Gültekin Tamgüney

Asymptomatic deer excrete infectious prions in faeces.

Infectious prion diseases—scrapie of sheep and chronic wasting disease (CWD) of several species in the deer family—are transmitted naturally within affected host populations. Although several possible sources of contagion have been identified in excretions and secretions from symptomatic animals, the biological importance of these sources in sustaining epidemics remains unclear. Here we show that asymptomatic CWD-infected mule deer (Odocoileus hemionus) excrete CWD prions in their faeces long before they develop clinical signs of prion disease. Intracerebral inoculation of irradiated deer faeces into transgenic mice overexpressing cervid prion protein (PrP) revealed infectivity in 14 of 15 faecal samples collected from five deer at 7–11 months before the onset of neurological disease. Although prion concentrations in deer faeces were considerably lower than in brain tissue from the same deer collected at the end of the disease, the estimated total infectious dose excreted in faeces by an infected deer over the disease course may approximate the total contained in a brain. Prolonged faecal prion excretion by infected deer provides a plausible natural mechanism that might explain the high incidence and efficient horizontal transmission of CWD within deer herds, as well as prion transmission among other susceptible cervids.

Transmission of Elk and Deer Prions to Transgenic Mice

ABSTRACT Chronic wasting disease (CWD) is a fatal prion disease in deer and elk. Unique among the prion diseases, it is transmitted among captive and free-ranging animals. To facilitate studies of the biology of CWD prions, we generated five lines of transgenic (Tg) mice expressing prion protein (PrP) from Rocky Mountain elk (Cervus elaphus nelsoni), denoted Tg(ElkPrP), and two lines of Tg mice expressing PrP common to white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus), denoted Tg(DePrP). None of the Tg(ElkPrP) or Tg(DePrP) mice exhibited spontaneous neurologic dysfunction at more than 600 days of age. Brain samples from CWD-positive elk, white-tailed deer, and mule deer produced disease in Tg(ElkPrP) mice between 180 and 200 days after inoculation and in Tg(DePrP) mice between 300 and 400 days. One of eight cervid brain inocula transmitted disease to Tg(MoPrP)4053 mice overexpressing wild-type mouse PrP-A in ∼540 days. Neuropathologic analysis revealed abundant PrP amyloid plaques in the brains of ill mice. Brain homogenates from symptomatic Tg(ElkPrP) mice produced disease in 120 to 190 days in Tg(ElkPrP) mice. In contrast to the Tg(ElkPrP) and Tg(DePrP) mice, Tg mice overexpressing human, bovine, or ovine PrP did not develop prion disease after inoculation with CWD prions from among nine different isolates after >500 days. These findings suggest that CWD prions from elk, mule deer, and white-tailed deer can be readily transmitted among these three cervid species.

Measuring prions by bioluminescence imaging

Prions are infectious proteins that cause fatal neurodegenerative diseases. Because astrocytic gliosis marked by the deposition of fibrils composed of GFAP is a prominent feature of prion disease, we asked whether GFAP might be used as a surrogate marker for prions. To interrogate this posit, we inoculated prions into transgenic (Tg) mice expressing luciferase (luc) under the GFAP gene (Gfap) promoter, denoted Tg(Gfap-luc) mice. Weekly noninvasive, bioluminescence imaging (BLI) detected an increase in light emitted from the brains of Tg(Gfap-luc) mice at ≈55 d after inoculation and ≈62 d before neurologic deficits appeared. To determine whether BLI could be used as a proxy bioassay for prion infectivity, we performed endpoint titrations of prions in Tg(Gfap-luc) mice. BLI bioassays were as or more sensitive than those determined by the onset of neurological dysfunction, and were completed in approximately half the time. Our studies argue that BLI is likely to be a suitable surrogate for measuring prion infectivity, and might be useful in the study of Tg mouse models for other neurodegenerative illnesses.

Salivary prions in sheep and deer.

Scrapie of sheep and chronic wasting disease (CWD) of cervids are transmissible prion diseases. Milk and placenta have been identified as sources of scrapie prions but do not explain horizontal transmission. In contrast, CWD prions have been reported in saliva, urine and feces, which are thought to be responsible for horizontal transmission. While the titers of CWD prions have been measured in feces, levels in saliva or urine are unknown. Because sheep produce ~17 L/day of saliva, and scrapie prions are present in tongue and salivary glands of infected sheep, we asked if scrapie prions are shed in saliva. We inoculated transgenic (Tg) mice expressing ovine prion protein, Tg(OvPrP) mice, with saliva from seven Cheviot sheep with scrapie. Six of seven samples transmitted prions to Tg(OvPrP) mice with titers of -0.5 to 1.7 log ID50 U/ml. Similarly, inoculation of saliva samples from two mule deer with CWD transmitted prions to Tg(ElkPrP) mice with titers of -1.1 to -0.4 log ID50 U/ml. Assuming similar shedding kinetics for salivary prions as those for fecal prions of deer, we estimated the secreted salivary prion dose over a 10-mo period to be as high as 8.4 log ID50 units for sheep and 7.0 log ID50 units for deer. These estimates are similar to 7.9 log ID50 units of fecal CWD prions for deer. Because saliva is mostly swallowed, salivary prions may reinfect tissues of the gastrointestinal tract and contribute to fecal prion shedding. Salivary prions shed into the environment provide an additional mechanism for horizontal prion transmission.

Neuroinvasion of α-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation

ABSTRACT α-Synuclein is a soluble, cellular protein that in a number of neurodegenerative diseases, including Parkinsons disease and multiple system atrophy, forms pathological deposits of protein aggregates. Because misfolded α-synuclein has some characteristics that resemble those of prions, we investigated its potential to induce disease after intraperitoneal or intraglossal challenge injection into bigenic Tg(M83+/−:Gfap-luc+/−) mice, which express the A53T mutant of human α-synuclein and firefly luciferase. After a single intraperitoneal injection with α-synuclein fibrils, four of five mice developed paralysis and α-synuclein pathology in the central nervous system, with a median incubation time of 229 ± 17 days. Diseased mice accumulated aggregates of Sarkosyl-insoluble and phosphorylated α-synuclein in the brain and spinal cord, which colocalized with ubiquitin and p62 and were accompanied by gliosis. In contrast, only one of five mice developed α-synuclein pathology in the central nervous system after intraglossal injection with α-synuclein fibrils, after 285 days. These findings are novel and important because they show that, similar to prions, α-synuclein prionoids can neuroinvade the central nervous system after intraperitoneal or intraglossal injection and can cause neuropathology and disease. IMPORTANCE Synucleinopathies are neurodegenerative diseases that are characterized by the pathological presence of aggregated α-synuclein in cells of the nervous system. Previous studies have shown that α-synuclein aggregates made of recombinant protein or derived from brains of patients can spread in the central nervous system in a spatiotemporal manner when inoculated into the brains of animals and can induce pathology and neurologic disease, suggesting that misfolded α-synuclein can behave similarly to prions. Here we show that α-synuclein inoculation into the peritoneal cavity or the tongue in mice overexpressing α-synuclein causes neurodegeneration after neuroinvasion from the periphery, which further corroborates the prionoid character of misfolded α-synuclein.

Chimeric elk/mouse prion proteins in transgenic mice

Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). Between codons 169 and 219, six residues distinguish ElkPrP from MoPrP: N169S, T173N, V183I, I202V, I214V and R219K. Using chimeric elk/mouse PrP constructs, we generated 12 Tg mouse lines and determined incubation times after intracerebral inoculation with the mouse-passaged RML scrapie or Elk1P CWD prions. Unexpectedly, one Tg mouse line expressing Elk3M(SNIVVK) exhibited incubation times of <70 days when inoculated with RML prions; a second line had incubation times of <90 days. In contrast, mice expressing full-length ElkPrP had incubation periods of >250 days for RML prions. Tg(Elk3M,SNIVVK) mice were less susceptible to CWD prions than Tg(ElkPrP) mice. Changing three C-terminal mouse residues (202, 214 and 219) to those of elk doubled the incubation time for mouse RML prions and rendered the mice resistant to Elk1P CWD prions. Mutating an additional two residues from mouse to elk at codons 169 and 173 increased the incubation times for mouse prions to >300 days, but made the mice susceptible to CWD prions. Our findings highlight the role of C-terminal residues in PrP that control the susceptibility and replication of prions.

Autocrine stimulation of rhadinovirus-transformed T cells by the chemokine CCL1/I-309

The rhadinovirus herpesvirus saimiri transforms human T lymphocytes to stable growth in culture. Besides the viral oncogenes stpC and tip, little is understood about the transformation process at the cellular level. To identify cellular factors that might contribute to growth transformation, we compared cellular gene expression in pairs of herpesvirus saimiri-transformed and nontransformed human T-cell clones. Using cDNA arrays and suppressive subtractive hybridization, we were able to identify the chemokine CCL1/I-309 as one of the few cellular genes that are strongly overexpressed in T cells after growth transformation with herpesvirus saimiri. The transformed T cells expressed CCR8, the receptor for CCL1, which rapidly induced intracellular calcium ion levels. Neutralizing antibodies to CCL1 led to reduced secretion of interferon-γ and tumor necrosis factor-α as well as to reduced proliferation rates in transformed T cells. Thus, we propose that growth transformation of human T cells with herpesvirus saimiri gives rise to an autocrine loop where the proliferation of transformed T cells is supported by the endogenous production of the chemokine CCL1.

Downregulation of p56 lck Tyrosine Kinase Activity in T Cells of Squirrel Monkeys (Saimiri sciureus) Correlates with the Nontransforming and Apathogenic Properties of Herpesvirus Saimiri in Its Natural Host

ABSTRACT Herpesvirus saimiri is capable of transforming T lymphocytes of various primate species to stable growth in culture. The interaction of the T-cellular tyrosine kinase p56 lck with the transformation-associated viral protein Tip has been shown before to activate the kinase and provides one model for the T-cell-specific transformation by herpesvirus saimiri subgroup C strains. In contrast to other primate species, squirrel monkeys (Saimiri sciureus) are naturally infected with the virus without signs of lymphoma or other disease. Although the endogenous virus was regularly recovered from peripheral blood cells from squirrel monkeys, we observed that the T cells lost the virus genomes in culture. Superinfection with virus strain C488 did not induce growth transformation, in contrast to parallel experiments with T cells of other primate species. Surprisingly, p56 lck was enzymatically inactive in primary T-cell lines derived from different squirrel monkeys, although the T cells reacted appropriately to stimulatory signals. The cDNA sequence revealed minor point mutations only, and transfections in COS-7 cells demonstrated that the S. sciureus lck gene codes for a functional enzyme. In S. sciureus, the tyrosine kinase p56 lck was not activated after T-cell stimulation and enzymatic activity could not be induced by Tip of herpesvirus saimiri C488. However, the suppression of p56 lck was partially released after administration of the phosphatase inhibitor pervanadate. This argues for unique species-specific conditions in T cells of S. sciureus which may interfere with the transforming activity and pathogenicity of herpesvirus saimiri subgroup C strains in their natural host.

A critical review of the prion hypothesis of human synucleinopathies

Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are neurodegenerative disorders which have been pathologically classified as synucleinopathies, since they are associated with pathognomonic deposits of misfolded alpha-synuclein in cells of the nervous system. Recently PD, DLB, and MSA were also suggested to be prion-like disorders. Much controversy exists regarding this analogy between synucleinopathies and prion diseases. Here, we discuss what characterizes prion diseases and in which way synucleinopathies may be considered prion-like or -unlike. We critically review recent clinical and in vivo evidence from transmission studies to animals in support of or questioning the prion hypothesis of human synucleinopathies. We conclude that, although PD, DLB, and MSA fulfill many criteria of prion-likeness, they also still fail some of these criteria.

A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization

The prion protein (PrP) is a cell surface protein that in disease misfolds and becomes infectious causing Creutzfeldt-Jakob disease in humans, scrapie in sheep, and chronic wasting disease in deer and elk. Little is known regarding the dimerization of PrP and its role in disease. We developed a bioluminescent prion assay (BPA) to quantify PrP dimerization by bimolecular complementation of split Gaussia luciferase (GLuc) halves that are each fused to PrP. Fusion constructs between PrP and N- and C-terminal GLuc halves were expressed on the surface of RK13 cells (RK13-DC cells) and dimerized to yield a bioluminescent signal that was decreased in the presence of eight different antibodies to PrP. Dimerization of PrP was independent of divalent cations and was induced under stress. Challenge of RK13-DC cells with seven different prion strains did not lead to detectable infection but was measurable by bioluminescence. Finally, we used BPA to screen a compound library for compounds inhibiting PrP dimerization. One of the most potent compounds to inhibit PrP dimerization was JTC-801, which also inhibited prion replication in RML-infected ScN2a and SMB cells with an EC50 of 370 nM and 220 nM, respectively. We show here that BPA is a versatile tool to study prion biology and to identify anti-prion compounds.