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

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

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

Diversity of plaque-associated myeloid cells subtypes in human alzheimer’s disease brain

Aims: Parenchymal microglia, as well other myeloid cells, have been postulated as a critical factor in Alzheimer´s disease (AD) pathogenesis since the identification of genetic risk factors related to their functions. However, the different phenotypes and the implication of the diverse immune cells in the human pathology have not been determined yet. In this work, we have further analyzed the phenotypic profile of the damage-associated myeloid cells in two AD vulnerable brain regions, the frontal cortex and hippocampus. Methods: Immunohistochemistry and image analysis approaches have been carried out in postmortem brain samples from patients with AD (Braak V-VI) and aged controls without neurological symptoms (Braak II). Results: Damage-associated microglial cells were clustered around amyloid plaques and expressed Iba1, TMEM119, CD68, Trem2 and CD45high. Moreover, AD brains exhibited parenchymal infiltration of CD163-positive monocyte-derived cells that invaded plaque near blood vessels. While the frontal cortex showed strong microglial activation similarly to that reported in amyloidogenic mice, the hippocampus of the same patients showed an attenuated microglial activation with a degenerative phenotype. Conclusions: These findings suggest the existence of different myeloid populations associated with Aβ plaques that correlates with disease severity. These results 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 of Spain grants PI18/01557 (AG), PI18/01556 (JV) co-financed by FEDER funds from EU, by Junta de Andalucia grants UMA18-FEDERJA-211(AG), P18-RT-2233(AG) and US-1262734(JV) co-financed by Programa Operativo FEDER 2014-2020, and by B1-2019_07 Universidad de Malaga. Campus de Excelencia Internacional Andalucia Tech (ESM)

Dissecting the microglial response in transgenic models of amyloidogenesis and tauopathy

Amyloid-beta (Abeta) peptide deposits and hyperphosphorylated tau protein (phospho-tau) accumulate in Alzheimer’s disease (AD) brains. These abnormal protein aggregates leads to glial activation, synaptic dysfunction, neuronal loss and cognitive decline. While microglial response has mostly been analyzed in relation to Abeta accumulation, little is still known about inflammatory processes associated with tau pathology. Microglial reactivity and defective glial responses have been involved in these proteinopathies. Our aim is to clarify the effects of Abeta and tau separately, in order to improve the comprehension of their differential contribution to neuroinflammation and neurodegeneration. We compared the progression of these processes in an amyloidogenic AD model (APPSL/PS1M146L) and two different models of tauopathy (ThyTau22 and hP301S) from 2 to 18 months of age. Accumulation of aggregated proteins was assessed using specific anti- Abeta and phospho-tau antibodies. Inflammatory response was studied using a battery of microglial markers (Iba1, CD45, CD68, Trem2 and Gal-3). In the hippocampus of these models, Tau and Abeta pathologies initiated as early as 2 months of age and increased progressively with aging. Neuritic plaques induced a strong microglial activation associated to plaques in APP/PS1 mice. Interestingly, inflammatory markers and microglial reactivity were barely increased in the hippocampus of ThyTau mice in contrast to not only APP/PS1, but also to P301S mice, which displayed a prominent microglial response. Deciphering the specific effects of Abeta, tau and their different toxic species, would indeed enable the development of novel therapeutic strategies and drugs targeting neuroinflammatory pathways related to these proteinopathies.Universidad de Málaga. Campus de excelencia Andalucía-Tech. Supported by PI18/01557 (AG) and PI18/01556 (JV) grants from ISCiii of Spain co-financed by FEDER funds from European Union, and by grant PPIT.UMA.B1.2017/26 (RS-V)

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

Comparing astroglial reactivity in two transgenic mouse models of tauopathy

Astrocytes are becoming crucial players in the pathology of neurodegenerative disorders, such as Alzheimer’s disease (AD). Astrocyte responses have been mainly analyzed in the context of amyloid-beta (Abeta) pathology, highlighting their role in the development/progression of amyloidosis and their relationship with the microglial response. Regarding tau pathology, some studies have reported that astrocytes respond to hyperphosphorylated tau (phospho-tau) and suggested their implication on tau transmission/elimination. Here, we aimed to analyze the astroglial reactivity to tau pathology in the hippocampus of two transgenic mouse models of tauopathy, ThyTau22 and P301S. Proteinopathy was assessed by western-blotting and immunohistochemistry using phospho-tau antibodies (AT8). Inflammatory markers (GFAP, Iba-1, CD45, TREM2) were analyzed by qPCR and immunohistochemistry for bright-field microscopy; glial-phospho-tau relationship was analyzed under confocal and transmission electron microscopy. P301S mice exhibited an intense reactive astrogliosis, increasing with aging in parallel to a strong phospho-tau pathology. ThyTau22 model showed a slighter astrocyte reactivity accompanied by a lesser accumulation of phospho-tau. Astrogliosis in P301S mice closely correlated with an acute DAM-like microglial activation, not observed in ThyTau22 hippocampus. Confocal and ultrastructural studies revealed that, in both models, astrocytic processes contained phospho-tau, especially those surrounding blood vessels. Our results support that astrocytes respond to tau pathology in the absence of Abeta. This reactivity highly correlates with phospho-tau pathology and markedly depends on microglial activation. Moreover, astrocytes may play a role in the elimination/spreading of phospho-tau species through the brain. Deciphering the mechanisms underlying these processes might help to develop therapies to slow down the progression of AD.Supported by Instituto de Salud Carlos III (ISCiii) of Spain, co-financed by FEDER funds from European Union through grants PI18/01557 (to AG), PI18/01556 (to JV), and by 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 FEDER2014-2020. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

Abeta from APP/PS1 Alzheimer mice hippocampus induced synaptic damage in vivo and in vitro

We aim to investigate the effects of Abeta from young APP/PS1 mouse model of Alzheimer`s disease (AD) on the synaptic integrity, as the loss of synapses strongly correlates with cognitive deficits in patients. Plaque-associated abnormal swellings of neuronal processes represent the first indicator of disease development and might compromise neuronal integrity and synaptic function. Here, we examined the synaptic nature of dystrophic neurites, and the reduction of both synapses and vesicles density in presynaptic terminals along with the progressive accumulation of autophagic structures and Abeta within hippocampal synaptosomes during the aging. We analysed both the direct synaptotoxic effect of plaques in the hippocampus of this model and also the repercussion of the soluble (S1) fraction in neuronal cultures. Hippocampal synapses were observed under both optic and electron microscopy. Synapses and vesicle density were quantified in periplaque and control (plaque-free) areas by electron microscopy. Primary neuronal cultures were incubated for 48 hours with 6-month-old APP/PS1 and wild-type S1 fractions. In addition, Abeta immunodepletion was carried out with different anti-Abeta antibodies and the levels of synaptic proteins were measured by Western-blot (WB). Both synapse number and synaptic-vesicles density were significantly decreased in young APP/PS1 mice, close to the Abeta deposits, in several hippocampal layers. Importantly, there was a correlation between the synaptic deficiencies and the distance to plaques, which presented oligomeric forms in their periphery. Some presynaptic elements were abnormally swollen, containing autophagic vesicles. In addition, we found by WB a decrease in several hippocampal synaptic markers as early as 4 months of age in this model, and also in neuronal cultures incubated with S1 fractions. Significantly, the neuronal reduction in VGLUT was reversed after Abeta immunodepletion. Plaque-associated oligomeric Abeta induced an early deleterious effect on synapses that correlates with memory deficits in young APP/PS1 mice. Moreover, soluble Abeta derived from these transgenic mice reduced synaptic protein content in vitro, which was restored after immunodepletion of Abeta species. Therefore, this model produced synaptotoxic Abeta and may represent a valuable tool to test novel treatments to protect synapses as an early therapeutic approach for 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