Colonoscopy and Subsequent Risk of Parkinson's Disease.

Background: Parkinson's disease (PD) is caused by the misfolding and aggregation of α-synuclein in neurons into toxic oligomers and fibrils that have prion-like properties allowing them to infect healthy neurons and to be transmitted to animal models of PD by injection or oral exposure. Given α-synuclein fibrils' potential transmission on the gut-brain axis, α-synuclein may be transmitted through colonoscopy procedures. Objective: This study examines a possible association between colonoscopy and PD. Methods: Longitudinal health insurance data of 250,000 individuals aged 50+ from 2004-2019 was analyzed. Cox proportional hazard and competing risk models with death as a competing event were estimated to calculate the risk of PD. Colonoscopy was categorized as never receiving colonoscopy, colorectal cancer (CRC) screening without or with biopsy, destruction or excision (BDE), and diagnostic colonoscopy without or with BDE. Results: We identified 6,422 new cases of PD among 221,582 individuals. The Cox model revealed a significantly increased risk of PD for patients who ever had a diagnostic colonoscopy without or with BDE (HR = 1.31; 95% CI: [1.23-1.40]; HR = 1.32 [1.22-1.42]) after adjustment for age and sex. After controlling for covariates and death, persons who ever underwent CRC screening had a 40% reduced risk of PD (CRHR = 0.60 [0.54-0.67]), while persons who underwent diagnostic colonoscopy had a 20% reduced risk of PD (CRHR = 0.81 [0.75-0.88]). Conclusions: Colonoscopy does not increase the risk of PD, after adjusting for death and covariates. Individuals who underwent only CRC screening had the lowest risk of PD, which may be a result of a more health-conscious lifestyle.

On-chip fabrication and in-flow 3D-printing of microgel constructs: from chip to scaffold materials in one integral process.

Bioprinting has evolved into a thriving technology for the fabrication of cell-laden scaffolds. Bioinks are the most critical component for bioprinting. Recently, microgels have been introduced as a very promising bioink, enabling cell protection and the control of the cellular microenvironment. However, the fabrication of the bioinks involves the microfluidic production of the microgels, with a subsequent multistep process to obtain the bioink, which so far has limited its application potential. Here we introduce a direct coupling of microfluidics and 3D-printing for the continuous microfluidic production of microgels with direct in-flow printing into stable scaffolds. The 3D-channel design of the microfluidic chip provides access to different hydrodynamic microdroplet formation regimes to cover a broad range of droplet and microgel diameters. After exiting a microtubing the produced microgels are hydrodynamically jammed into thin microgel filaments for direct 3D-printing into two- and three-dimensional scaffolds. The methodology enables the continuous on-chip encapsulation of cells into monodisperse microdroplets with subsequent in-flow cross-linking to produce cell-laden microgels. The method is demonstrated for different cross-linking methods and cell lines. This advancement will enable a direct coupling of microfluidics and 3D-bioprinting for scaffold fabrication.

Vaccination with structurally adapted fungal protein fibrils induces immunity to Parkinson's disease.

The pathological misfolding and aggregation of soluble α-synuclein into toxic oligomers and insoluble amyloid fibrils causes Parkinson's disease, a progressive age-related neurodegenerative disease for which there is no cure. HET-s is a soluble fungal protein that can form assembled amyloid fibrils in its prion state. We engineered HET-s(218-298) to form four different fibrillar vaccine candidates, each displaying a specific conformational epitope present on the surface of α-synuclein fibrils. Vaccination with these four vaccine candidates prolonged the survival of immunized TgM83+/- mice challenged with α-synuclein fibrils by 8% when injected into the brain to model brain-first Parkinson's disease or by 21% and 22% when injected into the peritoneum or gut wall, respectively, to model body-first Parkinson's disease. Antibodies from fully immunized mice recognized α-synuclein fibrils and brain homogenates from patients with Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Conformation-specific vaccines that mimic epitopes present only on the surface of pathological fibrils but not on soluble monomers, hold great promise for protection against Parkinson's disease, related synucleinopathies and other amyloidogenic protein misfolding disorders.

Sporadic Use of Antibiotics in Older Adults and the Risk of Dementia: A Nested Case-Control Study Based on German Health Claims Data.

BACKGROUND: Antibiotics for systemic use may increase the risk of neurodegeneration, yet antibiotic therapy may be able to halt or mitigate an episode of neurodegenerative decline. OBJECTIVE: To investigate the association of sporadic use of antibiotics and subsequent dementia risk (including Alzheimer's disease). METHODS: We used data from the largest public health insurance fund in Germany, the Allgemeine Ortskrankenkasse (AOK). Each of the 35,072 dementia cases aged 60 years and older with a new dementia diagnosis during the observation period from 2006 to 2018 was matched with two control-patients by age, sex, and time since 2006. We ran conditional logistic regression models for dementia risk in terms of odds ratios (OR) as a function of antibiotic use for the entire antibiotic group and for each antibiotic subgroup. We controlled for comorbidities, need for long-term care, hospitalizations, and nursing home placement. RESULTS: Antibiotic use was positively associated with dementia (OR = 1.18, 95% confidence interval (95% CI):1.14-1.22), which became negative after adjustment for comorbidities, at least one diagnosis of bacterial infection or disease, and covariates (OR = 0.93, 95% CI:0.90-0.96). Subgroups of antibiotics were also negatively associated with dementia after controlling for covariates: tetracyclines (OR = 0.94, 95% CI:0.90-0.98), beta-lactam antibacterials, penicillins (OR = 0.93, 95% CI:0.90-0.97), other beta-lactam antibacterials (OR = 0.92, 95% CI:0.88-0.95), macrolides, lincosamides, and streptogramins (OR = 0.88, 95% CI:0.85-0.92), and quinolone antibacterials (OR = 0.96, 95% CI:0.92-0.99). CONCLUSION: Our results suggest that there was a decreased likelihood of dementia for preceding antibiotic use. The benefits of antibiotics in reducing inflammation and thus the risk of dementia need to be carefully weighed against the increase in antibiotic resistance.

Patients with isolated REM-sleep behavior disorder have elevated levels of alpha-synuclein aggregates in stool.

Misfolded and aggregated α-synuclein is a neuropathological hallmark of Parkinson's disease (PD). Thus, α-synuclein aggregates are regarded as a biomarker for the development of diagnostic assays. Quantification of α-synuclein aggregates in body fluids is challenging, and requires highly sensitive and specific assays. Recent studies suggest that α-synuclein aggregates may be shed into stool. We used surface-based fluorescence intensity distribution analysis (sFIDA) to detect and quantify single particles of α-synuclein aggregates in stool of 94 PD patients, 72 isolated rapid eye movement sleep behavior disorder (iRBD) patients, and 51 healthy controls. We measured significantly elevated concentrations of α-synuclein aggregates in stool of iRBD patients versus those of controls (p = 0.024) or PD patients (p < 0.001). Our results show that α-synuclein aggregates are excreted in stool and can be measured using the sFIDA assay, which could support the diagnosis of prodromal synucleinopathies.

Stabilization of Monomeric Tau Protein by All D-Enantiomeric Peptide Ligands as Therapeutic Strategy for Alzheimer's Disease and Other Tauopathies.

Alzheimer's disease and other tauopathies are the world's leading causes of dementia and memory loss. These diseases are thought to be caused by the misfolding and aggregation of the intracellular tau protein, ultimately leading to neurodegeneration. The tau protein is involved in a multitude of different neurodegenerative diseases. During the onset of tauopathies, tau undergoes structural changes and posttranslational modifications and aggregates into amyloid fibrils that are able to spread with a prion-like behavior. Up to now, there is no therapeutic agent which effectively controls or reverses the disease. Most of the therapeutics that were developed and underwent clinical trials targeted misfolded or aggregated forms of tau. In the current manuscript, we present the selection and characterization of two all D-enantiomeric peptides that bind monomeric tau protein with a low nanomolar KD, stabilize tau in its monomeric intrinsically disordered conformation, and stop the conversion of monomers into aggregates. We show that the effect of the two all D-enantiomeric peptides is strong enough to stop ongoing tau aggregation in vitro and is able to significantly reduce tau fibril assembly in cell culture. Both compounds may serve as new lead components for the development of therapeutic agents against Alzheimer's disease and other tauopathies.

Quantitative detection of α-Synuclein and Tau oligomers and other aggregates by digital single particle counting.

The pathological hallmark of neurodegenerative diseases is the formation of toxic oligomers by proteins such as alpha-synuclein (aSyn) or microtubule-associated protein tau (Tau). Consequently, such oligomers are promising biomarker candidates for diagnostics as well as drug development. However, measuring oligomers and other aggregates in human biofluids is still challenging as extreme sensitivity and specificity are required. We previously developed surface-based fluorescence intensity distribution analysis (sFIDA) featuring single-particle sensitivity and absolute specificity for aggregates. In this work, we measured aSyn and Tau aggregate concentrations of 237 cerebrospinal fluid (CSF) samples from five cohorts: Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and a neurologically-normal control group. aSyn aggregate concentration discriminates PD and DLB patients from normal controls (sensitivity 73%, specificity 65%, area under the receiver operating curve (AUC) 0.68). Tau aggregates were significantly elevated in PSP patients compared to all other groups (sensitivity 87%, specificity 70%, AUC 0.76). Further, we found a tight correlation between aSyn and Tau aggregate titers among all patient cohorts (Pearson coefficient of correlation r = 0.81). Our results demonstrate that aSyn and Tau aggregate concentrations measured by sFIDA differentiate neurodegenerative disease diagnostic groups. Moreover, sFIDA-based Tau aggregate measurements might be particularly useful in distinguishing PSP from other parkinsonisms. Finally, our findings suggest that sFIDA can improve pre-clinical and clinical studies by identifying those individuals that will most likely respond to compounds designed to eliminate specific oligomers or to prevent their formation.

Ischemic stroke causes Parkinson's disease-like pathology and symptoms in transgenic mice overexpressing alpha-synuclein.

The etiology of Parkinson's disease is poorly understood and is most commonly associated with advancing age, genetic predisposition, or environmental toxins. Epidemiological findings suggest that patients have a higher risk of developing Parkinson's disease after ischemic stroke, but this potential causality lacks mechanistic evidence. We investigated the long-term effects of ischemic stroke on pathogenesis in hemizygous TgM83 mice, which express human α-synuclein with the familial A53T mutation without developing any neuropathology or signs of neurologic disease for more than 600 days. We induced transient focal ischemia by middle cerebral artery occlusion in 2-month-old TgM83+/- mice and monitored their behavior and health status for up to 360 days post surgery. Groups of mice were sacrificed at 14, 30, 90, 180, and 360 days after surgery for neuropathological analysis of their brains. Motor deficits first appeared 6 months after focal ischemia and worsened until 12 months afterward. Immunohistochemical analysis revealed ischemia-induced neuronal loss in the infarct region and astrogliosis and microgliosis indicative of an inflammatory response, which was most pronounced at 14 days post surgery. Infarct volume and inflammation gradually decreased in size and severity until 180 days post surgery. Surprisingly, neuronal loss and inflammation were increased again by 360 days post surgery. These changes were accompanied by a continuous increase in α-synuclein aggregation, its neuronal deposition, and a late loss of dopaminergic neurons in the substantia nigra, which we detected at 360 days post surgery. Control animals that underwent sham surgery without middle cerebral artery occlusion showed no signs of disease or neuropathology. Our results establish a mechanistic link between ischemic stroke and Parkinson's disease and provide an animal model for studying possible interventions.

Body-first Parkinson's disease and variant Creutzfeldt-Jakob disease - similar or different?

In several neurodegenerative disorders, proteins that typically exhibit an α-helical structure misfold into an amyloid conformation rich in ß-sheet content. Through a self-templating mechanism, these amyloids are able to induce additional protein misfolding, facilitating their propagation throughout the central nervous system. This disease mechanism was originally identified for the prion protein (PrP), which misfolds into PrPSc in a number of disorders, including variant Creutzfeldt-Jakob disease (vCJD) and bovine spongiform encephalopathy (BSE). More recently, the prion mechanism of disease was expanded to include other proteins that rely on this self-templating mechanism to cause progressive degeneration, including α-synuclein misfolding in Parkinson's disease (PD). Several studies now suggest that PD patients can be subcategorized based on where in the body misfolded α-synuclein originates, either the brain or the gut, similar to patients developing sporadic CJD or vCJD. In this review, we discuss the human and animal model data indicating that α-synuclein and PrPSc misfolding originates in the gut in body-first PD and vCJD, and summarize the data identifying the role of the autonomic nervous system in the gut-brain axis of both diseases.

Recurring Gastrointestinal Infections Increase the Risk of Dementia.

BACKGROUND: Gastrointestinal infections cause significant health problems, including those affecting the immune, musculoskeletal, and nervous system, and are one of the leading causes for death worldwide. Recent findings suggest that microbiota of the gastrointestinal tract contribute to dementia. OBJECTIVE: In this nested case-control study we investigated the role of common gastrointestinal infections on the subsequent risk of dementia. METHODS: We used a longitudinal sample of 202,806 individuals from health claims data of the largest German health insurer and applied a nested case-control design with 23,354 initial dementia cases between 2006 and 2014 and 23,354 matched controls. We used conditional logistic regression to compute odds ratios (ORs) for dementia and corresponding 95%confidence intervals (CIs), adjusting for potential confounders. RESULTS: The risk of dementia was increased in patients with recurring incidences of quarters with diagnosed gastrointestinal infections when compared to the unexposed population (one quarter: OR = 1.49, 95%CI = 1.40-1.58; two quarters: OR = 1.70, 95%CI = 1.51-1.91; three or more quarters: OR = 1.64, 95%CI = 1.40-1.93), adjusted for potential confounders. CONCLUSION: Our findings suggest that recurring gastrointestinal infections are associated with an increased risk of subsequent dementia.

Ligand-Induced Stabilization of the Native Human Superoxide Dismutase 1.

A common characteristic of familial (fALS) and sporadic amyotrophic lateral sclerosis (sALS) is the accumulation of aberrant proteinaceous species in the motor neurons and spinal cord of ALS patients-including aggregates of the human superoxide dismutase 1 (hSOD1). hSOD1 is an enzyme that occurs as a stable dimeric protein with several post-translational modifications such as the formation of an intramolecular disulfide bond and the acquisition of metal cofactors that are essential for enzyme activity and further contribute to protein stability. Some mutations and/or destabilizing factors promote hSOD1 misfolding, causing neuronal death. Aggregates containing misfolded wild-type hSOD1 have been found in the spinal cords of sALS as well as in non-hSOD1 fALS patients, leading to the hypothesis that hSOD1 misfolding is a common part of the ALS pathomechanism. Therefore, stabilizing the native conformation of SOD1 may be a promising approach to prevent the formation of toxic hSOD1 species and thus ALS pathogenesis. Here, we present the 16-mer peptide S1VL-21 that interferes with hSOD1 aggregation. S1VL-21 was identified by phage display selection with the native conformation of hSOD1 as a target. Several methods such as microscale thermophoresis (MST) measurements, aggregation assays, and cell viability assays revealed that S1VL-21 has a micromolar binding affinity to native hSOD1 and considerably reduces the formation of hSOD1 aggregates. This present work therefore provides the first important data on a potential lead compound for hSOD1-related drug development for ALS therapy.

Ageing promotes pathological alpha-synuclein propagation and autonomic dysfunction in wild-type rats.

Neuronal aggregates of misfolded alpha-synuclein protein are found in the brain and periphery of patients with Parkinson's disease. Braak and colleagues have hypothesized that the initial formation of misfolded alpha-synuclein may start in the gut, and then spread to the brain via peripheral autonomic nerves hereby affecting several organs, including the heart and intestine. Age is considered the greatest risk factor for Parkinson's disease, but the effect of age on the formation of pathology and its propagation has not been studied in detail. We aimed to investigate whether propagation of alpha-synuclein pathology from the gut to the brain is more efficient in old versus young wild-type rats, upon gastrointestinal injection of aggregated alpha-synuclein. Our results demonstrate a robust age-dependent gut-to-brain and brain-to-gut spread of alpha-synuclein pathology along the sympathetic and parasympathetic nerves, resulting in age-dependent dysfunction of the heart and stomach, as observed in patients with Parkinson's disease. Moreover, alpha-synuclein pathology is more densely packed and resistant to enzymatic digestion in old rats, indicating an age-dependent maturation of alpha-synuclein aggregates. Our study is the first to provide a detailed investigation of alpha-synuclein pathology in several organs within one animal model, including the brain, skin, heart, intestine, spinal cord and autonomic ganglia. Taken together, our findings suggest that age is a crucial factor for alpha-synuclein aggregation and complete propagation to heart, stomach and skin, similar to patients. Given that age is the greatest risk factor for human Parkinson's disease, it seems likely that older experimental animals will yield the most relevant and reliable findings. These results have important implications for future research to optimize diagnostics and therapeutics in Parkinson's disease and other age-associated synucleinopathies. Increased emphasis should be placed on using aged animals in preclinical studies and to elucidate the nature of age-dependent interactions.

Bioluminescence Imaging of Neuroinflammation in a Mouse Model of Parkinson's Disease.

In Parkinson's disease (PD) and related disorders pathological alpha-synuclein has been discussed to propagate via a prion-like mechanism in the CNS. The application of exogenous alpha-synuclein fibrils via injection to animal models of PD has been shown to be a useful method to study prion-like propagation of pathological alpha-synuclein and of transmission pathways that play a critical role in recapitulating characteristics of synucleinopathies. Using bigenic mice expressing mutant human alpha-synuclein in neurons and firefly luciferase in astrocytes we showed that transmission via the tongue and the peritoneum represent entrance points for pathological alpha-synuclein to invade the CNS. Here we present a method to quantify astrogliosis by bioluminescence imaging in an animal model of PD. This method allows noninvasive tracking of the neuroinflammatory process that often precedes neurological signs of disease and represents an alternative to behavioral or histological and biochemical analysis to detect disease.

Oral and intravenous transmission of α-synuclein fibrils to mice.

Parkinson's disease and related disorders are neuropathologically characterized by cellular deposits of misfolded and aggregated α-synuclein in the CNS. Disease-associated α-synuclein adopts a conformation that causes it to form oligomers and fibrils, which have reduced solubility, become hyperphosphorylated, and contribute to the spatiotemporal spreading of pathology in the CNS. The infectious properties of disease-associated α-synuclein, e.g., by which peripheral route and with which efficiency it can be transmitted, are not fully understood. Here, we investigated the potential of α-synuclein fibrils to induce neurological disease in TgM83+/- mice expressing the A53T mutant of human α-synuclein after oral or intravenous challenge and compared it to intraperitoneal and intracerebral challenge. Oral challenge with 50 µg of α-synuclein fibrils caused neurological disease in two out of eight mice in 220 days and 350 days, and challenge with 500 µg in four out of eight mice in 384 ± 131 days, respectively. Intravenous challenge with 50 µg of α-synuclein fibrils led to disease in 208 ± 20 days in 10 out of 10 mice and was in duration comparable to intraperitoneal challenge with 50 µg of α-synuclein fibrils, which caused disease in 10 out of 10 mice in 202 ± 35 days. Ten out of 10 mice that were each intracerebrally challenged with 10 µg or 50 µg of α-synuclein fibrils developed disease in 156 ± 20 days and 133 ± 4 days, respectively. The CNS of diseased mice displayed aggregates of sarkosyl-insoluble and phosphorylated α-synuclein, which colocalized with ubiquitin and p62 and were accompanied by gliosis indicative of neuroinflammation. In contrast, none of the control mice that were challenged with bovine serum albumin via the same routes developed any neurological disease or neuropathology. These findings are important, because they show that α-synuclein fibrils can neuroinvade the CNS after a single oral or intravenous challenge and cause neuropathology and disease.

Evidence for bidirectional and trans-synaptic parasympathetic and sympathetic propagation of alpha-synuclein in rats.

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.

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.

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.

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils.

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 ±17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.