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  Glial and vascular contributions to neurodegenerative diseases
    PP78
Transcriptional Profiling of Oxidative Stress in Alzheimer’s Disease
Zhaoqi Yan1,2, Andrew Mendiola1,2, Yu Yong1,2, Alexander Pico1, Katerina Akassoglou1,2,3
1Gladstone Institutes, San Francisco, USA, 2Center for Neurovascular Brain Immunology at Gladstone and UCSF, San Francisco, USA, 3Department of Neurology, Weill Institute for Neurosciences, University of California, San Francisco, USA
 Background: Alzheimer’s disease is the most common form of dementia; currently, there is no treatment for halting the disease progression. Cerebral vascular dysfunction is an early, independent, and additive predictor of cognitive decline. Blood-brain barrier (BBB) disruption is detected in mild cognitive impairment and early-stage AD and correlates with disease progression. Blood-brain barrier (BBB) disruption leads to oxidative stress and correlates progression of Alzheimer’s disease (AD). The blood coagulation factor fibrinogen induces oxidative stress and microglia-mediated spine elimination and cognitive impairment in AD mice. However, the transcriptomic landscape of oxidative stress-producing cells in AD is unclear. Methods: We recently developed Tox-seq for functional transcriptomic characterization of oxidative stress-producing central nervous system innate immune cells (Mendiola et al., Nat Immunol 2020). Here, we performed Tox-seq to determine the transcriptomic atlas of oxidative stress in a mouse model of AD.
Results: We identified a microglia subset, termed MgAD4, enriched in oxidative stress production in 5XFAD mice with a core oxidative stress gene signature. MgAD4 showed the highest expression for mouse and human AD microglia signatures with reduced expression of homeostatic markers. Comparison of oxidative stress signatures between 5XFAD, the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE) and fibrin-induced myeloid cell activation, revealed fibrin-induced signatures associated with oxidative stress production shared between autoimmune and neurodegenerative diseases.
Conclusions: Overall, our current work provides a molecular atlas of oxidative stress in AD and identifies potential convergent targets for neuroprotective intervention.
resulted from multiple overlap analysis. Target genes were labelled using immunofluorescence.
Results: Risk variants associated with PSP and FTD diagnosis were enriched in peaks of astrocytes (n=4,425), exclusively, while astrocyte- and microglia-driven signalling dominated in cellular immunity, synaptic and cytoskeleton pathways. In pseudotime analysis, astrocytes exhibited activation of immediate early response (IER) and homeostasis TFs (e.g., FOS, JUN, TFEB) at the expense of early differentiation candidates (e.g., LHX9, EMX2). Feature importance of a fitted boosting- tree identified the relevance of IER-related TFs. Tauopathy signatures confined with astrocytic pTau-correlated TF networks[3] comprised JUN/FOS and TFEB among others. TF target dysregulation of MAP3K8 (PSP/CBD: up) and CTSD (PSP: down) were validated at the protein level. Discussion: We integrated multimodal (epi-)genomic data to explore complex pathomechanisms and identify disease-specific TF signatures in 4R-tauopathies. Although microglia genetics are tightly linked with an Alzheimer’s phenotype [5,6], here a major genetic risk is attributed to astrocytes. TF signatures indicate neuropathological entities and pathogenetically relevant pathways (acquired immunity, homeostasis). Validation studies in larger cohorts, further assays and phenotypic information are desirable.
Conclusion: In this 4R-tauopathy cohort astrocytes harbour high genetic risks and contribute extensively to neuroinflammation and protein degradation. Molecular pTau signatures reflect distinctive pathomechanisms.
Background: Macular fibrosis is the end-stage complication of neovascular age-related macular degeneration (nAMD), the leading cause of blindness in the elderly in developed countries. Around 45% of nAMD develop macular fibrosis within 2 years leading to irreversible vision loss. We previously reported higher plasma levels of complement fragments C3a and C5a in nAMD patients with macular fibrosis(1). We aim to understand the role of the complement system in retinal fibrosis. Materials and Methods: RNAseq was conducted in a mouse model of subretinal fibrosis developed by us(2). The expression of complement C5b-9, C3a/C3aR and C5a/C5aR in fibrotic lesions was examined in nAMD donors and the mouse model. Primary retinal pigment epithelial (RPE) cells or bone marrow-derived macrophages (BMDMs) were treated with C3a, C5a or TGF-β. Expression of E-Cadherin, αSMA, collagen-1 and fibronectin or macrophage (CD11b, F4/80) markers was determined by qPCR, Western blot and immunocytochemistry. The effect of the complement pathway in subretinal fibrosis was examined using C3aR, C5a/C5aR antagonists or a C5 neutralising antibody.
Results: Inflammation-related pathways including the complement & coagulation cascades were enriched in subretinal fibrosis in the RNAseq study. Immunofluorescence revealed strongly C5b-9 expression at the lesion site. C3aR was detected in F4/80+ macrophages and C5aR in RPE cells. TGF-β treatment upregulated the expression of αSMA in both RPE and BMDMs. C3a but not C5a significantly upregulated the expression of αSMA, fibronectin and collagen-1 in BMDMs. C5a but not C3a increased αSMA and decreased E-Cadherin in RPE cells. Blocking C5 significantly reduced subretinal fibrosis, whereas blocking C3a or C5a/ C5aR only partially reduced retinal fibrosis.
Discussion/Conclusions: Complement activation contributes to the development of retinal fibrosis through the induction of macrophage- to-myofibroblast transition via the C3a/C3aR pathway and epithelial- to-mesenchymal transition of RPE via C5a/C5aR pathway. Targeting the complement system may be a novel approach to treating macular fibrosis.
  PP80
The role of the complement system
in neovascular age-related macular degeneration mediated retinal fibrosis
Heping Xu1,2, María Llorián-Salvador1, Karis Little1, Caijiao Yi2, Manon Szczepan1, Jian Liu2, Mei Chen1
1The Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, United Kingdom, 2Aier Institute of Optometry and Vision Science, Changsha, China
   PP79
Single-Nucleus Chromatin Accessibility Analysis and Machine Learning to Understand the Role of Astrocytes in 4R-Tauopathies
Nils Briel1,2, Viktoria C. Ruf1, Katrin Pratsch1,2, Sigrun Roeber1, Janina Mielke1, Jeannine Widmann1, Mario M. Dorostkar1, Otto Windl1,2, Thomas Arzberger1,2,3, Felix L. Struebing1,2, Jochen Herms1,2,4
1Center For Neuropathology, Ludwig-Maximilians-University, Munich, Germany, 2German Center for Neurodegenerative Diseases (DZNE), Site Munich, Munich, Germany, 3Department of Psychiatry and Psychotherapy, University Hospital Munich, Ludwig-Maximilians-
University, Munich, Germany, 4Munich Cluster of Systems Neurology (SyNergy), Munich, Germany
 Background: Molecular pathomechanisms in 4R-tauopathies like Progressive Supranuclear Palsy (PSP) and Corticobasal Degeneration (CBD) remain insufficiently characterized at single-cell resolution. Especially, characteristic astrocytic phospho-Tau (pTau) inclusion phenotypes (tufted astrocytes in PSP, astrocytic plaques in CBD) are associated with synapto- and neurodegeneration[1–3].
Methods: The Assay for Transposase-Accessible Chromatin using sequencing was applied on frontal cortices of 4 PSP, 4 CBD and 5 control cases. Celltype-resolved risk variant enrichment was computed with gchromVAR, cell-cell communication with CellChat. Transcription factor (TF) motif enrichment in astrocytes was assessed by pseudotime analysis, group-wise comparisons and a boosting-tree classifier, and finally compared with external PSP bulkRNA-seq data[4]. TF signatures
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