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  Glial and vascular contributions to neurodegenerative diseases
    PP59
Single cell transcriptomics of vascular cells from App knock-in mice reveal increased vascular tonus and activation of inflammatory pathways
Katrine Dahl Bjørnholm1, Michael Vanlandewijck2,3, Urban Lendahl2, Francesca Del Gaudio2, Helena Karlström1, Christer Betsholtz2,3, Per Nilsson1
1Karolinska Institutet, Department of Neurobiology Care Science and Society, Section of Neurogeriatrics, Solna, Stockholm, Sweden, 2Karolinska Institutet, Department of Medicine
Huddinge, Huddinge, Stockholm, Sweden, 3Uppsala University, Institute for Immunology, Genetics, and Pathology, Uppsala, Sweden
 Understanding blood brain barrier changes in Alzheimer’s diseases (AD) is important to unravel the contribution of the vasculature to the disease. Single cell RNA sequencing provides high resolution insight into cell populations responsible for the pathophysiological response of the vasculature. We have sequenced single cells obtained by a novel method for isolation of the microvasculature from an App knock-in mouse model of AD (AppNL-G-F), which exhibits severe Aβ plaque accumulation in the brain as well as significant cognitive dysfunction. 37386 cells were sequenced and analyzed from the AD and WT mice in total. In the present work we have focused on changes in endothelial cells transcriptomics. Here, several pathways were altered. Genes involved in vasomotor function were differentially expressed; Vasoconstriction genes (Edn1, Myh9) were upregulated, and genes involved in nitric oxide synthase function (Nostrin, KLF4, Mbd2) were downregulated in AD mice. Moreover, the endothelium of AD mice exhibited an increased expression of inflammatory genes including C1qa, Ccl4, Tgfb2, and Cxcl12.
In summary, we provide high resolution single cell transcriptomics specifically on the vascular niche from the AppNL-G-F mouse model of Alzheimer’s disease. Results from differential analysis of the endothelial cell portion suggest that the vasculature reacts to severe Aβ accumulation with increased vascular tonus and increased inflammatory response. This in turn can lower oxygen supply and decrease capillary blood flow, which might contribute to further deterioration of the clinical manifestations of AD, which warrants further investigation.
using optical coherence tomography (OCT). After OCT imaging, eyeball sections underwent immunohistochemical staining using Aβ-specific antibodies.
Results: ERG showed the amplitude of both a- and b-wave, especially in scotopic condition, was significantly declined with ages in AD mice. OCT analysis indicated that the retinal nerve fiber layer was thinner in 5xFAD at 3, 6, and 9 months of age than those in WT littermates but the inner plexiform layer was thicker. Immunohistochemical staining showed Aβ deposition and reactive gliosis in the retina including the ciliary body in AD mice, worsening over time.
Discussion: The present study demonstrated that the Aβ accumulates in the retina of AD mice and the ocular pathologies occurs in the inner retina at the early ages, progressing to the outer retina with age. Therefore, the retina harbors the earliest AD-specific signs functionally as well as structurally detectable. Further studies are required to validate clinically this retinal biomarker for early AD diagnosis.
Conclusion: It provides insight into retinal changes associated with early phase of AD and contributes to understanding mechanism of visual abnormalities in patients with AD.
  PP62
Phospholipase D3 deficiency compromises mitochondrial DNA degradation leading to lysosomal dysfunction and membrane lipid alterations
Zoë P. Van Acker1, Marine Bretou1, Ragna Sannerud1, Markus Damme2, Wim Annaert1
1VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium, 2Christian-Albrechts- University Kiel Institute of Biochemistry, Kiel, Germany
   PP60
Investigation of functional and structural changes of retina in a murine model of alzheimer’s disease
Chae-Eun Moon1,2, Kyusun Han1, Hae-Sol Shin1,3, Yejun Cho1, Jun Ki Lee1, Hyun Jin Kim1, Hong Kyung Kim1,3, Suk Ho Byeon1,2, Kyoung Yul Seo1,3, Hyung Keun Lee1, Yong Woo Ji1,4 1Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea, 2Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea, 3Korea Mouse Sensory Phenotyping Center, Yonsei
University College of Medicine, Seoul, Korea, 4Department of Ophthalmology, Yongin Severance Hospital, Yonsei University College of Medicine, Seoul, Korea
 Background: Recent clinical studies of Alzheimer’s disease (AD) have focused on developing retinal imaging as the retina is the only part of the central nervous system that can be imaged noninvasively using optical methods. However, the detailed underlying mechanism on retinal abnormalities in AD remain elusive. We aimed to investigate functional and structural changes of the retina in the early and late phase using a murine AD model overexpressing beta amyloid (Aβ) known to exhibit neurologic symptoms with age.
Materials and Methods: Full-field electroretinogram (ERG) was conducted at 3, 6, and 9 months of age to characterize retinal function in the 5xFAD and wild type (WT) mice (n=4/group). Subsequent examinations were performed on structural changes of the retina
Background: Phospholipase D3 (PLD3) is a single-pass type II membrane protein that is majorly localized to late endosomes/lysosomes (LE/Lys) where it functions as a 5'-3' exonuclease that degrades ssDNA. Whereas PLD3 has been identified as risk factor for late-onset Alzheimer’s disease (LOAD), knockout studies have resulted in conflicting data regarding its impact on the amyloid precursor protein metabolism. Moreover, the substrates for PLD3 in LE/Lys remain unexplored.
Materials and Methods: CRISPR/Cas9 gene editing was used to generate PLD3 knockout (KO) SH-SY5Y cells that were subsequently stably rescued with wild-type PLD3 and coding-variants (M6R & V232M). All cell lines were evaluated for morphological and functional alterations of the LE/Lys compartment, including lipid profiling and analysis of the nucleotide content of LE/Lys magnetically isolated from the different cell lines. We optimized a PLD3 activity assay and analyzed the LE/Lys nucleotide content as a means to identify PLD3 substrates. Results: We report that a PLD3 KO and LOAD-associated risk variants lead to a significant lysosomal pathology, comprising an increase in the size of LE/Lys, more LAMP1 immunoreactivity, a reduced catabolic activity, an increased propensity for leakage and a marked increase in cholesterol content and in storage lipids. At the ultrastructural level, this is accompanied with the appearance of multilamellar bodies, sometimes including mitochondrial remnants. This is suggestive for a defective mitophagy, which we confirm using mKeima assays. Furthermore, we provide support for a role of PLD3 in the homeostatic degradation of mtDNA. We argue that when PLD3’s role is compromised, this leads to a build-up of the lysosomal nucleotide content, impacting on the normal functioning of LE/Lys.
Conclusion: Collectively, our findings subscribe a key role for PLD3 exonuclease activity in lysosomal homeostasis. Its dysfunction, as occurs in LOAD, leads to the co-occurrence of lysosomal and mitochondrial defects.
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