Annalisa Cavallini

Conformation Determines the Seeding Potencies of Native and Recombinant Tau Aggregates

Background: Characteristics of seed-competent Tau are unknown. Results: Native Tau aggregates have a higher seeding potency than recombinant Tau aggregates. Recombinant Tau acquires the conformation and potency of native Tau aggregates by seeded assembly. Conclusion: Conformation determines the seeding potencies of Tau aggregates. Significance: Understanding the properties of seed-competent Tau gives insight into disease mechanisms. Intracellular Tau inclusions are a pathological hallmark of several neurodegenerative diseases, collectively known as the tauopathies. They include Alzheimer disease, tangle-only dementia, Pick disease, argyrophilic grain disease, chronic traumatic encephalopathy, progressive supranuclear palsy, and corticobasal degeneration. Tau pathology appears to spread through intercellular propagation, requiring the formation of assembled “prion-like” species. Several cell and animal models have been described that recapitulate aspects of this phenomenon. However, the molecular characteristics of seed-competent Tau remain unclear. Here, we have used a cell model to understand the relationships between Tau structure/phosphorylation and seeding by aggregated Tau species from the brains of mice transgenic for human mutant P301S Tau and full-length aggregated recombinant P301S Tau. Deletion of motifs 275VQIINK280 and 306VQIVYK311 abolished the seeding activity of recombinant full-length Tau, suggesting that its aggregation was necessary for seeding. We describe conformational differences between native and synthetic Tau aggregates that may account for the higher seeding activity of native assembled Tau. When added to aggregated Tau seeds from the brains of mice transgenic for P301S Tau, soluble recombinant Tau aggregated and acquired the molecular properties of aggregated Tau from transgenic mouse brain. We show that seeding is conferred by aggregated Tau that enters cells through macropinocytosis and seeds the assembly of endogenous Tau into filaments.

An unbiased approach to identifying tau kinases that phosphorylate tau at sites associated with Alzheimer disease.

Background: Abnormally hyperphosphorylated tau is present in neurofibrillary tangles in Alzheimer disease. Results: Key kinases that phosphorylate tau at Alzheimer disease-specific epitopes have been identified in a cell-based screen of kinases. Conclusion: GSK3α, GSK3β, and MAPK13 were the most active tau kinases. Significance: Findings identify novel tau kinases and novel pathways that may be relevant for Alzheimer disease and other tauopathies. Neurofibrillary tangles, one of the hallmarks of Alzheimer disease (AD), are composed of paired helical filaments of abnormally hyperphosphorylated tau. The accumulation of these proteinaceous aggregates in AD correlates with synaptic loss and severity of dementia. Identifying the kinases involved in the pathological phosphorylation of tau may identify novel targets for AD. We used an unbiased approach to study the effect of 352 human kinases on their ability to phosphorylate tau at epitopes associated with AD. The kinases were overexpressed together with the longest form of human tau in human neuroblastoma cells. Levels of total and phosphorylated tau (epitopes Ser(P)-202, Thr(P)-231, Ser(P)-235, and Ser(P)-396/404) were measured in cell lysates using AlphaScreen assays. GSK3α, GSK3β, and MAPK13 were found to be the most active tau kinases, phosphorylating tau at all four epitopes. We further dissected the effects of GSK3α and GSK3β using pharmacological and genetic tools in hTau primary cortical neurons. Pathway analysis of the kinases identified in the screen suggested mechanisms for regulation of total tau levels and tau phosphorylation; for example, kinases that affect total tau levels do so by inhibition or activation of translation. A network fishing approach with the kinase hits identified other key molecules putatively involved in tau phosphorylation pathways, including the G-protein signaling through the Ras family of GTPases (MAPK family) pathway. The findings identify novel tau kinases and novel pathways that may be relevant for AD and other tauopathies.

Short Fibrils Constitute the Major Species of Seed-Competent Tau in the Brains of Mice Transgenic for Human P301S Tau

The interneuronal propagation of aggregated tau is believed to play an important role in the pathogenesis of human tauopathies. It requires the uptake of seed-competent tau into cells, seeding of soluble tau in recipient neurons and release of seeded tau into the extracellular space to complete the cycle. At present, it is not known which tau species are seed-competent. Here, we have dissected the molecular characteristics of seed-competent tau species from the TgP301S tau mouse model using various biochemical techniques and assessed their seeding ability in cell and animal models. We found that sucrose gradient fractions from brain lysates seeded cellular tau aggregation only when large (>10 mer) aggregated, hyperphosphorylated (AT8- and AT100-positive) and nitrated tau was present. In contrast, there was no detectable seeding by fractions containing small, oligomeric (<6 mer) tau. Immunodepletion of the large aggregated AT8-positive tau strongly reduced seeding; moreover, fractions containing these species initiated the formation and spreading of filamentous tau pathology in vivo, whereas fractions containing tau monomers and small oligomeric assemblies did not. By electron microscopy, seed-competent sucrose gradient fractions contained aggregated tau species ranging from ring-like structures to small filaments. Together, these findings indicate that a range of filamentous tau aggregates are the major species that underlie the spreading of tau pathology in the P301S transgenic model. SIGNIFICANCE STATEMENT The spread of tau pathology from neuron to neuron is postulated to account for, or at least to contribute to, the overall propagation of tau pathology during the development of human tauopathies including Alzheimers disease. It is therefore important to characterize the native tau species responsible for this process of seeding and pathology spreading. Here, we use several biochemical techniques to dissect the molecular characteristics of native tau protein conformers from TgP301S tau mice and show that seed-competent tau species comprise small fibrils capable of seeding tau pathology in cell and animal models. Characterization of seed-competent tau gives insight into disease mechanisms and therapeutic interventions.

TRANSECTION OF TARGETED AXONAL PATHWAYS INHIBITS NETWORK SPREAD OF TAU PATHOLOGY IN A P301S MODEL OF TAU PROPAGATION

classified older individuals into disease stages using deciles of whole-brain PIB distribution volume ratio (DVR), assuming that those with lower whole-brain DVRs are at earlier stages. We identified epicenters by finding ROIs that met the following criteria: (1) the PIB DVR in the ROI is greater than the average of its neighbors on the MST, (2) in the successive stage average PIB DVR in the neighbors increases, and (3) the ROI is not near another epicenter on the MST. Results:At all stages of PIB progression we found a strong significant positive correlation of regional PIB DVR with the average PIB DVR in neighboring regions at the successive stage. Moreover, with each 5 or 10 percentile increase in whole brain DVR we found a progressively stronger positive correlation of regional PIB DVRwith increased PIB DVR in neighboring regions relative to the initial stage. Finally, we identified distinct epicenters of PIB accumulation in the left posterior cingulate, left rostral anterior cingulate, left putamen, right pars opercularis, right suparmarginal, right lateral orbitofrontal, and right pericalcarine cortex which this approach identified as arising independently from one another. Conclusions:PIB appears to arise from multiple epicenters based on patterns of normal brain connectivity.

CHARACTERISATION OF TAU SPECIES INVOLVED IN TAU SEEDING AND SPREAD IN CELLULAR AND ANIMAL MODELS

Background:The neurofibrillary tangle is a pathological hallmark of Alzheimer’s disease (AD) and primarily consists of hyperphosphorylated tau protein (pTau). pTau first appears in the entorhinal cortex in the presymptomatic stage, then gradually disseminates to the hippocampal region around the onset of clinical symptoms of AD. Halting this tau spread in the asympomatic stage is a promising therapeutic approach for AD. The exosome is a small vesicle of 50-100 nm in diameter, enriched in ceramide, and is suggested to contain neuropathogenic proteins, such as prion, a-synuclein, and recently tau proteins. A growing body of evidence suggests that microglia contribute to tauopathy-related pathogenesis in both human and animal models. We hypothesize that microglia transduce tau aggregates into nearby neuronal cells via exosomal secretion, and that inhibition of the exosome synthesis or secretory pathway reduces tau dissemination. Methods: Adeno-associated virus serotype 6 expressing FTDP-17-linked mutation of tau protein was stereotaxically injected into the entorhinal cortical region of C57BL/6 mice, and the animals were sacrificed at 7 and 28 days post injection. The brain specimens were examined for tau accumulation in the hippocampal region. The animals were also systemically treated with specific inhibitors of neutral sphingomyelinase-2 to block exosome synthesis, or colony stimulating factor 1 receptor (CSF1R) to deplete microglia. Results:We found that human tau propagate from entorhinal cortical neurons to dentate granular cells after AAV injection, and that this propagation is sensitive to the inhibition of exosome synthesis or microglial depletion. We also found that tau-containing exosomes isolated from microglia efficiently transduce tau protein to neurons in vitro and in vivo. Finally, these results were reproduced in P301S tau mice (PS19) treated with these compounds. Conclusions: These results demonstrate that exosome secretion from microglia play a significant role in propagation of tau protein from entorhinal cortex to hippocampal neurons. Our findings could lead to an entirely novel paradigm for delaying the progression of disease in AD and other tauopathies such as frontotemporal dementia and chronic traumatic encephalopathy.

IDENTIFICATION OF TAU SPECIES REQUIRED FOR SEEDING IN A CELL-BASED MODEL OF PATHOLOGICAL TAU PROPAGATION

P1-001 THE 3XTG-AD MOUSE MODEL OFALZHEIMER’S DISEASE EXHIBITS AGE-DEPENDENT IMPAIRED GLUCOSE TOLERANCE Milene Vandal, Phillip White, Isabelle St-Amour, Andre Marette, Frederic Calon, Laval University, Quebec, Quebec, Canada; Universite Laval, Quebec, Quebec, Canada; CHUL-Research Center, Quebec, Quebec, Canada; CHUL-Research Center, Quebec, Quebec, Canada. Contact e-mail: Milene.vandal@crchul.ulaval.ca

Chronic inhibition of the MEK/ERK pathway fails to delay disease progression in a transgenic animal model of tauopathy

Background: Neurofibrillary tangles (NFT), one of the hallmarks of Alzheimer’s disease (AD), are composed of paired helical filaments (PHF) of abnormally hyperphosphorylated tau. The accumulation of these proteinaceous aggregates in AD correlates with synaptic loss and severity of dementia. Identifying the kinases involved in the pathological phosphorylation of tau may identify novel targets for AD.Methods:We have used an unbiased approach to study the effect of 352 human kinases on their ability to phosphorylate tau at epitopes associated with early AD. The kinases were overexpressed together with the longest form of human tau in human neuroblastoma cells. Levels of total and phosphorylated tau (epitopes pS202, pT231, pS235 and pS396/404) were measured in cell lysates using AlphaScreen assays. Results: GSK3a, GSK3b and MAPK13 were found to be the most active tau kinases, phosphorylating tau at all 4 epitopes. Pathway analysis of all the ‘hits’ suggests the Ras family of GTPases (MAPK family) to be a key regulator of tau phosphorylation. Pathway analysis on subgroups of kinases reveals several mechanisms involved in regulating tau expression levels by inhibition or activation of translation.Conclusions: The findings identify novel tau kinases and novel pathways that may be relevant for AD and other tauopathies.

Dramatic increases in phosphorylation of tau only on specific epitopes correlates with decline in motor function in aged P301S mice

P1-348 DRAMATIC INCREASES IN PHOSPHORYLATION OF TAU ONLY ON SPECIFIC EPITOPES CORRELATES WITH DECLINE IN MOTOR FUNCTION IN AGED P301S MICE Philip G. Szekeres, Sarah J. Glover, Viktor Lakics, Claire V. Cella, Robert Purcell, Tracey K. Murray, Annalisa Cavallini, Isabelle Lavenir, Michel Goedert, Peter Davies, Michael L. Hutton, Michael J. O’Neill, Eli Lilly, Windlesham, United Kingdom; MRC, Laboratory of Molecular Biology, Cambridge, United Kingdom; Albert Einstein College of Medicine, New York, NY, USA. Contact e-mail: szekeresp@lilly.com

Investigation of GSK-3 alpha and GSK-3 beta phosphorylation of tau in primary neuronal cultures and neuroblastoma cells

P1-332 INVESTIGATION OF GSK-3 ALPHA AND GSK-3 BETA PHOSPHORYLATION OF TAU IN PRIMARY NEURONAL CULTURES AND NEUROBLASTOMA CELLS Suchira Bose, Annalisa Cavallini, Sarah Glover, Amanda Bell, Samantha Sargent, Rachel Angers, Viktor Lakics, Peter Davies, Michael Hutton, Philip Szekeres, Eli Lilly & Co Ltd, Windlesham, Surrey, United Kingdom; MRC Laboratory of Molecular Biology, Cambridge, United Kingdom; AECOM, The Feinstein Institute for Medical Research, Manhasset, NY, USA. Contact e-mail: bose_suchira@lilly.com