- Email: [email protected]
LOSS OF OLIGODENDROCYTES IS AN EARLY AMYLOID-INDEPENDENT EVENT IN THE PROGRESS OF DEMENTIA
Poster Presentations: Sunday, July 24, 2016
and in vivo. Conclusions: Suppressing pathogenic intracellular calcium signaling during early disease stages is strategic and effective therapeutic approach to prevent the progression of AD. P1-141
LOSS OF OLIGODENDROCYTES IS AN EARLY AMYLOID-INDEPENDENT EVENT IN THE PROGRESS OF DEMENTIA
Kai-Hei Tse, Karl Herrup, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Contact e-mail: [email protected] Background: DNA damage and neuronal cell cycle re-entry are commonly observed during neurodegeneration in Alzheimer’s disease (AD). White matter (WM) degeneration is a recognized co-morbidity, but little is known about the underlying neurobiology. We hypothesize that oligodendrocyte loss (OL) occurs early in the process of dementia that is independent of amyloid. Methods: A cross sectional dataset of MRI data from healthy controls, individuals with mild cognitive impairment (MCI) or subjects with AD (n ¼ 507) was obtained from the National Alzheimer’s Coordinating Center and Spearman’s correlational analysis was performed. Additional specimens from healthy controls, individuals with plaque-free dementia and others diagnosed as AD came from NeuroBioBank (n ¼ 8 per group). These cases were examined by immunofluorescence. Results: Gray matter (GM) and white matter (WM) volume declined with age as well as with MMSE. In MCI, the GM reduction was highly correlated with a reduced hippocampal volume (R ¼ 0.42) and increased white matter hyperintensities (WMH; R ¼ 0.49). We found no correlation with WM volume (R ¼ 0.13). These findings, especially the MCI cohort, implicate myelin lesions as an early event in the onset of dementia. By immunocytochemistry, the number of actively myelinating oligodendrocytes (myelin regulatory factor [MyRF]-positive), mature OLs (myelin basic protein positive) or Olig2-positve OLs were signifcantly reduced in plaque-free dementia as well as in AD (p < 0.005). Moreover, 70% of the Olig2+ cells co-labelled with gH2A.X in the plaque-free dementia group, indicative of DNA damage (p ¼ 0.006). In all dementia groups, 25% of the postmitotic MyRF+ cells stained with the cell cycle marker, cyclin D (p < 0.05); caspase-3 staining was found in the same areas. Conclusions: WMH in the aging population is an early correlate of neuronal loss and cognitive decline. The presence of OLs double stained for cyclin D and caspase-3 suggests that, just as in neurons, aberrant cell cycle re-entry is associated with OL cell death. Finally, the correlation of these events with dementia, but not with plaques implicates an amyloid independent mechanism as the root cause of the white matter abnormalities. P1-142
INVESTIGATING THE EFFECT OF MULTIPLE GENES ON CELLULAR PROCESSES IMPLICATED IN LATE ONSET ALZHEIMER’S DISEASE
Anna N. Barrett1, Melanie L. Dunstan1, Rhian S. Thomas1, Emma J. Kidd1, Lesley Jones1, Julie Williams2, 1Cardiff University, Cardiff, United Kingdom; 2MRC Centre for Neuropsychiatric Genetics & Genomics, Institute of Psychological Medicine and Clinical Neurosciences, Cardiff University, Cardiff, United Kingdom. Contact e-mail: Barretta5@cardiff. ac.uk Background: The number of genetic loci associated with late-onset
Alzheimer’s disease (LOAD) has increased from 1 to over 20 in the
P457
past seven years. The largest LOAD genome wide association study (GWAS) to date, performed by the International Genomics of Alzheimer’s Project (IGAP), identified 11 novel risk loci and confirmed 8 previously reported genome-wide associations in addition to APOE 1. These loci do not appear to be random and point towards defects in specific biological processes/pathways that contribute to LOAD development. Endocytic processes, thought to be disrupted in the early stages of LOAD development, are implicated in the loci identified via GWAS2. We hypothesise that loss of function of BIN1, CD2AP and PICALM disrupts these processes, resulting in increased production and/or reduced clearance of bamyloid (Ab). To investigate this hypothesis further, we aim to create co-knockdown models of BIN1, CD2AP and PICALM. Methods: Endogenous gene expression of BIN1, CD2AP and PICALM will be knocked down in H4 human neuroglioma cells using targeting siRNA. Control cultures will receive non-targeting siRNA. Knockdown will be quantified and confirmed by comparison with controls, via western blot followed by Image J analysis. Initially the effects of the co-knockdown on cell viability will be investigated with the use of MTS viability assays. To determine the effects of knockdown on transport pathways, levels of APP, Ab, sAPPa, sAPPb and b-CTF are to be quantified using high-sensitivity ELISAs. The effect of gene knockdown on receptor-mediated and fluid-phase endocytosis will be analysed by flow cytometry. Any effects of the co-knockdown on cellular cholesterol levels will also be studied with the use of filipin staining and cholesterol quantification assays. Results: Results to be presented at AAIC 2016. Conclusions: The creation of these unique disease models will hopefully elucidate further their combined effect on cell viability, APP processing and endocytosis and potentially further our understanding of LOAD pathogenesis. 1. Lambert et al. Nat. Gen. 2013, 45, 1452. 2. Jones et al, Alz & Dem. 2015, 11, 658. P1-143
INVESTIGATING THE ROLE OF BIN1, AN ALZHEIMER’S DISEASE RISK GENE, IN ENDOCYTOSIS AT THE BLOOD-BRAIN BARRIER
Anna L. Burt1, Rhian S. Thomas1, Melanie L. Dunstan1, Rebecca Sims2, Julie Williams2, 1Cardiff University, Cardiff, United Kingdom; 2MRC Centre for Neuropsychiatric Genetics & Genomics, Institute of Psychological Medicine and Clinical Neurosciences, Cardiff University, Cardiff, United Kingdom. Contact e-mail: [email protected] Background: Genome-wide association studies (GWAS) high-
light a prominent role for endocytosis in late-onset Alzheimer’s disease (LOAD) aetiology, demonstrating the association of several endocytosis-related genes, including BIN1 and PICALM, with disease (1). Integration of GWAS with expression data corroborates significant enrichment of endocytic pathways in LOAD (2). Recently, a central role for PICALM in Ab transcytosis at the blood-brain barrier (BBB) was identified, implicating this process in LOAD (3). Similarly, BIN1 plays an important role in clathrin-mediated endocytosis (CME), an essential component of transcytosis, and is currently the most significantly associated LOAD susceptibility gene after APOE (1). This study investigates the role of BIN1 in endocytosis at the BBB. Methods: The hCMEC/D3 human brain endothelial cell line was used as an in vitro model of endocytosis. BIN1 protein was depleted using targeting siRNA, with siRNA directed against GFP used for control cultures. Cells
and in vivo. Conclusions: Suppressing pathogenic intracellular calcium signaling during early disease stages is strategic and effective therapeutic approach to prevent the progression of AD. P1-141
LOSS OF OLIGODENDROCYTES IS AN EARLY AMYLOID-INDEPENDENT EVENT IN THE PROGRESS OF DEMENTIA
Kai-Hei Tse, Karl Herrup, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Contact e-mail: [email protected] Background: DNA damage and neuronal cell cycle re-entry are commonly observed during neurodegeneration in Alzheimer’s disease (AD). White matter (WM) degeneration is a recognized co-morbidity, but little is known about the underlying neurobiology. We hypothesize that oligodendrocyte loss (OL) occurs early in the process of dementia that is independent of amyloid. Methods: A cross sectional dataset of MRI data from healthy controls, individuals with mild cognitive impairment (MCI) or subjects with AD (n ¼ 507) was obtained from the National Alzheimer’s Coordinating Center and Spearman’s correlational analysis was performed. Additional specimens from healthy controls, individuals with plaque-free dementia and others diagnosed as AD came from NeuroBioBank (n ¼ 8 per group). These cases were examined by immunofluorescence. Results: Gray matter (GM) and white matter (WM) volume declined with age as well as with MMSE. In MCI, the GM reduction was highly correlated with a reduced hippocampal volume (R ¼ 0.42) and increased white matter hyperintensities (WMH; R ¼ 0.49). We found no correlation with WM volume (R ¼ 0.13). These findings, especially the MCI cohort, implicate myelin lesions as an early event in the onset of dementia. By immunocytochemistry, the number of actively myelinating oligodendrocytes (myelin regulatory factor [MyRF]-positive), mature OLs (myelin basic protein positive) or Olig2-positve OLs were signifcantly reduced in plaque-free dementia as well as in AD (p < 0.005). Moreover, 70% of the Olig2+ cells co-labelled with gH2A.X in the plaque-free dementia group, indicative of DNA damage (p ¼ 0.006). In all dementia groups, 25% of the postmitotic MyRF+ cells stained with the cell cycle marker, cyclin D (p < 0.05); caspase-3 staining was found in the same areas. Conclusions: WMH in the aging population is an early correlate of neuronal loss and cognitive decline. The presence of OLs double stained for cyclin D and caspase-3 suggests that, just as in neurons, aberrant cell cycle re-entry is associated with OL cell death. Finally, the correlation of these events with dementia, but not with plaques implicates an amyloid independent mechanism as the root cause of the white matter abnormalities. P1-142
INVESTIGATING THE EFFECT OF MULTIPLE GENES ON CELLULAR PROCESSES IMPLICATED IN LATE ONSET ALZHEIMER’S DISEASE
Anna N. Barrett1, Melanie L. Dunstan1, Rhian S. Thomas1, Emma J. Kidd1, Lesley Jones1, Julie Williams2, 1Cardiff University, Cardiff, United Kingdom; 2MRC Centre for Neuropsychiatric Genetics & Genomics, Institute of Psychological Medicine and Clinical Neurosciences, Cardiff University, Cardiff, United Kingdom. Contact e-mail: Barretta5@cardiff. ac.uk Background: The number of genetic loci associated with late-onset
Alzheimer’s disease (LOAD) has increased from 1 to over 20 in the
P457
past seven years. The largest LOAD genome wide association study (GWAS) to date, performed by the International Genomics of Alzheimer’s Project (IGAP), identified 11 novel risk loci and confirmed 8 previously reported genome-wide associations in addition to APOE 1. These loci do not appear to be random and point towards defects in specific biological processes/pathways that contribute to LOAD development. Endocytic processes, thought to be disrupted in the early stages of LOAD development, are implicated in the loci identified via GWAS2. We hypothesise that loss of function of BIN1, CD2AP and PICALM disrupts these processes, resulting in increased production and/or reduced clearance of bamyloid (Ab). To investigate this hypothesis further, we aim to create co-knockdown models of BIN1, CD2AP and PICALM. Methods: Endogenous gene expression of BIN1, CD2AP and PICALM will be knocked down in H4 human neuroglioma cells using targeting siRNA. Control cultures will receive non-targeting siRNA. Knockdown will be quantified and confirmed by comparison with controls, via western blot followed by Image J analysis. Initially the effects of the co-knockdown on cell viability will be investigated with the use of MTS viability assays. To determine the effects of knockdown on transport pathways, levels of APP, Ab, sAPPa, sAPPb and b-CTF are to be quantified using high-sensitivity ELISAs. The effect of gene knockdown on receptor-mediated and fluid-phase endocytosis will be analysed by flow cytometry. Any effects of the co-knockdown on cellular cholesterol levels will also be studied with the use of filipin staining and cholesterol quantification assays. Results: Results to be presented at AAIC 2016. Conclusions: The creation of these unique disease models will hopefully elucidate further their combined effect on cell viability, APP processing and endocytosis and potentially further our understanding of LOAD pathogenesis. 1. Lambert et al. Nat. Gen. 2013, 45, 1452. 2. Jones et al, Alz & Dem. 2015, 11, 658. P1-143
INVESTIGATING THE ROLE OF BIN1, AN ALZHEIMER’S DISEASE RISK GENE, IN ENDOCYTOSIS AT THE BLOOD-BRAIN BARRIER
Anna L. Burt1, Rhian S. Thomas1, Melanie L. Dunstan1, Rebecca Sims2, Julie Williams2, 1Cardiff University, Cardiff, United Kingdom; 2MRC Centre for Neuropsychiatric Genetics & Genomics, Institute of Psychological Medicine and Clinical Neurosciences, Cardiff University, Cardiff, United Kingdom. Contact e-mail: [email protected] Background: Genome-wide association studies (GWAS) high-
light a prominent role for endocytosis in late-onset Alzheimer’s disease (LOAD) aetiology, demonstrating the association of several endocytosis-related genes, including BIN1 and PICALM, with disease (1). Integration of GWAS with expression data corroborates significant enrichment of endocytic pathways in LOAD (2). Recently, a central role for PICALM in Ab transcytosis at the blood-brain barrier (BBB) was identified, implicating this process in LOAD (3). Similarly, BIN1 plays an important role in clathrin-mediated endocytosis (CME), an essential component of transcytosis, and is currently the most significantly associated LOAD susceptibility gene after APOE (1). This study investigates the role of BIN1 in endocytosis at the BBB. Methods: The hCMEC/D3 human brain endothelial cell line was used as an in vitro model of endocytosis. BIN1 protein was depleted using targeting siRNA, with siRNA directed against GFP used for control cultures. Cells