Animal models may offer important insights into disease progression and potential treatments of prodromal Parkinson’s Disease (PD). Recent advancements in the development of both genetic and α-synuclein seeding models have driven discoveries specific to the prodromal phase of PD. Drs. Van Den Berge, Borghammer, and Yamakado provide a picture of the current state of the field.
Drs. Van Den Berge & Borghammer:
Since the discovery that decreased dopamine results in Parkinson’s Disease (PD) motor symptoms, many animal models have focused on recapitulating loss of dopamine in the substantia nigra with neurotoxins. This has led to insights into dopaminergic pathways in the brain as well as the development of levodopa and other symptomatic treatments for motor features. However, in recent decades, we have come to realize that PD is in fact a multi-system disorder affecting multiple neuronal systems in the brain and peripheral organs. Importantly, the peripheral nervous system (PNS) seems to be involved prior to the central nervous system (CNS) in many patients. This translates into a heterogeneous prodromal disease phase with a broad range of non-motor symptoms such as constipation, hyposmia, sleep disorders including REM sleep behavior disorder (RBD), and orthostatic hypotension. These prodromal symptoms may occur up to 20 years prior to the occurrence of motor symptoms (when > 50% of dopamine is lost in the brain). To date, there is still no cure for PD. Since the presence of non-motor symptoms in the early disease stage may provide a large window for intervention, we need comprehensive animal models that mimic peripheral pathology and prodromal symptoms as observed in human prodromal PD to identify new disease-modifying treatment targets.
What are the available animal models for constipation, hyposmia, sleep disorders, or other prodromal Parkinson’s symptoms?
Drs. Van Den Berge & Borghammer:
Prodromal PD symptoms may be linked to the accumulation of α-synuclein pathology in peripheral organs such as the gut (constipation), heart and autonomic ganglia (orthostatic hypotension), skin (dry skin or excessive sweating), or lower brainstem structures (sleep disorders). Therefore, modeling a certain prodromal PD symptom in animals can be done by induction of α-synuclein pathology in the associated organ or structure. The local induction of pathology can be achieved by administration of (1) recombinant α-synuclein seeds, (2) a neurotoxin, (3) overexpression of α-synuclein using viral vectors or (4) transgenic models, or a combination thereof. α-synuclein can spread trans-synaptically along the autonomic connectome, thereby affecting multiple organs1. Consequently, the animal usually shows a broad range of prodromal symptoms within some months after induction of α-synuclein pathology in a certain organ.
In 2010 and 2014, two seminal papers provided the first animal evidence for the plausibility of Braak’s gut-first hypothesis2, 3. Since then, several studies have seeded the upper gastrointestinal tract to model prodromal PD using animals with a focus on prodromal gut dysfunction1, 4, 5. In parallel, alternative possible initiation sites have been explored to model other non-motor symptoms. Targeted seeding of the autonomic ganglia in mice can mimic orthostatic hypotension, hyposmia, and constipation without the presence of motor symptoms6. And, targeted seeding of lower brainstem structures can model prodromal anxiety, depression, and RBD. More specifically, targeted seeding of the sublaterodorsal tegmental nucleus in the middle of the brainstem induced early RBD-like behavior with later depression, hyposmia, gut dysmotility and eventually motor dysfunction7.
Furthermore, bacterial artificial chromosome (BAC) transgenic rodents, which express human α-synuclein, exhibit prodromal symptoms, such as RBD-like dysfunction without atonia and hyposmia, and later dopaminergic degeneration8, 9. Similarly, BAC transgenic mice overexpressing a mutant A30P α-synuclein exhibit early accumulation of α-synuclein in the gut, associated with gut dysmotility and molecular dysregulations in the gut, prior to motor dysfunction10. Finally, a wide range of environmental toxins (such as rotenone2 or amyloid-producing bacteria11) have been used to induce enteric inflammation and to recapitulate clinical neuroinflammation and prodromal gut dysfunction12.
Many rodent models exhibit single non-motor symptoms of PD13, and these mice are useful in developing symptomatic therapies targeting them. However, there are few animal models in which multiple non-motor symptoms precede dopaminergic (DA) neuronal loss or motor symptoms and are accompanied by α-synuclein aggregation, as is observed in PD cases. One of the most successful models is mice injected with α-synuclein preformed fibrils (PFFs) to the gastrointestinal (GI) tract. They exhibit GI dysfunction, followed by increased anxiety and DA neuronal loss, although olfactory dysfunction appears later5. Mice injected with PFFs into the RBD-responsible region also show RBD-like behaviors followed by decreased olfaction, GI dysfunction, and motor deficits7. In a genetic model, bacterial artificial chromosome (BAC) transgenic mice expressing human α-synuclein under its native promoter show hyposmia and RBD, followed by DA neuronal loss8. A unique model is the VMAT2 deficiency model, which shows increased α-synuclein aggregation and progressive DA neuronal loss along with decreased olfaction and increased anxiety14.
What have we learned from animal models?
Drs. Van Den Berge & Borghammer:
These novel models, which aim to reproduce prodromal PD, address molecular and cellular mechanisms involved in the initiation of pathology and spreading to specific brain regions and organs. It is important to note that modeling prodromal PD is not absolute and overlapping phenotypes across different PD models occur throughout disease progression. Nevertheless, the temporal pattern of appearance of prodromal symptoms seems to be associated with the site of α-synuclein pathology induction. For example, disease initiation in the gut will lead to gut dysfunction prior to other autonomic disturbances and vice versa when disease is initiated in the autonomic ganglia. Furthermore, all peripheral seeding models do appear to induce similar distribution of pathology in the CNS regardless of their peripheral initiation site. This is not surprising since body-to-brain propagation of pathology is trafficked along parasympathetic and sympathetic nerves that enter the brain bilaterally. Upon bilateral invasion of pathology into the lower brainstem structures, the pathology further progresses bilaterally in the brain in a predictable fashion (Braak’s stages of PD). In contrast, models with unilateral disease initiation in the brain are characterized by a predominant unilateral spread of pathology and neurodegeneration. The pattern of pathology in those models is dependent on the brain structure where pathology was initiated12. These observations may shed light into the hitherto unexplained conundrum of asymmetric motor symptoms in PD15. In short, if the first pathology in some patients starts in one hemisphere and then spreads according to the neuronal network, the resulting asymmetry in distribution of pathology will lead to initial asymmetric loss of dopamine cells and therefore asymmetric motor symptoms. Finally, controlled experimental conditions in rodent models have enabled investigations on the causality and interactions of pathology trigger factors such as the gut microbiome, intestinal permeability and inflammation12.
The phenotype of animal models simply depends on the injection site in the α-synuclein propagation model or viral model, and on the promoter used in the genetic model. To faithfully reflect the entire natural history of human PD, including the prodromal phase, it is necessary to understand the pathogenesis and reproduce it accurately. Such a model, which is not yet available, would answer the following questions:
- Can interventions for prodromal symptoms, particularly for GI or sleep dysfunction, alter the natural course of the disease?
- What role do inflammation and immune system activation, which are believed to do harm in the symptomatic phase, play in the prodromal phase?
- Can therapeutics that failed in previous preclinical or clinical trials be effective if administered in the prodromal phase?
What are we failing to model adequately?
Dr. Van Den Berge & Dr. Borghammer:
The traditional dopamine lesion models focus on one single PD feature, namely the dopamine depletion and associated motor symptoms at late disease-stage. Thanks to the knowledge gained from the wealth of dopamine lesion studies, we are now able to treat PD motor symptoms quite well. However, if we want to find a cure for PD, we should focus on deciphering the multi-factorial complexity of PD at the early prodromal disease stage. In this stage, the dopamine system shows minimal or no damage and early intervention could delay, or, ideally, stop further disease progression. In the past decade, we made a lot of progress recapitulating human prodromal PD with cardinal disease features that are not represented in the traditional dopamine lesion models, such as α-synuclein pathology initiation and spreading, multi-organ involvement, and a pre-motor disease phenotype. These novel prodromal PD animal models have provided an excellent platform to explore the body-brain link in PD pathogenesis from a causal and mechanistic point of view. However, fundamental disease aspects remain to be addressed for a more accurate recapitulation of human PD:
- First, prodromal symptoms seem to occur in a specific order in human PD, which may depend on the disease initiation site15, 16. Striatal and intramuscular seeding have been popular to investigate PD using animal models, however, those targets are likely not representative of the initiation site in human PD. Future animal studies could explore more realistic disease initiation targets (such as the amygdala, olfactory bulb or gut) to model the sequential occurrence of prodromal symptoms as observed in human PD subtypes12, 15, 16.
- Second, age is the largest risk factor of PD. However, most studies use young animals, thereby ignoring the age-related failing of cellular systems and other mechanisms at play. Importantly, treatment validation studies directly comparing young and old animals have shown treatment is less effective in older animals. It is possible that the almost exclusive use of young animals in preclinical PD research may contribute to the continued disappointing results in translational treatment validation studies.
- Third, the inclusion of other disease contributing factors, such as mitochondrial dysfunction, calcium dysregulation, lysosomal dysfunction, gut microbiome, and inflammation need to be further explored in conjunction with α-synuclein propagation models to unravel the interactions between these many potentially disease-causing factors. It seems increasingly likely that a personalized “cocktail” of drugs targeting different pathogenic mechanisms simultaneously may be needed to achieve the goal of disease-modification in PD.
The entire natural history of PD, from the prodromal phase to the symptomatic phase in which motor symptoms appear, has not been fully reproduced. Genetic models focus on the onset of the disease, but rarely lead to DA neuron death and motor symptoms. In contrast, some of the α-synuclein propagation models show robust propagation and DA neuron death, but skip the most important initial pathological event, i.e., the formation of α-synuclein aggregates. To fill the gap in these models, we should address the following questions:
- What environmental or genetic factors produce the α-synuclein aggregates with high seeding activity, as in human PD?
- What drives the disease progression in the prodromal phase?
- Prodromal PD models have advanced significantly in recent years leading to new insights and understanding but there are still many outstanding questions.
- New models leverage α-synuclein seeding and genetics approaches to replicate prodromal symptoms similar to those seen in humans.
- These models have helped connect the timeline of pathology to the disease progression.
- Further efforts are necessary to fully mirror the motor symptom progression, realistic disease initiation sites, the risk factor of age, and other disease contributing factors.
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