Search

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.

Aims: The lack of reliable models of Alzheimer´s disease (AD) has impeded the development of effective therapies. Glial cells have a key role in AD pathology, but this cannot be properly modeled using available animal models, so we hypothesized that cells derived from Alzheimer's patients can serve as a better platform for studying the disease. In this sense, human pluripotent stem cells (hPSC) allow the generation of different types of neural cells, which can be used for disease modeling, identification of new targets and drugs development. Methods: We have a collection of hiPSCs derived from patients with sporadic forms of AD stratified based on APOE genotype. We have differentiated these cells towards neural lineage to obtain astrocytes to assess intrinsic differences between those derived from AD patients or healthy controls. Results: We have implemented a serum-free approach and generated neural precursors and astrocytes from all the lines tested. Cells are different at the phenotypic level, suggesting intrinsic differences in neural cells derived from AD patients. Conclusions: Human pluripotent stem cell-derived methodology can be used to elucidate the pathogenic pathways associated with neurodegeneration and to identify new therapeutic targets susceptible to modulation, contributing to the development of new effective drugs against AD. Supported by Instituto de Salud Carlos III of Spain PI18/01557, PI21/00915 (to AG), PI18/01556 (to JV), and CIBERNED (CB06/05/1116 to AG and CB06/05/0094 to JV); by Junta de Andalucia UMA18-FEDERJA-211 (to AG), PY18-RT-2233 (to AG) and US-1262734 (to JV), Consejeria de Salud PI-0276-2018 (to JAGL) and Programa Operativo de Empleo Juvenil SNGJ4-11 to LCP. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

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).

Acknowledgments: This study was 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 CIBERNED (CB06/05/1116 to AG and CB06/05/0094 to JV); by Junta de Andalucia through Consejería de Economía y Conocimiento grants UMA18-FEDERJA-211 (to AG), PY18-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). Oligodendrocytes (OLs) are highly specialized cells of the central nervous system responsible for myelin production and metabolic support of neurons. Defects in OLs are crucial in several neurodegenerative diseases including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Scarce access to primary samples and lack of efficient protocols to generate OLs from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. To promote the conversion of hPSCs into OLs, we have screened for a number of transcription factors (TFs) previously reported to be involved in OL generation. We found that the overexpression of SOX10 was sufficient to generate O4+ oligodendrocyte precursor cells (OPCs) from hPSCs only 10 days after SOX10 induction. Generated OPCs expressed mature OL proteins as MBP or MOG. At the transcriptome level, generated OPCs resembled primary OPCs. To test the functionality of generated OPCs, O4+ cells were co-cultured together with neurons, finding that O4+ cells were able to myelinate the neurons. Moreover, O4+ cells were injected intracerebrally in newborn shiverer RAG2-/- mice, finding that generated OLs extended within the corpus callosum and generated functional myelin, demonstrating the functionality of generated cells also in vivo. The protocol also describes an alternative for viral transduction, by incorporating an inducible SOX10 in the safe harbor locus AAVS1, yielding ~100% pure OPCs. O4+ OPCs can be co-cultured with maturing hPSC-derived neurons in 96/384-well- format plates, allowing the screening of pro-myelinating compounds. We have developed a novel methodology for a fast (20 days from hPSC stage) and efficient generation of functional OLs, which allow testing of compounds involved in myelination. This technology will allow further studies to better understand human OL biology and the screening of potential compounds involved in myelination in a human setting. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Parenchymal microglia are the brain-resident immune cells capable of responding to damage. Though the role of microglial cells in the development/progression of AD is still unknown, a dysfunctional response has recently gained support since the identification of genetic risk factors related to microglial. In this sense, we reported an attenuated microglial activation in the hippocampus of AD patients, including a degenerative process of the microglial population in the dentate gyrus. On the other hand, it is also known that others myeloid components could also be involved in the neurodegenerative process. However, the implication of the diverse immune 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 the hippocampus of AD brains. For this purpose, immunohistochemistry and image analysis approaches have been carried out in non-demented controls and AD cases. Damage-associated myeloid cells from Braak II and Braak VI individuals were clustered around amyloid plaques and expressed Iba1, TMEM119, CD68, Trem2 and CD45high. A subset of these cells also expressed ferritin. However, and even though some Braak II individuals accumulated CD45-positive plaques, only AD patients exhibited parenchymal infiltration of CD163-positive cells, along with a decrease of the resident microglial marker TMEM119. Moreover, a negative correlation was observed between CD163 and TMEM119 intensities in Braak VI patients, showing a functional cooperation among these different myeloid populations. Taken together, these findings suggest the existence of different populations of amyloid-associated myeloid cells in the hippocampus during disease progression. The differential contribution of these myeloid populations to the pathogenesis remains to be elucidated. The dynamic of the myeloid molecular phenotypes associated to AD pathology needs to be considered for guarantee clinical success. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Microglial cells, the immune cells of the brain, and the neuroinflammatory process associated, have been postulated as a critical factor in AD pathogenesis, since the identification of genetic risk factors related to microglial function. However, the microglial role in the development/progression of AD has not been determined yet. In this sense, we have previously reported a limited activation and microglial degeneration in the hippocampus of AD patients in contrast to the proinflammatory view based on findings in amyloidogenic models. Here, we have further analyzed the functional/phenotypic profile displayed by microglial cells in other vulnerable brain region of AD patients, the frontal cortex. Immunohistochemistry and image analysis approaches were performed in the frontal cortex of post mortem samples from controls (Braak 0-II) and AD patients (Braak V-VI) including familial cases. Microglia of Braak V-VI individuals were observed forming clusters and showed, both plaque (Iba1+/TMEM119+/P2ry12-/CD45high/Trem2+) and inter-plaque (Iba1+/ TMEM119+/P2ry12-/CD45high/Trem2-) microglial activation, similar that observed in amyloidogenic mice. By contrast, homeostatic and ramified microglial cells of non-demented Braak II cases presented Iba1+/P2ry12+/TMEM119+/CD45low/Trem2- profile. Furthermore, different microglial responses were observed between sporadic and familial AD cases. These different microglial phenotypes associated with AD pathology show the heterogeneity and complexity of the microglial phenotypes and suggest different functional states of these glial cells in a region-specific manner. These data need to be considered for better understand the immunological mechanisms underlying AD progression. Modulating brain inflammatory responses might be a promising avenue to prevent cognitive dysfunction in AD patients. ISCiii:PI18/01557(AG)-PI18/01556(JV);Junta Andalucia:UMA18-FEDERJA211(AG). All cofinanced by FEDER funds (European-Union). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Aims: Cognitive decline in Alzheimer's disease (AD) is highly related to synaptic/neuronal loss. Tau hyperphosphorylation destabilizes microtubules leading to axonal transport failure and generation of dystrophic neurites, thus contributing to synaptic dysfunction. The effect of microtubule stabilization on amyloid-beta pathology has not been assessed in vivo yet. This study evaluated the effect of the microtubule-stabilizing agent, Epothilone D (EpoD) in the pathology of an amyloidogenic mouse model. Methods: APP751SL/PS1M146L mice (3-month-old) were treated weekly with intraperitoneal injections of EpoD (2 mg/kg) or vehicle for 3 months. For memory performance, animals were tested on the objectrecognition, Y-maze and Morris water maze. Hippocampal proteinopathies were quantified by image analysis after immunostaining. Somatostatin (SOM)-numerical density was calculated by stereology. APPswe-N2a cells were treated with EpoD 100nM for 12/24 hours. Protein levels were analysed by Western/dot-blot. Results: EpoD-treated mice improved their performance of cognitive tests, while hippocampal phospho-tau and Ab (especially oligomers) accumulation decreased, together with synaptic/neuritic pathology. Remarkably, EpoD exerted a neuroprotective effect on SOM-interneurons, a highly AD-vulnerable GABAergic subpopulation. Conclusions: EpoD improved microtubule dynamics and axonal transport in an AD-like context, reducing tau and Ab accumulation and promoting neuronal and cognitive protection, underlining the cross-talk between cytoskeleton pathology and proteinopathy. Therefore, microtubule-stabilizing drugs could be candidates for slowing AD at both tau and Ab pathologies. Supported by PI18/01557 (to AG) and PI18/01556 (to JV) grants from ISCiii of Spain, co-financed by FEDER funds (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.