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.

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.

Alpha-synuclein aggregates are the hallmark pathology of Parkinson's disease, which can propagate in a stereotypical pattern along the brain-body axis. Parkinson's disease patients not only display heterogeneous symptoms but also show variable patterns of alpha-synuclein pathology and affected neuronal systems during the disease course, complicating early and accurate diagnosis. Emerging data from post-mortem and imaging studies strongly suggest that disease heterogeneity could, at least in part, be explained by variable disease onset site, i.e. brain or body. This has led to the recently hypothesized formulation of two Parkinson's disease-subtypes, a body-first subtype where pathogenic alpha-synuclein arises in the body and spreads to the brain, and a brain-first subtype where pathogenic alpha-synuclein arises in the brain and spreads to the body. From a preclinical perspective, several animal models have been adapted or developed to reproduce Parkinson's disease-like pathology in the brain or periphery aiming to address the site of disease onset. Here, we review the current rodent and primate models that aim to reproduce Parkinson's disease pathology development and spreading in the brain and/or body and discuss the value and shortcomings of these models for the development of potential future applications in clinical trials and personalized medicine.

Aging is the biggest risk factor for developing Parkinson's disease (PD), the second most common neurodegenerative disorder. Several animal models have been developed to explore the pathophysiology underlying neurodegeneration and the initiation and spread of alpha-synuclein-related PD pathology, and to investigate biomarkers and therapeutic strategies. However, bench-to-bedside translation of preclinical findings remains suboptimal and successful disease-modifying treatments remain to be discovered. Despite aging being the main risk factor for developing idiopathic PD, most studies employ young animals in their experimental set-up, hereby ignoring age-related cellular and molecular mechanisms at play. Consequently, studies in young animals may not be an accurate reflection of human PD, limiting translational outcomes. Recently, it has been shown that aged animals in PD research demonstrate a higher susceptibility to developing pathology and neurodegeneration, and present with a more disseminated and accelerated disease course, compared to young animals. Here we review recent advances in the investigation of the role of aging in preclinical PD research, including challenges related to aged animal models that are limiting widespread use. Overall, current findings indicate that the use of aged animals may be required to account for age-related interactions in PD pathophysiology. Thus, although the use of older animals has disadvantages, a model that better represents clinical disease within the elderly would be more beneficial in the long run, as it will increase translational value and minimize the risk of therapies failing during clinical studies. Furthermore, we provide recommendations to manage the challenges related to aged animal models.

Pathogenic alpha-synuclein (asyn) aggregates are a defining feature of neurodegenerative synucleinopathies, which include Parkinson's disease, Lewy body dementia, pure autonomic failure and multiple system atrophy. Early accurate differentiation between these synucleinopathies is challenging due to the highly heterogeneous clinical profile at early prodromal disease stages. Therefore, diagnosis is often made in late disease stages when a patient presents with a broad range of motor and non-motor symptoms easing the differentiation. Increasing data suggest the clinical heterogeneity seen in patients is explained by the presence of distinct asyn strains, which exhibit variable morphologies and pathological functions. Recently, asyn seed amplification assays (PMCA and RT-QuIC) and conformation-specific ligand assays have made promising progress in differentiating between synucleinopathies in prodromal and advanced disease stages. Importantly, the cellular environment is known to impact strain morphology. And, asyn aggregate pathology can propagate trans-synaptically along the brain-body axis, affecting multiple organs and propagating through multiple cell types. Here, we present our hypothesis that the changing cellular environments, an asyn seed may encounter during its brain-to-body or body-to-brain propagation, may influence the structure and thereby the function of the aggregate strains developing within the different cells. Additionally, we aim to review strain characteristics of the different synucleinopathies in clinical and preclinical studies. Future preclinical animal models of synucleinopathies should investigate if asyn strain morphology is altered during brain-to-body and body-to-brain spreading using these seeding amplification and conformation-specific assays. Such findings would greatly deepen our understanding of synucleinopathies and the potential link between strain and phenotypic variability, which may enable specific diagnosis of different synucleinopathies in the prodromal phase, creating a large therapeutic window with potential future applications in clinical trials and personalized therapeutics.

Alpha-synucleinopathies include Parkinson’s disease, dementia with Lewy bodies, pure autonomic failure and multiple system atrophy. These are all progressive neurodegenerative diseases that are characterized by pathological misfolding and accumulation of the protein alpha-synuclein (αsyn) in neurons, axons or glial cells in the brain, but also in other organs. The abnormal accumulation and propagation of pathogenic αsyn across the autonomic connectome is associated with progressive loss of neurons in the brain and peripheral organs, resulting in motor and non-motor symptoms. To date, no cure is available for synucleinopathies, and therapy is limited to symptomatic treatment of motor and non-motor symptoms upon diagnosis. Recent advances using passive immunization that target different αsyn structures show great potential to block disease progression in rodent studies of synucleinopathies. However, passive immunotherapy in clinical trials has been proven safe but less effective than in preclinical conditions. Here we review current achievements of passive immunotherapy in animal models of synucleinopathies. Furthermore, we propose new research strategies to increase translational outcome in patient studies, (1) by using antibodies against immature conformations of pathogenic αsyn (monomers, post-translationally modified monomers, oligomers and protofibrils) and (2) by focusing treatment on body-first synucleinopathies where damage in the brain is still limited and effective immunization could potentially stop disease progression by blocking the spread of pathogenic αsyn from peripheral organs to the brain.