y FIS PI15/0095, cofinanciado por los fondos FEDER de la Unión Europea, y por Junta de Andalucia Proyecto de Excelencia CTS385 2035. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Decoding damage-associated microglia in post mortem hippocampus of Alzheimer’s disease patients

The relationship between Alzheimer’s disease (AD) and neuroinflammation has become stronger since the identification of several genetic risk factors related to microglial function. Though the role of microglial cells in the development/progression of AD is still unknown, a dysfunctional response has recently gained support. In this sense, we have reported an attenuated microglial activation associated to amyloid plaques in the hippocampus of AD patients, including a prominent degenerative process of the microglial population in the dentate gyrus, which was in contrast to the exacerbated microglial response in amyloidogenic models. This microglial degeneration could compromise their normal role of surveying the brain environment and respond to the damage. Here, we have further analyzed the phenotypic profile displayed by the damage-associated microglial cells by immunostaining and qPCR in the hippocampus of postmortem samples of AD patients (Braak V-VI) and control cases (Braak 0-II). Damage-associated microglial cells of Braak V-VI individuals were clustered around amyloid plaques and expressed Iba1, CD68, Trem2, TMEM119 and CD45high. A subset of these cells also expressed ferritin. On the contrary, these microglia down-regulated homeostatic markers, such as Cx3cr1 and P2ry12. The homeostatic and ramified microglial cells of non-demented Braak II cases were characterized by Iba1, CX3CR1, P2ry12, TMEM119 and CD45low expression. The dynamic of the microglial molecular phenotypes associated to AD pathology needs to be considered for better understand the disease complexity and, therefore, guarantee clinical success. Correcting dysregulated brain inflammatory responses might be a promising avenue to prevent/slow cognitive decline.Universidad de Málaga. Campus de excelencia Internacional-Andalucía Tech. Supported by PI18/01557 (AG) and PI18/01556 (JV) grants from ISCiii of Spain co-financed by FEDER funds from European Union

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

Tau pathology and astroglial reactivity: a comparative study of two mouse models of tauopathy

Objectives: Astrocytes are becoming crucial players in the context of neurodegenerative proteinopathies, such as Alzheimer’s disease (AD). Astroglial response has been mainly analyzed in amyloidogenic scenarios, but less is known about their involvement in tauopathies. Here, we aimed to analyze astroglial reactivity to hyperphosphorylated-tau (ptau) in the hippocampus of two transgenic mouse models of tauopathy, ThyTau22 and P301S (2- to 12/18-months). Methods: Proteinopathy was assessed by western-blotting and immunohistochemistry (AT8). Neuroinflammation was analyzed by qPCR and bright-field immunohistochemistry, glial-ptau relationship by confocal and transmission electron microscopy. Results: P301S mice exhibited an intense reactive astrogliosis, increasing progressively with aging accordingly to a strong ptau accumulation, whereas ThyTau22 model showed slighter astrocytosis related to lesser proteinopathy. P301S astrogliosis correlated with an acute DAM-like microglial activation, not observed in ThyTau22 hippocampus. In both models, reactive astrocytes contained ptau, especially around vessels. Conclusions: Our results support that astrocytes respond to ptau in the absence of Abeta. This reactivity correlates with tau pathology and depends on microglial DAM-like activation. In addition, reactive astrocytes may play a role in the elimination/spreading of ptau species through the brain. Deciphering the mechanisms underlying these processes might allow the development of strategies to slow down the progression of AD and other tauopathies.Supported by Instituto de Salud Carlos III of Spain, co-financed by FEDER funds from European Union, through grants PI18/01557 (to AG),PI18/01556 (to JV), and Junta de Andalucia through Consejería de Economía y Conocimiento grants UMA18-FEDERJA-211 (AG), P18-RT-2233 (AG) and US-1262734 (JV) co-financed by Programa Operativo FEDER 2014-2020. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Analyzing hippocampal synaptic damage and glial response in a mouse model of tauopathy

Tau pathology is highly related to synaptic and neuronal loss, leading to cognitive decline and dementia in Alzheimer’s disease (AD) and other tauopathies. Tau transgenic mice are widely used to investigate the specific contribution of this protein to AD since they reproduce the synaptic and cognitive dysfunction in parallel to an age-dependent accumulation of hyperphosphorylated forms of tau (phospho-tau). The aim of this study was to investigate the progression of tau aggregation and analyze its relationship with microglial activation and synaptic damage within the hippocampus of a transgenic tau model. 2, 6, 9, 12 and 18 month-old THY-Tau22 transgenic and WT mice were analyzed. Tau pathology was assessed by western blotting and immunohistochemistry (AT8, AT100). Confocal microscopy was used to study microglial/phospho-tau relationship, and Thioflavin-S staining to evidence fibrillar aggregates. Levels of general (Synaptophysin) and subtype-specific (ChAT, VGAT, VGLUT-1) synaptic proteins were determined by WB and immunohistochemistry. Inflammatory markers were assessed by quantitative PCR (CD45, CD68, TREM2), immunohistochemistry (Iba-1) and image analysis. Tau pathology was detectable in the hippocampus from 2 months of age and increased progressively during aging. Presynaptic protein levels were significantly decreased from 9-12 months compared to age-matched WT mice. Even though some inflammatory markers were slightly increased in the hippocampus, microglial reactivity was found to be generally attenuated and some cells even exhibited reduction in their prolongations and a clear degenerative phenotype at advanced ages similar to that seen in the hippocampus of AD patients. Finally, this model could be a relevant tool to further understand the specific role of tau in both microglial response and synaptic pathology in AD.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Disentangling the contribution of tau and abeta pathologies in transgenic models of Alzheimer's disease

AIMS: Amyloid-beta (Abeta) deposits and intraneuronal hyperphosphorylated tau are major pathological hallmarks of Alzheimer’s disease (AD). The coexistence of these aggregates in AD brains leads to synaptic dysfunction, neuronal loss and cognitive decline. Failures in protein homeostasis, along with defective glial responses, have been identified as pathological mechanisms linked to this disorder. Thus, our main objective is to better understand the differential impact of Abeta- and tau-aggregates to these processes in the hippocampus of AD models. METHODS: We analyzed APP- (APPSL/PS1M146L) and Tau- (ThyTau22 and hP301S) based models from 2 to 18 months of age. Tau and Abeta pathologies were assessed by western blotting and immunohistochemistry. Confocal microscopy was used to study microglia/aggregates relationship. Levels of synaptic proteins, autophagy and inflammatory markers were determined by quantitative PCR, WB and immunohistochemistry. RESULTS: Tau and Abeta pathologies initiated as early as 2 months of age and increased progressively with aging. Even though only APP/PS1 hippocampus showed dystrophic neurites positive to proteostatic and presynaptic markers, their protein levels were altered in both types of models from 6-9 months compared to age-matched WT mice. Inflammatory markers and microglial reactivity were barely increased in the hippocampus of ThyTau mice in contrast to P301S and APP/PS1 mice which displayed a prominent microglial response. CONCLUSIONS: Clarifying the effects of Abeta and tau separately would indeed enable the development of novel therapeutic strategies and drugs targeting pathways related to these proteinopathies. Supported by grants FIS PI15/00796 and PI15/00957 co-financed by FEDER funds from European Union, by Junta de Andalucia Proyecto de Excelencia CTS385 2035 and by grant PPIT.UMA.B1/2017.26Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec