- Email: [email protected]
P1-142: A new neurotrophic support culture technique for primary neurons
T248
Poster Presentations P1
was expressed and treated with the proteasome inhibitor, cytoplasmic inclusions positive for both anti-phosphorylated TDP-43 and anti-ubiquitin antibodies were observed. The expression of delta187-192 caused the formation of intranuclear inclusions after proteasome inhibition. Furthermore, when the double-deletion mutant (delta78-84 and 187-192) was expressed, cytoplasmic inclusions positive for both anti-phosphorylated TDP-43 and anti-ubiquitin were observed without proteasome inhibition. These all mutants had no exon skipping activity of CFTR exon 9, suggesting that loss of function in TDP-43 may lead to its accumulation in cells. Conclusions: We successfully established the cellular models recapitulating the pathological lesions found in FTLD-U and ALS. P1-141
MODELING THE “DENDRITIC HYPOTHESIS” OF ALZHEIMER PATHOGENESIS: A NEUROINFORMATICS APPROACH FOR ELUCIDATING PART OF THE AMYLOID BETA CASCADE
Thomas M. Morse1, Pradeep G. Mutalik2, Kei-Hoi Cheung2, Perry L. Miller1,2, Gordon M. Shepherd1, 1Yale Univ. Sch. of Med., New Haven, CT, USA; 2Yale Univ. Sch. of Med., Med. Informatics, New Haven, CT, USA. Contact e-mail: [email protected] Background: The amyloid beta cascade hypothesis posits that an increase in the production of ABeta1-42 is the single event from which all other Alzheimer’s disease pathology (ABeta plaques, tau tangles, cell death, and clinical dementia) follows. It is likely, however, that early cognitive deficits in Alzheimer’s are the result of electrophysiological deficits in cortical neurons. Our “Dendritic Hypothesis” posits that the second step in the progression of Alzheimer’s disease is the disruption of dendritic integrative properties and perturbations of neuronal electrical homeostasis, resulting in failure of specific synapses. Published experimental evidence suggests that ABeta1-42 blocks A-type potassium channels in CA1 pyramidal cells. Several authors have further suggested that the block of this hyperpolarizing potassium channel may lead to excessive depolarization of the cell and subsequent Ca2⫹ influx excitotoxicity. Methods: In a biologically realistic multicompartmental CA1 model obtained from our neuronal simulation database, ModelDB, we applied an A-type current block of 46.5% in dendrite and 31.5% in soma (based on measurements in membrane patch recordings by Chen C., 2005) and examined how the block of the A-type current disrupts the integrative properties of the dendrites in this cortical pyramidal neuron model. Results: At 100 nM ABeta, (used in Chen’s experiments), the fine distal dendrites and distal portions of the oblique dendrites receive back propagating action potential peak depolarizations at many times the values of the healthy cell, suggesting that these would be the first sites of excitotoxicity. We modeled a smaller concentration block of A-type channels to predict which synapses on the cell would be first to experience an inappropriate spread of depolarization from back-propagating action potentials. Conclusions: The Abeta A-type potassium channel block first leads to impairment of neuronal signaling that may underlie the earliest cognitive deficits since back-propagating action potentials are widely thought to be important for maintaining synapses through LTP and LTD, and then as others have suggested, excitotoxicity, as the ABeta concentration increases. The study demonstrates the effectiveness of a neuroinformatics approach to give insights by integrating diverse types of data from different sources for the early to late functional pathogenesis of Alzheimer’s disease. P1-142
A NEW NEUROTROPHIC SUPPORT CULTURE TECHNIQUE FOR PRIMARY NEURONS
Yazi D. Ke1, Lars M. Ittner1, Peter Gunning2, Jurgen Gotz1, Thomas Fath2, 1Alzheimer’s & Parkinson’s Disease Laboratory, Brain & Mind Research Institute, University of Sydney, Sydney, Australia; 2 Oncology Research Unit, The Children’s Hospital at Westmead, Sydney, Australia. Contact e-mail: [email protected]
Background: Primary neuronal cultures provide are widely used to study molecular and cellular mechanisms. They were employed in analyzing dendritic spine morphology, synaptic plasticity and dissecting pathomechanisms in neurodegeneration. These cultures are often limited by low and short survival rate of cells. Different procedures have been developed to overcome these problem, including hippocampal slice cultures, mixed cultures or co-cultures with both tissue explants and astroglial cells. However, culturing at low density is often not possible or dependent on sophisticated methods. Methods: We have established an easy and robust technique for culturing hippocampal neurons at low-cell density for long-term survival. This was achieved by culturing embryonic (E16.5) murine hippocampal neurons, surrounded by a spatially separated ring of cortical cells for neurotrophic support. Both cell types were prepared from the same embryo providing sufficient material for numerous experiments. Results: Utilizing this technique, we achieved long-term surviving primary hippocampal cultures at low cell density (⬍10 cells/mm2). Moreover, the neurons developed mature spines and a dense axonal network. We also used the technique to successfully culture primary substantia nigra neurons with neurotrophic support of striatal cells. Both types of neurons were used to study the spatial distribution of cellular cargos and markers. Since only low cell numbers are needed, this approach allows furthermore for the comparative study of single embryos from different genetic backgrounds. Conclusions: This new ring support system enables the long-term culture of hippocampal neurons at low cell density, suitable for studies of cellular morphology and mechanisms. P1-143
CSF OUTFLOW MODELS: HUMAN ARACHNOID MEMBRANE AS A PATHWAY FOR A-BETA CLEARANCE
Deborah M. Grzybowski, David W. Holman, Shelley A. Glimcher, Martin Lubow, The Ohio State University, Columbus, OH, USA. Contact e-mail: [email protected] Background: The arachnoid membrane (AM), though a CNS brain barrier, is responsible for the absorption of cerebrospinal fluid and the removal of toxic metabolites. Localization of LRP-1 and RAGE in the AM may provide explanation for altered rates of CSF movement, leading to CSF stagnation. The outflow of CSF through the human AM was modelled using cell culture (in-vitro) and whole tissue (ex-vivo) perfusion as an acceptable option for examining hydrodynamic disorders. Methods: Human AG tissue was harvested within 24-hours post-mortem. AG cells were isolated and grown on filter inserts or tissue was fit into an Ussing chamber. Cells and tissue were perfused at physiologic pressure with fluorescent microparticles and fixed under experimental pressure. Fixed tissue was processed for TEM or cryo-sectioned and stained for visualization. Results: The ex-vivo perfusion model results show that the in vivo properties of the AM are maintained, including uni-directionality, particle transport, and ultrastructural barrier characteristics. Ex vivo perfusion results showed average hydraulic conductivity (Lpave) in the physiologic direction (basal to apical) was 1.05⫾0.15ul/min with average perfusion pressure(Pave) across the membrane of 5.88⫾0.22mmHg (n⫽20) which was statistically higher (p⬍0.001) than Lpave⫽0.11⫾0.03ul/min for perfusion in the nonphysiologic direction at Pave⫽6.14⫾0.23mmHg (n⫽3). In vitro AM perfusion permeability results showed uni-directional flow in the physiologic direction. The Lpave for AG cells perfused non-physiologically was 4.49⫾0.53ul/min/mmHg/cm2 (n⫽17) with Pave⫽3.15mmHg, which was statistically higher(p⬍0.001) than Lpave⫽0.28⫾0.16ul/min/mmHg/cm2 for cells perfused physiologically, with Pave⫽3.33mmHg (n⫽6). Conclusions: Arachnoid membrane perfusion results in both in vitro and ex vivo models showed uni-directional, physiologic flow. Electron microscopy showed large intracellular vacuoles and extra-cellular cisternal spaces which may represent two distinct mechanisms by which arachnoidal cells move fluid: trans-cellular transport via intra-cellular vacuoles and paracellular transport via extra-cellular cisterns. CSF outflow is a critical element of CSF circulation and of CNS maintenance, which can be successfully modelled by engineered ex vivo and in vitro perfusion systems
Poster Presentations P1
was expressed and treated with the proteasome inhibitor, cytoplasmic inclusions positive for both anti-phosphorylated TDP-43 and anti-ubiquitin antibodies were observed. The expression of delta187-192 caused the formation of intranuclear inclusions after proteasome inhibition. Furthermore, when the double-deletion mutant (delta78-84 and 187-192) was expressed, cytoplasmic inclusions positive for both anti-phosphorylated TDP-43 and anti-ubiquitin were observed without proteasome inhibition. These all mutants had no exon skipping activity of CFTR exon 9, suggesting that loss of function in TDP-43 may lead to its accumulation in cells. Conclusions: We successfully established the cellular models recapitulating the pathological lesions found in FTLD-U and ALS. P1-141
MODELING THE “DENDRITIC HYPOTHESIS” OF ALZHEIMER PATHOGENESIS: A NEUROINFORMATICS APPROACH FOR ELUCIDATING PART OF THE AMYLOID BETA CASCADE
Thomas M. Morse1, Pradeep G. Mutalik2, Kei-Hoi Cheung2, Perry L. Miller1,2, Gordon M. Shepherd1, 1Yale Univ. Sch. of Med., New Haven, CT, USA; 2Yale Univ. Sch. of Med., Med. Informatics, New Haven, CT, USA. Contact e-mail: [email protected] Background: The amyloid beta cascade hypothesis posits that an increase in the production of ABeta1-42 is the single event from which all other Alzheimer’s disease pathology (ABeta plaques, tau tangles, cell death, and clinical dementia) follows. It is likely, however, that early cognitive deficits in Alzheimer’s are the result of electrophysiological deficits in cortical neurons. Our “Dendritic Hypothesis” posits that the second step in the progression of Alzheimer’s disease is the disruption of dendritic integrative properties and perturbations of neuronal electrical homeostasis, resulting in failure of specific synapses. Published experimental evidence suggests that ABeta1-42 blocks A-type potassium channels in CA1 pyramidal cells. Several authors have further suggested that the block of this hyperpolarizing potassium channel may lead to excessive depolarization of the cell and subsequent Ca2⫹ influx excitotoxicity. Methods: In a biologically realistic multicompartmental CA1 model obtained from our neuronal simulation database, ModelDB, we applied an A-type current block of 46.5% in dendrite and 31.5% in soma (based on measurements in membrane patch recordings by Chen C., 2005) and examined how the block of the A-type current disrupts the integrative properties of the dendrites in this cortical pyramidal neuron model. Results: At 100 nM ABeta, (used in Chen’s experiments), the fine distal dendrites and distal portions of the oblique dendrites receive back propagating action potential peak depolarizations at many times the values of the healthy cell, suggesting that these would be the first sites of excitotoxicity. We modeled a smaller concentration block of A-type channels to predict which synapses on the cell would be first to experience an inappropriate spread of depolarization from back-propagating action potentials. Conclusions: The Abeta A-type potassium channel block first leads to impairment of neuronal signaling that may underlie the earliest cognitive deficits since back-propagating action potentials are widely thought to be important for maintaining synapses through LTP and LTD, and then as others have suggested, excitotoxicity, as the ABeta concentration increases. The study demonstrates the effectiveness of a neuroinformatics approach to give insights by integrating diverse types of data from different sources for the early to late functional pathogenesis of Alzheimer’s disease. P1-142
A NEW NEUROTROPHIC SUPPORT CULTURE TECHNIQUE FOR PRIMARY NEURONS
Yazi D. Ke1, Lars M. Ittner1, Peter Gunning2, Jurgen Gotz1, Thomas Fath2, 1Alzheimer’s & Parkinson’s Disease Laboratory, Brain & Mind Research Institute, University of Sydney, Sydney, Australia; 2 Oncology Research Unit, The Children’s Hospital at Westmead, Sydney, Australia. Contact e-mail: [email protected]
Background: Primary neuronal cultures provide are widely used to study molecular and cellular mechanisms. They were employed in analyzing dendritic spine morphology, synaptic plasticity and dissecting pathomechanisms in neurodegeneration. These cultures are often limited by low and short survival rate of cells. Different procedures have been developed to overcome these problem, including hippocampal slice cultures, mixed cultures or co-cultures with both tissue explants and astroglial cells. However, culturing at low density is often not possible or dependent on sophisticated methods. Methods: We have established an easy and robust technique for culturing hippocampal neurons at low-cell density for long-term survival. This was achieved by culturing embryonic (E16.5) murine hippocampal neurons, surrounded by a spatially separated ring of cortical cells for neurotrophic support. Both cell types were prepared from the same embryo providing sufficient material for numerous experiments. Results: Utilizing this technique, we achieved long-term surviving primary hippocampal cultures at low cell density (⬍10 cells/mm2). Moreover, the neurons developed mature spines and a dense axonal network. We also used the technique to successfully culture primary substantia nigra neurons with neurotrophic support of striatal cells. Both types of neurons were used to study the spatial distribution of cellular cargos and markers. Since only low cell numbers are needed, this approach allows furthermore for the comparative study of single embryos from different genetic backgrounds. Conclusions: This new ring support system enables the long-term culture of hippocampal neurons at low cell density, suitable for studies of cellular morphology and mechanisms. P1-143
CSF OUTFLOW MODELS: HUMAN ARACHNOID MEMBRANE AS A PATHWAY FOR A-BETA CLEARANCE
Deborah M. Grzybowski, David W. Holman, Shelley A. Glimcher, Martin Lubow, The Ohio State University, Columbus, OH, USA. Contact e-mail: [email protected] Background: The arachnoid membrane (AM), though a CNS brain barrier, is responsible for the absorption of cerebrospinal fluid and the removal of toxic metabolites. Localization of LRP-1 and RAGE in the AM may provide explanation for altered rates of CSF movement, leading to CSF stagnation. The outflow of CSF through the human AM was modelled using cell culture (in-vitro) and whole tissue (ex-vivo) perfusion as an acceptable option for examining hydrodynamic disorders. Methods: Human AG tissue was harvested within 24-hours post-mortem. AG cells were isolated and grown on filter inserts or tissue was fit into an Ussing chamber. Cells and tissue were perfused at physiologic pressure with fluorescent microparticles and fixed under experimental pressure. Fixed tissue was processed for TEM or cryo-sectioned and stained for visualization. Results: The ex-vivo perfusion model results show that the in vivo properties of the AM are maintained, including uni-directionality, particle transport, and ultrastructural barrier characteristics. Ex vivo perfusion results showed average hydraulic conductivity (Lpave) in the physiologic direction (basal to apical) was 1.05⫾0.15ul/min with average perfusion pressure(Pave) across the membrane of 5.88⫾0.22mmHg (n⫽20) which was statistically higher (p⬍0.001) than Lpave⫽0.11⫾0.03ul/min for perfusion in the nonphysiologic direction at Pave⫽6.14⫾0.23mmHg (n⫽3). In vitro AM perfusion permeability results showed uni-directional flow in the physiologic direction. The Lpave for AG cells perfused non-physiologically was 4.49⫾0.53ul/min/mmHg/cm2 (n⫽17) with Pave⫽3.15mmHg, which was statistically higher(p⬍0.001) than Lpave⫽0.28⫾0.16ul/min/mmHg/cm2 for cells perfused physiologically, with Pave⫽3.33mmHg (n⫽6). Conclusions: Arachnoid membrane perfusion results in both in vitro and ex vivo models showed uni-directional, physiologic flow. Electron microscopy showed large intracellular vacuoles and extra-cellular cisternal spaces which may represent two distinct mechanisms by which arachnoidal cells move fluid: trans-cellular transport via intra-cellular vacuoles and paracellular transport via extra-cellular cisterns. CSF outflow is a critical element of CSF circulation and of CNS maintenance, which can be successfully modelled by engineered ex vivo and in vitro perfusion systems