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  Glial and vascular contributions to neurodegenerative diseases
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Transcriptomic changes in astrocytes and fibroblasts from an App knock-in ADmouse model (AppNLGF)
Hao Li1, Katrine Dahl Bjørnholm1, Helena Karlström1, Christer Betsholtz2,3, Michael Vanlandewijck2,3, Per Nilsson1 1Department of Neurobiology, Care sciences, and Society, Division of Neurogeriatrics, Karolinska Institutet, Stockholm, Sweden, 2Department of Medicine, Huddinge, Karolinska
Institutet , Stockholm, Sweden, 3Department of Immunology, Genetics, and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden
Alzheimer disease (AD) exhibits aberrant protein aggregation in the brain, but our understanding of the vascular response to these aggregates is limited [1]. Amyloid Precursor Protein (App) knock-in mice (AppNLGF) recapitulate several aspects of AD including amyloid beta (Aβ) plaque formation, neuroinflammation and synaptic loss. We investigated the potential vasculature impairment in this mouse model by performing CD31 panning to isolate the vasculature recovering several main vascular components. However, the pool of perivascular fibroblasts (FB) and astrocytes were limited.
For the specific isolation of astrocytes, established microbeads-based purifications will be used. However, no such isolation procedures are established for FB cells. Thus, we have crossed App knock-in mice with reporter mice [2] that specifically express eGFP in the FB cells (driven by the promoter for pericyte-derived growth factor alpha receptor (Pdgfra) [3]. Dissected brains were homogenized followed by single cell FACS into a Smart-seq3 compatible lysis buffer. RNA was reverse transcribed to cDNA, fragmented by the Tn5 transposase and finally sequenced on an Illumina NextSeq2000. Cluster analysis, gene expression comparisons and pathway analysis are ongoing.
Based on these results, validation of target genes will be performed to find the molecular questions that drive brain vascular dysfunction (ischemic damage [4], oxidative stress [5], cytokines [6], inflammasome, etc.). We will use immunohistochemical staining and develop RNA- scope probes to characterize the corresponding mRNA distribution and compare the data with protein/mRNA distribution in brain sections from control and AD patient brains to determine the molecular underpinnings of vasculature changes in AD to discover possible biomarkers and drug targets.
Background: Both healthy aging and neuroinjury increase neuro- inflammatory processes in the brain. Neuroinflammatory processes, moreover, induce synapse loss in aging and in Alzheimer’s and related dementing diseases[1,2]. A direct connection between aging, neuroinflammation, and structural outcome following traumatic brain injury (TBI) could have important implications related to the increased risk of dementia in TBI victims. We used genetic and pharmacological manipulations to determine how structural and behavioral outcomes are impacted by cellular and humoral neuroinflammation following TBI. Materials and Methods: Adult (3-6 mo) and aged (12-24 mo) male and female C57/BL/6J and C3KO[3] mice with impaired complement activation received a focal brain injury (controlled cortical impact) or sham procedure[4]. Microglia were depleted in a subset of mice using a colony stimulating factor 1 receptor (CSF1R) inhibitor[5]. Tissue was collected at 3, 7, and 30 days post-injury. Learning and memory was assessed using the Morris Water Maze[6]. Synapses were analyzed using SEQUIN[7,8].
Results: At all times post-injury, wild-type mice showed significant loss of excitatory synapses in the ipsilateral cortex and hippocampus. Pretreatment with a CSF1R inhibitor, however, largely prevented injury- induced synapse loss. Genetic inhibition of complement activation similarly protected synapses. C3KO animals, moreover, were protected from neurobehavioral impairment relative to controls. We furthermore assessed synapse loss as a predictor of functional outcome following TBI. Lastly, we assessed the interaction between healthy aging and neuroinflammation as determinants of trauma-induced synapse loss and functional impairment.
Discussion: Inhibition of neuroinflammation mitigated trauma- induced synaptopathy and behavioral deficits. Thus neuroinflammatory mechanisms are key contributors to structural and functional outcome following TBI. Synapse loss may represent a shared pathomechanism connecting acute neuroinjury to long-term neurodegeneration. This is being tested in ongoing studies.
Conclusions: These findings
neuroinflammation following TBI and suggest potential therapeutic approaches to alleviating secondary injury and subsequent neurodegeneration.
highlight the critical role of
  PP74
Preclinical steps for improved compound delivery in neurodegeneration using BP-EVs nanocarriers
Aida Serra1, Cristina Lorca1, Xavier Gallart-Palau2
1IMDEA-Food Research Institute, Campus of International Excellence UAM+CSIC , Madrid, Spain, 2Biomedical Research Institute of Lleida Dr. Pifarré Foundation (IRBLLEIDA) - +Pec
Proteomics Research Group (+PPRG) - Neuroscience Area – University Hospital Arnau de Vilanova (HUAV), Lleida, Spain
 Background: Compound delivery within the Central Nervous System (CNS) is challenging due to the impermeable function of the blood brain barrier. Usually, only a very small portion of the total plasma circulating compound reaches strategic areas within the CNS. This fact alters the normal efficiency of CNS-oriented compounds and is linked to the requirement of using higher dosages with exacerbated undesired effects. Extracellular vesicles (EVs) have proven unparallel ability to improve compound delivery within the CNS [1]. However, reliable, safe and highly available sources of EVs at low cost remain scarce [2]. Material and Methods: EVs from certain food industry by-products were screened for the presence of nanocarrier-oriented EVs by Transmission Electron Microscopy and Nanoparticle Tracking Analysis. These vesicles were then characterized by systems biology technologies including mass spectrometry proteomics and lipidomics [3]. Oral and intravenous capacity to reach the central nervous system was evaluated by in vivo fluorescent imaging in mice and toxicity was measured in vivo and in vitro.
Results and Discussion: Our results indicate that BP-EVs show a predominant exosome-like profile(s) in terms of size, structural features and biochemical profile. Furthermore, these vesicles have ability to reach the brain in vivo with signaling peaks that follow 4-24h post- oral administration. BP-EVs also remain within CNS tissues beyond 24h post-oral administration. Brain targeting capacity of BP-EVs increases the bioavailability of these vesicles by ~25x compared to standard vias of administration. Furthermore, preliminary results indicate that these vesicles have excellent ability to accommodate therapeutic compounds that have vastly shown low CNS bioavailability coupled to promising therapeutic capacity in neurodegeneration.
Conclusions: Collectively, the findings discussed here demonstrate that certain food-industry by-products are an excellent source of nanocarrier oriented EVs for compound delivery within the CNS with highly promising application in neurodegenerative conditions.
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Neuroinflammation mediates synaptic pruning following traumatic brain injury
Sydney J. Reitz1, Akshata A. Korgaonkar1, Andrew D.
Sauerbeck1, Terrance T. Kummer1
1Department of Neurology, Washington University In St. Louis, St. Louis, USA
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