14 Dec 2018

Researchers have strengthened the case that certain medical procedures can transfer amyloid pathology between people. John Collinge and Sebastian Brandner at University College London previously reported finding amyloid plaques and cerebral amyloid angiopathy (CAA) in the brains of seven young to middle-aged adults who had died from prion disease. As children, they had been treated with human growth hormone extracted from the pituitary glands of human cadavers; the data then implied that the extracts contained Aβ seeds as well as prions. In the December 13 Nature, researchers led by Collinge now provide direct evidence for this. The authors report that archived vials of the same growth hormone extract given to patients accelerated amyloidosis in transgenic mice. “I was amazed we could seed so easily from this material, which had sat as a dry powder at room temperature for 30 or 40 years. It shows the resistance of these seeds to degradation,” Collinge said in a Nature press briefing.

Some patients treated with pituitary extracts developed prion disease and amyloidosis.
The same extracts accelerated amyloid deposition in transgenic mice.
This confirms the presence of long-lasting Aβ seeds in these extracts.

Other scientists said the finding supports the transmission hypothesis proposed in the 2015 paper. “These two articles combined provide a very strong case for transmission of Alzheimer’s pathology in a prion-like infectious manner in human beings,” Claudio Soto at the University of Texas Medical School, Houston, wrote to Alzforum (full comment below). In an accompanying editorial, Tien-Phat Huynh and David Holtzman at Washington University, St. Louis, wrote, “These results provide strong evidence that the Aβ pathology previously reported in people who died of CJD after receiving cadaveric human growth hormone was indeed caused by their treatment.”

Not everyone agreed, however. “It is a long way from the data presented in this paper to the idea that Aβ pathology can be transmitted between people by medical procedures,” John Trojanowski at the University of Pennsylvania, Philadelphia, wrote to Alzforum. He noted that the APP mutations in these transgenic mice are rare in humans, and that the same kind of seeding might not happen in most people. "We studied the U.S. cadaver growth hormone cohort and found no PD or AD in more than 6,000 subjects," Trojanowski said (Irwin et al., 2013). More recently, he recently argued that multiple system atrophy should not be considered a prion disease, and said the same arguments apply to AD (Wenning et al., 2018).

The practice of obtaining growth hormone from cadavers ended in 1985, after some recipients developed the fatal prion disorder Creutzfeldt-Jakob disease (CJD). In the U.K., clinicians treated a total of 1,883 people with the extract. So far, 80 of them have contracted CJD. Notably, all of these patients received at least one treatment with growth hormone purified by the Hartree-modified Wilhelmi procedure (HWP) (Swerdlow et al., 2003). Unlike other methods, HWP does not include a size-exclusion chromatography step, which may help filter out protein aggregates. When Collinge and colleagues analyzed postmortem brain samples from eight of the patients treated with HWP preparations, they found extensive amyloidosis and CAA in four of them, while three more had occasional plaques (Sep 2015 news).

To confirm the presence of Aβ seeds, Collinge obtained archived vials from three HWP batches used to treat the eight patients, as well as from two additional HWP batches, and compared them to 11 non-HWP batches that had also been administered to the same group. Co-author Dominic Walsh at Brigham and Women’s Hospital, Boston, tested each vial for the presence of Aβ40, Aβ42, and tau using sensitive ELISAs and electrochemiluminescent assays. All HWP preparations were positive for these proteins, whereas all non-HWP preparations were negative.

Were these peptides able to seed amyloidosis, however? First author Silvia Purro injected samples from two of the HWP vials into the right parietal lobe of six-week-old APP NL-F knock-in mice. These animals carry humanized APP bearing the Swedish and Iberian mutations, and begin depositing amyloid in the hippocampus and cerebral cortex at six months. However, APP NL-F mice treated with either of the HWP samples developed more extensive amyloidosis. Eight months after injection, they sported plaques in the cerebellum, where control transgenics injected with vehicle had none. They also displayed extensive vascular amyloid, whereas control transgenics had almost no CAA at that age.

Transgenic mice that received the HWP preparation deposited almost as much amyloid as those injected with homogenates from AD brain. On the other hand, mice that received recombinant human growth hormone, or extracts from healthy human postmortem brains, looked like controls. Wild-type mice injected with the HWP batches did not develop plaques; mice expressing human wild-type APP were not tested.

These findings are not isolated to U.K. samples. Charles Duyckaerts and Stéphane Haïk at Hôpital Pitié-Salpêtrière, Paris, noted that they have found Aβ and tau contaminants in French cadaver-derived human growth hormone preparations (Duyckaerts et al., 2018). They suggested comparing the amount of Aβ in preparations from France and the U.K. and correlating this with the average incubation period before amyloidosis appeared in patients. “This will help determine the effect of the inoculum dose on the transmission of amyloid pathology,” they wrote to Alzforum (full comment below).

Researchers puzzled over why the tau that is also present in these preparations did not seed tangles in human patients. “It’s possible the tau is not in a seed-competent form,” suggested Lary Walker at Emory University, Atlanta. To test this, Collinge plans to repeat these experiments in mouse models of tauopathy. Researchers speculated that the CJD patients might have developed tau tangles and full-blown Alzheimer’s disease if they had lived longer. They also noted that CAA by itself can be fatal, causing cerebral hemorrhages.

Other studies have found that Aβ aggregates can be transmitted by other medical procedures, such as dural grafts and perhaps even neurosurgery (Jan 2016 news; Feb 2018 news; Hervé et al., 2018). How concerned should physicians and indeed brain surgery patients be? Walker noted that the medical community improved sterilization procedures for neurosurgical tools in the 1970s, when the risks of prion contamination became widely known, and no new cases of prion disease have been reported as a result of surgeries performed since then. “The procedures appear to be effective,” he said.

Even so, Collinge believes the issue deserves more study. Ronald Melki at the French National Center for Scientific Research in Gif–sur–Yvette and Luc Bousset at the Université Paris-Saclay recently analyzed how well various cleansers removed aggregated protein from different surfaces. They found that the detergent sodium dodecyl sulfate (SDS) worked well to dislodge and break up aggregates of Aβ42, tau, and α-synuclein from glass, plastic, and steel. Other solvents, such as sodium hydroxide, the alkaline cleaning solution Hellmanex, and the detergent TFD4, performed poorly. Aggregates clung most stubbornly to aluminum surfaces, resisting cleaning with all reagents (Fenyi et al., 2018).

Mathias Jucker at the German Center for Neurodegenerative Disease in Tubingen, suggested that surgical instruments should be designated for use on either young or old patients to prevent cross-contamination. Because of the long incubation period for protein aggregation diseases, young people would be at the greatest risk from surgical tools (full comment below).

Are researchers who work with protein aggregates in danger? Walker thought this unlikely. “There’s no evidence whatsoever for transmission of Aβ through routine laboratory handling,” he told Alzforum. Nonetheless, Melki said his lab routinely uses biosafety level 2 or 3 procedures as a precaution when working with aggregated proteins. This means handling materials in a ventilated safety cabinet, wearing protective clothing, and taking extra care with contaminated sharps.—Madolyn Bowman Rogers

Comments

- Mathias Jucker
  Hertie Institute for Clinical Brain Research, University of Tübingen, and DZNE Tübingen
- Posted: 14 Dec 2018
This is an important control and confirmation of the previous human transmission observations by this group. I am not surprised that Aβ in the hormone extracts seeded cerebral amyloidosis in these mice, since it is exactly what transmission studies in mouse models had predicted (Meyer-Lühmann et al., 2006; Eisele et al., 2010). APP transgenic mouse models can be rightly criticized for many things, but their amyloid deposition aspect has nearly always translated faithfully to human observation.

From my lab’s perspective, having done many transmission studies in mouse models, the present results do not change anything; however, for others who did not believe that such transmission to humans is real, it may be yet another wakeup call.

What are the real-word implications of this? It may be a good idea to use separate sets of medical surgical instruments for young and for aged patients. Given the long incubation time, this may minimize the risk of transmission. I was struck that Purro et al. report Aβ concentrations in the hormone extracts almost as high as in AD brain extract. This is somewhat unexpected. It makes me curious about the nature of the Aβ species in the hormone extracts, since the seeding of the hormone extract was much less than that of the AD tissue.

I believe Aβ assemblies in AD brain behave like prions. Lary Walker and I summarized the state of current knowledge in a very recent review (Jucker and Walker, 2018).

References:

Meyer-Luehmann M, Coomaraswamy J, Bolmont T, Kaeser S, Schaefer C, Kilger E, Neuenschwander A, Abramowski D, Frey P, Jaton AL, Vigouret JM, Paganetti P, Walsh DM, Mathews PM, Ghiso J, Staufenbiel M, Walker LC, Jucker M. Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. Science. 2006 Sep 22;313(5794):1781-4. PubMed.

Eisele YS, Obermüller U, Heilbronner G, Baumann F, Kaeser SA, Wolburg H, Walker LC, Staufenbiel M, Heikenwalder M, Jucker M. Peripherally applied Abeta-containing inoculates induce cerebral beta-amyloidosis. Science. 2010 Nov 12;330(6006):980-2. PubMed.

Jucker M, Walker LC. Propagation and spread of pathogenic protein assemblies in neurodegenerative diseases. Nat Neurosci. 2018 Oct;21(10):1341-1349. Epub 2018 Sep 26 PubMed.

View all comments by Mathias Jucker

- Neil Cashman
  UBC
- Posted: 14 Dec 2018
Purro et al. have published an important study. There is much scientific value in this work, but I will focus on the nature of the “proteopathic seeds” that template Aβ amyloidosis, and why this question is critical to human health.

Three sobering studies from Mathias Jucker and Lary Walker directly address the magnitude of the problem. First, initial work revealed that AD brain homogenate inoculated into mutant APP mice can induce a spreading Aβ amyloidosis; this seeding activity is sensitive to Aβ immune-depletion and formic acid treatment (Meyer-Luehmann et al., 2006). Thus, the seeds must be non-covalent aggregates of Aβ, but likely not the free Aβ peptides detected in the HWP vials by Purro et al.

Second, the most efficient Aβ-seeding species activity is small and soluble—i.e., oligomeric (Langer et al., 2011). Finally, stainless steel wires provide an excellent fomite in transmission of Aβ amyloidosis to mice (Eisele et al., 2009), in a manner that is sensitive to plasma sterilization, but not boiling.

Given the frequency of preclinical AD in aging, the suggestion that the most difficult species of aggregated Aβ to detect and sterilize is also the most “seeding” in animal models poses major challenges—to develop assays and techniques to obviate the unwitting transmission of Aβ proteopathic seeds in iatrogenic settings, particularly via neurosurgical instruments.

References:

Meyer-Luehmann M, Coomaraswamy J, Bolmont T, Kaeser S, Schaefer C, Kilger E, Neuenschwander A, Abramowski D, Frey P, Jaton AL, Vigouret JM, Paganetti P, Walsh DM, Mathews PM, Ghiso J, Staufenbiel M, Walker LC, Jucker M. Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. Science. 2006 Sep 22;313(5794):1781-4. PubMed.

Langer F, Eisele YS, Fritschi SK, Staufenbiel M, Walker LC, Jucker M. Soluble Aβ seeds are potent inducers of cerebral β-amyloid deposition. J Neurosci. 2011 Oct 12;31(41):14488-95. PubMed.

Eisele YS, Bolmont T, Heikenwalder M, Langer F, Jacobson LH, Yan ZX, Roth K, Aguzzi A, Staufenbiel M, Walker LC, Jucker M. Induction of cerebral beta-amyloidosis: intracerebral versus systemic Abeta inoculation. Proc Natl Acad Sci U S A. 2009 Aug 4;106(31):12926-31. PubMed.

View all comments by Neil Cashman

- Charles Duyckaerts
  University of Paris VI
- Posted: 14 Dec 2018
This paper proves that the hGH preparation contained Aβ contaminants that have seeding properties, and confirms the concept of iatrogenic CAA. We had also found Aβ and tau contaminants in the French hGH preparations (Duyckaerts et al., 2018). It will be interesting to compare the amounts of Aβ that have been injected in the French and British cases, and to evaluate the effect on the incubation period. This will help determine the effect of the inoculum dose on the transmission of amyloid pathology.

Quite intriguing is the absence of Alzheimer-type tau pathology in the injected cases. The authors suggest that it may be a question of time. We have published a case of symptomatic iatrogenic CAA (Hervé et al., 2018) in which the incubation period was longer than four decades; no tau pathology was detected. It is, for us, very unlikely that incubation time was the key determinant of the absence of tau seeding. In our view, it also suggests that Aβ accumulation does not cause initiation of tau pathology in this context.

I think that evidence is now sufficient to be quite sure that Aβ pathology has been transmitted. Precautions that have been taken to prevent the prion risk in neurosurgery are probably sufficient to prevent the risk of transmission of Aβ pathology, but new studies are required to test their effectiveness specifically on Aβ aggregates.

References:

Duyckaerts C, Sazdovitch V, Ando K, Seilhean D, Privat N, Yilmaz Z, Peckeu L, Amar E, Comoy E, Maceski A, Lehmann S, Brion JP, Brandel JP, Haïk S. Neuropathology of iatrogenic Creutzfeldt-Jakob disease and immunoassay of French cadaver-sourced growth hormone batches suggest possible transmission of tauopathy and long incubation periods for the transmission of Abeta pathology. Acta Neuropathol. 2018 Feb;135(2):201-212. Epub 2017 Nov 22 PubMed.

Hervé D, Porché M, Cabrejo L, Guidoux C, Tournier-Lasserve E, Nicolas G, Adle-Biassette H, Plu I, Chabriat H, Duyckaerts C. Fatal Aβ cerebral amyloid angiopathy 4 decades after a dural graft at the age of 2 years. Acta Neuropathol. 2018 May;135(5):801-803. Epub 2018 Mar 5 PubMed.

View all comments by Charles Duyckaerts

- Claudio Soto
  University of Texas Medical School at Houston
- Posted: 14 Dec 2018
I find this article quite interesting. In this follow-up study, the authors examined aliquots of human-derived growth hormone used decades ago and found they indeed contained Aβ. More importantly, they showed that when these materials were injected into mouse models of Alzheimer’s disease, the animals developed accelerated amyloid deposition in the brain parenchyma and around cerebral vessel walls. This data indicate that the vials used for human treatment indeed contained Aβ material that was competent to seed the accumulation of pathological amyloid. Therefore, this study, added to the previous one, strongly suggests that the Aβ deposition seen in patients was induced in a manner reminiscent to prions.

I believe these two articles combined provide a very strong case for transmission of Alzheimer’s pathology in a prion-like infectious manner in human beings. However, the question still remains whether transmission of Alzheimer’s pathology is restricted to these rare conditions (use of human-derived products for treatment) or can occur under more common medical practices, such as for example blood transfusion.

As a precautionary note, I believe we should think seriously of implementing tools to screen medical equipment used in brain surgery or that can in some way act as vectors to transmit amyloid.

View all comments by Claudio Soto

- David Harris
  Boston University School of Medicine
- Posted: 18 Dec 2018
This is an important study, which provides further support for the hypothesis that Aβ pathology can be transmitted by iatrogenic means. Several correlative studies, including a previous one from this group, showed that patients who had received cadaverically derived human growth hormone (c-hGH) or dura mater grafts, some of whom subsequently developed iatrogenic Creutzfeldt-Jakob disease, displayed parenchymal or vascular Aβ deposition. However, it was not possible to conclude with certainty that Aβ seeds in the c-hGH preparations or grafts were actually the cause of the Aβ pathology. In this paper, Purro and colleagues provide one further link in the chain of proof by showing that several archived, >30-year-old c-hGH preparations used to treat patients do, in fact, contain Aβ peptides based on biochemical assays, as well as seeding activity based on transmission to transgenic mice expressing a mutant APP gene.

As the authors point out, their results do not suggest that Alzheimer’s disease is contagious, or is transmissible by conventional means like blood transfusion, a fear that has sometimes been stoked by the popular press. Importantly, it should be kept in mind that the appearance of amyloid pathology in the brains of patients exposed to c-hGH or dura mater grafts does not equate with clinically apparent Alzheimer’s disease. However, this paper, in conjunction with other published studies, argues that care should be exercised to prevent the possible transmission of Aβ or tau pathology as a result of contamination of surgical instruments.

Prion-like propagation has been offered as a mechanism for the spread of misfolded proteins within the CNS in a number of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and tauopathies. It is important to note that this concept remains a hypothesis that rests largely on cell culture experiments, as well as artificial seeding experiments in animals, but lacks proof in natural disease settings. The paper by Purro et al. does not directly address this question, since it deals with an iatrogenic form of Aβ pathology, rather than a naturally occurring form that would develop during the course of Alzheimer’s disease.

View all comments by David Harris

- Ronald Melki
  CNRS
- Posted: 19 Dec 2018
I am not surprised by these findings. A recent paper reported propagation of Aβ aggregates after dura mater grafting (Hervé et al., 2018).

I have no doubt that growth hormone from diseased people, purified as it used to be, contributed to the transmission of diseases other than CJD. Luckily, it looks as if few batches were contaminated. While a number of people were certainly exposed to pathogenic protein assemblies, very few developed disease. We should thank the capacity of our bodies to clear such pathogenic protein aggregates.

I think tools used in neurosurgery may transmit disease if they are not cleaned thoroughly. Some colleagues may argue that there is no clear evidence for that. The evidence is difficult to bring. We have every year over 150,000 cases of PD and over 300,000 case of AD in France. Let’s imagine that we have 10 or 100 or even 1,000 surgical procedures that led to contamination of patients—how would we detect those in the midst of hundreds of thousands of new cases? The situation was different for CJD, with an average number of 25 to 30 cases per year in France. When all of sudden within two years, the Ministry of Health noticed a jump to 70 cases, we started worrying and obtained an answer. For AD and PD we simply cannot detect changes unless we all of a sudden had a massive increase in case number. Fortunately this will not happen.

I am convinced that pathogenic protein assemblies found in humans and animal models are pathogenic, as are the Aβ, tau, and α-syn assemblies we generate de novo. I therefore assessed the efficiency of cleaning methods to lower the risk of exposure of my co-workers to those assemblies we manipulate on a daily basis. Our studies show that detergent such as SDS are good at cleaning and capable of disaggregating the aggregates. Our paper contains a roster for selecting the best cleaning procedure depending on the protein aggregate, the contaminated material nature etc. … I further implemented working conditions in my lab that minimize risk.

References:

Hervé D, Porché M, Cabrejo L, Guidoux C, Tournier-Lasserve E, Nicolas G, Adle-Biassette H, Plu I, Chabriat H, Duyckaerts C. Fatal Aβ cerebral amyloid angiopathy 4 decades after a dural graft at the age of 2 years. Acta Neuropathol. 2018 May;135(5):801-803. Epub 2018 Mar 5 PubMed.

View all comments by Ronald Melki