Alzheimer's Disease and Tau: Pathogenic Mechanisms and Therapeutic Approaches

Abstracts

Etiopathogenesis of Hyperphosphorylation of Tau and Potential Therapeutic Targets
Khalid Iqbal, PhD, New York State Institute for Basic Research in Developmental Disabilities

Alzheimer’s disease (AD) is a multifactorial disorder. However, independent of the etiology, AD is histopathologically characterized by the presence of numerous neurofibrillary tangles of abnormally hyperphosphorylated tau and Aβ core-neuritic (senile) plaques. The tangles and not Aβ plaques are required for the clinical expression of the disease, the dementia. Besides AD, hyperphosphorylated tau is also a hallmark of a family of neurodegenerative disorders called tauopathies. The activity of protein phosphatase-2A (PP2A), which is the major regulator of phosphorylation of tau, is compromised in the brains of cases with AD, Down syndrome and Guam amyotrophic lateral sclerosis Parkinsonism dementia complex (Guam ALSPD). My lab has been conducting research on finding how hyperphosphorylated tau can lead to neurodegeneration and how different etiological factors can lead to tau pathology. In my talk I will discuss our studies that show the possible molecular mechanisms by which hyperphosphorylation of tau may be involved in neurodegeneration in different tauopathies, and what therapeutic opportunities are available based on these findings.
 

Amyloid-β and Tau: the Trigger and Bullet for Alzheimer’s Disease Pathogenesis
George S. Bloom, PhD, University of Virginia

The most conspicuous histopathological features of Alzheimer’s disease (AD) brain include two types of poorly soluble aggregates: extracellular amyloid plaques made from amyloid-β (Aβ) peptides and intraneuronal neurofibrillary tangles assembled from the neuron-specific protein, tau. The behavioral symptoms of AD result from synaptic dysfunction among neurons that mediate memory and cognition, and the death of those neurons. Growing evidence indicates that soluble forms of Aβ and tau work together, independently of their accumulation into plaques and tangles, to drive healthy neurons into the diseased state, and that trademark toxic properties of Aβ require tau. One example of tau-dependent Aβ toxicity is ectopic cell cycle re-entry (CCR) of neurons, which ironically leads to neuron death and may account for as much as 90% of neuron death in AD. Brain insulin resistance is also an AD hallmark and potentially fundamental driver of AD symptoms, but the mechanisms by which it causes the synaptic dysfunction and neuron death that underlie memory and cognitive impairment had remained unknown. We now demonstrate that CCR follows Aβ oligomer (AβO) induced activation of mechanistic target of rapamycin complex 1 (mTORC1) at the plasma membrane and mTORC1-dependent tau phosphorylation, and is blocked in cultured neurons by lysosomal mTORC1 activation by insulin. Because AβOs also cause neuronal insulin resistance, our data suggest that AβOs dysregulate normal mTOR signaling by activating mTORC1 at the cell surface while preventing insulin from stimulating lysosomal mTORC1. These dual effects of AβOs provide a mechanistic basis for classifying AD as type 3 diabetes.
 

Microglia: A Reintroduction
Richard M. Ransohoff, MD, VP and Senior Research Fellow, Neuroimmunology and Adjunct Professor of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University

Microglia arise during primitive hematopoiesis and initially enter the developing murine brain around E10.5, before other glia and prior to neuronal differentiation. During development, microglia interact extensively with neurons, helping to establish neuronal populations through influences on survival, apoptosis and corpse-clearance. As neuron-to-neuron contacts are forming during early postnatal life, microglia refine neuronal network properties by synaptic pruning.
 
Neuron-microglial and microglial-neuron dialogs are mediated by contact-dependent and soluble signals. As one example, fractalkine (CX3CL1) is a transmembrane chemokine expressed in the CNS solely by neurons, which also release soluble fractalkine to signal at a distance. The fractalkine receptor (CX3CR1) is restricted to microglia among CNS cells. Perturbation of fractalkine/fractalkine receptor signaling results in altered microglial function, with consequences observed from mid-gestation through aging and neurodegeneration. Using mice deficient for fractalkine or its receptor provides a wealth of insights into microglial function in the intact CNS and may have direct clinical relevance, given a common hypomorphic CX3CR1 allele in humans. Examination of the mechanism underlying fractalkine regulation of microglial responses may yield information useful for translating model results to clinical application.
 

Regulation of Tau Secretion
Nicole Leclerc, PhD, Université de Montréal

Recent studies have demonstrated that human tau protein can be secreted by neurons, an event linked to the propagation of tau pathology in Alzheimer brain. We recently examined whether tau secretion by cortical neurons would be modified upon insults. Both starvation and inhibition of lysosomal function, two insults known to take place in Alzheimer’s disease, significantly increased tau secretion. However, the most important increase of tau secretion was observed when both insults were superimposed. In such conditions, the pattern of tau secretion differed from that of control neurons. As a result of these treatments, at least two tau species were released. Furthermore, in all conditions tested, an important pool of secreted tau was dephosphorylated. Collectively, our results demonstrate that insults such as nutrient deprivation and lysosomal dysfunction observed in neurodegenerative diseases could result in an increase of tau secretion and propagation of tau pathology in the brain. The question to be answered is whether the different tau species that are released by neurons exert distinct toxic effects and/or whether they contribute distinctly to the propagation of tau pathology. It is crucial to unravel the distinct contribution of tau species to these events to develop therapeutic strategies to prevent tau-induced neuronal dysfunction and to abrogate the spreading of tau pathology in Alzheimer brain.
 

Insights Into Human Tau Pathology from the Prion Model
Marc I. Diamond, MD, University of Texas Southwestern Medical Center

Neurodegenerative diseases linked to protein amyloids could be caused by trans-cellular propagation of protein aggregates. In this model, protein assemblies escape a cell, enter a vulnerable cell, and create new pathology by acting as a template, or "seed," for further aggregation. Prion diseases progress by this mechanism. To quantify seeding activity, and to test the role of proteopathic seeds in the development of pathology, we have developed a FRET-based biosensor cell assay that quantifies seeding activity. This identifies seeds in human and mouse brain far in advance of standard histopathology. Prion diseases have tremendous clinical and neuropathological diversity that has been linked to specific amyloid structures, or "strains," which replicate faithfully over decades in animal hosts. Like prionopathies, tauopathies are clinically and neuropathologically heterogeneous, and feature intraneuronal accumulation of tau amyloid assemblies that are rich in beta sheet structure. We have described essential strain characteristics of tau prions, in which unique conformations propagate indefinitely in cells and in animals, and create unique patterns of neuropathology. We have linked human tauopathies to distinct groups of tau prion strains. We find that after intracerebral inoculation, strain conformation defines the structural and biochemical properties of the induced aggregates, neuronal pathology, and patterns of spread. Our results suggest a very proximal role for proteopathic seeds in the pathogenesis of tauopathies, with unique strains determining patterns of pathology and clinical phenotype. We hypothesize this could ultimately enable syndromic classification of tauopathies based entirely on aggregate structure.
 
Coauthors: Sarah K. Kaufman^1,2, Jennifer L. Furman¹, PhD, Kelly Del Tredici, MD, PhD³, Heiko Braak, MD³, Ann C. McKee, MD⁴, Nigel J. Cairns, PhD⁵, Lea T. Grinberg, MD, PhD⁶, William Seeley, MD⁶
 
1 Center for Alzheimer's and Neurodegenerative Diseases, University of Texas, Southwestern Medical Center, Dallas, Texas, United States
2 Program in Neuroscience, Washington University in St. Louis School of Medicine, St. Louis, Missouri, United States
3 Department of Neurology, University of Ulm, Germany
4 Department of Neurology and Pathology Boston University School of Medicine Boston, Massachusetts, United States
5 Department of Neurology, Washington University in St. Louis School of Medicine, St. Louis, Missouri, United States
6 Department of Neurology, UCSF, San Francisco, California, United States
 

Tau Pathology Spreading and Propagation
Michael Hutton, Eli Lilly and Co Ltd

Neurofibrillary pathology composed of the microtubule associated protein tau is a characteristic feature of Alzheimer’s Disease as well as multiple rare tauopathies such as FTLD-tau and Progressive Supranuclear Palsy. In each case, the density and distribution of the tau lesions correlates with the extent of neuronal loss and with the clinical progression of the disorder. Moreover multiple mutations in the tau (MAPT) gene have been shown to cause FTD linked to chromosome 17 establishing that tau dysfunction is sufficient to cause neurodegeneration. These mutations impact tau in different ways but significantly all are predicted to enhance tau aggregation suggesting that protein misfolding is a critical component of the neurodegenerative cascade. More recently, research from several laboratories has suggested, both in vitro and in vivo, that misfolded tau proteins can be released from neurons at synaptic terminals and can then enter recipient cells. The misfolded tau is then able to template its structure onto endogenous tau leading to seeded tau aggregation and the resultant propagation of neurofibrillary pathology. However, despite considerable research in this area over the past 5 years significant uncertainty remains regarding the mechanism of tau pathology spreading and the tau species involved in this process. To address these questions, we have established multiple cell-based and in vivo models that have allowed us to examine both the process of tau pathology spreading and the tau species involved. These studies have demonstrated that, in these model systems, tau pathology appears to spread only through anatomically connected regions and that large tau assemblies are required for spreading, templating and pathology propagation in vivo. Characterization of the various tau species that underpin propagation in our model suggests that the most efficient structures are short filaments that contain hyperphosphorylated tau proteins.
 

Monoclonal Antibodies as Therapies for Tau Pathology
Peter Davies, PhD, Litwin-Zucker Center for Research on Alzheimer’s Disease, Feinstein Institute for Medical Research

Monoclonal antibodies are increasingly popular therapeutics, although applications to diseases of the central nervous system have been limited. Targeting extracellular deposits of  beta-amyloid with specific antibodies appeared to be an attractive idea, but the notion that the intracellular deposits of tau that make up the neurofibrillary tangles of Alzheimer’s disease and related disorders could also be targets for antibody therapy seemed very speculative. However, numerous such studies are now underway, and it seems probable that human testing of such therapy is imminent.
Unlike the situation with beta-amyloid, which has a limited number of antibody epitopes to target, the 441 amino acids of tau, plus numerous post-translational modifications and conformational changes provide an almost unlimited number of potential targets for monoclonal antibodies. Tau sequence, conformational and phosphoepitope antibodies have all been tested in mouse models. It is not yet clear which type of antibody shows the most consistent efficacy in mice, although experience suggests that differences between these classes might be dependent on dosage.
The issue of how antibodies to an intracellular pathology can be efficacious is still controversial. Most mechanistic studies assume contact between antibody and target in the extracellular space. Once bound to antibody, there are two routes for removal of tau: sequestration and degradation by glial cells through antibody Fc receptors, and exit from CNS into the circulation. Recent results suggest that CNS-derived tau does appear in blood after treatment with some antibodies, but do not exclude glial-mediated clearance, which seems almost certain.
Supported by NIA R37 AG022102
 

Cistauosis: A Common Early Disease Mechanism in Alzheimer’s and Traumatic Brain Injury that can Be Blocked by Antibody
Kun Ping Lu, MD, PhD¹

A major neuropathological hallmark of Alzheimer’s disease (AD) and chronic traumatic encephalopathy (CTE) is tangles made of phosphorylated tau (P-tau). A major environmental risk factor for these diseases is traumatic brain injury (TBI), but tauopathy is not detectable acutely or subacutely after TBI. Therefore, how TBI leads to tauopathy and how to stop it are still unknown. We identify a unique proline isomerase, Pin1 that inhibits tauopathy development in AD by converting P-tau from cis to trans. By creating antibodies able to distinguish these two P-tau conformations, we discover that cis P-tau is an early pathogenic tau conformation leading to tauopathy and memory loss in AD and a novel drug target (Cell 2012, 149: 232). We further extend these studies to TBI and find robust cis p-tau after sport- and military-related TBI in humans and mice. Acutely after TBI in mice and stress in vitro, neurons prominently produce cis p-tau, which disrupts axonal microtubule network and mitochondrial transport, spreads to other neurons, and leads to apoptosis. This process, termed “cistauosis”, appears long before other tauopathy, and is effectively blocked by cis P-tau antibody, but enhanced by trans antibody. Treating TBI mice with cis antibody not only blocks early cistauosis, but also effectively prevents TBI-related widespread tauopathy and neuronal loss, as well as behavioral and psychological defects (Nature 2015, 523: 431). Thus, cistauosis is a common early disease mechanisms in TBI, AD and CTE, and cis antibody may be further developed for early diagnosis and treatment of these devastating diseases.
 
Coauthors: Asami Kondo, PhD1, Koorosh Shahpasand, PhD¹, Onder Albayram, PhD¹, Yu-Min Lin, PhD¹, Rebekah Mannix, MD², William Meehan, MD², and Xiao Zhen Zhou, MD¹
1 Division of Translational Therapeutics, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massuchussetts, United States
2 Micheli Center for Sports Injury Prevention, Children’s Hospital Boston, Harvard Medical School, Boston, Massuchussetts, United States
 

Development of PET Imaging Biomarkers for PHF-Tau: [18F]-T807 and [18F]-T808
Hartmuth C. Kolb, PhD, Johnson & Johnson, Janssen R&D

Background: There are two main neuropathologic hallmarks in Alzheimer’s Disease (AD), senile neuritic plaques containing ß-amyloid protein, and neurofibrillar protein aggregates containing hyperphosphorylated tau protein.
 
Methods: We have developed PET imaging agents for these pathological hallmarks, using a competitive autoradiography assay based on human AD brain sections. Brain uptake of [F18]-labeled tracers was measured in rodents and non-human primates. First in human studies were conducted to compare tracer uptake and retention with the established Braak staging patterns of tau pathology in AD.
 
Results: We have discovered several novel, small molecule tau binding compounds that show high selectivity for native tau aggregates in human AD brain sections over β-amyloid. The most promising compounds, [18F]-T807 and [18F]-T808, have a high brain uptake/fast clearance in rodents and primates. White matter distribution is very low. First in human studies revealed good agreement with Braak staging.
 
Conclusions: We have identified several small heterocyclic compounds that bind to human PHF-tau. In vitro and in vivo studies confirm that the lead candidates [F18]-T807 and T808 bind to PHF-tau selectively over β-amyloid, show good PK and metabolic properties, and exhibit excellent brain uptake/washout kinetics in rodents, non-human primates and in humans, in agreement with the known Tau pathology in AD.
 
Disclaimer: This work was conducted at Siemens MI, Biomarker Research. The Tau imaging assets now belong to Avid Radiopharmaceuticals. T807 (AV-1451) is an investigational drug. The presentation is not sanctioned and/or endorsed by Avid/Lilly or Johnson & Johnson.
 

26S Proteasome Dysfunction and Cognitive Impairment Caused by Aggregated Tau Accumulation can be Attenuated by PKA-Mediated Phosphorylation of Proteasomes
Natura Myeku, PhD¹

The ubiquitin proteasome system (UPS) selectively degrades misfolded proteins including those implicated in the pathogenesis of neurodegenerative diseases. We investigated the effects of abnormal tau accumulation on proteasome function in the brains of a mouse model of tauopathy and in a cross to a UPS reporter mouse (line Ub-G76V-GFP). Accumulation of insoluble tau correlated with a progressive decrease in the peptidase activity of brain 26S proteasomes, while levels of ubiquitinated proteins and undegraded Ub-G76V-GFP increased. After affinity purification, the 26S proteasomes from mice with tauopathy were less active in hydrolyzing ubiquitinated proteins, small peptides (by the 20S core particle), and ATP (by the 19S ATPases). Tau was physically associated with these defective proteasomes, and 26S proteasomes from normal mice incubated with oligomers or fibrils generated from recombinant tau displayed less hydrolyzing capacity for ubiquitinated proteins, peptides, and ATP.  The loss of proteasome activity was attenuated by administering an agent which activates cAMP/PKA signaling and proteasome subunit phosphorylation. Enhancing proteasome function led to reduced levels of aggregated tau and improved cognitive performance in early stages of tauopathy. Thus, proteasome function and proteolysis by the UPS decreases with worsening tauopathy, and stimulating proteasome activity through cAMP/PKA is a promising therapeutic strategy.
 
Coauthor: Karen E. Duff PhD¹
1 Department of Pathology and Cell Biology, Taub Institute for Alzheimer's Disease Research, Columbia University, New York, NY, USA
 

TAU Cleavage to Aggregation Prone Fragments: Therapeutic Effects of cAMP and IU1
Magdalena Kiprowska, MS (PhD soon)¹

TAU is a highly soluble protein. Under non-pathological conditions TAU is turned over by the ubiquitin/proteasome pathway (UPP) and the autophagy/lysosome pathway. In brains of AD patients, TAU accumulates as aggregates in neurofibrillary tangles (NFTs). One of the critical aspects of TAU pathology is its abnormal cleavage causing it to switch from a soluble monomer to a non-soluble form characteristic of NFTs. This abnormal TAU cleavage occurs early in the development of TAU pathology. The conditions that cause TAU to be cleaved to oligomerization- and aggregation-prone fragments are poorly established. We demonstrate in rat primary cerebral cortical neuronal cultures that impairment of the UPP and mitochondria trigger caspase and calpain activation respectively, leading to TAU cleavage to aggregation-prone fragments. These data are important since UPP and mitochondrial dysfunction occur early in AD pathogenesis. The dual vulnerability of TAU to caspase and calpain cleavage needs to be addressed therapeutically to maintain TAU integrity and prevent TAU pathology. We focused on preserving UPP and mitochondrial function. Increasing neuronal cAMP levels does both, while IU1 potentially stimulates degradation via the UPP by selectively inhibiting the de-ubiquitinating enzyme Usp14. Our results demonstrate that raising cAMP levels with the lipophilic peptide PACAP27 rescues TAU cleavage and neuronal viability, while IU1 does not. We established that IU1 inhibits mitochondrial function and E1-dependent ubiquitin activation. These data support the use of PACAP27 as a novel therapeutic strategy to preserve TAU integrity and prevent TAU cleavage, oligomerization and aggregation relevant to Alzheimer’s disease.
 
Coauthors: Maria Figueiredo-Pereira, PhD¹
1 Hunter College and Graduate Center, CUNY, New York, New York, United States