Research Process of Parkinson’s Disease

Dopamine neuron damage in the substantia nigra (SN) of the midbrain is the main pathological characteristic of Parkinson’s disease (PD). Moreover, the most important pathological marker of PD, Lewy bodies, can also be found in many surviving cells. Lewy bodies are aggregated from α-synuclein, but α-synuclein is not detected in some PD patients’ SN.
The substantia nigra (SN) is named because of the accumulation of dopamine neurons in this area, and the dopamine neurons in this area are rich in neuromelanin, which makes this area darker in color. This area is part of the basal ganglia (BG), which is a sub-area of the middle brain. From the perspective of human or rodent brains, this area is very small, but it is of great importance for animal locomotion. Despite its small size, SN can be divided into two regions, the substantia nigra pars compacta (SNc) and substantia nigra pars reticulata (SNr), of which SNc is rich in dopamine neurons, and is also the hardest hit area of PD. SNr receives and processes the input signals from BG and other brain areas. The dopamine neurons in SNc are responsible for providing dopamine to the striatum according to the information from SNr, thus regulating movement. However, as the occurrence of PD is due to dopamine hyposecretion caused by the death of dopamine neurons in this area, this leads to obstacles in the movement process controlled by the thalamus .

Figure 2. Main lesion locations and pathological characteristics of PD. Note: the green zone represents the entire middle brain, while SN is just near the dotted line to which the arrow points. Source: Fundamental Neuroscience 2013；https://www.alztennessee.org

 

Drug and Therapy Development for Parkinson’s Disease (PD)

When Dr. Parkinson first discovered PD in 1817, he only found symptoms. Later anatomical studies found that the lesions in the SN to be the main cause of PD onset. In the 20th century, Lewy bodies formed by aggregation of pathogenesis-related proteins were discovered. After World War II, the development of science and technology laid the foundation for the discovery of dopamine. Subsequently, dopamine analogues began to be used in the treatment of PD. These drugs served the main purpose of strengthening dopamine function - some were proven effective while some others failed. Although they cannot cure the disease, the research has greatly deepened our qualitative understanding of dopamine, an important neurotransmitter.

To be clear, the common disadvantage of these dopamine analog drugs is that their effect will be greatly reduced after a period of use, and many of them showed significant side effects. Therefore, the researchers developed various therapies towards Lewy bodies and α-synuclein, a component of Lewy bodies. There are three main ideas: 1. Immunotherapy blocks the synapses and protein from diffusing; 2. Inhibits synuclein aggregation; 3. Enhances synuclein degradation and clearance. However, there is no current method working to produce effective drugs. In addition, there are innumerable links among the various signaling pathways in the brain. So, many researcher programs have begun to develop drugs targeting mitochondria, endoplasmic reticula, autophagy, and calcium ion signaling.

Gene therapy works primarily by using inactivated viral vectors to package neurotrophic factors or other important protein genes. In short, most FDA-approved PD drugs are still focused on regulation of dopamine function. In such case, gene therapy expresses the corresponding proteins to promote the recovery of dopamine synthesis and regulate the metabolism of dopamine, such as glial cell line-derived neurotrophic factors (GDNF) and neurturin (NRTN). However, the good results in animal experiments cannot be well reproduced in clinical trials. Despite this difficulty, there are still different gene therapy approaches in development.

There is a special, non-pharmacological treatment approach for PD, known as deep brain stimulation (DBS) therapy. Electrodes are inserted into the brain to stimulate the cerebral activity in the corresponding brain area to achieve the effect of relieving symptoms. However, this method is only effective for specific PD patients. With the development of DBS technology, this method has become increasingly effective, while the degree of invasiveness has gradually decreased, which has led to increasing acceptance by patients. However, the cost remains relatively high and the therapeutic benefit is only effective in improving symptoms – its efficacy is gradually lost as the disease progresses. Therefore, the development of drugs and therapies that effectively mitigate the pathogenic proteins and lesions of PD is still the top priority for treatment.

Figure 3. Development history of PD treatment
Source: 10.1038/nrd.2018.136

(1) Gene-related Animal Models of Parkinson’s Disease (PD)

An animal model is essential for investigating the mechanisms of PD. Here, we first introduce PD models made by gene editing methods. Since α-synuclein is the protein most directly related to PD onset, the development of corresponding transgenic mice is also the earliest such model. The corresponding gene of α-synuclein is called SNCA, also known as Park1. The three Park1 mutations of familial PD (A30P, A53T and E46K) and the abnormal increase in the copy number of this gene are the main causes of PD. The transgenic mice developed for the above-mentioned causes of PD have been widely used in experiments. In transgenic mice which express human mutant α-synuclein, some exhibit significantly reduced dopamine in the striatum, and in some strains there appear to be inclusion bodies similar to Lewy body precursors, and the symptoms of PD also appear. However, very few substantia nigra (SN) neurons are found dead in almost all gene-edited mice models targeting α-synuclein.

LRRK2 is a gene related to autophagy, which is associated with late-onset PD. However, it is difficult to obtain PD phenotypes in gene-edited animals that manipulate this gene alone, whether they are generated via overexpression, knockout, or knock-in, or in rat or mouse strains. Although LRRK2 knockout mice will not produce Lewy bodies, they can display α-synuclein aggregation to a certain extent, and some strains can also have movement disorders (dyskinesia).

PINK1 is another gene related to late-onset PD. The synuclein overexpression in the SNc of PINK1 knockout mice can cause the death of many dopamine neurons in this area. On the other hand, if the PINK1 gene is directly knocked out, the dopamine in the striatum will be reduced, but it will not cause the death of dopamine neurons. After E3 ligase PARKIN is knocked out in mice, there will not be any change except a slight decrease of dopamine expression in some mice.

DJ-1 is associated with early-onset PD, but the PD phenotypes in the human body cannot be reproduced in most of the related genetically modified mice. However, in a DJ-1 knockout mouse with C57 background, the researchers found an obvious loss of dopamine neurons in the early-onset SNc area - this change will increase with age and cause mild motor deficits. If the phenotype of the mouse can be repeated, this strain may also be a model worthy of using in PD research.

ATP13A2 is a lysosome-related ATPase. At present, there are very few studies on this gene, but it still has a great potential for PD research.

Figure 4. PD model-related genes
Source: 10.1016/j.cell.2015.01.019

 

Based on the PD research publications from 1990 to 2018, rats and mice account for 85% of the number of papers published. Especially, There has been rapid accumulation of studies based on mouse models, primarily because of relatively simple gene editing in mice. In recent years, with the widespread application and maturity of CRISPR gene editing technology, the gene-editing rat models of PD has also been significantly developed.

Although the gene editing PD model generally does not show obvious phenotypes, in this case, researchers can cross it with relevant genetic mutation or functional abnormality models. The addition of these externally introduced genes have effects on the various phenotypes of PD, so as to providing assistance for the research on the function and treatment of PD.

There are many genes related to familial PD. Cyagen has developed a series of PD models for these important genes related to pathology. You may find your desired models from our following resources:

1) Cyagen Knockout Catalog Models:

 

 

2) Custom Model Design

From strategy design through to delivery of research-ready custom models, Cyagen offers complete outsourcing for all your animal model needs.

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Surgery/Drug Induced Parkinson’s Disease (PD) Animal Model

As mentioned earlier, chemical induction plays an important role in PD modeling because it can induce a severe PD phenotype within a short period of time. However, a chemically induced PD model has an obvious disadvantage: dopamine neurons die at an excessively fast rate. In normal humans, it generally takes more than ten years from the death process of dopamine neurons to the onset of PD, while the number of neurons in a chemically induced PD model that have died in a few days reaches the number of neurons that have died in humans over the course of decades. Moreover, there is no appearance of Lewy bodies in the models with the most severe neuron and dopamine loss.

MPTP was the first chemical substance used to establish a PD model. Interestingly, MPTP is highly toxic to non-human primates (NHP) and mice, but much less toxic to rats. Whether in NHP or mice experiments, MPTP can cause a severe loss of SN neurons and serious reduction of striatal dopamine. The above-mentioned damage is quite a good simulation of this pathological phenotype of PD, but this substance cannot induce formation of Lewy bodies.

Compared with MPTP, 6-OHDA can be used for PD rat modeling. However, 6-OHDA cannot pass through the blood-brain barrier (BBB), so, in most cases, it is injected directly into the brain. A large number of neurons will die within two or three days after the injection, thus reducing more than 90% of the dopamine in NAc. Like MPTP, 6-OHDA has a specific effect on dopamine neurons, with mild injury to other types of neuron, but it does not induce the production of Lewy bodies.

The long-term use of rotenone can reduce dopamine in the substantia nigra (SN) striatum of rats, and can also produce many symptoms analogous to those in human PD. More importantly, rotenone can also induce neurons to produce an initial α-synuclein polymer that has the potential to become Lewy bodies.

Paraquat may not only reproduce some of motor symptoms of PD, but also has a significant inhibitory effect on SN dopamine neurons and striatal dopamine, and it can also induce the production of Lewy bodies.

In addition, METH (methamphetamine) and MDMA (methylenedioxymethamphetamine) are also substances often used for PD-related chemical modeling.

Figure 6. Chemically induced PD mouse models (Tara Spires-Jones and Shira Knafo, 2012)

 

Behavioral Experiment

The method used to assess movement and exercise ability can usually be used for the detection of PD. Figure 7 shows the mainstream movement testing methods.

1）Cylindrical barrel test: Mice are placed in a transparent cylinder. The mice put their forepaws on the cylindrical barrel wall to explore. Their gesture, forelimb preference and standing duration while touching indicate their movement and balance ability.

2）Autonomous motion roller: The autonomous roller wheel may be placed in a breeding cade or in a special experimental facility with the mice. It can be used to detect the initial movement state through the whole process of the animal's active movement and can study the animal's movement skills through the speed of the roller.

Normally, in order to estimate the degree of the lateral lesion and the effectiveness of subsequent interventions, a rotometer is used to measure the animals’ rotation bias after the use of a psychoactive or movement-stimulating drug.

The rotating rod in the rolling facility is a beam that can rotate at a fixed or accelerated speed. The mice are placed on the rotating rod in the middle, and their motor coordination can be measured in accordance with the incubation period of falling.

3）Balance beam test: The mice are placed on the beam, and their ability to move on the beam is considered to be a manifestation of their balance ability. The frequency and time of sideslips of the mice’s paws can be used to characterize their foot strength and motor coordination.

A gait instrument is used, or the mice’s paws are dipped in ink or paint so that they will leave a string of footprints when running down the corridor to the target box. The stride length, the base width, and the overlap between the forelimbs and hind paws can be measured to reveal gait indices.

In these experiments, the rolling facility is undoubtedly the most widely and easily used device for PD research. It is equivalent to the application of the Morris water maze in AD research.

Figure 7. PD-related behavioral research methods
Source: Tara Spires-Jones and Shira Knafo, 2012

Parkinson’s disease (PD) is primarily caused by the death of dopaminergic neurons in the SNc of the midbrain, which results in insufficient dopamine secretion in this area and affects the normal function of the downstream striatum. Therefore, the imaging studies of PD focus on the SN and striatum of the midbrain. The level of dopamine expression and secretion and whether the projection relationship between the two brain areas is smooth are key pathological indicators of PD. The genetic similarity between both mice/rats and humans is over 90%. Considering economic and experimental cycles, both mice and rats remain the ideal model species for PD research because the projection relationship between their brain areas are very similar to that of humans.

Figure 8. Main pathological characteristics of PD (macro)
Source: Somayaji. M & Sulzer. D 2017

 

In the brain anatomy of PD patients, it can be clearly found that the black neurons in the substantia nigra compact area (SNc) of the midbrain are reduced, accompanied by the death of these dopamine neurons, and the glial cells proliferate in large numbers instead of the original neurons. There are 30-60% fewer dopamine neurons in this area in PD patients than in the control subjects of the same age. From an intracellular perspective, Lewy bodies are the most important feature of PD. As can be seen from Figure 9A-C, the cells in SNc are filled with black Lewy bodies, and there are also Lewy bodies in the prefrontal area in Figure 9D, but less severe than in SNc. Also, the Lewy bodies in the cells are smaller and accompanied by pores. However, it is exceedingly difficult to simulate Lewy bodies in a mouse model. Therefore, in current animal experiments, we usually just need to detect the aggregation of α-synuclein in order to regard this type of animal as a model of PD.

Figure 9. Main pathological characteristics of PD
(Parkinson’s Disease Pathogenesis and Clinical Aspects, 2018)

 

In addition to animal model generation, Cyagen has established a range of supporting services, such as animal breeding, embryo/sperm cryopreservation, histology, and transcriptome profiling. Outsourcing these services to Cyagen is a more cost-effective way of conducting your research. All these services are performed by experienced specialists following standardized procedures, so we can guarantee delivery of high quality services and data acquisition with unbeatable price and turnaround.

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Signaling Pathways for Parkinson’s Disease (PD)

Mitochondrial integrity destruction and protein heterolocation exist in the PD signaling pathways. The latter is abnormal due to changes in the function of the ubiquitin-proteasome system (UPS) and autophagy-mediated degradation pathways.

Due to genetic factors, the combination of redox stress, energy deficiency, protein aggregation, and inflammation induced by mitochondrial DNA triggers the gradual accumulation of cellular metabolites, and eventually results in cell death – which manifests as impaired dopamine system pathways and the loss of some sensory neurons (e.g., olfactory neurons).

The mutation of α-nucleoprotein or the overexpression of SNCA can interfere with the aggregation and fusion of vesicles, thereby destroying the transport pathway from the endoplasmic reticulum (ER) to the Golgi apparatus. Similar mutations may also disrupt the release of synaptic vesicles. SNCA is a structurally unfolded protein, and its mutants tend to aggregate. It recruits other proteins into the polymer aggregate and may cover the aggregate and ubiquitinated cargo protein. All of this requires a functional E3 ubiquitin ligase, including Parkin.

The mutations in Parkin, UCHL1 and SNCA all interfere with ubiquitin protease system (UPS), which may cause stress response and neuronal apoptosis. Under normal circumstances, proteasome dysfunction triggers other cell repair mechanisms, such as activating chaperone-mediated autophagy (CMA), transferring abnormal proteins to the lysosome through the LAMP2a receptor. However, proteins such as mutated SNCA or LRRK2 with a gain-of-function mutation may cause CMA dysfunction, thus rendering it impossible to complete this repair process.

Certain mutated LRRK2 may also cause a pathological change in mitochondria, leading to increased degradation of mitochondria. In this process, the important proteins that maintain the survival of mitochondria are ubiquitinated. In this process, Pink1 binds to Parkin, which is further involved in the ubiquitination process of the outer membrane proteins (OMP) of damaged mitochondrial fragments, the ubiquitination of these important membrane proteins will eventually promote the degradation of mitochondria. In addition, LRRK2 is also a regulator of mitochondrial fission/fusion.

Both excessive division and mitochondrial dysfunction are related to the increase of reactive oxygen species (ROS). DJ-1 acts as a redox sensor and antioxidant in the mitochondria, responsible for maintaining mitochondrial energy balance and redox homeostasis. Other mutations associated with sporadic PD include many lysosomal-related enzymes, glucosylceramide synthase (GBA1)-galactosidase A (GLA), sphingomyelin phosphodiesterase (SMPD1), and Niemann Pick disease type 1 (NPC1) - they are also related to lysosomal-related diseases.

Changes in the above pathways are important phenotypic testing items in PD research and are often used to verify experimental causality. In the PD-related genes shown in the figure below, the white background means recessive, the red background means dominant, and gray background means PD risk genes.

Figure 10. PD signaling pathways
Source: 10.1242/dmm.039396

With more than 15 years’ experience in genetic engineering, Cyagen offers a complete range of services to support Parkinson’s  disease research, including: custom genetically engineered models, ready-to-use catalog models, drug development models, colony management, and phenotype analysis - allowing researchers worldwide to create, manage, and monitor models with ease.

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● TurboKnockout^® Gene Targeting Mice
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Ready-to-Use Knockout Catalog Mouse Models

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● Metabolic Disease Models
● CDX/Syngeneic Models
● Immunodeficient Mice

In addition to animal model generation, Cyagen has established a range of supporting services, such as animal breeding, embryo/sperm cryopreservation, histology, and transcriptome profiling. Outsourcing these services to Cyagen is a more cost-effective way of conducting your research. All these services are performed by experienced specialists following standardized procedures, so we can guarantee delivery of high quality services and data acquisition with unbeatable price and turnaround.

Animal Model Supporting Services

- Breeding & Model Management
- Cryopreservation & Cryorecovery
- Phenotype Analysis
- Surgical Model Services

Contact us with your project details to request your complimentary model generation project consultation, strategy, and quote.