- Email: [email protected]
Three-dimensional telomere analysis in Alzheimer's disease (AD)
P274
Poster Presentations: P2
Prague, First Faculty of Medicine and General University Hospital, Institute of Medical Biochemistry and Laboratory Diagnostics, Prague, Czech Republic.
Ontario, Canada; 2University of Manitoba, Winnipeg, Manitoba, Canada; 3 U of Manitoba, Winnipeg, Manitoba, Canada; 4U of Manitoba, Winnipeg, Manitoba, Canada.
Background: Humoral immunity related to neuron- and disease-specific cytoskeletal proteins in patients with Alzheimer’s disease (AD) is largely unknown. Methods: Antibodies against three targets [tau, light (NFL) and heavy (NFH) neurofilaments] were measured using ELISA in 80 paired serum and CSF samples from 25 patients with AD fulfilling NINCDSADRDA criteria, 13 patients with other dementias (OD), 25 cognitively normal elderly controls (NC), 17 patients with neuroinflammatory diseases (IC). We estimated intrathecal synthesis of all antibodies according to formula (CSF anti-neurocytoskeletal IgG /serum anti-neurocytoskeletal IgG)/ (CSF total IgG /serum total IgG). Results: The AD patients had significantly higher intrathecal anti-tau antibodies than in the OD group (p ¼ 0.005) or the IC group (P ¼ 0.0003) and higher intrathecal anti-NFH antibodies than in the OD group (P ¼ 0.046), the NC group (p ¼ 0.04) or the IC group (P ¼ 0.0007), but not intrathecally synthesized anti-NFL antibodies. All three anti-neurocytoskeletal antibodies in serum or CSF did not differ among four groups with the exceptions for the IC group having them elevated in CSF. Conclusions: These new findings suggest some hints of specific alterations in humoral anti-neurocytoskeletal immunity and selectivity in AD. It may have diagnostic and immunotherapeutic implications.
Background: Telomeres are linear TTAGGG repeats capping human chromosomes maintaining chromosomal integrity. Telomeres shorten progressively with each cell division and with age. The aims of our study was to analyze the three-dimensional (3D) architecture of telomeres in AD patients compared to age-matched normal controls in cells obtained from buccal swaps. 3D analysis allows for quantification of telomere numbers, length and aggregates. Buccal swaps were chosen because cells derive from the neuroectoderm from which brain tissue also originates, and they can be collected non-invasively. Methods: Twenty six patients with AD diagnosed and staged by standard procedures (mild ¼ 14, moderate ¼ 7, and severe ¼ 6) and 26 cognitively normal age-matched controls were included in the study. Cells were obtained from buccal swaps using sterile Epicentre Biotechnologies swabs, smeared on VWR micro slides and frozen at -20 C. Quantitative fluorescence in situ hybridization (Q-FISH) technique was used for telomere numbers and length analysis in 30 interphase cells/person. Digital images were taken using Zeiss AxioImager Z1 with a cooled AxioCam HR B&W, DAPI, Cy3 filters in combination with a Planapo 63x/1.4 oil objective lens. Images were acquired by using AXIOVISION 4.8 (Zeiss) in multichannel mode followed by constrained iterative deconvolution. For every fluorochrome, 120 images stacks were acquired with a sampling distance of 200 nm along the z, and 102 nm in the XY axis. Quantitation of 3D nuclear telomeric signals was performed using TeloView. Differences in telomere intensity between AD patients and normal controls, were analyzed by Fisher exact test (number of telomeres) and Chi-Square (telomere length). Results: Patients with mild to severe AD had significantly less number of telomeres and shorter telomeres than the control subjects (range from P<.001 to P<.0001). There was no difference in telomere aggregates between AD patients and controls. Conclusions: Smeared buccal swap cells are suitable for telomere 3D analysis in AD and normal controls. Subjects with AD, at any stage of the disease had less and shorter telomeres in their buccal cells when compared to their age-matched controls. No differences in aggregates distinguish the findings from the ones in cancer patients. Validation in a larger sample is warranted.
P2-031
PROTEIN KINASE ACTIVITY PROFILING ON CEREBROSPINAL FLUID TO FIND DIAGNOSTIC BIOMARKERS FOR ALZHEIMER’S DISEASE
Andrea F.N. Rosenberger1, Jeroen Hoozemans1, Riet Hilhorst2, Philip Scheltens3, Wiesje Van der Flier3, Saskia M. van der Vies1, 1Vrije University Medisch Centrum, Amsterdam, Netherlands; 2PamGene International BV, Hertogenbosch, Netherlands; 3VU University Medical Center, Amsterdam, Netherlands. Background: Alzheimer’s disease (AD) pathology starts decades before clinical signs appear. Hence there is a need to identify molecular biomarkers that reflect AD pathology and can be used for early diagnosis. Human cerebrospinal fluid (CSF) is in direct contact with the brain and thus an optimal source for the discovery of biomarkers. Protein kinases are key enzymes that regulate signal transduction pathways in the cell and control cellular processes including cell metabolism, cell growth and cell division. Kinases are involved in many aspects of AD pathogenesis and therefore have a high potential to be used as diagnostic biomarkers and drug targets for patients with AD. Although kinases are primarily localized intracellular, it has been shown that different kinases are present in the extracellular space and in CSF. Recently, it was shown that extracellular amyloid beta undergoes phosphorylation by protein kinases in human CSF, indicating that there is protein kinase activity in CSF. Methods: This study explores the potential to utilise protein kinase activity in CSF as a diagnostic tool for AD. For the detection of protein kinase activities a peptide-based microarray assay was employed. The peptide microarray contained 144 peptides derived from known kinase substrate sequences covalently attached to a porous matrix, through which a protein solution is constantly pumped up and down. Fluorescently labelled anti-phosphoserine and/or anti-phosphothreonine antibodies are used to detect phosphorylated peptides on the chip, which generates a kinase activity profile of the sample. Samples of human CSF derived from patients with AD or subjective memory complaints (SMC) were analysed. Results: The results show that using a peptide array, ATP dependent protein kinase activities are detectable in human CSF. Protein kinase activity profiling is a fast method and requires very little sample without the necessity of pretreatment. Furthermore, different phosphorylation profiles can be identified between patients with AD or SMC. Conclusions: These results open the way to investigate the potential of protein kinase activity profiling as a biomarker assay for AD diagnosis. P2-032
THREE-DIMENSIONAL TELOMERE ANALYSIS IN ALZHEIMER’S DISEASE (AD)
Angeles Garcia1, Elisabeth McAvoy1, Cornelia Willing2, Shubha Mathur3, Aleksandra Glogowska4, Sabine Mai4, 1Queen’s University, Kingston,
P2-033
CHARACTERIZATION OF BRAIN PLASTICITY IN ALZHEIMER’S DISEASE PATIENTS AND HEALTHY SUBJECTS
Anna-Katharine Brem1, Lukas Schilberg2, Catarina Freitas2, Natasha Atkinson2, Leonie Asboth2, Christina Carbone2, Ilya Vidrin2, Alvaro Pascual-Leone2, 1Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States; 2Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts, United States. Background: A wealth of in vitro and in vivo evidence demonstrates alterations in neuroplasticity and impaired synaptic function in Alzheimer’s disease (AD). There is a critical need for an assay of neuroplasticity that can act as a surrogate marker of the pathological changes in AD. Theta-burst stimulation (TBS) modulates neuronal responses to TMS, which can be assessed with EEG/EMG. Intermittent (iTBS) or continuous (cTBS) theta-burst stimulation lead to an enhancement or suppression of cortical activity and are considered an index of LTP- and LTD-like induction of synaptic plasticity. Glutamatergic and GABAergic mediators, which are involved in TBS-effects, have been shown to be dysfunctional in AD patients. Here we provide pilot data in support of the feasibility of this method to assess neuroplastic changes in AD patients. Methods: We applied iTBS and cTBS in mild AD patients and elderly healthy controls (HC) over the left primary motor cortex (M1). MRI-navigated single-pulse TMS (120% RMT) was applied before (baseline) and repeatedly after TBS (over a time-period of 75 min). EMG was recorded from the right first dorsal interosseus muscle (FDI). cTBS consisted of a continuous train of 600 pulses (3 pulses at 50 Hz repeated at 200 ms intervals, at 80% AMT), iTBS consisted of 20 intermittent 2-second trains repeated every 10 seconds (600 pulses, 3 pulses at 50 Hz repeated at 200 ms intervals, at 80% AMT). Results: Cortical responses in AD
Poster Presentations: P2
Prague, First Faculty of Medicine and General University Hospital, Institute of Medical Biochemistry and Laboratory Diagnostics, Prague, Czech Republic.
Ontario, Canada; 2University of Manitoba, Winnipeg, Manitoba, Canada; 3 U of Manitoba, Winnipeg, Manitoba, Canada; 4U of Manitoba, Winnipeg, Manitoba, Canada.
Background: Humoral immunity related to neuron- and disease-specific cytoskeletal proteins in patients with Alzheimer’s disease (AD) is largely unknown. Methods: Antibodies against three targets [tau, light (NFL) and heavy (NFH) neurofilaments] were measured using ELISA in 80 paired serum and CSF samples from 25 patients with AD fulfilling NINCDSADRDA criteria, 13 patients with other dementias (OD), 25 cognitively normal elderly controls (NC), 17 patients with neuroinflammatory diseases (IC). We estimated intrathecal synthesis of all antibodies according to formula (CSF anti-neurocytoskeletal IgG /serum anti-neurocytoskeletal IgG)/ (CSF total IgG /serum total IgG). Results: The AD patients had significantly higher intrathecal anti-tau antibodies than in the OD group (p ¼ 0.005) or the IC group (P ¼ 0.0003) and higher intrathecal anti-NFH antibodies than in the OD group (P ¼ 0.046), the NC group (p ¼ 0.04) or the IC group (P ¼ 0.0007), but not intrathecally synthesized anti-NFL antibodies. All three anti-neurocytoskeletal antibodies in serum or CSF did not differ among four groups with the exceptions for the IC group having them elevated in CSF. Conclusions: These new findings suggest some hints of specific alterations in humoral anti-neurocytoskeletal immunity and selectivity in AD. It may have diagnostic and immunotherapeutic implications.
Background: Telomeres are linear TTAGGG repeats capping human chromosomes maintaining chromosomal integrity. Telomeres shorten progressively with each cell division and with age. The aims of our study was to analyze the three-dimensional (3D) architecture of telomeres in AD patients compared to age-matched normal controls in cells obtained from buccal swaps. 3D analysis allows for quantification of telomere numbers, length and aggregates. Buccal swaps were chosen because cells derive from the neuroectoderm from which brain tissue also originates, and they can be collected non-invasively. Methods: Twenty six patients with AD diagnosed and staged by standard procedures (mild ¼ 14, moderate ¼ 7, and severe ¼ 6) and 26 cognitively normal age-matched controls were included in the study. Cells were obtained from buccal swaps using sterile Epicentre Biotechnologies swabs, smeared on VWR micro slides and frozen at -20 C. Quantitative fluorescence in situ hybridization (Q-FISH) technique was used for telomere numbers and length analysis in 30 interphase cells/person. Digital images were taken using Zeiss AxioImager Z1 with a cooled AxioCam HR B&W, DAPI, Cy3 filters in combination with a Planapo 63x/1.4 oil objective lens. Images were acquired by using AXIOVISION 4.8 (Zeiss) in multichannel mode followed by constrained iterative deconvolution. For every fluorochrome, 120 images stacks were acquired with a sampling distance of 200 nm along the z, and 102 nm in the XY axis. Quantitation of 3D nuclear telomeric signals was performed using TeloView. Differences in telomere intensity between AD patients and normal controls, were analyzed by Fisher exact test (number of telomeres) and Chi-Square (telomere length). Results: Patients with mild to severe AD had significantly less number of telomeres and shorter telomeres than the control subjects (range from P<.001 to P<.0001). There was no difference in telomere aggregates between AD patients and controls. Conclusions: Smeared buccal swap cells are suitable for telomere 3D analysis in AD and normal controls. Subjects with AD, at any stage of the disease had less and shorter telomeres in their buccal cells when compared to their age-matched controls. No differences in aggregates distinguish the findings from the ones in cancer patients. Validation in a larger sample is warranted.
P2-031
PROTEIN KINASE ACTIVITY PROFILING ON CEREBROSPINAL FLUID TO FIND DIAGNOSTIC BIOMARKERS FOR ALZHEIMER’S DISEASE
Andrea F.N. Rosenberger1, Jeroen Hoozemans1, Riet Hilhorst2, Philip Scheltens3, Wiesje Van der Flier3, Saskia M. van der Vies1, 1Vrije University Medisch Centrum, Amsterdam, Netherlands; 2PamGene International BV, Hertogenbosch, Netherlands; 3VU University Medical Center, Amsterdam, Netherlands. Background: Alzheimer’s disease (AD) pathology starts decades before clinical signs appear. Hence there is a need to identify molecular biomarkers that reflect AD pathology and can be used for early diagnosis. Human cerebrospinal fluid (CSF) is in direct contact with the brain and thus an optimal source for the discovery of biomarkers. Protein kinases are key enzymes that regulate signal transduction pathways in the cell and control cellular processes including cell metabolism, cell growth and cell division. Kinases are involved in many aspects of AD pathogenesis and therefore have a high potential to be used as diagnostic biomarkers and drug targets for patients with AD. Although kinases are primarily localized intracellular, it has been shown that different kinases are present in the extracellular space and in CSF. Recently, it was shown that extracellular amyloid beta undergoes phosphorylation by protein kinases in human CSF, indicating that there is protein kinase activity in CSF. Methods: This study explores the potential to utilise protein kinase activity in CSF as a diagnostic tool for AD. For the detection of protein kinase activities a peptide-based microarray assay was employed. The peptide microarray contained 144 peptides derived from known kinase substrate sequences covalently attached to a porous matrix, through which a protein solution is constantly pumped up and down. Fluorescently labelled anti-phosphoserine and/or anti-phosphothreonine antibodies are used to detect phosphorylated peptides on the chip, which generates a kinase activity profile of the sample. Samples of human CSF derived from patients with AD or subjective memory complaints (SMC) were analysed. Results: The results show that using a peptide array, ATP dependent protein kinase activities are detectable in human CSF. Protein kinase activity profiling is a fast method and requires very little sample without the necessity of pretreatment. Furthermore, different phosphorylation profiles can be identified between patients with AD or SMC. Conclusions: These results open the way to investigate the potential of protein kinase activity profiling as a biomarker assay for AD diagnosis. P2-032
THREE-DIMENSIONAL TELOMERE ANALYSIS IN ALZHEIMER’S DISEASE (AD)
Angeles Garcia1, Elisabeth McAvoy1, Cornelia Willing2, Shubha Mathur3, Aleksandra Glogowska4, Sabine Mai4, 1Queen’s University, Kingston,
P2-033
CHARACTERIZATION OF BRAIN PLASTICITY IN ALZHEIMER’S DISEASE PATIENTS AND HEALTHY SUBJECTS
Anna-Katharine Brem1, Lukas Schilberg2, Catarina Freitas2, Natasha Atkinson2, Leonie Asboth2, Christina Carbone2, Ilya Vidrin2, Alvaro Pascual-Leone2, 1Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States; 2Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts, United States. Background: A wealth of in vitro and in vivo evidence demonstrates alterations in neuroplasticity and impaired synaptic function in Alzheimer’s disease (AD). There is a critical need for an assay of neuroplasticity that can act as a surrogate marker of the pathological changes in AD. Theta-burst stimulation (TBS) modulates neuronal responses to TMS, which can be assessed with EEG/EMG. Intermittent (iTBS) or continuous (cTBS) theta-burst stimulation lead to an enhancement or suppression of cortical activity and are considered an index of LTP- and LTD-like induction of synaptic plasticity. Glutamatergic and GABAergic mediators, which are involved in TBS-effects, have been shown to be dysfunctional in AD patients. Here we provide pilot data in support of the feasibility of this method to assess neuroplastic changes in AD patients. Methods: We applied iTBS and cTBS in mild AD patients and elderly healthy controls (HC) over the left primary motor cortex (M1). MRI-navigated single-pulse TMS (120% RMT) was applied before (baseline) and repeatedly after TBS (over a time-period of 75 min). EMG was recorded from the right first dorsal interosseus muscle (FDI). cTBS consisted of a continuous train of 600 pulses (3 pulses at 50 Hz repeated at 200 ms intervals, at 80% AMT), iTBS consisted of 20 intermittent 2-second trains repeated every 10 seconds (600 pulses, 3 pulses at 50 Hz repeated at 200 ms intervals, at 80% AMT). Results: Cortical responses in AD