- Email: [email protected]
SELECTIVELY ENHANCING PATHOLOGICAL FORMS OF TAU VIA THE AUTOPHAGY PATHWAY
P306
Poster Presentations: P1
pathological tau can transfer between cells and seed the misfolding of cytosolic soluble tau into aggregates. We have developed cell-based models to dissect the mechanisms behind the propagation of tau pathology and assess potential therapeutic strategies. Methods: Filamentous tau preparations were added to neuronal and non-neuronal cells expressing soluble tau. Their uptake and the subsequent seeded aggregation of endogenous tau was studied using flow cytometry, immunofluorescence and SDS-PAGE-Western blot. Biochemical assays were used to determine structural properties of the filamentous tau preparations. A series of tau mutants was engineered to study the role of primary structure and phosphorylation state of tau in seeded aggregation and propagation in cells. Results: Hyperphosphorylated and non-phosphorylated, native and synthetic filamentous tau preparations efficiently entered cells and seeded the self-perpetuating formation of hyperphosphorylated filamentous tau composed of endogenous protein, in a process that was time- and concentration- dependent. Native filamentous tau seeded with a much higher potency than synthetic filamentous tau. Biochemical analyses revealed conformational differences between the filament preparations, which could account for the difference in seeding potency. The ability of tau to form filaments was necessary for seeded aggregation to occur; however, alteration of tau phosphorylation at pathologically associated sites did not affect this process. Conclusions: We developed a cell-based model of pathological tau propagation, whereby the addition of minute quantities of exogenous filamentous tau induced the formation of intracellular hyperphosphorylated filamentous tau by direct interaction with endogenous soluble tau. Using this model we have been able to identify structural requirements of seed-competent tau. We present evidence that filamentous tau can adopt multiple conformations, which determines its efficiency at seeding aggregation of soluble tau in cells. This is reminiscent of protein ’strains,’ such as have been identified for prion protein.
P1-005
SELECTIVELY ENHANCING PATHOLOGICAL FORMS OF TAU VIA THE AUTOPHAGY PATHWAY
Adrianne Chesser1, Zhinian Lei2, Gail V.W. Johnson2, 1University of Rochester School of Medicine, Rochester, New York, United States; 2 University of Rochester Medical Center, Rochester, New York, United States. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is the leading cause of neurodegeneration worldwide. Unfortunately, there are limited therapeutic options, due in part to an incomplete understanding of disease pathogenesis. One pathological hallmark of AD is the intracellular accumulation of hyperphosphorylated and truncated forms of the microtubule associated protein tau. These pathological forms of tau are unable to appropriately interact with microtubules, facilitatiing neuronal damage in AD. In addition, there are data to indicate that pathological tau, particularly in its oligomeric form, causes toxicity. Impairment of protein degradative systems is likely a contributing factor to the accumulation and aggregation of tau in AD. Therefore interventions that result in the selective upregulation of pathways that result in the clearance pathological tau without further diminishing the pool of available functional tau are of significant interest. Methods: Mouse cortical cells that inducibly express a single form of tau-unmodified, D421 truncation, phosphomimetic or phosphonull at T231 or S262-were established. Using these lines, degradation profiles for each form of tau were established both in the basal condition and in the presence of inhibitors or activators of the different protein degradation pathways. Primary neurons were used for complementary studies with phosphoepitope antibodies. Results: Particular pathological forms of tau are preferentially cleared via the autophagy pathway. Additionally, activating the autophagy pathway leads to enhanced clearance of these potentially disease-mediating forms. Importantly, this process is specific. Treatment with trehalose, an activator of non mTor-dependent autophagy, is effective, while rapamycin is not. This may relate to the involvement of adaptor proteins that help chaperone pathological tau to autophagy. In particular, the adaptor protein NDP52 appears to be important. Expression of NDP52 is signaled by Nrf2, a transcription factor that can be activated by phytochemicals found
in common foods. Treating primary neurons with a Nrf2 agonist enhances clearance of AD-specific phosphoforms of tau. Conclusions: The ability to specifically target pathological tau to degradation will facilitate removing toxic species from the cell while preserving the pool of normal tau that is critical for neuronal function. The use of Nrf2 agonists for this effect offers an exciting novel therapeutic target.
P1-006
INTRAVENOUSLY INJECTED HUMAN APOLIPOPROTEIN A-I RAPIDLY ENTERS THE CENTRAL NERVOUS SYSTEM VIA THE CHOROID PLEXUS IN MICE
Sophie Stukas1, Jerome Robert1, Mike Lee1, Iva Kulic1, Nicole DeValle2, Michael Carr3, Jianjia Fan3, Dhananjay Namjoshi3, Kalistyne Lemke2, Michael Oda2, Cheryl Wellington3, 1University of British Columbia, Vancouver, British Columbia, Canada; 2Children’s Hospital of Oakland Research Institute, Oakland, California, United States; 3The University of British Columbia, Vancouver, British Columbia, Canada. Contact e-mail: [email protected] Background: Cerebrovascular dysfunction contributes significantly to the pathoetiology of Alzheimer’s disease (AD). Midlife vascular risk factors, such as hypertension, cardiovascular disease, diabetes, and dyslipidemia, increase the relative risk for AD. These comorbidities are all characterized by low and/or dysfunctional high-density lipoproteins (HDL), which itself is a suggested risk factor for AD. In addition to lipid transport, HDL enhances vasorelaxation, reduces inflammation and oxidative stress, and promotes endothelial cell survival and integrity. In mouse models of AD, apolipoprotein (apo) A-I, the primary protein component of HDL, reduces neuroinflammation, suppresses cerebrovascular Ab deposition, and protects cognitive function, making it an intriguing therapeutic target. However, how apoA-I, which is made only in the liver and intestine yet is present in cerebrospinal fluid (CSF), enters the central nervous system (CNS) is unknown. Methods: Endogenous levels of apoA-I in brain, liver, CSF, and plasma were measured by immunoblotting. Recombinant, fluorescently tagged, lipid-free human (h) apoA-I was injected into the tail vein of wild-type mice at doses ranging from 7.5-120mg/kg and tissue was collected 0.5-24h post injection. hapoA-I levels were measured by ELISA. Results: Steady state levels of endogenous murine apoA-I in CSF and brain are approximately 0.01% and 10-15% of its levels in plasma and liver, respectively. hapoA-I delivered via intravenous injection localizes to the choroid plexus within 0.5h and accumulates in a saturable, dose-dependent manner in brain. hapoA-I accumulates in the brain for up to 2h, after which it is turned over with a half-life of w133 minutes, 3 times longer than its 40-45 half0-life in plasma, liver, and kidney. In vitro, hapoA-I is taken up, specifically binds to, and is actively transported across confluent monolayers of primary human choroid epithelial cells. Conclusions: As apoA-I mRNA is undetectable in murine brain, apoA-I in the CNS is derived from the circulation. Following intravenous injection, hapoA-I rapidly and strongly localizes to the choroid plexus and can be transported across choroidal epithelial cells in vitro. These results suggest that plasma apoA-I gains access to the CNS primarily via the blood-CSF barrier.
P1-007
NEURON-TO-NEURON TRANSMISSION OF ALPHA-SYNUCLEIN
Jakob Domert1, Emelie Severinsson1, Lotta Agholme1, Chris Sackmann1, Sangeeta Nath2, Jessica Sigvardsson3, Lars Lannfelt4, Joakim Bergstr€om4, Martin Ingelsson4, Martin Hallbeck5, 1Link€oping University, Linkoping, Sweden; 2Link€oping University, Linkoping, Sweden; 3Bioarctic, Stockholm, Sweden; 4Uppsala University, Uppsala, Sweden; 5Link€oping University, Linkoping, Sweden. Contact e-mail: [email protected] Background: The progression of Alzheimer’s disease and Parkinson’s disease follows a hierarchical anatomical pattern. Recent evidence indicates that this could be caused by a prion-like spread of the disease with aggregates of b-amyloid and a-synuclein respectively, propagating from
Poster Presentations: P1
pathological tau can transfer between cells and seed the misfolding of cytosolic soluble tau into aggregates. We have developed cell-based models to dissect the mechanisms behind the propagation of tau pathology and assess potential therapeutic strategies. Methods: Filamentous tau preparations were added to neuronal and non-neuronal cells expressing soluble tau. Their uptake and the subsequent seeded aggregation of endogenous tau was studied using flow cytometry, immunofluorescence and SDS-PAGE-Western blot. Biochemical assays were used to determine structural properties of the filamentous tau preparations. A series of tau mutants was engineered to study the role of primary structure and phosphorylation state of tau in seeded aggregation and propagation in cells. Results: Hyperphosphorylated and non-phosphorylated, native and synthetic filamentous tau preparations efficiently entered cells and seeded the self-perpetuating formation of hyperphosphorylated filamentous tau composed of endogenous protein, in a process that was time- and concentration- dependent. Native filamentous tau seeded with a much higher potency than synthetic filamentous tau. Biochemical analyses revealed conformational differences between the filament preparations, which could account for the difference in seeding potency. The ability of tau to form filaments was necessary for seeded aggregation to occur; however, alteration of tau phosphorylation at pathologically associated sites did not affect this process. Conclusions: We developed a cell-based model of pathological tau propagation, whereby the addition of minute quantities of exogenous filamentous tau induced the formation of intracellular hyperphosphorylated filamentous tau by direct interaction with endogenous soluble tau. Using this model we have been able to identify structural requirements of seed-competent tau. We present evidence that filamentous tau can adopt multiple conformations, which determines its efficiency at seeding aggregation of soluble tau in cells. This is reminiscent of protein ’strains,’ such as have been identified for prion protein.
P1-005
SELECTIVELY ENHANCING PATHOLOGICAL FORMS OF TAU VIA THE AUTOPHAGY PATHWAY
Adrianne Chesser1, Zhinian Lei2, Gail V.W. Johnson2, 1University of Rochester School of Medicine, Rochester, New York, United States; 2 University of Rochester Medical Center, Rochester, New York, United States. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is the leading cause of neurodegeneration worldwide. Unfortunately, there are limited therapeutic options, due in part to an incomplete understanding of disease pathogenesis. One pathological hallmark of AD is the intracellular accumulation of hyperphosphorylated and truncated forms of the microtubule associated protein tau. These pathological forms of tau are unable to appropriately interact with microtubules, facilitatiing neuronal damage in AD. In addition, there are data to indicate that pathological tau, particularly in its oligomeric form, causes toxicity. Impairment of protein degradative systems is likely a contributing factor to the accumulation and aggregation of tau in AD. Therefore interventions that result in the selective upregulation of pathways that result in the clearance pathological tau without further diminishing the pool of available functional tau are of significant interest. Methods: Mouse cortical cells that inducibly express a single form of tau-unmodified, D421 truncation, phosphomimetic or phosphonull at T231 or S262-were established. Using these lines, degradation profiles for each form of tau were established both in the basal condition and in the presence of inhibitors or activators of the different protein degradation pathways. Primary neurons were used for complementary studies with phosphoepitope antibodies. Results: Particular pathological forms of tau are preferentially cleared via the autophagy pathway. Additionally, activating the autophagy pathway leads to enhanced clearance of these potentially disease-mediating forms. Importantly, this process is specific. Treatment with trehalose, an activator of non mTor-dependent autophagy, is effective, while rapamycin is not. This may relate to the involvement of adaptor proteins that help chaperone pathological tau to autophagy. In particular, the adaptor protein NDP52 appears to be important. Expression of NDP52 is signaled by Nrf2, a transcription factor that can be activated by phytochemicals found
in common foods. Treating primary neurons with a Nrf2 agonist enhances clearance of AD-specific phosphoforms of tau. Conclusions: The ability to specifically target pathological tau to degradation will facilitate removing toxic species from the cell while preserving the pool of normal tau that is critical for neuronal function. The use of Nrf2 agonists for this effect offers an exciting novel therapeutic target.
P1-006
INTRAVENOUSLY INJECTED HUMAN APOLIPOPROTEIN A-I RAPIDLY ENTERS THE CENTRAL NERVOUS SYSTEM VIA THE CHOROID PLEXUS IN MICE
Sophie Stukas1, Jerome Robert1, Mike Lee1, Iva Kulic1, Nicole DeValle2, Michael Carr3, Jianjia Fan3, Dhananjay Namjoshi3, Kalistyne Lemke2, Michael Oda2, Cheryl Wellington3, 1University of British Columbia, Vancouver, British Columbia, Canada; 2Children’s Hospital of Oakland Research Institute, Oakland, California, United States; 3The University of British Columbia, Vancouver, British Columbia, Canada. Contact e-mail: [email protected] Background: Cerebrovascular dysfunction contributes significantly to the pathoetiology of Alzheimer’s disease (AD). Midlife vascular risk factors, such as hypertension, cardiovascular disease, diabetes, and dyslipidemia, increase the relative risk for AD. These comorbidities are all characterized by low and/or dysfunctional high-density lipoproteins (HDL), which itself is a suggested risk factor for AD. In addition to lipid transport, HDL enhances vasorelaxation, reduces inflammation and oxidative stress, and promotes endothelial cell survival and integrity. In mouse models of AD, apolipoprotein (apo) A-I, the primary protein component of HDL, reduces neuroinflammation, suppresses cerebrovascular Ab deposition, and protects cognitive function, making it an intriguing therapeutic target. However, how apoA-I, which is made only in the liver and intestine yet is present in cerebrospinal fluid (CSF), enters the central nervous system (CNS) is unknown. Methods: Endogenous levels of apoA-I in brain, liver, CSF, and plasma were measured by immunoblotting. Recombinant, fluorescently tagged, lipid-free human (h) apoA-I was injected into the tail vein of wild-type mice at doses ranging from 7.5-120mg/kg and tissue was collected 0.5-24h post injection. hapoA-I levels were measured by ELISA. Results: Steady state levels of endogenous murine apoA-I in CSF and brain are approximately 0.01% and 10-15% of its levels in plasma and liver, respectively. hapoA-I delivered via intravenous injection localizes to the choroid plexus within 0.5h and accumulates in a saturable, dose-dependent manner in brain. hapoA-I accumulates in the brain for up to 2h, after which it is turned over with a half-life of w133 minutes, 3 times longer than its 40-45 half0-life in plasma, liver, and kidney. In vitro, hapoA-I is taken up, specifically binds to, and is actively transported across confluent monolayers of primary human choroid epithelial cells. Conclusions: As apoA-I mRNA is undetectable in murine brain, apoA-I in the CNS is derived from the circulation. Following intravenous injection, hapoA-I rapidly and strongly localizes to the choroid plexus and can be transported across choroidal epithelial cells in vitro. These results suggest that plasma apoA-I gains access to the CNS primarily via the blood-CSF barrier.
P1-007
NEURON-TO-NEURON TRANSMISSION OF ALPHA-SYNUCLEIN
Jakob Domert1, Emelie Severinsson1, Lotta Agholme1, Chris Sackmann1, Sangeeta Nath2, Jessica Sigvardsson3, Lars Lannfelt4, Joakim Bergstr€om4, Martin Ingelsson4, Martin Hallbeck5, 1Link€oping University, Linkoping, Sweden; 2Link€oping University, Linkoping, Sweden; 3Bioarctic, Stockholm, Sweden; 4Uppsala University, Uppsala, Sweden; 5Link€oping University, Linkoping, Sweden. Contact e-mail: [email protected] Background: The progression of Alzheimer’s disease and Parkinson’s disease follows a hierarchical anatomical pattern. Recent evidence indicates that this could be caused by a prion-like spread of the disease with aggregates of b-amyloid and a-synuclein respectively, propagating from