Thursday, December 3, 2015

Transmission of Soluble and Insoluble α-Synuclein to Mice

Journal of Neuropathology & Experimental Neurology:
December 2015 - Volume 74 - Issue 12 - p 1158–1169
doi: 10.1097/NEN.0000000000000262
Original Articles
Transmission of Soluble and Insoluble α-Synuclein to Mice
Jones, Daryl Rhys PhD; Delenclos, Marion PhD; Baine, AnnMarie T.; DeTure, Michael PhD; Murray, Melissa E. PhD; Dickson, Dennis W. MD; McLean, Pamela J. PhD
 Supplemental Author Material
The neurodegenerative synucleinopathies, which include Parkinson disease, multiple-system atrophy, and Lewy body disease, are characterized by the presence of abundant neuronal inclusions called Lewy bodies and Lewy neurites. These disorders remain incurable, and a greater understanding of the pathologic processes is needed for effective treatment strategies to be developed. Recent data suggest that pathogenic misfolding of the presynaptic protein, α-synuclein (α-syn), and subsequent aggregation and accumulation are fundamental to the disease process. It is hypothesized that the misfolded isoform is able to induce misfolding of normal endogenous α-syn, much like what occurs in the prion diseases. Recent work highlighting the seeding effect of pathogenic α-syn has largely focused on the detergent-insoluble species of the protein. In this study, we performed intracerebral inoculations of the sarkosyl-insoluble or sarkosyl-soluble fractions of human Lewy body disease brain homogenate and show that both fractions induce CNS pathology in mice at 4 months after injection. Disease-associated deposits accumulated both near and distal to the site of the injection, suggesting a cell-to-cell spread via recruitment of α-syn. These results provide further insight into the prion-like mechanisms of α-syn and suggest that disease-associated α-syn is not homogeneous within a single patient but might exist in both soluble and insoluble isoforms.
© 2015 by American Association of Neuropathologists, Inc.
Prion-like propagation of human brain-derived alpha-synuclein in transgenic mice expressing human wild-type alpha-synuclein
Maria E. Bernis1, Affiliated with Julius T. Babila1, Affiliated with Sara Breid1, Affiliated with Katharina Annick Wüsten1, Affiliated with Ullrich Wüllner1, 2 and Affiliated with Gültekin Tamgüney1Email author Affiliated with Acta Neuropathologica Communications20153:75 DOI: 10.1186/s40478-015-0254-7
© Bernis et al. 2015
Received: 6 November 2015
Accepted: 6 November 2015
Published: 26 November 2015
Introduction Parkinson’s disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases that are characterized by the intracellular accumulation of alpha-synuclein containing aggregates. Recent increasing evidence suggests that Parkinson’s disease and MSA pathology spread throughout the nervous system in a spatiotemporal fashion, possibly by prion-like propagation of alpha-synuclein positive aggregates between synaptically connected areas. Concurrently, intracerebral injection of pathological alpha-synuclein into transgenic mice overexpressing human wild-type alpha-synuclein, or human alpha-synuclein with the familial A53T mutation, or into wild-type mice causes spreading of alpha-synuclein pathology in the CNS. Considering that wild-type mice naturally also express a threonine at codon 53 of alpha-synuclein, it has remained unclear whether human wild-type alpha-synuclein alone, in the absence of endogenously expressed mouse alpha-synuclein, would support a similar propagation of alpha-synuclein pathology in vivo.
Here we show that brain extracts from two patients with MSA and two patients with probable incidental Lewy body disease (iLBD) but not phosphate-buffered saline induce prion-like spreading of pathological alpha-synuclein after intrastriatal injection into mice expressing human wild-type alpha-synuclein. Mice were sacrificed at 3, 6, and 9 months post injection and analyzed neuropathologically and biochemically. Mice injected with brain extracts from patients with MSA or probable iLBD both accumulated intraneuronal inclusion bodies, which stained positive for phosphorylated alpha-synuclein and appeared predominantly within the injected brain hemisphere after 6 months. After 9 months these intraneuronal inclusion bodies had spread to the contralateral hemisphere and more rostral and caudal areas. Biochemical analysis showed that brains of mice injected with brain extracts from patients with MSA and probable iLBD contained hyperphosphorylated alpha-synuclein that also seeded aggregation of recombinant human wild-type alpha-synuclein in a Thioflavin T binding assay.
Conclusions Our results indicate that human wild-type alpha-synuclein supports the prion-like spreading of alpha-synuclein pathology in the absence of endogenously expressed mouse alpha-synuclein in vivo.
We show that human wild-type alpha-synuclein by itself, in the absence of endogenously expressed mouse alpha-synuclein, supports a prion-like mechanism in the spreading of pathological alpha-synuclein, which does not necessitate the presence of fulminant Lewy body-type pathology. In our experiments sarkosyl-soluble pathogenic alpha-synuclein species, which need to be further characterized, were readily transmitted between neurons supporting the concept that the appearance of additional detergent-insoluble alpha-synuclein species and Lewy body pathology may represent a late cellular event in mature synucleinopathies [19, 34, 41]. Consequentially, potential therapeutic strategies targeting the interneuronal spread of soluble pathogenic alpha-synuclein in synucleinopathies such as PD or MSA may be promising but effective only early in the disease process.
Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Keywords Parkinson’s disease Multiple system atrophy Incidental Lewy body disease alpha-synuclein Synucleinopathy Prion-like
Tuesday, December 1, 2015
Sorting Out Release, Uptake and Processing of Alpha-Synuclein During Prion-Like Spread of Pathology
O.01: Transgenic mice expressing human wild-type a-synuclein develop neuropathology after inoculation with brain homogenates from patients with multiple system atrophy or aged subjects without neurological disorder
Maria Eugenia Bernis1, Julius Tachu Babila1, Sara Breid1, Ullrich W€ullner1,2, and G€ultekin Tamg€uney1 1German Center for Neurodegenerative Diseases (DZNE); Bonn, Germany; 2Department of Neurology; University of Bonn; Bonn, Germany
Multiple system atrophy (MSA) and Parkinson’s disease are synucleinopathies that are defined by the presence of aggregated and hyperphosphorylated a-synuclein (a-syn) within cells of the central nervous system (CNS). Recent findings suggest that pathological a-syn may spread prion-like within the nervous system. We investigated prion-like propagation of pathological a-syn in Tg(SNCA)1Nbm/J mice that do not express mouse but low levels of human wt a-syn and do not naturally develop any pathology or neurodegenerative disease.
We inoculated brain homogenate from 2 patients with MSA, from 2 aged control subjects without neurological disorder, or saline intrastriatally into Tg(SNCA)1Nbm/J mice. Challenged mice were sacrificed at 90, 180, and 270 d post inoculation and were analyzed biochemically and immunohistochemically for pathological a-syn.
Brain homogenates from MSA or aged control subjects but not saline triggered progressive accumulation of aggregated a-syn in neurons of inoculated mice. Aggregates of a-syn were hyperphosphorylated and costained for p62 that targets proteins for degradation. Aggregates of pathological a-syn were first observable in the ipsilateral brain hemisphere and over time in the contralateral hemisphere and in more rostral and caudal areas.
Our findings show that brain homogenate from MSA patients but not saline induces pathological changes in the CNS of Tg(SNCA) 1Nbm/J mice. Our data support that pathological a-syn may propagate prion-like along neuronal networks. Furthermore, human wt a-syn supports propagation of pathological a-syn. Intriguingly, brain homogenate from aged control subjects without neurological disorder equally induced synucleinopathy in brains of Tg(SNCA)1Nbm/J mice suggesting that aged human brains can contain pathological a-syn.
O.02: Experimental transmissibility of mutant SOD1 motor neuron disease
Jacob Ayers, Susan Fromholt, Morgan Koch, Adam DeBosier, Ben McMahon, Guilian Xu, and David Borchelt University of Florida; Gainesville, FL USA
By unknown mechanisms, the symptoms of amyotrophic lateral sclerosis (ALS) seem to spread along anatomical pathways to engulf the motor nervous system. The rate at which symptoms spread is one of the primary drivers of disease progression. One mechanism by which ALS symptoms could spread is by a prion-like propagation of a toxic misfolded protein from cell to cell along anatomic pathways. Proteins that can transmit toxic conformations between
S1 Prion, 9:S1–S10, 2015 ISSN: 1933-6896 print / 1933-690X online
cells often can also experimentally transmit disease between individual organisms. To survey the ease with which motor neuron disease (MND) can be transmitted, we injected spinal cord homogenates prepared from paralyzed mice expressing mutant superoxide dismutase 1 (SOD1-G93A and G37R) into the spinal cords of genetically vulnerable SOD1 transgenic mice. From the various models we tested, one emerged as showing high vulnerability. Tissue homogenates from paralyzed G93A expressing mice induced MND in 6 of 10 mice expressing low levels of G85R-SOD1 fused to yellow fluorescent protein (G85R-YFP mice) by 3– 11 months, and produced widespread spinal inclusion pathology. Importantly, second passage of homogenates from G93A!G85R-YFP mice back into newborn G85R-YFP mice, induced disease in 4 of 4 mice by 3 months of age. Homogenates from paralyzed mice expressing the G37R variant were among those that transmitted poorly regardless of the strain of recipient transgenic animal injected, a finding suggestive of strain-like properties that manifest as differing abilities to transmit MND. Together, our data provide a working model for MND transmission to study the pathogenesis of ALS.
Tuesday, September 1, 2015
Evidence for α-synuclein prions causing multiple system atrophy in humans with parkinsonism
Wednesday, September 2, 2015
Clinically Unsuspected Prion Disease Among Patients With Dementia Diagnoses in an Alzheimer’s Disease Database
Tuesday, June 30, 2015
PRION2015 Alzheimer’s disease
Tuesday, November 26, 2013
Transmission of multiple system atrophy prions to transgenic mice
Sunday, February 10, 2013
Parkinson's Disease and Alpha Synuclein: Is Parkinson's Disease a Prion-Like Disorder?
Wednesday, September 21, 2011
PrioNet Canada researchers in Vancouver confirm prion-like properties in Amyotrophic Lateral Sclerosis (ALS)
Wednesday, January 5, 2011
David W. Colby1,* and Stanley B. Prusiner1,2
Friday, September 3, 2010
Alzheimer's, Autism, Amyotrophic Lateral Sclerosis, Parkinson's, Prionoids, Prionpathy, Prionopathy, TSE
... a TSE is found that is linked to Alzheimer’s disease.
Self-Propagative Replication of Ab Oligomers Suggests Potential Transmissibility in Alzheimer Disease
Received July 24, 2014; Accepted September 16, 2014; Published November 3, 2014
iatrogenic, what if ???
BSE101/1 0136
From: . Dr J S Metiers DCMO
4 November 1992
1. Thank you for showing me Diana Dunstan's letter. I am glad that MRC have recognised the public sensitivity of these findings and intend to report them in their proper context. 'This hopefully will avoid misunderstanding and possible distortion by the media to portray the results as having more greater significance than the findings so far justify.
2. Using a highly unusual route of transmission (intra-cerebral injection) the researchers have demonstrated the transmission of a pathological process from two cases one of severe Alzheimer's disease the other of Gerstmann-Straussler disease to marmosets. However they have not demonstrated the transmission of either clinical condition as the "animals were behaving normally when killed". As the report emphasises the unanswered question is whether the disease condition would have revealed itself if the marmosets had lived longer. They are planning further research to see if the conditions, as opposed to the partial pathological process, is transmissible.
what are the implications for public health?
3. The route 'of transmission is very specific and in the natural state of things highly unusual. However it could be argued that the results reveal a potential risk, in that brain tissue from these two patients has been shown to transmit a pathological process. Should therefore brain tissue from such cases be regarded as potentially infective? Pathologists, morticians, neuro surgeons and those assisting at neuro surgical procedures and others coming into contact with "raw" human brain tissue could in theory be at risk. However, on a priori grounds given the highly specific route of transmission in these experiments that risk must be negligible if the usual precautions for handling brain tissue are observed.
BSE101/1 0137
4. The other dimension to consider is the public reaction. To some extent the GSS case demonstrates little more than the transmission of BSE to a pig by intra-cerebral injection. If other prion diseases can be transmitted in this way it is little surprise that some pathological findings observed in GSS were also transmissible to a marmoset. But the transmission of features of Alzheimer's pathology is a different matter, given the much greater frequency of this disease and raises the unanswered question whether some cases are the result of a transmissible prion. The only tenable public line will be that "more research is required’’ before that hypothesis could be evaluated. The possibility on a transmissible prion remains open. In the meantime MRC needs carefully to consider the range and sequence of studies needed to follow through from the preliminary observations in these two cases. Not a particularly comfortable message, but until we know more about the causation of Alzheimer's disease the total reassurance is not practical.
J S METTERS Room 509 Richmond House Pager No: 081-884 3344 Callsign: DOH 832 llllYc!eS 2 92/11.4/1.2
>>> The only tenable public line will be that "more research is required’’ <<<
>>> possibility on a transmissible prion remains open<<<
O.K., so it’s about 23 years later, so somebody please tell me, when is "more research is required’’ enough time for evaluation ?
Self-Propagative Replication of Ab Oligomers Suggests Potential Transmissibility in Alzheimer Disease
Received July 24, 2014; Accepted September 16, 2014; Published November 3, 2014
*** Singeltary comment PLoS ***
Alzheimer’s disease and Transmissible Spongiform Encephalopathy prion disease, Iatrogenic, what if ?
Posted by Terry S. Singeltary Sr. on 05 Nov 2014 at 21:27 GMT
Thursday, October 1, 2015
Alzheimergate, re-Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy, Singeltary Submission to Nature
Wednesday, September 2, 2015
Clinically Unsuspected Prion Disease Among Patients With Dementia Diagnoses in an Alzheimer’s Disease Database
March 1989
INACTIVATION OF SCRAPIE-LIKE AGENTS Some implications for the use of bovine material in sterile medical devices in the UK
Sunday, November 22, 2015
*** Effect of heating on the stability of amyloid A (AA) fibrils and the intra- and cross-species transmission of AA amyloidosis ***
Transmission of Creutzfeldt-Jakob disease to a chimpanzee by electrodes contaminated during neurosurgery.
Gibbs CJ Jr, Asher DM, Kobrine A, Amyx HL, Sulima MP, Gajdusek DC. Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
Stereotactic multicontact electrodes used to probe the cerebral cortex of a middle aged woman with progressive dementia were previously implicated in the accidental transmission of Creutzfeldt-Jakob disease (CJD) to two younger patients. The diagnoses of CJD have been confirmed for all three cases. More than two years after their last use in humans, after three cleanings and repeated sterilisation in ethanol and formaldehyde vapour, the electrodes were implanted in the cortex of a chimpanzee. Eighteen months later the animal became ill with CJD. This finding serves to re-emphasise the potential danger posed by reuse of instruments contaminated with the agents of spongiform encephalopathies, even after scrupulous attempts to clean them.
Tuesday, May 26, 2015
*** Minimise transmission risk of CJD and vCJD in healthcare settings ***
Last updated 15 May 2015
Terry S. Singeltary Sr.