Infectious prion diseases 1 – scrapie of sheep 2 and chronic wasting disease (CWD) of several species in the deer family 3,4 – are transmitted naturally within affected host populations. Although several possible sources of contagion have been identified in excretions and secretions from symptomatic animals 5–8, 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 feces long before they develop clinical signs of prion disease. Intracerebral (i.c.) inoculation of irradiated deer feces into transgenic (Tg) mice overexpressing cervid PrP revealed infectivity in 14 of 15 fecal samples collected from 5 deer at 7–11 months before the onset of neurological disease. Although prion concentrations in deer feces were considerably lower than in brain tissue from the same deer collected at the disease terminus, the estimated total infectious dose excreted in feces by an infected deer over the disease course may approximate the total contained in brain tissue. Prolonged fecal 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 3,4,9, as well as prion transmission between susceptible deer species.

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 ϳ540 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.

The conversion of endogenous alpha-synuclein (asyn) to pathological asyn-enriched aggregates is a hallmark of Parkinson’s disease (PD). These inclusions can be detected in the central and enteric nervous system (ENS). Moreover, gastrointestinal symptoms can appear up to 20 years before the diagnosis of PD. The dual-hit hypothesis posits that pathological asyn aggregation starts in the ENS, and retrogradely spreads to the brain. In this study, we tested this hypothesis by directly injecting preformed asyn fibrils into the duodenum wall of wild-type rats and transgenic rats with excess levels of human asyn. We provide a meticulous characterization of the bacterial artificial chromosome (BAC) transgenic rat model with respect to initial propagation of pathological asyn along the parasympathetic and sympathetic pathways to the brainstem, by performing immunohistochemistry at early time points post-injection. Induced pathology was observed in all key structures along the sympathetic and parasympathetic pathways (ENS, autonomic ganglia, intermediolateral nucleus of the spinal cord (IML), heart, dorsal motor nucleus of the vagus, and locus coeruleus (LC)) and persisted for at least 4 months post-injection. In contrast, asyn propagation was not detected in wild-type rats, nor in vehicle-injected BAC rats. The presence of pathology in the IML, LC, and heart indicate trans-synaptic spread of the pathology. Additionally, the observed asyn inclusions in the stomach and heart may indicate secondary anterograde propagation after initial retrograde spreading. In summary, trans-synaptic propagation of asyn in the BAC rat model is fully compatible with the “body-first hypothesis” of PD etiopathogenesis. To our knowledge, this is the first animal model evidence of asyn propagation to the heart, and the first indication of bidirectional asyn propagation via the vagus nerve, i.e., duodenum-to-brainstem-to-stomach. The BAC rat model could be very valuable for detailed mechanistic studies of the dual-hit hypothesis, and for studies of disease modifying therapies targeting early pathology in the gastrointestinal tract.Electronic supplementary materialThe online version of this article (10.1007/s00401-019-02040-w) contains supplementary material, which is available to authorized users.

In chronic wasting disease (CWD) of cervids and scrapie of sheep, prions appear to be transmitted horizontally. Oral exposure to prion-tainted blood, urine, saliva and feces has been suggested as the mode of transmission for CWD and scrapie among herbivores susceptible to these prion diseases. To explore the transmission of prions through feces, uninoculated Syrian hamsters (SHa) were cohabitated with or exposed to the bedding of SHa orally infected with Sc237 prions. Incubation times of ~140 days and 80–100% prion infection rate in exposed animals suggested transmission by feces, probably via coprophagy. We measured the disease-causing isoform of the prion protein (PrPSc) in feces by the conformation-dependent immunoassay and titrated the irradiated feces intracerebrally in transgenic mice overexpressing SHaPrP. Feces collected from infected SHa in the first 7 days after oral challenge harbored ~60 ng/g of PrPSc and prion titers of ~106.6 ID50 units/g. The excretion of infectious prions continued at lower levels throughout the asymptomatic phase of the incubation period, most likely by shedding prions from infected Peyer’s patches. Our findings suggest that horizontal transmission among herbivores may occur through the consumption of feces or foodstuff tainted with prions from feces of CWD-infected cervids and scrapie-infected sheep.

Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrPC) to a misfolded, pathogenic isoform (PrPSc). Prion inoculation experiments in mice expressing homologous PrPC molecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrPC, our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.

IntroductionParkinson’s disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases that are characterized by the intracellular accumulation of alpha-synuclein containing aggregates. Recent increasing evidence suggests that Parkinson’s disease and MSA pathology spread throughout the nervous system in a spatiotemporal fashion, possibly by prion-like propagation of alpha-synuclein positive aggregates between synaptically connected areas. Concurrently, intracerebral injection of pathological alpha-synuclein into transgenic mice overexpressing human wild-type alpha-synuclein, or human alpha-synuclein with the familial A53T mutation, or into wild-type mice causes spreading of alpha-synuclein pathology in the CNS. Considering that wild-type mice naturally also express a threonine at codon 53 of alpha-synuclein, it has remained unclear whether human wild-type alpha-synuclein alone, in the absence of endogenously expressed mouse alpha-synuclein, would support a similar propagation of alpha-synuclein pathology in vivo.ResultsHere we show that brain extracts from two patients with MSA and two patients with probable incidental Lewy body disease (iLBD) but not phosphate-buffered saline induce prion-like spreading of pathological alpha-synuclein after intrastriatal injection into mice expressing human wild-type alpha-synuclein. Mice were sacrificed at 3, 6, and 9 months post injection and analyzed neuropathologically and biochemically. Mice injected with brain extracts from patients with MSA or probable iLBD both accumulated intraneuronal inclusion bodies, which stained positive for phosphorylated alpha-synuclein and appeared predominantly within the injected brain hemisphere after 6 months. After 9 months these intraneuronal inclusion bodies had spread to the contralateral hemisphere and more rostral and caudal areas. Biochemical analysis showed that brains of mice injected with brain extracts from patients with MSA and probable iLBD contained hyperphosphorylated alpha-synuclein that also seeded aggregation of recombinant human wild-type alpha-synuclein in a Thioflavin T binding assay.ConclusionsOur results indicate that human wild-type alpha-synuclein supports the prion-like spreading of alpha-synuclein pathology in the absence of endogenously expressed mouse alpha-synuclein in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0254-7) contains supplementary material, which is available to authorized users.

Leptin is produced predominantly in adipose tissue but has recently also been found in gastric mucosa. It has been shown that the oral application of leptin induces neuronal activity in the brain stem of rodents. The objective of the present study was to identify this hormone in human saliva and to examine the production and stability of salivary leptin. We have demonstrated production of leptin in salivary glands and oral mucosa by RT-PCR, its storage by immunocytochemistry, and the release of the peptide by RIA . Chromatographic analysis and immunoblotting confirmed the identity of leptin. There is a strong linear correlation (r 2 ‫؍‬ 0.78) between leptin concentrations from simultaneously collected saliva and plasma samples (n ‫؍‬ 61). Stimulation of saliva flow increases total leptin secretion up to 3-fold (P < 0.001). As to the stability of leptin in gastric fluid, we found the peptide was not degraded above pH 3. S INCE THE DISCOVERY of leptin in 1994, research activities have been directed at understanding the regulation and actions of this peptide hormone. Leptin is the product of the obese gene with a single-chain structure and a molecular mass of 16 kDa. It is produced by differentiated adipocytes (1, 2) as well as in the placenta (3, 4). More recently, storage and secretion of leptin has also been demonstrated in the stomach (5-7). The signal function of leptin on the central nervous system has been the major area of research (8 -12). Leptin influences food intake (e.g. by suppressing neuropeptide Y in the hypothalamus [11], and stimulates energy expenditure and thermogenesis (e.g., by interaction with the adrenal cortex [13,14]). However, specific receptors for leptin have been found ubiquitously in the body (e.g., thyroid gland, adrenal glands, lung, placenta, kidney, liver, and endothelial cells [15][16][17]). This suggests a peripheral role for leptin. Interestingly, a specific leptin receptor was recently identified in the gastric mucosa (6) and in other parts of the gastrointestinal tract (18). To date, the sources of leptin as a gastrointestinal receptor ligand have been only partially investigated.It was therefore an objective of the present study to identify and characterize the presence of leptin in saliva. A second aim was to investigate the regulation of leptin in the salivary glands and its stability under gastric conditions. Materials and Methods Sample materialPlasma and saliva were collected simultaneously from healthy adult males (n ϭ 23; age 28 -80 yr; body mass index [BMI] 20 -50 kg/m 2 ) and females (n ϭ 25; age 22-85 yr; BMI 19.5-54 kg/m 2 ) and healthy adolescents (male: n ϭ 13; female: n ϭ 5; age 10 -18 yr; BMI: 15-39.9 kg/m 2 ) recruited in our hospital, who gave informed consent of participation. None of the volunteers took medication or contraceptives and all participants had fasted for at least 2 h. The study was approved by the Ethics Review Board of the University of Erlangen.Plasma was collected with S-Monovettes, saliva was collected with the Salivette device (both Sarst...

ABSTRACTα-Synuclein is a soluble, cellular protein that in a number of neurodegenerative diseases, including Parkinson's 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.