Sunday, July 9, 2017

Scientists uncover the structure of tau filaments from Alzheimer's disease

Scientists uncover the structure of tau filaments from Alzheimer's disease

5 Jul 2017

Researchers at the MRC Laboratory of Molecular Biology (LMB) have, for the first time, revealed the atomic structures of one of the two types of the abnormal filaments which lead to Alzheimer's disease. Understanding the structures of these filaments will be key in developing drugs to prevent their formation.

The researchers, whose study is published today in Nature, believe the structures they have uncovered could also suggest how tau protein may form different filaments in other neurodegenerative diseases.

Alzheimer’s, the most common neurodegenerative disease, is characterised by the existence of two types of abnormal ‘amyloid’ forms of protein which form lesions in the brain. Tau forms filaments inside nerve cells and amyloid-beta forms filaments outside cells. Tau lesions appear to have a stronger correlation to the loss of cognitive ability in patients with the disease.

Almost thirty years ago, scientists at the LMB (including Michel Goedert, one of the senior authors on this paper) identified tau protein as an integral component of the lesions found in Alzheimer’s and a range of other neurodegenerative diseases. But, until now, scientists have been unable to identify the atomic structure of the filaments.

The researchers extracted tau filaments from the brain of a patient who had died with Alzheimer's disease. The filaments were then imaged using cryo-electron microscopy (cryo-EM). Senior author Sjors Scheres and colleagues developed new software in order to calculate the structure of the filaments in sufficient detail to deduce the arrangement of the atoms inside them.

Sjors Scheres said: “It’s very exciting that we were able to use this new technique to visualise filaments from a diseased brain as previous work depended on artificial samples assembled in the laboratory. Amyloid structures can form in many different ways, so it has been unclear how close these lab versions resembled those in human disease.

“Knowing which parts of tau are important for filament formation is relevant for the development of drugs. For example, many pharmaceutical companies are currently using different parts of tau in tests to measure the effect of different drugs on filament formation; this new knowledge should significantly increase the accuracy of such tests."

Fellow senior author Michel Goedert said: “We have known for almost three decades that the abnormal assembly of tau protein into filaments is a defining characteristic of Alzheimer's disease. In 1998, the dysfunction of tau protein was shown to be sufficient for neurodegeneration and dementia. In 2009, the prion-like properties of assembled tau were identified. These properties allow the abnormal form to convert previously normal forms.

“Until now the high-resolution structures of tau or any other disease-causing filaments from human brain tissue have remained unknown. This new work will help to develop better compounds for diagnosing and treating Alzheimer's and other diseases which involve defective tau.”

Dr Rob Buckle, chief science officer at the MRC, which funded the research, said: “This ground-breaking work is a major contribution to our understanding of Alzheimer's disease. Nearly thirty years ago scientists at the LMB were the first to discover that tau protein plays a key role in the disease. Knowing the basic structure of these filaments in diseased tissue is vital for the development of drugs to combat their formation.

“This research opens up new possibilities to study a range of other diseases where the accumulation of abnormal protein filaments plays a role, including Parkinson’s disease, motor neuron disease and prion diseases.”

The work was funded by the MRC, the European Union, US National Institutes of Health and the Department of Pathology and Laboratory Medicine, Indiana University School of Medicine.

Cryo-EM structures of tau filaments from Alzheimer’s disease

Anthony W. P. Fitzpatrick, Benjamin Falcon, Shaoda He, Alexey G. Murzin, Garib Murshudov, Holly J. Garringer, R. Anthony Crowther, Bernardino Ghetti, Michel Goedert & Sjors H. W. Scheres

AffiliationsContributionsCorresponding authors Nature (2017) doi:10.1038/nature23002 Received 24 February 2017 Accepted 05 June 2017 Published online 05 July 2017 Article tools


Abstract• Accession codes• References• Author information• Extended data figures and tables

Alzheimer’s disease is the most common neurodegenerative disease, and there are no mechanism-based therapies. The disease is defined by the presence of abundant neurofibrillary lesions and neuritic plaques in the cerebral cortex. Neurofibrillary lesions comprise paired helical and straight tau filaments, whereas tau filaments with different morphologies characterize other neurodegenerative diseases. No high-resolution structures of tau filaments are available. Here we present cryo-electron microscopy (cryo-EM) maps at 3.4–3.5 Å resolution and corresponding atomic models of paired helical and straight filaments from the brain of an individual with Alzheimer’s disease. Filament cores are made of two identical protofilaments comprising residues 306–378 of tau protein, which adopt a combined cross-β/β-helix structure and define the seed for tau aggregation. Paired helical and straight filaments differ in their inter-protofilament packing, showing that they are ultrastructural polymorphs. These findings demonstrate that cryo-EM allows atomic characterization of amyloid filaments from patient-derived material, and pave the way for investigation of a range of neurodegenerative diseases.

At a glance

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Neurodegeneration: Taming tangled tau

David S. Eisenberg & Michael R. Sawaya Affiliations Corresponding authors Nature (2017) doi:10.1038/nature23094

Published online 05 July 2017

Article tools Citation Rights & permissions Article metrics

The protein tau forms abnormal filamentous aggregates called tangles in the brains of people with neurodegeneration. Structures of two such filaments offer pathways to a deeper understanding of Alzheimer's disease.


P154 Development of an in vitro amplification assay for misfolded proteins in for misfolded proteins in Alzheimer’s diseases (AD) and Parkinson’s disease (PD)

Ms Susana Margarida Silva Correia1
1National Reference Center For Tse, Department Of Neurology, Georg-august University , Göttingen, Germany

A characteristic feature of major neurodegenerative diseases leading to dementia is the progressive accumulation of protein aggregates in the brain in a self-propagation-manner with a regional pattern specific to each disease. The concept of protein misfolding was initially thought to play a crucial role mainly in prion diseases, recent studies identified similar characteristics for amyloid beta, tau and alpha synuclein in various models. The term “prion-like“ protein propagation is now widely used to address the mechanisms which might play a role in amyloidopathies or tauopathies such as Alzheimer’s disease (AD) and alpha synucleinopathies, such as Parkinson’s disease (PD) or Lewy body dementia (LBD).

We established the RT-QuIC for the amplification of prion protein scrapie (PrPSc) in human brain tissue and cerebrospinal fluid (CSF) of CJD-patients (Schmitz M et al, 2016). The technique bears a huge diagnostic and analytical potential also for other misfolded proteins, which are showing a prion-like mechanism of protein misfolding.

The aim of the study is the implementation of the amyloid beta and alpha synuclein QuIC in diagnostic of neurodegenerative diseases. In particular, we aim to improve the diagnostic of AD, PD and LBD. The implementation of the amyloid beta and alpha synuclein QuIC in human disease diagnostic requires the analysis of a huge cohort of patients (available from our biobank) consisting of healthy controls, patients with alternative diagnosis as well as of patients with AD and PD.

The aim of the study is to develop the RT-QuIC to create a novel diagnostic-tests for other neurodegenerative diseases such as AD and PD. In a first step, we plan to produce recombinant amyloid beta and alpha synuclein and to test systematically different substrates in our QuIC-amplification assay. After validation of the most suitable substrates, we will applicate the RT-QuIC for the detection of amyloid beta and alpha synuclein aggregates in CSF from AD- and PD-patients.


P188 Misfolded PrP is not always associated with formation of p-tau

Dr Debbie Brown1, Mr Declan King1, Dr Rona Barron1, Professor Pedro Piccardo1
1Roslin Institute, Edinburgh, United Kingdom

The conversion of cellular prion protein into a misfolded isoform is central to the development of prion diseases. Amyloid beta (Aβ) and hyperphosphorylated tau (p-tau) participate in the pathogenesis of Alzheimer´s disease (AD). Additionally several prion diseases in humans accumulate p-tau in the brain and therefore we hypothesise that proteins that participate in the pathogenesis of one disease may play a role in other disorders to establish complex proteinopathies, a mechanism that could explain the phenotypic variability observed in prion diseases. To explore this possibility we analysed p-tau accumulation in mouse models with varying degrees of PrP deposits in the brain. We used animals inoculated with :- mouse-adapted prion agents, typical and atypical bovine spongiform encephalopathy agents along with mice inoculated with wild-type (Wt) /mutant (101L) recombinant PrP fibrils and mice overexpressing 101L PrP. We observed that p-tau is consistently present in animals with prion infectivity (i.e. models that transmit disease upon serial passage). However, p-tau is not observed in non-prion mice inoculated with recombinant PrP fibrils or mice overexpressing PrP, both of which form large amyloid plaques in the absence of disease. The data suggest that p-tau is not necessarily associated with deposition of misfolded PrP, but that the interaction between the prion agent and host regulates the formation of p-tau and may contribute to the heterogeneous phenotype of prion diseases.


P17 Induction of transmissible tau pathology by traumatic brain Injury

Dr Elisa R Zanier1, Ilaria Bertani1, Dr Maria Antonietta Chiaravalloti1, Dr Eliana Sammali1, Dr Francesca Pischiutta1, Dr Gloria Vegliante1, Dr Fabrizio Ortolano2, Dr Nino Stocchetti2,3, Dr Maria-Grazia De Simoni1, Dr Roberto Chiesa1
1Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy, 2Università degli Studi di Milano, Milano, Italy, 3Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico di Milano, Milano, Italy

Aims: The aim of this study was to test whether traumatic brain injury (TBI), a risk factor for Alzheimer’s disease and chronic traumatic encephalopathy, induces a tau pathology that spreads in the brain in a prion-like manner, causing functional and histopathological abnormalities.

Methods: C57BL/6J mice were subjected to focal TBI by single controlled cortical impact, and the presence of pathological tau was investigated by immunohistochemistry and Western blot using antibodies specific for different phosphorylated tau isoforms. The emergence of self-propagating tau isoform was investigated by inoculating 10% brain homogenates from TBI mice and humans into the brain of naïve C57BL/6J mice.

Results: Hyperphosphorylated tau was detected in the injured brain hemisphere at 3 months post-TBI, and in the ipsi and contralateral hemispheres at 12 months, indicating progressive spreading of tauopathy. Mice inoculated with TBI brain homogenates developed memory deficits detectable by the novel object recognition task at 4, 8 and 12 months after inoculation. Immunohistochemistry at 12 months post-inoculation showed hyperphosphorylated tau in the injected area and remote brain regions.

Conclusions: Results establish that a single focal TBI induces a tau pathology in wild-type mice that progressively spreads from the site of injury to other brain regions. They also show the emergence of self-propagating tau isoforms in the brains of TBI mice and humans, which can be transmitted to wild-type mice like bona fide prions, inducing memory dysfunction. These data suggest that inhibiting propagation of tau may become a treatment strategy in TBI.

=====WOW AMAZING...TSS=====


Amyloid-β accumulation in human growth hormone related iatrogenic CJD patients in the UK

Saturday, June 17, 2017

PRION 2017 P115 α- Synuclein prions from MSA patients exhibit similar transmission properties as PrPSc prions

Thursday, July 6, 2017


TUESDAY, JUNE 20, 2017

Prion 2017 Conference Transmissible prions in the skin of Creutzfeldt-Jakob disease patients


*** First evidence of intracranial and peroral transmission of Chronic Wasting Disease (CWD) into Cynomolgus macaques

FRIDAY, JUNE 16, 2017

PRION 2017 P55 Susceptibility of human prion protein to in vitro conversion by chronic wasting disease prions

MONDAY, JUNE 19, 2017

PRION 2017 P20 Descriptive epidemiology of human prion diseases in Japan: a prospective 16-year surveillance study

Japan Prion Disease Increasing Annually to 2.3 patients per 1 million populations in 2014

TUESDAY, JUNE 13, 2017

PRION 2017 CONFERENCE ABSTRACT Chronic Wasting Disease in European moose is associated with PrPSc features different from North American CWD


Chronic Wasting Disease CWD TSE Prion to Humans, who makes that final call, when, or, has it already happened?

MONDAY, JUNE 19, 2017

PRION 2017 CONFERENCE ABSTRACT P61 vCJD strain properties in a Spanish mother and son replicate as those of a young UK case

TUESDAY, JULY 04, 2017





Thursday, June 29, 2017


Wednesday, May 24, 2017

PRION2017 CONFERENCE VIDEO UPDATE 23 – 26 May 2017 Edinburgh UPDATE 1

Subject: PRION2017 CONFERENCE VIDEO UPDATE 23 – 26 May 2017 Edinburgh

*see archives of previous Prion Conferences, the ones that are still available, scroll down towards bottom in this link.


National Prion Disease Pathology Surveillance Center Cases Examined(1) (May 18, 2017)

Terry S. Singeltary Sr.

Wednesday, June 7, 2017

Characterization of tau prion seeding activity and strains from formaldehyde-fixed tissue

Characterization of tau prion seeding activity and strains from formaldehyde-fixed tissue

  • Sarah K. Kaufman,
  • Talitha L. Thomas,
  • Kelly Del Tredici,
  • Heiko Braak and
  • Marc I. DiamondEmail author
Acta Neuropathologica CommunicationsNeuroscience of Disease20175:41
DOI: 10.1186/s40478-017-0442-8
Received: 11 May 2017
Accepted: 11 May 2017
Published: 7 June 2017


Tauopathies such as Alzheimer’s disease (AD) feature progressive intraneuronal deposition of aggregated tau protein. The cause is unknown, but in experimental systems trans-cellular propagation of tau pathology resembles prion pathogenesis. Tau aggregate inoculation into mice produces transmissible pathology, and tau forms distinct strains, i.e. conformers that faithfully replicate and create predictable patterns of pathology in vivo. The prion model predicts that tau seed formation will anticipate neurofibrillary tau pathology. To test this idea requires simultaneous assessment of seed titer and immunohistochemistry (IHC) of brain tissue, but it is unknown whether tau seed titer can be determined in formaldehyde-fixed tissue. We have previously created a cellular biosensor system that uses flow cytometry to quantify induced tau aggregation and thus determine seed titer. In unfixed tissue from PS19 tauopathy mice that express 1 N,4R tau (P301S), we have measured tau seeding activity that precedes the first observable histopathology by many months. Additionally, in fresh frozen tissue from human AD subjects at early to mid-neurofibrillary tangle stages (NFT I-IV), we have observed tau seeding activity in cortical regions predicted to lack neurofibrillary pathology. However, we could not directly compare the same regions by IHC and seeding activity in either case. We now describe a protocol to extract and measure tau seeding activity from small volumes (.04 mm3) of formaldehyde-fixed tissue immediately adjacent to that used for IHC. We validated this method with the PS19 transgenic mouse model, and easily observed seeding well before the development of phospho-tau pathology. We also accurately isolated two tau strains, DS9 and DS10, from fixed brain tissues in mice. Finally, we have observed robust seeding activity in fixed AD brain, but not controls. The successful coupling of classical IHC with seeding and strain detection should enable detailed study of banked brain tissue in AD and other tauopathies.


Propagation of tau aggregation along neuronal networks may mediate the progressive accumulation of pathology observed in tauopathy patients. To measure tau seeding activity in well-characterized human brains, it will be necessary to analyze formaldehyde-fixed tissues. We now present a method for extracting tau seeding activity from miniscule amounts of fixed tissue (approximately .04 mm3) to permit direct comparison with tissues stained by IHC.
We first tested this method in PS19 mice that overexpress full-length human tau (1 N,4R) containing the P301S mutation. We drop-fixed brain samples that had been embedded either in paraffin or PEG and sectioned them coronally for microscopy. We analyzed adjacent 50 μm sections using standard IHC to detect phospho-tau or 1 mm circular punch biopsies of tissue for seeding assays. We homogenized punch biopsies by water-bath sonication in closed tubes, and assayed them in a cellular FRET bioassay system as described previously [1013].
Tau seeding activity tracked the development of pathology more efficiently than IHC, with a lower degree of inter-animal variation, and a higher dynamic range. This was perfectly comparable to previously obtained results using fresh frozen tissue [13]. In addition, we detected seeding activity relatively early in the course of disease (1–2 months) and it steadily increased over time. Next, we tested brain tissues from animals previously inoculated with two distinct tau prion strains. We recovered these strains from fixed mouse brain tissue as accurately as we had previously from fresh frozen tissue. Finally, we tested the extraction method in fixed human brain tissue with documented AT8-positive tau pathology, including AD, and readily detected tau seeding activity in cases archived for up to 27 years in formaldehyde.

Seeding activity

Our laboratory previously detected tau seeding activity in fresh frozen brain tissue from mouse tauopathy models and human AD cases[1113]. However, fresh frozen samples are much more difficult to obtain than fixed tissue sections, must be carefully stored at−80 °C, and are very challenging to dissect precisely to isolate specific brain regions. The assay described here accurately quantifies tau seeding from fixed tissue sections over three log orders of signal. Remarkably, in a mouse model from which we sampled tissue at different time points, fixed tissue seeding proved comparable to seeding activity detected in fresh frozen tissue. Thus, we expect that this assay will enable assessment of tau seeding activity in a range of fixed tissues at a similar level of sensitivity to fresh frozen samples.
Moreover, we detected seeding activity in a small sample of human tauopathy cases that were collected and stored in formalin for over 20 years prior to this study. We observed lower seeding activity in these human samples than in PS19 mice, probably because of the overexpression of an aggregation-prone form of tau in this mouse model. However, the length of fixation may affect the level of seeding observed in samples. Further, differences in seeding activity observed between patients at Braak stage III and V likely reflect differences in the level of tau aggregate burden between these patients, cell loss, or ghost-tangle formation at later disease stages. Given the early detection of seeding activity relative to AT8 staining in PS19 mice, we anticipate that this assay could represent a more sensitive metric of tau pathology. Additional studies in a large number of well-characterized human tissue samples will help address these important questions, and provide additional insight into the progression of seeding activity in human tauopathies.
Earlier work described a dose-dependent increase in tau seeding activity in the PS19 mouse tauopathy model [13]. However, the regional specificity possible with fresh frozen tissue was limited to gross dissection. We now have reliably isolated and characterized punch biopsies as small as 1 mm diameter x 50 μm (or ~ .04 mm3). When we quantified the level of seeding activity at increasing ages vs. the tau pathology observed in adjacent tissue slices using anti-tau AT8 staining, we easily detected tau seeding activity, even in fixed tissue sections with a minimal AT8 signal. For example, when PS19 mice were inoculated with tau strains, we induced strong AT8 pathology with DS9, whereas DS10 produced a weak signal. In both cases, the pathology spread from the site of inoculation to connected regions, as described elsewhere [22]. The fixed tissue seeding assay more readily detected the spread of tau pathology in this propagation model. Furthermore, we readily detected seeding activity in DS10 inoculated mice despite the relatively subtle AT8 staining phenotype induced by this strain (mossy fiber dots). Consequently seeding activity can serve as an important measure of tau pathology when routine AT8 staining reports otherwise minimal pathology. The combination of precise quantification of seeding activity with the ability to sample brain tissue to 1 mm resolution indicates that this method could help define the seeding activity in human brain with remarkably high accuracy.

Detection of tau strains in formaldehyde-fixed tissue

Prior experimental work indicates that distinct tau aggregate conformations may underlie different patterns of pathology, rates of progression, and disease phenotypes observed in distinct tauopathies [2722]. Distinct tau strains are associated with different tauopathies [22], and inoculation of unique tau strains produces different patterns and tau pathology rates of progression [16]. We observed that fixed tissue from mice inoculated with DS9 and DS10 produced strain phenotypes identical to the original strains upon inoculation into LM1 biosensor cells. Thus, tau strains are stable upon fixation. We anticipate that formaldehyde-fixed tissues will serve as an invaluable resource to examine the role of strain composition in tauopathies.
Studies that use traditional IHC techniques to detect tau pathology have provided important insights into the progression and anatomy of macromolecular accumulations of tau assemblies. However, these methods cannot discriminate among distinct strains, nor can they detect submicroscopic tau assemblies. The present assay measures tau pathology based on seeding activity and is also sensitive to strain composition. We anticipate that punch biopsies taken from tissue sections will be useful to measure strain identity with high anatomical precision. By carefully comparing seeding activity and strain composition with standard neuropathology, it should be possible to add new dimensions to analyses of tissue samples from a range of neurodegenerative diseases. In turn, this will facilitate more widespread testing of the putative role of tau prion activity in human tauopathies.
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Terry S. Singeltary Sr.