September 1, 2005 (Vol. 25, No. 15)
Finding Faulty Proteins
Transmissible spongiform encephalopathies (TSEs), the diseases caused by proteinaceous infectious particles, or prions, comprise a group of fatal neurodegenerative diseases that take their name from the holes they cause in the brains of susceptible animals and humans.
Animal forms of prion diseases occur in sheep and goats (scrapie), mule deer and elk (chronic wasting disease; CWD), and cows (bovine spongiform encephalopathy, or mad cow disease). In humans, prion diseases occur randomly or rarely in genetic forms.
Human forms of these brain-destroying diseases include Kuru, a disease restricted to the Fore people of New Guinea and transmitted thorough the now-banned practice of ritual cannibalism, in which relatives consumed the tissues, including the brain, of deceased family members.
Creutzfeldt-Jakob Disease (CJD) is a human TSE that occurs worldwide; 1015% of CJD cases are inherited; others result from inadvertent transmission through corneal transplantation, growth hormone derived from human pituitaries or contaminated surgical instruments.
Confirming the Disease
Currently available tests for prion diseases identify the abnormally-folded prion protein in brains of infected or dead animals through either antibody- or Western blot-based technologies. To date, no tests can identify the presence of the disease-causing proteins in easily accessible blood or tissues.
Disease can be confirmed only upon the appearance of outward symptoms and/or the availability of post-mortem brain and nervous symptom tissues which are examined immunohistochemically.
Within the last few years, cases of vCJD, or variant CJD, have been reported in Europe and Asia originating from transfusions. The appearance of these vCJD infections has been a driving force in the search for diagnostic tests that can find evidence of prion disease in accessible tissues and blood.
All these diseases involve changes in the conformation of prion precursor protein (PrPc), a protein normally expressed in mammalian cells, particularly in neurons, as a plasma membrane glycoprotein.
The inability to detect disease until animals are either sick or dead has seriously complicated control of infected herds. The inadvertent introduction of the bovine disease into the human food supply through an animal feed supplement fed to cows containing meat and bone meal from dead sheep prompted the British government to ban the use of animal-derived food supplements in 1988.
While this step contained the epidemic, the sick cows that sporadically appear in herds cause considerable alarm, as the disease is transmissible to humans through meat consumption.
Apparently absorbed through the intestinal tract upon ingestion of infected meat, abnormal prion particles migrate until they reach the brain to recruit normal prion particles into the disease-causing contorted forms that aggregate, accumulate, and cause brain destruction.
Mass slaughter of potentially infected animals causes considerable economic hardship; between 1996 and 2000, as the British government attempted to eradicate BSE-infected cattle, over five million cattle were killed with financial losses nearing $3 billion. Currently, a definitive diagnosis of BSE can be made only through post-mortem testing.
The unusual etiology of these diseases has fueled a long-standing scientific debate. Stanley B. Prusiner, M.D., of the University of California, San Francisco, first proposed that the causative agents were misfolded protein particles. This idea was considered highly unlikely based on what was understood about infectivity, that is, that a nucleic acid, either DNA or RNA, was required to ensure the replication of the infectious agent in a host.
Studies in Dr. Prusiners and other laboratories showed that the infectious agent in scrapie lacked any kind of nucleic acid, as would be expected if the infectious agent were bacterial or viral, and consisted only of protein. Brain extracts prepared from infected animals in which all nucleic acid had been destroyed could cause infection in healthy animals.
Further experimentation confirmed that a protease-resistant form of a normal brain protein could both cause and transmit this group of diseases. Ongoing research efforts are aimed at further verification and extension of the prion hypothesis and in developing rapid and reliable means of testing for evidence of the disease in animals and animal products.
The concept that proteins could be infectious received further support from studies that identified specific genetic mutations associated with disease development in families with inherited prion diseases. Characterizing the mutations that caused warped prions allowed the development of transgenic mice expressing both the normal prion protein and/or a mutated form.
Animals carrying the mutated gene developed scrapie and the homogenized brain tissue from such animals could transmit the disease to healthy animals. More recent studies have concentrated on developing formal proof that spongiform encephalopathies are caused solely by conformationally altered prion proteins and on developing methods for detection and diagnosis of these diseases.
It’s the Shape that Matters
Instead of containing alpha helices, the well-behaved normal spirals forming part of the normal prion protein, the scrapie form contains untwisted structures, beta strands that form beta sheets. Ultimately, the abnormal structures can also alter the normal protein and cause it to accumulate as amyloid material in neurons.
While it remains unclear how the accumulation of abnormal protein actually destroys nerve cells, the specific areas of the proteins involved in helix formation suggest sites for therapeutic interventions.
Giuseppe Legname, M.D., associate adjunct professor of neurology at UCSFs Institute for Neurodegenerative Diseases, reported that he and his colleagues formed synthetic infectious prions in vitro that polymerized into the amyloid form.
When injected into transgenic mice that overexpressed the normal prion protein, some of these synthetic prion forms caused neurological disease between 380 and 660 days following inoculation, while control mice injected with saline remained healthy.
Serial transmission of the brain extract from the sick mice caused the disease in other, disease-free mice. These scientists identified at least two new prion types with differing biochemical and neuropathological characteristics.
Dr. Legname explains that while we managed to make infectious prions in vitro, we dont yet know how to control the process. We understand the required ingredients and how to control the reaction, but we have no idea how many prion strains might be infective. We are now trying to identify and characterize their different features.
That process of characterization, the UCSF group believes, will yield an understanding of how the folding and structure of the amyloid formed by infectious prions sets off the neuron-destroying aggregation of this material. Ultimately, he says, what we want is to discover how to impede this pathological process.
Developing Prion Tests: Bioconformatics
The need to identify infected animals and animal products quickly and efficiently has created an opportunity for novel detection and diagnostics to find infectious prions in easily accessible body tissues and fluids.
According to Alan S. Rudolph, CEO of Adlyfe (Rockville, MD), a privately held company developing novel tests prion-detecting tests, Bionconformatics, or detection and quantitation of biological material based on conformation, is a wonderful target for the detection and development of therapeutics.
Scientists at Adlyfe and Georgetown University have developed diagnostics based on the changes that occur in the prion protein during the progression to a TSE.
The company claims, for example, that its BSE-pronucleon test can detect infectious prions in living animals in preclinical, presymptomatic, and clinical stages of infection, with results in a little as 30 minutes.
Cindy S. Orser, Ph.D., the companys CSO and the developer of the test technology, says that the abnormally folded proteins can be quantitated in tissue and blood using a test detecting PrP protein conformation as it changes from an alpha-helical to the abnormal beta sheet structure.
The idea behind the technology is to use small peptides that mimic the misfolding to detect it. When the peptide associates with a misfold, it changes shape itself. The peptide is labeled on each end with a fluorophore that emits a signal as the shape change occurs, giving us a quantitative readout of the amount of beta sheet present, Dr. Orser explains.
She further points out that the test is highly sensitive. The peptides exist as an ensemble so there are lots of them around when the change of shape occurs. The other peptides participate and amplify the fluorescence shift signal.
What is so unusual about this test is that there is no antibody needed, and no requirement for proteinase k digestion. Little sample preparation is required and we only need 100 microliters of plasma to do the test.
Right now, Dr. Orser, notes, the tests biggest applications are in Europe and Japan for blood screening. There are three individuals in the U.K. who have died from vCJD, who received blood transfusions from people who subsequently died from CJD variants.
And, while the prion target was the proof of principle for Adlyfe, its ultimate goal, she says, is to demonstrate that this platform will also work in other neurological diseases where proteins change form and lead to clinical symptoms.
Rudolph says that the company received its original funding through research contracts with DARPA and the NIH as part of biodefense initiatives. We raised over $6 million in non-dilutive capital through these contracts. We are now evolving toward the commercialization phase during which collaborations with diagnostic companies are a key part of our development path.
Since no regulatory precedent for a prion blood test exists, Rudolph expects that Adlyfe will be relying on experienced partners to move the test through the regulatory process.
Making More of Less
Because the amount of abnormal prion protein present outside the nervous system is likely to be very small, other diagnostic methods will most likely depend on amplification schemes for detection.
Scientists from Serono International (Geneva Switzerland) reported amplification of misfolded prion proteins using a method conceptually analogous to DNA amplification by PCR. The method depends upon the tiny amounts of misfolded protein present in samples and their recruitment of normal prion proteins into abberant forms.
Claudio Soto, Ph.D., and his colleagues at Serono reported that five amplification cycles converted 97% of the normal form to the abnormal form, and believe that the technology can be applied to the detection of many proteins caused by misfolded proteins.
