Detection of infectious prions in urine

Abstract

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). The mechanism of prion transmission is unknown. In this study, we attempted to detect prions in urine of experimentally infected animals. PrP(Sc) was detected in approximately 80% of the animals studied, whereas no false positives were observed among the control animals. Semi-quantitative calculations suggest that PrP(Sc) concentration in urine is around 10-fold lower than in blood. Interestingly, PrP(Sc) present in urine maintains its infectious properties. Our data indicate that low quantities of infectious prions are excreted in the urine. These findings suggest that urine is a possible source of prion transmission.

Figure 1. Identification of the experimental conditions to process urine samples for PrP^Sc detection by PMCA

Urine from several hamsters at the symptomatic stage of the disease (and uninfected controls) produced by i.p. inoculation of 263K prions was collected with metabolic cages. Twelve ml of urine was pooled and processed as schematically illustrated in panel A. First, urine was centrifuged at a low speed (5,000 × g for 20 min) to remove debris. The supernatant was collected and divided in 2 groups: one was dialyzed overnight against PBS at 4°C using a membrane with 30,000 Da cutoff; the other sample was left untreated. Both samples were divided into two identical aliquots, one of which was supplemented with 1 volume of 20% sarkosyl and the other one was left untreated. After 30 min incubation at 4°C, all the samples were centrifuged at 100,000 × g for 1h at 4°C. Pellet of each sample was resuspended directly in 80 ul of 10% normal hamster brain homogenate and subjected to 96 cycles of PMCA (30 min incubation at 37°C followed by a pulse of 30 s sonication, as described in Methods). Then, 8 µl of this sample were diluted into 72µl of normal brain homogenate and a new round of 96 PMCA cycles was performed. This process was repeated several times. After each round of PMCA, 20 µl of the sample was used for detection of PrP^Sc by western blot after PK digestion (50 ug/ml for 1h at 37°C), using 3F4 antibody. Panel B shows the results of serial rounds 5 and 6 of PMCA. No signal was observed before the fifth round. Sx: samples from sick animals; Cx: samples from control animals. All samples were treated with PK before electrophoresis, except the normal brain homogenate (NBH) in which –PK is indicated.

Figure 2. PrP^Sc detection in urine of sick hamsters by PMCA

Three ml of urine from 5 clinically sick hamsters (infected i.p. with HY prions) and 5 control animals was collected using metabolic cages. The samples were processed as described in figure A1. Briefly, urine was centrifuged at 5,000 × g for 20 min to remove debris. The supernatant was collected and subjected to a high speed centrifugation at 100,000 × g for 1h at 4°C to precipitate PrP^Sc. Pellet was resuspended directly in 80 ul of 10% normal hamster brain homogenate. Samples were subjected to serial rounds of 96 cycles of PMCA, as described [10,11]. 20 µl of the sample was used for detection of PrP^Sc by western blot after PK digestion. The figure shows only the rounds 5, 6, 7 and 8, since the first four rounds of PMCA did not show signal in any of the samples. More than 7 rounds of PMCA do not show any more positive signals. Sx: samples from sick animals; Cx: samples from control animals. All samples were treated with PK before electrophoresis, except the normal brain homogenate (NBH) in which –PK is indicated.

Figure 3. Urinary PrP^Sc is infectious

To assess whether PrP^Sc amplified from urine maintain the infectious properties, we inoculated intra-cerebrally 5 wild type hamsters with the sample S4 after 7 serial rounds of PMCA. As controls, groups of hamsters were inoculated with equivalent quantities of brainderived HY PrP^Sc and PMCA-generated PrP^Sc starting from HY brain. The later was generated as previously described (9). Briefly a 10⁴ dilution of HY brain was diluted into healthy hamster brain homogenate and subjected to 48 PMCA cycles. Thereafter, an aliquot of the amplified material was diluted 10-fold into healthy hamster brain homogenate and amplified again. This procedure was repeated to reach a 10²⁰ dilution of brain inoculum in order to eliminate any brain derived PrP^Sc. A negative control group was included consisting of normal urine samples subjected to the same procedure of serial PMCA amplification. The onset of clinical signs was monitored as described in Methods and animals were considered sick when they reach clinical level 4, characterized by extensive behavioral problems including tremor of the head, ataxia, wobbling gait, head bobbing, irritability, aggressiveness, jerks of the head and body and spontaneous backrolls. At this time, animals were sacrificed to avoid further pain and this is the number provided in the graph.

Figure 4. Pathological and biochemical features of the disease produced by inoculation of PrP^Sc amplified from urine

As positive control we used animals inoculated with brain-derived HY prions and as negative control we analyzed brains of uninfected hamsters. A: spongiform degeneration was assessed by hematoxilin-eosin staining in diverse areas of the brain, including medulla, hippocampus and superior colliculus. B: PrP^Sc accumulation was studied by histological staining with anti-PrP 3F4 antibody as described in methods. C: Reactive astrocytes were visualized by immunological staining with antibodies against glial fibrillar acidic protein (GFAP). D: PrP^Sc accumulation was evaluated by western blot after PK digestion. Different quantities of brain homogenate (dilutions 1:25, 1:50 and 1:100 with respect to the brain) were loaded into the gel. All samples were treated with PK, except for the normal brain homogenate (NBH) were –PK is indicated. Results shown in panels A–D are representative of several animals analyzed.