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MEDICAL BIOLOGY: EXTRA-NEURAL PRION PROTEIN IN BRAIN DISEASE

The following points are made by M. Glatzel et al (New Engl. J. Med. 2003 349:1812):

1) Prion diseases are characterized by degeneration of central nervous tissue associated with the replication of a transmissible agent, a prion.(1) Prions are mainly composed of an abnormal, partially protease-resistant conformer (PrPSc) of a cellular protein (PrPC).(2) Although tissue damage occurs only in the central nervous system, the accumulation of PrPSc and prion infectivity are not necessarily confined to neural tissue in all prion diseases. In scrapie in sheep, chronic wasting disease in deer, and numerous animal models of prion diseases, prions invade the lymphoreticular system.(3) PrPSc has also been reported in the skeletal muscle of mice with experimentally induced prion disease.(4)

2) The most common prion disease in humans is Creutzfeldt–Jakob disease, which has been classified as sporadic, familial, iatrogenic, or variant. The causation of sporadic Creutzfeldt–Jakob disease is unclear, whereas biochemical, histopathological, and epidemiologic evidence suggests that the variant form results from the transmission of bovine spongiform encephalopathy prions to humans.(5) PrPSc accumulates in tonsils and other lymphoreticular organs of patients with variant Creutzfeldt–Jakob disease, whereas the sporadic form of the disease is not thought to target the lymphoreticular system. The allegedly unique involvement of the lymphoreticular system in variant Creutzfeldt–Jakob disease serves as a diagnostic criterion to distinguish variant from sporadic Creutzfeldt–Jakob disease and is one of the principal pieces of evidence that these forms represent two distinct disease entities.

3) In summary: In patients with sporadic Creutzfeldt–Jakob disease, pathologic disease-associated prion protein (PrPSc) has been identified only in the central nervous system and olfactory-nerve tissue. Understanding the distribution of PrPSc in Creutzfeldt–Jakob disease is important for classification and diagnosis and perhaps even for prevention. The authors report they used a highly sensitive method of detection -- involving the concentration of PrPSc by differential precipitation with sodium phosphotungstic acid, which increased the sensitivity of Western blot analysis by up to three orders of magnitude -- to search for PrPSc in extraneural organs of 36 patients with sporadic Creutzfeldt–Jakob disease who died between 1996 and 2002. The authors identified extraneural deposition of PrPSc in spleen and muscle samples from approximately one third of patients who died with sporadic Creutzfeldt–Jakob disease. The authors suggest that extraneural PrPSc appears to correlate with a long duration of disease.

References (abridged):

1. Prusiner SB. Prions. Proc Natl Acad Sci U S A 1998;95:13363-13383

2. Prusiner SB, McKinley MP, Bowman KA, et al. Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 1983;35:349-358

3. Aguzzi A, Montrasio F, Kaeser PS. Prions: health scare and biological challenge. Nat Rev Mol Cell Biol 2001;2:118-126

4. Bosque PJ, Ryou C, Telling G, et al. Prions in skeletal muscle. Proc Natl Acad Sci U S A 2002;99:3812-3817

5. Bruce ME, Will RG, Ironside JW, et al. Transmissions to mice indicate that `new variant' CJD is caused by the BSE agent. Nature 1997;389:498-501

New Engl. J. Med. http://www.nejm.org

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ON PRIONS AND PRION DISEASE

The following points are made by Nigel M. Hooper (Current Biology 2002 12:R248):

1) The prion protein (PrP) appears to be the sole causative agent of the fatal neurodegenerative spongiform encephalopathies, such as Creutzfeldt–Jakob (CJD) disease in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals [1] . A key, possibly unique, feature of prion diseases is their transmissibility from one organism to another. Although it has been known for many years that prion diseases are transmissible, the molecular mechanisms involved in transmission from one cell to another have remained unclear. Recently Kanu et al. [2] , using prion infected and uninfected cell lines, demonstrated that cell contact is required for the effective transmission of the disease.

2) During disease progression, the normal cellular form of PrP (PrPC) undergoes a conformational change from a predominantly alpha-helical form to an abnormal isoform (PrPSc) which has mainly beta-sheet structure. Although the structure of PrPC has been determined [3–5], the structure of PrPSc awaits elucidation. PrPSc can, however, be distinguished experimentally from PrPC by its relative resistance to protease digestion. PrPC is a glycosylated cell surface protein, attached to the outer leaflet of the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor. Cells expressing PrPC can be converted to stable production of PrPSc upon exposure to subcellular preparations containing PrPSc, such as homogenates from the brains of prion-infected animals. The extent of infection of cells in culture by such subcellular preparations is usually poor, however, and the molecular mechanisms involved in the initial conversion of the cellular PrPC by the exogenous PrPSc are not fully understood.

3) In summary: Co-culture of prion-infected cells with uninfected target cells has recently been found to lead to rapid and efficient transmission of infectivity to the target cells by a process that is dependent on direct cell contact.

References (abridged):

1. Prusiner S.B. (1998) Prions. Proc. Natl. Acad. Sci. U.S.A., 95:13363-13383.

2. Kanu, N., Imokawa, Y., Drechsel, D.N., Williamson, R.A., Birkett, C.R., Bostock, C.J. and Brockes, J.P. (2002). Transfer of scrapie prion infectivity by cell contact in culture.

3. Donne D.G., Viles J.H., Groth D., Mehlhorn I., James T.L., Cohen F.E., Prusiner S.B., Wright P.E. and Dyson H.J. (1997) Structure of the recombinant full-length hamster prion protein PrP (29–231): the N terminus is highly flexible. Proc. Natl. Acad. Sci. U.S.A., 94:13452-13457

4. Zahn R., Liu A., Luhrs T., Riek R., von Schroetter C., Lopez Garcia F., Billeter M., Calzolai L., Wider G. and Wuthrich K. (2000) NMR solution structure of the human prion protein. Proc. Natl. Acad. Sci. U.S.A., 97:145-150

5. Knaus K.J., Morillas M., Swietnicki W., Malone M., Surewicz W.K. and Yee V.C. (2001) Crystal structure of the human prion protein reveals a mechanism for oligomerization. Nat. Struct. Biol., 8:770-774

Current Biology http://www.current-biology.com

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ON NEURODEGENERATIVE DISEASES AND PRIONS

Prions are a class of poorly understood proteins implicated in a number of exotic human neurological diseases and in some common animal diseases such as sheep scrapie and bovine spongiform encephalopathy in cattle ("mad cow disease"). Spongiform encephalopathies are a type of brain disease found in humans and animals and are characterized by macroscopic vacancies produced by the disease process (the brain has a sponge-like appearance). What is remarkable about prions is that they behave as infectious agents, but they are 100 times smaller than viruses and their mechanism of replication is unclear. One human disease in which prions have been strongly implicated is *Creutzfeldt-Jakob disease (CJD), which appears to have a genetic basis in about 15% of the cases. All the prion diseases are apparently associated with the accumulation in the brain of an abnormal *protease-resistant isoform of the prion protein. In other words, an abnormal variant of the normal prion protein is somehow copied or produced by the disease process, which can be initiated by introducing infectious prion into the system.

The term "amyloid" ("starch-like") refers to a variety of polypeptide molecules defined by their properties, particularly by their tendency to arrange in a twisted *beta-pleated fibrillar structure. Amyloid is in general a proteinaceous material, deposits of which have been classically noted to occur in the brains of *Alzheimer's disease and older *Down syndrome patients, and to a much lesser degree, in association with normal aging. Amyloid material consists primarily of a highly aggregated 42-amino acid polypeptide called "beta-amyloid".

In general, a "somatic mutation" is a mutation occurring in non-germ cells, which means the mutation is not transmitted to the next generation. In contrast, a "germ-line mutation" is a mutation occurring in germ cells, and is thus transmitted to the next generation.

In this context, the term "sporadic" means non-familial.

In 1997 Stanley B. Prusiner was awarded the Nobel Prize in Physiology or Medicine for his discovery of prions, an entirely new genre of disease-causing agents.

The following points are made by Stanley B. Prusiner (New England J. Med. 2001 344:1516):

1) The author points out that Alzheimer's disease is the most common neurodegenerative disorder, with approximately 4 million people in the US having this disease. In the US at present, approximately 1 million people have *Parkinson's disease. Much less common are *frontotemporal dementia (40,000 people), *Huntington's disease (30,000 people), *amyotrophic lateral sclerosis (20,000 people), and *spinocerebellar ataxia (12,000 people), and prion diseases (400 people). Among persons who are 60 years of age, the prevalence of Alzheimer's disease is approximately 1 in 10,000, but among those who are 85 years of age, the prevalence is greater than 1 in 3. These data suggest that by 2025, there will be more than 10 million cases of Alzheimer's disease in the US, and by 2050, the number will approach 20 million. The current annual cost associated with Alzheimer's disease in the US is estimated at $200 billion. Age is also the most important risk factor for Parkinson's disease, with at least 50 percent of persons who are 85 years of age having at least one symptom or sign of parkinsonism.

2) The author points out that nearly all neurodegenerative disorders involve abnormal processing of neuronal proteins. The aberrant mechanism can involve a misfolding of proteins, altered *post-translational modification of newly synthesized proteins, abnormal *proteolytic cleavage, anomalous *gene splicing, improper gene expression, or diminished clearance of degraded protein. Misprocessed proteins often accumulate because the cellular mechanisms for removing them are ineffective, and the particular protein that is improperly processed determines the malfunction of distinct sets of neurons and thus the clinical manifestation of the disease.

3) The author points out that prions are infectious proteins. In mammals, prions replicate by recruiting normal cellular prion protein and stimulating its conversion to the disease-causing prion isoform ("scrapie prion"; Prp[supSC]). A major feature that distinguishes prions from viruses is that scrapie prion can be encoded by a chromosomal gene resulting from a mutation of the normal prion gene. Limited proteolysis of scrapie prion produces a smaller and protease-resistant molecule of approximately 142 amino acids which polymerizes into amyloid.

4) The author points out that the polypeptide chains of normal prion and scrapie prion are identical in composition but differ in their 3-dimensional folded structures. Normal prion is rich in spiral-like formations of amino acids (alpha-helices) and has little flattened strands (beta-sheets) of amino acids, whereas scrapie prion is less rich in alpha-helices and has much more beta-sheet domains. There is evidence that normal prion has 3 alpha-helices and 2 short beta-strands; in contrast, a plausible model suggests that scrapie prion may have only 2 alpha-helices and more beta-strands. This structural transition from alpha-helices to beta-sheet in prion protein is apparently the fundamental event underlying prion disease.

5) The author points out that four new concepts have emerged from studies of prions:

a) Prions are the only known example of infectious pathogens that are devoid of nucleic acid. All other infectious agents have genomes composed of either RNA or DNA that direct the synthesis of their progeny.

b) Prion diseases may be manifested as infectious, genetic, or sporadic disorders. No other group of illnesses with a single cause has such a wide spectrum of clinical manifestations.

c) Prion diseases result from the accumulation of scrapie prion, which has a substantially different molecular conformation from that of its precursor, normal prion.

d) Scrapie prion can have a variety of conformations, each of which seems to be associated with a specific disease. How a particular conformation of scrapie prion is imparted to normal prion during replication in order to produce a nascent scrapie prion with that conformation is unknown. The factors that determine the site in the central nervous system where a particular scrapie prion is deposited are also not known.

6) The author tabulates the known pathogenic features of known prion diseases as follows:

a) *Kuru: Human hosts, "Fore" people in New Guinea. Infection occurs via ritualistic cannibalism.

b) Creutzfeldt-Jakob disease:

Via medical procedures (iatrogenic): Human hosts. Infection from prion-contaminated human *growth hormone, *dura mater grafts, etc.

*New variant: Human hosts. Possible infection from bovine prions.

Familial: Human hosts. Results from germ-line mutations in the prion gene.

Sporadic: Human hosts. Results from somatic mutation or spontaneous conversion of normal prion into scrapie prion.

c) *Gerstmann-Straeussler-Scheinker disease: Human hosts. Results from germ-line mutations in the prion gene.

d) *Fatal familial insomnia: Human hosts. Results from germ-line mutations in the prion gene.

e) *Scrapie: Sheep hosts. Results from infection in genetically susceptible sheep.

f) Bovine spongiform encephalopathy: Cattle hosts. Results from infection with prion-contaminated meat and bone meal.

g) Transmissible mink encephalopathy: Mink hosts. Results from infection with prion from sheep or cattle.

h) Chronic wasting disease: Mule, deer, elk hosts. Mechanism of pathogenesis unknown.

i) Feline spongiform encephalopathy: Cat hosts. Results from infection with prion-contaminated beef.

j) Exotic *ungulate encephalopathy: Greater kudu, nyala, oryx hosts. Results from infection with prion-contaminated meat and bone meal.

7) Concerning the general paradigm that virtually all neurodegenerative disorders involve abnormal processing of neuronal proteins, the author states: "It is tempting to speculate that abnormal processing of neuronal proteins also occurs in other diseases of the central nervous system, such as schizophrenia, bipolar disorders, autism, and narcolepsy. Most cases of these diseases are sporadic, but a substantial minority appear to be familial."

8) The author concludes: "Over the past two decades, remarkable progress has been made in elucidating the causes of neurodegenerative diseases, and the time has come to intensify the search for drug targets and for compounds that interrupt the disease processes. Drugs that block the mishandling of a particular protein may be most effective for certain disorders; for others, drugs that enhance the clearance of an aberrant protein or fragment may prove most useful. Regardless of the therapeutic approach, accurate, early detection of neurodegeneration will be extremely important so that drugs can be given before substantial damage to the central nervous system has occurred. However, the enormity of these tasks -- developing useful diagnostic tests and discovering effective therapies --should not be underestimated."

New Engl. J. Med. http://www.nejm.org

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Notes:

Creutzfeldt-Jakob disease (CJD): Until 30 years ago, Creutzfeldt-Jakob disease was an obscure form of dementia unknown to most physicians. The name is now familiar to the medical community as the major prion disease in humans.

protease: In general, any enzyme that cleaves proteins, usually by hydrolysis.

beta-pleated: In general, protein chains fold into alpha-helices or beta-sheet structures. The beta-sheet is a protein structure where the peptide is extended and stabilized by hydrogen bonding between NH and CO groups of different polypeptide chains or of separate regions of the same chain.

Alzheimer's disease: There are various forms of dementia produced by various causes. Alzheimer-type dementia (Alzheimer's disease) is apparently related to what appear to be specific cellular and histological degenerative processes, with loss of cells from several specific brain areas, the brain showing moderate to marked atrophy. Memory loss is the most prominent early symptom.

Down syndrome: A birth defect marked by mental retardation and many physical defects, the syndrome arising from an extra chromosome 21 (trisomy 21).

Parkinson's disease: A neurological disorder first described by James Parkinson (1817) and associated with degeneration of a specific small region of the brain and a resultant loss of projection to several important brain centers. One must distinguish "parkinsonism" from Parkinson's disease. Parkinsonism is a syndrome (a complex of symptoms; in this context, a complex of various movement symptoms) that may be caused by Parkinson's disease, but which may also be caused by infectious, vascular, pharmacological, toxic, metabolic, structural, and various degenerative disorders.

frontotemporal dementia: Dementia associated with loss of functions associated with the frontotemporal lobes of the brain.

Huntington's disease: (Huntington's chorea) First described by George Huntington (1850-1916), the disease attacks specific regions of the brain (e.g., caudate nucleus and putamen), and leads to insanity and eventual death.

amyotrophic lateral sclerosis: A progressive disease of motor neurons (spinal cord nerve cells that control voluntary muscles). 50 percent of patients die within 3 years of the first symptoms.

spinocerebellar ataxia: In general, an ataxia is an inability to coordinate muscle activity during voluntary movement. Spinocerebellar ataxia is the most common hereditary ataxia. The spinocerebellar degenerative disorders are a group of diseases involving neurons in several nervous system structures, including the spinal cord and cerebellum.

post-translational: In this context, translation is protein synthesis, the process during which polypeptides are synthesized in accordance with RNA code.

proteolytic: In general, "proteolysis" is the enzyme-catalyzed degradation of protein by hydrolysis of one or more peptide bonds.

gene splicing: In this context, the production of new genes by an abnormal replication process that combines fragments of DNA in new arrangements.

Kuru: This disease is similar to Creutzfeldt-Jakob disease, and is a human spongiform encephalopathy. Kuru occurs only in the eastern highlands of New Guinea, occurs more frequently in women than in men, which apparently coincides with the customs surrounding cannibalism in a society where the remains of dead relatives are handled and eaten primarily by children and women. After cannibalism was outlawed, the incidence of the disease decreased, and the current consensus is that cannibalism was the primary mode of transmission of the pathological agent.

growth hormone: A vertebrate polypeptide hormone that regulates growth. In general, hormones are signaling molecules secreted into the blood stream by endocrine cells and acting on target cells that possess receptors for the hormone.

dura mater: In this context, a thick protective membrane that surrounds the brain. There is also a dura mater surrounding the spinal cord.

New variant: In 1996, the new variant of Creutzfeldt-Jakob disease (new-variant CJD) was recognized in the UK population, primarily in younger people, the new disease with distinctive pathological characteristics similar to those seen in macaque monkeys infected with the agent of bovine spongiform encephalopathy.

Gerstmann-Straeussler-Scheinker disease: A slowly progressive neurodegenerative genetic disease similar to Creutzfeldt-Jakob disease and transmissible to experimental animals. The disease is much rarer than Creutzfeldt-Jakob disease and has an earlier onset.

Fatal familial insomnia: insomnia: Another familial form of Creutzfeldt-Jakob disease. This very rare disease is difficult to transmit to experimental animals. The age of onset varies widely, the course of the disease averaging 13 months. (Note: All the human spongiform encephalopathies are invariably fatal.)

Scrapie: Susceptibility to scrapie varies among different breeds of sheep, with goats 100 percent susceptible. The disease is transmissible to laboratory monkeys, mice, and hamsters.

ungulate: In general, a hoofed mammal.

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