Consensus is building among researchers that misfolded Aβ peptides can transmit their bad behavior when transferred from human to mouse or animal to animal—but could this transmission happen between people? According to a small study published in Nature on September 9, the possibility is real. Drawing on autopsy tissue, researchers led by John Collinge and Sebastian Brandner at University College London reported the presence of abundant Aβ plaques in the brain’s parenchyma and blood vessels in a handful of people who received injections of human growth hormone extracted from cadavers (c-HGH) decades ago. The patients had all died between the ages of 36 and 51 of Creutzfeldt-Jakob Disease (CJD), which they contracted from the injections. None had developed symptoms of Alzheimer’s disease or cerebral amyloid angiopathy (CAA). Moreover, their brains lacked the telltale tau pathology that defines AD. Even so, the presence of Aβ pathology in such a young group of people set off alarm bells. Other researchers called for further investigation into the possibility that, like prions, perhaps Aβ seeds can indeed spread from one person to another under certain conditions.
“This is a pathogenic principle we discovered in animals, and this study suggests it could happen in humans,” said Mathias Jucker of the German Center for Neurodegenerative Diseases and Hertie Institute for Clinical Brain Research in Tübingen, who was not involved in the study. “The conclusion of transmission is not yet proven, but surely this finding warrants heightened concern and further study,” he said. Jucker published a paper on the same day in Nature Neuroscience. It revealed that so-called Aβ seeds remained able to induce amyloidosis even after lying dormant for six months in a mouse brain. These findings add to mounting evidence that such seeds are robust. They also allude to the extraordinarily long incubation period of Alzheimer’s disease. Studies indicate that amyloid plaque and tau tangle pathology start to manifest 25 or more years before the emergence of cognitive symptoms.
Stealth Seeds. Besides prions, an unknown fraction of human growth hormone extracts isolated from pituitary glands may also have contained Aβ seeds. Recipients of these treatments later developed both CJD and Aβ deposition. [Courtesy of Mathias Jucker and Lary Walker, News & Views, Nature 2015.]
As misfolded versions of normal proteins, prions corrupt properly folded proteins and propagate pathology. The prion protein PrP is the infectious agent in prion diseases in animals and humans, in whom it can cause CJD. Some people contracted a variant of this fatal neurodegenerative disease by eating contaminated meat, and fears of an impending epidemic rocked the United Kingdom and ravaged its beef industry at the turn of this century. CJD can also arise sporadically or due to mutations that render PrP prone to misfolding.
Transplants from donors with CJD or exposure to contaminated surgical equipment represent another route. Called iatrogenic CJD (iCJD), this version of the disease afflicted 226 people who had received intramuscular injections of c-HGH derived from pituitary glands of cadavers. Most of these patients had received the injections as children to help them grow taller. Scores of pituitary glands excised from the brains of organ donors were pooled and served as the source for the hormone, which was partially purified with the biochemical methods of the day. In the United Kingdom alone, an estimated 400,000 pituitary glands were used for this purpose. An estimated 30,000 patients received repeated injections of this preparation from 1959 until 1985, when cases of CJD surfaced and manufacturers switched to recombinant production (see Will, 2003; and Brown et al., 2012).
Athletes and body builders have long used HGH to boost performance. Despite the International Olympic Committee’s ban on doping with HGH, some athletes continued to purchase the cadaveric form of the hormone on the black market long after the more expensive, recombinant version had supplanted it for medical purposes (see Deyssig and Frisch, 1993; Sonksen, 2001; Holt and Sonksen, 2008). Pituitary glands illegally taken from cadavers may have provided a source for the hormone, as one Russian media report of a pituitary-gland-smuggling operation documented in 2000 (see Moscow Times, 2000).
Other neurodegenerative disease-associated proteins, including Aβ, tau, and α-synuclein, have also been shown to propagate and cause pathology in a mode that some researchers call “prion-like.” For example, Aβ seeds extracted from human AD brain or transgenic AD mouse brains and transferred to mice expressing human APP induced AD pathology in those recipient mice (see Jul 2009 news and Oct 2011 news). Given the similarities with prions, researchers have wondered whether Aβ or other neurodegenerative-disease proteins could also spread via c-HGH injections or other procedures. In 2013, researchers led by John Trojanowski at the University of Pennsylvania in Philadelphia concluded that such transmission was unlikely. Sifting through death certificates from nearly 800 people who had died since receiving the c-HGH injections from the U.S. National Hormone and Pituitary Program (NHPP) about 30 years earlier, they found no evidence of increased rates of death due to AD or PD in this cohort (see Feb 2013 news).
For the current study, first author Zane Jaunmuktane and colleagues examined whether c-HGH recipients who later contracted CJD also harbored AD pathology. By 2012, 65 out of 1,800 people in the United Kingdom who had been previously treated with c-HGH had developed CJD. In the United Kingdom, a majority of patients with prion disease have been recruited into the National Prion Monitoring Cohort (NPMC) study, including 22 c-HGH recipients who developed iCJD in recent years. The researchers conducted in-depth autopsies on eight of these patients, who had developed iCJD an average of 25 years after c-HGH treatment.
In addition to the expected CJD pathology, Aβ deposits riddled the brain parenchyma of four of the patients. An additional two patients had smaller, focal Aβ deposits, and one had a small amount; only one of the eight patients’ brains was devoid of Aβ pathology. Three of the four patients with substantial amyloidosis also had cerebral amyloid angiopathy (CAA), a condition marked by damage to brain vessels that can trigger bleeding or strokes. Genetic testing indicated that none of the eight people harbored mutations in any of 16 genes associated with early onset AD, CAA, or other neurodegenerative diseases, and none carried the ApoE4 allele.
“These are highly unusual findings, as you wouldn’t expect this level of Aβ deposition at this age,” Collinge told reporters at a press briefing. For example, one neuropathology study of people without CJD reported that just 10 out of 290 people aged 36 to 51 had comparable Aβ pathology (see Braak and Braak, 1997). Still, the small sample size precludes definitive statements about whether the frequency of Aβ pathology in these eight patients was truly abnormal, commented Trojanowski.
To determine whether pituitary glands could have been the source of Aβ contamination, the researchers examined autopsies from 49 people with cerebral Aβ pathology, and found AD pathology in the pituitary glands of seven. This indicated that pituitary glands were a potential source of Aβ seeds. Both autopsy and amyloid PET studies have shown that the prevalence of brain AD pathology rises with age (e.g., Price et al., 2009; Villemagne et al, 2013). Many pituitary donors were elderly, suggesting that, unbeknownst to investigators at the time, the prevalence of amyloid pathology among them may have been significant.
One alternative potential explanation for the Aβ pathology in the present study is that CJD pathology somehow could have triggered amyloid deposition. A connection between CJD and Aβ has been reported in some studies but not others (see Tousseyn et al., 2015; Hainfellner et al., 1998). To investigate this, the researchers looked to autopsy data from more than 100 people in the NPMC who suffered from forms of CJD not transmitted though c-HGH, such as sporadic or variant CJD and inherited prion diseases. They found no significant Aβ pathology in the 19 patients who had been between 36 and 51 at death. Two of the 35 patients between the ages of 52 and 60 harbored significant Aβ pathology, but they carried the ApoE4 allele, which is known to bring on Aβ at an earlier age. This suggested to the authors that CJD, at least in its other forms, does not hasten Aβ deposition.
Furthermore, the researchers found no spatial overlap between CJD pathology and Aβ pathology in the brains of their iCJD cohort. While these findings do not prove that the patients acquired Aβ pathology directly from the c-HGH stocks rather than as a consequence of CJD, they support that conclusion, Collinge said.
Marc Diamond of the University of Texas Southwestern Medical Center in Dallas agreed that transmission of Aβ from contaminated c-HGH extracts was the likeliest explanation for the results. “The implication, of course, is that many other proteins besides PrP could potentially be infectious,” Diamond wrote. Nevertheless, he downplayed potential risks. “I still highly doubt that in standard medical practice it would be possible to transmit pathology efficiently between individuals. Since we are no longer doing these pooled tissue treatments, which in retrospect seem particularly foolhardy, I would not imagine that we will be seeing a high number of “infected” cases of AD, CAA, or PD,” Diamond wrote to Alzforum.
Importantly, tau pathology was absent from all eight of the iCJD patients autopsied by Collinge and colleagues. The researchers speculated that perhaps the patients would have developed tau pathology in later years, had they not succumbed to CJD in midlife. CSF biomarker studies suggest that Aβ changes precede tau, and human mutant APP transgenic mice develop CSF tau changes subsequent to CSF Aβ changes.
In a joint comment to Alzforum, Dominic Walsh and Dennis Selkoe of Brigham and Women’s Hospital in Boston cautioned against equating the presence of Aβ deposition with future AD. “This paper does not report the transmission of AD or AD pathology, rather it documents the detection of Aβ deposits in individuals at an age when normally such deposits are extremely rare,” they wrote. “The fact that the brains of these individuals did not contain neurofibrillary tangles indicates that these individuals did not have AD, and even if they had lived longer, it is uncertain that they would have developed AD.”
While the U.K. researchers cannot know whether the patients would have developed AD, they concluded that the severity of CAA in these patients alone was cause for concern, as they would have been at elevated risk for cerebral hemorrhages had they lived longer. Walsh and Selkoe agreed that potential fallout from CAA was more troubling than Aβ deposits.
What of the thousands of c-HGH recipients who did not develop iCJD? According to Collinge, approximately 1,500 people who received the injections in the United Kingdom are still alive. They were not formally contacted by the respective authority, the U.K. Department of Health, about the results of this study prior to its publication. “Many of these individuals unfortunately are going to find out about this from the media,” Collinge said in the press briefing. He added he would like to monitor these people for the emergence of AD pathology via PET scans or CSF biomarkers. Collinge invited concerned patients to contact his center; however, no plan for a formal study is yet in place. Approximately 1,800 patients in France and 7,700 patients in the United States also received c-HGH injections.
The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) retains records of recipients in the United States, but officials at NIDDK were unavailable for comment. Information for patients regarding risks associated with c-HGH injections can be found on the agency’s website.
Collinge told reporters that his lab is seeking permission to access archived batches of the c-HGH extracts that were injected into patients. He would like to test these batches for the presence of infectious Aβ seeds, however, he noted that the properties of such seeds are poorly understood and no sufficiently sensitive biochemical assay exists to date. The tried-and-true way to test an Aβ-containing mixture for infectivity is to inject animals and wait, sometimes as long as a year, for Aβ pathology to emerge.
Researchers are busy developing in vitro assays to obtain quicker results. Claudio Soto of the University of Texas in Houston has developed research-grade assays to detect Aβ oligomers in the CSF of AD patients, as have several other labs (see Mar 2014 news), but results are not broadly reproduced and it is not clear how those species relate to the Aβ seeds that propagate amyloid pathology. Similar to assays scientists developed to detect prions, Soto’s so-called protein-misfolding cyclic amplification (PMCA) assay works by allowing misfolded proteins to aggregate, then breaking them up into particles that seed further aggregates until detection becomes possible. Soto told Alzforum that his lab is working on expanding the assay to other sample types, including blood, and that he would test old c-HGH lots for the seeds if given access. Collinge said the development of such in vitro assays are also an active area of research in his lab.
A seed assay could be used to test whether Aβ seeds can reside outside the brain. If this is true, it would raise the question of whether recipients of transplant organs or blood products from donors unwittingly harboring AD pathology could potentially contract Aβ seeds.
What about Aβ seeds that stick to surgical equipment or to electrodes placed inside the brain, for example in the course of deep-brain-stimulation surgery or invasive EEG monitoring? Prion proteins reportedly have been transmitted in this way (see Belay et al., 2013; Thomas et al., 2013). Collinge told reporters he had previously developed a combination of enzymes and detergents that could destroy prions, but that the product was never manufactured for use in surgical settings, much to his disappointment. He said a similar product would likely destroy Aβ seeds as well.
According to previous work from Jucker’s lab, Aβ seeds are hardy. They retain their ability to trigger Aβ pathology in mice after a two-year soak in formaldehyde, and vanishingly small amounts of infectious particle can trip off the cascade (see Sep 2014 news). Like prions, Aβ seeds have a fondness for metal surfaces and are resistant to boiling (see Eisele et al., 2009; Meyer-Luehmann et al., 2006).
In his Nature Neuroscience paper, Jucker added further evidence of the potency of Aβ seeds. First author Lan Ye and colleagues injected 2.5 microliters of Aβ seed-containing tissue extract into two different mouse strains: APP23 mice that express human APP harboring an AD-associated mutation, and mice expressing no APP. One month later, the APP23 mice had higher levels of human Aβ than they had previously, indicating an early seeding response. The seeds stagnated in the mouse strain devoid of APP, as they had no template to corrupt and could not propagate. After 30 days, the seeds were undetectable by standard ELISA methods and immunoblotting, however, when the researchers employed the highly sensitive, bead-based platform Simoa, they were able to detect residual amounts of seed even after six months of dormancy in the APP-null mice. Despite their minuscule concentration, these seeds came alive again once researchers injected them into APP23 mice. Even after languishing in APP-null mice for half a year, the seeds ultimately triggered AD pathology upon secondary transmission into APP23 recipients.
Jucker’s latest finding indicates that Aβ seeds could be even more tenacious than prions, which reportedly lose their infectivity in mice sooner. Soto was surprised by the seeds’ persistence; however, he noted that newer, more sensitive detection methods may show prions to be more persistent than previously thought, as well.
What does all this say about precautions that should be taken during surgical and/or transplant procedures? All researchers and commentators interviewed for this story emphasized that much more research is needed to determine where Aβ seeds reside, whether they are truly capable of transmitting AD, and under what circumstances. The incubation period of up to 30 years makes these questions particularly difficult to answer. The present study could prompt researchers to systematically investigate the possibility of AD transmission, and to develop safety tools, such as assays for Aβ seeds and methods to destroy them. “Right now we don’t know if or how AD can be transmitted,” Soto said, “but it is better to be safe than sorry.”—Jessica Shugart
- Aβ the Bad Apple? Seeding and Propagating Amyloidosis
- Seeds of Destruction—Prion-like Transmission of Sporadic AD?
- In Case You Wondered: Neurodegenerative Diseases Are Not Contagious
- Test Uses 'Seeding' to Detect Aβ Oligomers in Cerebrospinal Fluid
- Bad Seeds—Potent Aβ Peptides Instigate Plaques, Won’t Be Fixed
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