Tau seeding without tauopathy.

Neurodegenerative tauopathies such as Alzheimer's disease (AD) are caused by brain accumulation of tau assemblies. Evidence suggests tau functions as a prion, and cells and animals can efficiently propagate unique, transmissible tau pathologies. This suggests a dedicated cellular replication machinery, potentially reflecting a normal physiologic function for tau seeds. Consequently, we hypothesized that healthy control brains would contain seeding activity. We have recently developed a novel monoclonal antibody (MD3.1) specific for tau seeds. We used this antibody to immunopurify tau from the parietal and cerebellar cortices of 19 healthy subjects without any neuropathology, ranging 19 to 65 years. We detected seeding in lysates from the parietal cortex, but not in the cerebellum. We also detected no seeding in brain homogenates from wildtype or human tau knockin mice, suggesting that cellular/genetic context dictates development of seed-competent tau. Seeding did not correlate with subject age or brain tau levels. We confirmed our essential findings using an orthogonal assay, real-time quaking-induced conversion, which amplifies tau seeds in vitro. Dot blot analyses revealed no AT8 immunoreactivity above background levels in parietal and cerebellar extracts and ∼1/100 of that present in AD. Based on binding to a panel of antibodies, the conformational characteristics of control seeds differed from AD, suggesting a unique underlying assembly, or structural ensemble. Tau's ability to adopt self-replicating conformations under nonpathogenic conditions may reflect a normal function that goes awry in disease states.

A synthetic heparinoid blocks Tau aggregate cell uptake and amplification.

Tau aggregation underlies neurodegeneration in Alzheimer's disease and related tauopathies. We and others have proposed that transcellular propagation of pathology is mediated by Tau prions, which are ordered protein assemblies that faithfully replicate in vivo and cause specific biological effects. The prion model predicts the release of aggregates from a first-order cell and subsequent uptake into a second-order cell. The assemblies then serve as templates for their own replication, a process termed "seeding." We have previously observed that heparan sulfate proteoglycans on the cell surface mediate the cellular uptake of Tau aggregates. This interaction is blocked by heparin, a sulfated glycosaminoglycan. Indeed, heparin-like molecules, or heparinoids, have previously been proposed as a treatment for PrP prion disorders. However, heparin is not ideal for managing chronic neurodegeneration, because it is difficult to synthesize in defined sizes, may have poor brain penetration because of its negative charge, and is a powerful anticoagulant. Therefore, we sought to generate an oligosaccharide that would bind Tau and block its cellular uptake and seeding, without exhibiting anticoagulation activity. We created a compound, SN7-13, from pentasaccharide units and tested it in a range of assays that measured direct binding of Tau to glycosaminoglycans and inhibition of Tau uptake and seeding in cells. SN7-13 does not inhibit coagulation, binds Tau with low nanomolar affinity, and inhibits cellular Tau aggregate propagation similarly to standard porcine heparin. This synthetic heparinoid could facilitate the development of agents to treat tauopathy.

Anatomic survey of seeding in Alzheimer's disease brains reveals unexpected patterns.

Tauopathies are heterogeneous neurodegenerative diseases defined by progressive brain accumulation of tau aggregates. The most common tauopathy, sporadic Alzheimer's disease (AD), involves progressive tau deposition that can be divided into specific stages of neurofibrillary tangle pathology. This classification is consistent with experimental data which suggests that network-based propagation is mediated by cell-cell transfer of tau "seeds", or assemblies, that serve as templates for their own replication. Until now, seeding assays of AD brain have largely been limited to areas previously defined by NFT pathology. We now expand this work to additional regions. We selected 20 individuals with AD pathology of NFT stages I, III, and V. We stained and classified 25 brain regions in each using the anti-phospho-tau monoclonal antibody AT8. We measured tau seeding in each of the 500 samples using a cell-based tau "biosensor" assay in which induction of intracellular tau aggregation is mediated by exogenous tau assemblies. We observed a progressive increase in tau seeding according to NFT stage. Seeding frequently preceded NFT pathology, e.g., in the basolateral subnucleus of the amygdala and the substantia nigra, pars compacta. We observed seeding in brain regions not previously known to develop tau pathology, e.g., the globus pallidus and internal capsule, where AT8 staining revealed mainly axonal accumulation of tau. AT8 staining in brain regions identified because of tau seeding also revealed pathology in a previously undescribed cell type: Bergmann glia of the cerebellar cortex. We also detected tau seeding in brain regions not previously examined, e.g., the intermediate reticular zone, dorsal raphe nucleus, amygdala, basal nucleus of Meynert, and olfactory bulb. In conclusion, tau histopathology and seeding are complementary analytical tools. Tau seeding assays reveal pathology in the absence of AT8 signal in some instances, and previously unrecognized sites of tau deposition. The variation in sites of seeding between individuals could underlie differences in the clinical presentation and course of AD.

Loss of a Negative Regulator of mTORC1 Induces Aerobic Glycolysis and Altered Fiber Composition in Skeletal Muscle.

The conserved GATOR1 complex consisting of NPRL2-NPRL3-DEPDC5 inhibits mammalian target of rapamycin complex 1 (mTORC1) in response to amino acid insufficiency. Here, we show that loss of NPRL2 and GATOR1 function in skeletal muscle causes constitutive activation of mTORC1 signaling in the fed and fasted states. Muscle fibers of NPRL2 knockout animals are significantly larger and show altered fiber-type composition, with more fast-twitch glycolytic and fewer slow-twitch oxidative fibers. NPRL2 muscle knockout mice also have altered running behavior and enhanced glucose tolerance. Furthermore, loss of NPRL2 induces aerobic glycolysis and suppresses glucose entry into the TCA cycle. Such chronic activation of mTORC1 leads to compensatory increases in anaplerotic pathways to replenish TCA intermediates that are consumed for biosynthetic purposes. These phenotypes reveal a fundamental role for the GATOR1 complex in the homeostatic regulation of mitochondrial functions (biosynthesis versus ATP) to mediate carbohydrate utilization in muscle.