Early Diagnosis of Parkinson’s Disease: Utility of Animal Models

Abstract

The effectiveness of the treatment strategies for Parkinson’s disease (PD) is highly dependent on the time of therapeutic intervention. This makes early diagnosis of PD an essential factor for its treatment; however, the complexities of the symptoms make it difficult to diagnose at an early stage. Moreover, by the time the symptoms start to appear, the disease has already been propagated in the patients. Even for the researchers, it is difficult to understand the important early diagnostic biomarkers due to the unavailability of the patients at the early stage, that is, before the manifestation of visible symptoms. The solution to this problem appears to develop animal models and monitor them from the early days to discover the diagnostic biomarkers. In this chapter, we shall discuss the use of animal models in the research intended to discover early diagnostic biomarkers for PD and why it is important to use animal models.

1. Introduction

The number of Parkinson’s disease (PD) cases per year is rising worldwide. In the coming two decades, it is estimated to outpace Alzheimer’s disease in terms of casualties [1]. The economic burden of PD is also very high, as it can be understood by the fact that the US alone spends more than $50 billion annually on PD [2]. The disease goes unnoticed in the earlier stages as neurons are highly arborized and redundant; therefore, when degeneration of dopaminergic neurons starts, other neurons compensate for this loss. Consequently, motor symptoms appear when nearly half of the dopaminergic neurons have degenerated [2, 3]. PD’s effects on the central nervous system are prolonged, and the neuronal damage cannot be reversed, making the disease’s symptoms and progression inexorable. By the time PD is diagnosed, individuals have difficulty coordinating their bodies due to tremors, bradykinesia, stiffened limbs or trunk, and poor balance. Also, moving, speaking, swallowing, and other ordinary functions can become problematic as the symptoms progress. Apart from obvious motor-related symptoms, there are a few non-motor symptoms such as behavioral changes, sleep-related disorders, cognitive impairment, constipation etc. that severely impact the healthy well-being of the individuals. Interestingly, some non-motor symptoms such as hyposmia, REM sleep behavior disorder (RBD), constipation, etc., follow motor symptoms over many years [4, 5, 6, 7, 8]. The multifactorial nature of PD makes it difficult for clinical diagnosis as the symptoms and causes are not universal among all patients. Particularly early diagnosis of PD is challenging because the symptoms at an early stage overlap with other diseases and normal aging. There is a need to identify biomarkers that can be helpful in early and accurate diagnosis, which must be aimed to prevent PD progression. In this chapter, we first discuss the different aspects of PD diagnosis, followed by challenges in early diagnosis. We also discuss the existing animal models used in PD research. Later we shall focus on various diagnostic markers and the utility of animal models. We conclude by stating the importance of animal models in PD research intended to discover early diagnostic biomarkers.

2. Different aspects of PD diagnosis

There are different aspects to diagnose PD, such as (1) Non-motor symptom assessment, (2) Brain imaging, and (3) Molecular markers-based diagnosis—e.g., metabolome analysis, miRNA-based analysis, genome sequencing.

2.1 Non-motor symptoms assessment

It has been shown that a few non-motor symptoms appear before the inception of the motor symptoms, e.g., Smell loss or hyposmia is a common phenomenon in PD (75–95%), however, a study reports ∼25% of the normal population faces smell loss at later stages of life [9, 10, 11]. Constipation associated problems have been reported in PD, but only 15–20% of patients suffer from this problem [6, 7]. RBD is often reported in PD patients. It has been reported that ∼67% of patients with RBD complaints whose nigrostriatal dopaminergic system is damaged, develop PD within 4 years [4, 5]. Although these non-motor symptoms help in PD diagnosis, their accuracy is very low. As these symptoms are not exclusive to PD, there is a need to identify a set of important non-motor symptoms that can accurately predict PD condition.

2.2 Brain imaging

With advancements in radiology, one could think of having non-invasive imaging (MRI, PET etc.) techniques to identify degenerated regions in the brain, such as substantia nigra, but the depletion is gradual which will go undetected in the early stages. Moreover, performing MRIs are too expensive to be carried out for a healthy population. Despite that, if someone wants to deploy imaging for early diagnosis, then it is necessitated to build some machine learning models (e.g., Deep learning methods such as Convolution Neural Networks etc.) for analyzing the whole brain regions that can be used for early diagnosis and predict the disease condition better than the existing models [12].

2.3 Molecular markers-based diagnosis

Metabolome-based analysis has been thriving in the last decade, and it is now extensively used for diagnosing PD using various sample matrices, such as Cerebrospinal fluid metabolome, Blood metabolome, Tissue metabolome, Fecal metabolome, Urine metabolome. Although the initial metabolome results seem promising, it has some serious impediments to deal with, as the metabolome varies demographically and individually. Moreover, differences in genotype, presence of other diseases, lifestyle, diet, past medical records, and use of dissimilar tools and techniques for analysis enormously impact the results and conclusions. The reproducibility and validity of the results can be improved by standardizing the protocols, taking large samples, including various demographic populations in one study, and joint analysis with other methods. Among all, blood biomarkers are the most straightforward and cost-effective way of diagnosing a disease, but it does not seem to keep up to the mark in the case of PD. Heretofore, not a single biomarker has been found that can be employed universally in diagnosing the disease at the early stages. Many laboratories have conducted experiments relating to identification of circulating miRNAs, and they have come with a few novel miRNAs that are beneficial for early diagnosis of PD, but the results vary among the laboratories. This might be because of the difference in genotyping, symptoms, small sample size, demographic constraints, implementation of different protocols etc. Since miRNA can be collected aptly, if we improvise our approach, we can expect beneficial results. There is a dearth of rigorous standardization of the techniques, and one must address the above-mentioned challenges to improve the outcome. Nevertheless, different body fluids biomarkers such as α-synuclein aggregation or the formation of toxic tau isoforms are also considered hallmarks in PD. But they are deposited at the later stages of PD; therefore, they cannot be detected in the early stages and hence are not helpful for early diagnosis. There is a need to identify a biomarker that can be detected in the early stages of the disease. Scientists also contemplate a few miRNAs found in CSF as potential biomarkers, but sampling CSF is cumbersome and costly, and it also may lead to some untoward circumstances. Apart from CSF, it is required to identify miRNA from other body fluids, which can be collected easily and help diagnose the disease early [13, 14].

Few researchers have come up with machine learning (ML) models using various features to predict the disease condition. Several models focused on pre-motor symptoms of the disease and can predict the disease condition with acceptable accuracy. There is a need to tune the models with more data and play with other parameters and features, deploy other ML algorithms to improve the accuracy of the disease early diagnosis [15].

3. Challenges in early diagnosis

PD begins much before the motor symptoms occur, also known as the Preclinical phase of the disease that may last several years. Symptoms of this phase include hyposmia, depression, anxiety, and sleep difficulties along with autonomic nervous system disorders such as digestion, breathing, salivation, bladder malfunction, excessive sweating, and sexual dysfunction [16]. Clinical PD begins with onset of motor symptoms such as resting tremors, bradykinesia, limb rigidity, and coordination issues. Symptoms usually proceed gradually, often starting on one side of the body, such as reduced one-sided arm swing and intermittent tremors. Cognitive impairment (a non-motor symptom) appears often after the motor symptoms [17]. Many patients with PD develop dementia, however the time frame varies significantly from one individual to another. Dementia is a primary reason for PD patients to enter long-term care facilities [18]. However, it has been observed that the onset of motor symptoms i.e. clinical PD starts much earlier than it can be diagnosed with current diagnostic criteria. Hitherto, no biomarkers have been found which will assist accurate diagnosis of these conceptual phases of pre-diagnostic PD with high sensitivity and specificity [19].

Furthermore, it should be noted that perception of a PD case on early diagnosis may be significantly influenced by their demographics, their family history, or a genetically identical state [20]. However, there is also the possibility of entering in disease-modifying therapy trials associated with gene targeting. Along with the fact that different mutations for PD have been reported, exhibiting a varying degree of penetrance in various populations. As a result, while some populations are susceptible to one mutation, the same mutation may not be active as a risk factor for the development or progression of the disease in other populations. Genetic testing of unselected PD cohorts revealed that up to 10% of cases with Glucocerebrosidase gene A (GBA) mutations had type 1 GD (Gaucher’s disease), which is known to dramatically enhance the risk of acquiring PD (and is considerably high in few populations, like Ashkenazi Jewish demography) [21, 22]. Leucine-rich repeat kinase 2 (LRRK2) gene mutations are present in a substantial number of PD patients in several groups, including Ashkenazi Jewish and Berber Arab communities [23, 24, 25]. Similarly, it has been discovered that Han Chinese people have a substantial association with GAK gene mutation [26].

Despite various studies, PD diagnosis and misclassification in routine clinical practice are frequent, with error rates between 15–24% [27, 28, 29]. Approximately 10% of cases that were diagnosed with PD by neurologists had alternate pathologies (like multiple system atrophy, tauopathies and progressive supranuclear palsy), despite the adoption of strict clinical diagnostic criteria [30]. A recent meta-analysis of 11 clinicopathological investigations revealed a shared accuracy in clinical diagnosis of PD cases of only 81% which is a reason why various forms of secondary parkinsonism and tremor diseases such as essential tremor, are frequently misdiagnosed as PD [31]. Early diagnostic separation of PD from atypical parkinsonian disorders presents the toughest challenge even for qualified professionals. Diseased defined by neuronal deposition of phosphorylated tau aggregates like tauopathies and progressive supranuclear palsy are among the parkinsonism disorders including multiple system atrophy which are pathologically characterized by presence of cytoplasmic glial inclusions formed due to aggregation of α-synuclein in oligodendrocytes. All these conditions can be exceedingly difficult to identify from one another and from PD in the early stages of the disease [32, 33]. According to clinicopathological research clinical diagnostic mistakes account for 7 to 35% of cases [34, 35, 36]. It is important to optimize the clinical biomarkers that can be used to differentiate between PD and these main subtypes of atypical degenerative parkinsonism.

To address this need, various cross-sectional case-control studies have strived to define the prognostic value of non-genetic, genetic risk and prodromal clinical factors (biomarkers identified from studies on animal models) to establish the probability of translation to clinically identified PD. Some of the studies which have been initiated to better characterize this using a variety of risk factors or markers and prodromal features are:

• The Parkinson’s Associated Risk Study (PARS) is a multicentre study in the United States that compares older individuals with and without hyposmia and involves annual physical exams and twice-yearly dopamine transporter SPECT scans [37].

• The Prospective Validation of Risk Factors for the Development of Parkinsonian Syndromes (PRIPS) study focused on early diagnosis of PD by analyzing risk factors such as age, male gender, family history, hyposmia, subtle motor impairment and enlarged substantia nigra hyperechogenicity. The study was performed on 1847 individuals from three European nations aged over 50 years [38].

• The Tübingen Evaluation of Risk Factors for Early Detection of Neurodegeneration (TREND) study highlight the prodromal markers by examining 698 individuals aged between 50 and 85 years with selected prodromal markers (SPM) with no neurodegeneration. Individuals with SPM show higher prevalence of various prodromal symptoms making SPMs as better diagnostic markers [39].

• The Parkinson’s Progression Markers Initiative (PPMI) is a global cohort, having at least 50 data collection sites, started back in 2010. It has now more than 4000 participants, the major involved countries include North America, Australia, Europe, and Israel. PPMI is interested in collecting various data that comprises of motor assessments, non-motor assessments, brain imaging, cognitive impairments, blood samples, genetic and various omics data which are publicly available. The main aim of PPMI includes finding novel biomarkers, new and better treatments for PD [40].

These cohorts offer valuable pre-diagnosis information from at-risk subjects who may experience PD in the course of study period or who have recently received a diagnosis. Studies based on these cohorts pay particular attention to the emergence of prodromal symptoms and epidemiologic traits based on biofluids or biopsies. However, a basic barrier in research on neurodegenerative diseases is the inability to characterize disease at molecular level and infer disease progression from these parameters [41]. Animal models are therefore necessary since they are the only way to directly and longitudinally analyze any disease-relevant tissue. Additionally, by standardizing and closely observing living conditions, they enable the assessment of environmental and behavioral influences on etiologically complicated disorders like PD.

In addition to identification of novel biomarkers for early and accurate diagnosis, better diagnostic methods are needed to identify PD earlier in the course of disease progression. By the time a patient exhibits clinical symptoms and is diagnosed with PD, neurons and autonomic nervous system functions have been lost. An earlier diagnosis may provide a therapeutic window to slow or alleviate PD before onset of motor deficits (Figure 1). Even with the varying methodologies, there is a clear indication with promising results which can detect cases with strong manifestation of “pre-diagnostic” PD via clinical, imaging, and other risk markers. As many of these cohorts mature, the numbers of “high-risk” individuals “converting” to established PD provide proof of concept and will assist to establish the optimum approach to “early” detection. We must redraft our strategy and amalgamate the above-mentioned approaches to early diagnose the disease with high accuracy, sensitivity, and specificity. If PD is detected at the early stages, disease progress can be slowed or at best can be halted, consequently reducing the economic burden and improve the quality of life.

Figure 1.

Parkinson’s disease symptoms during the course of a human’s life, both prodromal and clinical. Diagram illustrating the progression of life from early to old age and the related brain health curves in PD cases compared to the healthy condition. The distinctive motor phenotypes that show up in late stages of the disease are the basis for the current clinical diagnosis of PD. Contrary to the prodromal phase, which might last between 10 and 15 years, the length of the preclinical phase is unclear, as shown by the dotted arrow. A prodromal diagnosis of PD might be made by following altered molecular trajectories from preclinical to clinical stages, which would increase the positive effects of neuroprotective lifestyle modifications or available therapeutic alternatives.

Early PD diagnosis will be beneficial in many terms such as (i) Early diagnosis will give ample time to the patient, the caregivers, the family and the clinicians to understand the disease and to decide the course of treatment and plan their future goals accordingly, (ii) It will help the patient to modify their lifestyle such that progression of the symptoms can be halted, a few non-drug treatments might be a possibility in that situation, (iii) Early diagnosis will improve the chance of cure for the disease, (iv) The classic drugs will be more effective in the patients whose symptoms have been detected at the early stages (v) Early diagnosis will reduce the economic burden on both the patient and the state.

4. Animal models in Parkinson’s disease

The animal model of PD has been extensively used to study the pathophysiology of PD progression and design new therapeutic intervention. Several models have been developed by various methods in model animals (rodents, non-human primates and non-mammalians) to recapitulate clinical phenotypic features and parkinsonian manifestations (Table 1). Highly reproducible models have occurred in rodent such as rats and mice due to their short life of span, low maintenance, and easy handling. There are two ways to culminate the PD model in animals: one is the systemic injection of PD-inducing drugs and the local administration of the drug (intracerebral, intracerebroventricular, intrastriatal, Intra-SNpc. Etc.) with the use of the stereotaxic instrument.

Model	Treatment	Rodents		Non-human primates	Non-mammalian species			Advantages	Disadvantages	References (PMID)
		Rat	Mice	Monkey	Zebrafish	Drosophila	Nematode
Pharmacological model	Reserpine	✓	✓	✗	✗	✗	✗	Approx. 85% loss in DA neurons	No pathological characters	25203719, 26514557
	Haloperidol	✓	✓	✓	✓	✗	✗	Induce motor symptoms	No pathological characters	29634484, 25203719, 19940105
Neurotoxin model	6-OHDA	✓	✓	✓	✓	✗	✗	Behavioral studies	Special skill required, lack of lewy bodies	24333330, 28130746, 29809058
	MPTP	✓	✓	✓	✓	✗	✗	Mimics PD biochemical features	Reproducibility is difficult	29809058, 29515360, 28978077
Pesticide model	Rotenone	✓	✗	✗	✓	✓	✗	Replicate all biochemical hallmarks of PD	Mortality is high, other deleterious effects	26013581, 29209747, 29809058
	Paraquat	✓	✓	✗	✗	✗	✗	Selective for SNc dopaminergic neurons, leads to a 50% loss with multiple doses	High mortality rates	24483602, 20079141, 29809058
α-Synuclein model	α-Synuclein	✓	✓	✓	✗	✗	✓	Formation of lewy bodies, used for evaluation of neuroprotective stratergies	No DA neuron loss in SNpc	27658420, 25565982, 25954517
Genetic model	LRRK-2	✗	✓	✗	✗	✓	✗	Evalute role of LRRK-2 in PD, DA neurotoxicity	Nuclear abnormalities, No lewy odies	23799078, 24957201
	PINK1	✗	✓	✗	✗	✗	✗	No DA neurotoxicity	Lack of lewy bodies and neurodegeneration	25037286, 25954517
	PARKIN	✗	✓	✗	✗	✗	✓	Dose dependent DA cell death	No significant DA anormalities	20126261, 24423640
	DJ-1	✗	✓	✗	✗	✗	✗	Understand ubqiuitin protease system	Further evaluation required to support this model	23019375, 31484320

Table 1.

Advantages and disadvantages of various PD models in different animals.

Over the last decade, the advent of the genetic era of PD gives out phenomenal insight into the genetic model of PD. These models are solemnly based on mammalian and non-mammalian transgenic models that propagate disease-causing mutation considered to be a monogenetic form of familial PD. Neurotoxic, herbicides, and transgenic models have their characteristics and limitations, which must be taken very carefully chosen to be our model. Here, in particular, we discuss the neurotoxin-based model in rodent animals. There are many chemicals are being used in the development of PD such as MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) a prodrug that readily crosses the BBB due to its lipophilic nature. It is bound to complex 1 of electron transporter and reduces the conversion ATP from molecular oxygen, which consequently embarks the production of ROS, lipid peroxidation, irregular homeostasis in iron metabolism, and leads to cell death. MPTP has all hallmarks of PD in primates compared to non-primates. Therefore, MPTP could be the most suitable compound for PD development in Primates. Another, drug that has been used in the PD model is 6-hydroxy dopamine, it does not cross the BBB and consider non-systemic. So, it has to be injected directly into the SN region of the midbrain. Apart from the neurotoxin model herbicide and insecticide drugs are also used frequently in the development of PD such as Paraquat, and Rotenone. Transgenic models are also available in the form of monogenetic mutation. α-synuclein is abundantly expressed at 1% of total cytosolic brain protein. Overexpression of α-synuclein and its role in dopaminergic neurons has to be observed in the genetic model of PD. Mutation in α-synuclein, and LRRK2 leads to autosomal dominant disease while PINK, Parkin, and DJ1 are probably considered for Autosomal recessive disease.

4.1 Neurotoxin-based model in PD

The emergence of technology and advancement of biological research revealed the reduced levels of Dopamine in the striatum region of PD in human patients opened the window of research and treatment interventions. There are several models of PD in animals that have been characterized by the low amount of Dopamine in the SN region and striatum. Many neurotoxins and herbicide compounds ameliorate ATP production subsequently leading to cell death. Deprivation of dopamine-producing neurons is directly proportional to the less amount of dopamine in the SN region. This vital characteristic seems to be a cardinal hallmark of PD in the various animal models.

4.1.1 MPTP-based PD model

MPTP is a non-toxic, lipophilic compound that readily crosses the BBB. After entering the brain, especially in Astrocyte cells it metabolizes by monoamine oxidase B into MPP⁺ (1-methyl-4-phenylpyridinium ion). MPP⁺ enters in dopamine-producing neurons with the help of Dopamine transporter in substantia nigra pars compacta (SNpc) region. Active MPP+ binds to Complex1 of ETC and reduces ATP production [42]. This suggests that mitochondria are a preferential target of neurotoxicity. Administration of MPTP in primates through bilateral carotid injection causes L-DOPA responsive Parkinsonian syndrome characterized by all the clinical manifestations of PD which showed the best model of PD in Primates [43]. Intraperitoneally administration of MPTP in mice at certain doses (four MPTP every 2 hours. Over a day) gives the similar kind of lesions and phenotypic characters as in primates [44]. MPTP model has certain limitations such as, administration of MPTP in an animal model fails to mimic the progressive nature of PD [45]. Long term chronic treatments of MPTP may overcome this issue however, the smaller doses for long term resulted in the recovery of motor behavior deficit in animals when the treatment was discontinued. Another limitation of this model is that SNpc lesions are rarely accompanied by the formation of Lewy bodies [46].

4.1.2 6-Hydroxydopamine-based PD model

6-Hydroxydopamine (6-OHDA) is a chemical compound that is also known as oxidopamine or structurally known as 2, 4,5 trihydroxy phenethylamine is the first-ever generated PD model in animals [47]. This neurotoxin destroys the dopaminergic neurons in the SNpc region. 6-OHDA was a noble chemical compound that had a neurotoxic effect on catecholaminergic pathways [48]. Due to its lack of lipophilic nature 6-OHDA does not penetrate BBB hence it has to be directly administered stereotactically to a specific region of the brain such as SNpc or striatum. 6-OHDA effectively destroys the dopaminergic neurons in 12 hrs in the SNpc region while striatum-based neurons are conventionally lost within 2–3 days [49]. This kind of degeneration replicates the PD phenotypes. 6-OHDA agonistically binds to DA & NAT transporter respectively that facilitate to move inside of the cell where it auto-oxidizes in the cytoplasm, therefore, generating intracellular oxidative stress [50, 51]. 6-OHDA infused in neurons thus elevated cytotoxic molecules that are produced by an enzymatic and non-enzymatic process in which intrinsic trace element like Mg, and Fe is completely involved in cellular homeostasis [50, 52]. Moreover, 6OHDA generates H₂O₂ by the oxidation process in which it is highly toxic to cellular environments. Aside of its toxicity, it plays a vital role in free radicals’ formation, ROS species, and quinone intermediate products [53]. Dopamine is a neurotransmitter and is metabolized into 6-OHDA. It acts as a neurotoxin and therefore produces lesions in the nigrostriatal pathway. In spite of that, 6-OHDA does not promote protein aggregation of alpha-synuclein protein with other fibrils, thus Lewy neurites inclusion bodies are not produced in 6-OHDA-based animal models [49].

4.2 Herbicides-based PD model

Several reports suggest that farmers who are exposed to herbicides such as Rotenone and Paraquat suffered with symptoms similar to familial PD [54]. This finding suggests the role of this chemical in the development of the PD model in animals.

4.2.1 Paraquat-based PD model in animals

Unlike MPTP, Paraquat (PQ) does not cross BBB, but it has a similar structure to MPP+ (an active metabolite of MPTP). Due to its structural resemblance, it behaves like MPP+. PQ acts through the involvement of the redox cycle and subsequently induces oxidative stress. Therefore, it produced Reactive oxygen species, particularly: superoxide radical, peroxide, and hydrogen radicals that lead to damage to lipid molecules, protein, DNA, and RNA. A recent study proposed dilemmatic evidence on paraquat exposure in rats. One report mentioned arbitrary statements chronic systemic injection of paraquat in mice reduced the motor activity and subsequently loss of the tyrosine hydroxylase-positive striatal fibers and SNpc neurons. On the other hand, Cory-Slechta et al. reported that prolonged treatment of PQ does not have any effect on the nigrostriatal DA region in mice model [55].

4.2.2 Rotenone-based PD model in animals

Rotenone is a naturally occurring herbicide/insecticide. Its half-life is generally 3–5 days depending on the exposure to natural light. Like MPTP neurotoxin, Rotenone has similar chemical properties and crosses the BBB readily and uniformly inhibits the complex-1 of ETC [56] In this retro-aspect, MPTP inhibits dopaminergic neurons due to the dependence of the DAT transporter in dopaminergic neurons while Rotenone inhibits complex 1 selectively in the nigrostriatal dopaminergic pathway. Thus, Rotenone seems to recapitulate all kinds of PD hallmarks such as systemic complex 1 inhibition, inflammation, ubiquitin-α-synuclein aggregation in nigral cells that look like Lewy bodies in PD, oxidative stress, and GI problems [57]. Behaviorally, rotenone-treated rats have hypokinetic characteristics along with flexed posture similar to stooped posture in human PD patients. Few rats have severe rigidity and few have spontaneously shaken which is similar to a resting tremor. The existing beauty of this model is that like paraquat, it also introduced α-synuclein aggregation and Lewy body-like formation. The limitation of this model is that it does augment the DA oxidation but the evidence is narrow about the depletion of dopaminergic neurons in the nigrostriatal system [58].

4.3 Genetic model of PD

According to Cedric Bardy (2020), 85% of the PD population are sporadic and the remaining are familial PD [59]. Familial PD is generally based on genetic defects that are counted as autosomal dominant (AD) or autosomal recessive (AR). α-synuclein gene (SNCA) and LRRK2 are experimentally proven to be involved in AD in Parkinson’s Disease. α-synuclein is a small (14kD) protein, present abundantly in brain tissue, while a lesser tone of protein is present in the heart, muscle, and other tissue. Currently, its peculiar role is not clear but plays an important role in the membrane, vesicular dynamics, and intracellular trafficking within the ER/Golgi network. The identification of α-synuclein mutation was the first to be involved in familial PD thereafter many researchers try to overexpress in Drosophila and yeast resulting in that hampers the ER-Golgi network trafficking and toxic α-synuclein leads to neuropathology and amyloid aggregation in nigral cells which are the key features of familial PD [60, 61]. Mutations in α-synuclein create a high propensity for protein misfolding. α-synuclein exists in various structures including oligomers, protofibrils, fibrils, and, filaments. The amalgamation of filamentous and fibril structures seems to be a more toxic form [62]. Mashliah et al. (2000) developed the first-ever model using mutated SNCA (A53T, A30P, and E46K) and observed the inclusion kind of bodies in the hippocampus, SNpc, and neocortex region but they do not have any evidence of α-synuclein inclusion bodies like LB in human patients. Meanwhile, the same group had done another experiment to confirm the previous findings but unfortunately, the result was the same no dopaminergic neuron degeneration has been observed in mice [63]. Another group developed a double mutant (A30P, A53T) model in mice and apparent neurotrophy was reported. This key feature was retorted motor activity and promotes neuronal aggregates [63].

LRRK2 (leucine-rich repeat kinase 2) is a multidomain having 286 Kd protein. It is also known as dardarin and PARK8. One part of the dardarin protein that enriches the protein building block amino acid is known as leucine. LRRK2 is a large multimeric protein that is localized to an outer membranous structure. LRRK2 protein plays various roles in the cell but neuronal outgrowth and guidance [64]. Mutation in LRRK2 is associated with autosomal dominant PD with varying occurrence in the population [65]. The most common mutation is G2019S has a low frequency of 1% of sporadic PD patients while 4% of familial PD. The risk of PD in the person of LRRK2 G2019S is age-dependent: 28% at 59 years old, 51% at 69 years old 74% at 79 years old [66]. The two most important mutant model G2019S and R 1441C/G have failed to recapitulate the PD hallmarks. Przedborski, S.; et al., use BCA-R 1441C mutant mice to show motor deficit and axonal pathology in the striatum, however, loss of DA neurons in SNpc and alpha-synuclein is not seen clearly [67]. Another team developed the LRRK2 model using a viral vector-like Herp simplex virus (HSV) and an adenoviral vector. Transfection of G2019S is more effective than WTR1441C in stimulating neuronal pathology and Lewy body aggregation [68]. In Addition, infusion of HSV-LRRK2-G2019S in mouse striatum achieved 50% DA neuronal loss in the SN region [67, 69]. So, the LRRK2 model could provide a good platform to understand the neuropathology, mechanism of neuronal loss in the mid-brain region, and their function in PD.

5. Diagnostic biomarkers for PD

Despite decades of research, PD is currently diagnosed primarily on motor symptoms. Majority of dopaminergic neurons are degenerated by the time PD is confirmed, complicating treatment. Due to intersection of symptoms between PD and other atypical parkinsonian disorder the misdiagnosis rates by the clinicians for PD are quite high. Misdiagnosis and delayed diagnosis undermine disease-modifying therapy. Therefore, identification and quantification of biomarkers are vital for evaluating individual physiological and clinical responses, supporting therapeutic decisions, defining treatment and management programs, and managing causes of individual or group changes. Although motor and non-motor symptoms are visible clinically, the brain pathology in humans can only be established by evaluating post-mortem tissue samples or body fluids. The use of humans as a PD model for the identification of early diagnostic biomarkers is complicated by the fact that we do not know the time of PD onset, which may span between 10 and 15 years [18]. This can be circumvented by utilizing animal models, as we can monitor them from the moment of neurotoxic injection until the onset of symptoms, as well as identify the molecular alterations preceding the onset of symptoms. In addition, the identification of markers from non-invasive or minimally invasive techniques necessitates the use of animal models for early diagnostics. Consequently, there is a huge demand for experimental models to enhance our comprehension. To date, however, only a handful of putative biomarkers have been tested in clinical settings.

5.1 Non-invasive biomarkers

Two main techniques fluorodopa positron emission tomography (F-DOPA PET) and dopamine transporter single-photon emission computed tomography (DAT-SPECT) are used to measure the neurochemical differences dopamine system [70, 71]. Another technique called the susceptibility-weighted imaging (SWI) also works with high sensitivity and specificity on Nigrosome-1 (N1) cluster in differentiating PD from control and other non-PD parkinsonism [72]. An important prodromal PD marker is RBD which occurs at a high risk of 45% in early prodromal stages and 76% in late prodromal stages. As per a report on RBD cohort, 39.7% of RBD patients were found to develop PD or dementia with Lewy bodies. Another study on the same cohort, demonstrated the conversion of prodromal stages into PD with high sensitivity (81.3%) and a specificity (67.9%) [73, 74].

5.2 Invasive biomarkers

Biomarkers obtained via invasive technique are blood-based biomarkers which include α-synuclein, Extracellular Vesicles, miRNAs and inflammation related biomarkers. α-synuclein is a promising biomarker that is a key protein found in the Lewy bodies. Its malformation and aggregation due to both post translational modification and genetic factors in PD is a good indicator of PD pathogenesis [75]. This pathogenic protein is mainly transported from cells to cells through extracellular vesicles which makes them a candidate to use as a biomarker. A study performed by Majbour et al. using Oligomeric α-synuclein/total α-synuclein in CSF was not able to classified PD from DATATOP cohort whereas Oligomeric α-synuclein/total α-synuclein, phosphorylated was able to distinguish PD from healthy controls by sensitivity and specificity of 79% and 67% respectively [76]. Another promising candidate are the miRNAs, which are a class of non-coding RNA and the combination of different biomarkers can readily differentiate PD from healthy cases. For example, a study performed by using MiR-19a, miR-19b, miR-24, miR-30c, miR-34b, miR-133b, and miR-205 from CSF classified PD from control cases with AUC of 0.98 [77]. In various studies it been stated that inflammation is a major deriver of PD and certain kinds of cytokines like TNF α, IL-1, IL-4, IL-6, and IL-10 are highly expressed in PD patients. In a recent study IFN-γ, IL-10, and TNF-α obtained from blood serum distinguished patient with cognitive impairment, postural instability and PD have high expression levels than in the control samples [78]. In recent times, gut-inflammation related biomarkers have also been discovered which are found to be linked with severe constipation and motor phenotype which includes high expression of TRL4, CD3+ T cells, and cytokines in colonic biopsies of PD patients [79].

Combination of above mentioned biomarkers can increase the sensitivity of prediction accuracy. Developing a system to aggregate the diverse types and intensities of these biomarkers into a single set of criteria is difficult. Plasma aggregated α-synuclein and various ESWAN imaging indicators were integrated in the prediction model of a cohort research, and it was revealed that it has a sensitivity and specificity of 0.80 and 0.80, respectively, for predicting PD [80]. Analysis of age with the combination of CSF oligomeric/total α-synuclein ratio and β-glucocerebrosidase activity distinguished PD cases from non-dementia cases with 82% sensitivity and 71% specificity [81]. Matsusue et al. used combination of imaging methods, including NM-MRI and DAT-SPECT, demonstrated a good diagnosis accuracy with an AUC of 0. 935 [82].

6. Utility of animal models

As discussed in the earlier sections, PD is idiopathic with multiple genetic and environmental risk factors. In humans, motor symptoms are noticeable at advanced disease stages; the pathology starts much earlier than the diagnosing time. In these situations, animal models can play a vital role where we can induce such types of disease pathology and monitor the disease from the beginning. Motor and non-motor symptoms are easily detected, but brain pathology is only possible with post-mortem brain tissue. The heterogeneous nature of PD in terms of etiology & pathology demands a range of animal models [83]. Therefore, it is crucial to deepen our understanding through various experimental models to scale up the limited available treatment options. We need a diverse range of animal models to recapitulate different aspects of PD in humans. Thus studying PD with an appropriate animal model is very important to understand the biology of disease in every aspect. Humans share most of the genetic information with different animals. There is about 96% genetic similarity between a human and a chimpanzee and 90% between humans and rats. Mouse shares about 85% of their genome with humans regarding protein-coding genes, while fruit fly shares about 61% of genetic information with humans. The handling and ease of propagation of these small animals make them valuable research tools.

Three categories of PD models are used in research so far. This includes rodents, non-human primates, and non-mammalian species [84]. By 2018 the major percentage of animal models used for PD is a rodent (85%), followed by non-human primates (10%) and non-mammalian species (5%). In another study, out of 1851 papers screened for PD, 996 used a mouse model followed by 805 rat model. Others include Drosophila-43, C. elegans-14, non-human primates-69, Chinese Hamster-6, Yeast-17, E.coli-6, Zebrafish-24, and others 27 [85]. These animal models and their advantages and disadvantages have been discussed briefly in the Table 1. The array of animal models available today ranges from small worms (nematodes) to flies (drosophila) to rodents (mice and rats) and primates (monkey and chimpanzee). The worms and flies model can be used to study individual pathological pathways. Still, when it comes to getting closer to the relevance of the human disease features, we need to switch to higher-order animals like rodents (mice or rats) or Primates (chimpanzees, gorillas, orangutans, etc.) [86].

Three different modeling pathologies are most common in the case of PD. These include Nigrostriatal tract degeneration, outside the nigrostriatal tract neuron dysfunction, and Lewy body pathology. Here we have summarized the different models and their respective modeling pathologies along with other features like Mitochondrial Dysfunction, Oxidative stress, Motor deficit, Cognitive deficit, Autophagy, Proteasomal Dysfunction, Neuroinflammation, Response to L-DOPA, Sensory abnormalities, Somnolescent alterations, Psychiatric Changes and Organ system dysfunction based on the latest review by Joana Lama (Figure 2) [87].

Figure 2.

Occurrence of PD pathologies in different animal models.

Small animal models and rodents partially replicate human PD’s clinical and pathological features [88, 89]. In one of the studies, it has been reported that most transgenic rats show no dopaminergic neuron loss [90]. Rodent models have been widely used to model selective pathological pathways in PD like how the α-synuclein aggregate and spread, mitochondria damage and malfunction, faulty degradation of misfolded proteins, and immune system activation in PD state [91, 92]. As primates (chimpanzees, orangutans, etc.) are closer to humans than rodents, they classify as good models for identifying critical pathological events in humans than small animals. Higher-order animal models have played a key role in understanding PD so far [93]. In large animal models like the rhesus monkey, by expressing mutant α-synuclein in the fertilized embryos, the obtained progeny after 2.5 years show age-dependent non-motor symptoms like anxiety, cognitive defects, poor dexterity, and finger coordination [94]. Monkeys with stereotaxically injected Lentiviral vectors carrying A53T mutant (A53T α-synuclein) in substantia nigra at differing ages disclosed that aging is the major factor that promotes neuropathology in non-human primate brain [95]. Thus, large transgenic animals provide us with critical information regarding neuropathology and disease pathogenesis which is difficult to understand from rodent models. With the latest gene-editing technology like CRISPR Cas9, large animal models for PD, like non-human primates, can be easily created with a genetic mutation in one of the critical PD genes to understand the disease’s pathology better [93].

All the models discussed above have contributed significantly to understanding PD. But none of the models completely replicate PD in humans exactly. Neurotoxic models are an excellent candidate because they replicate the nigrostriatal neurodegeneration and motor dysfunction but lack proper Lewy body formation. While transgenic animal models show Lewy body pathogenesis, but lacks the loss of dopaminergic neurons [96]. Many mutation studies have been performed in cell lines, but those results seem inconsistent. After the discovery that reduced dopamine levels was responsible for the motor symptoms in PD, most of the animal models focused on mimicking this dopamine loss through the use of neurotoxins. These animal studies led to the discovery of pathways related to the DA loss in substantia nigra and L-Dopa drug development for PD related motor symptoms treatment. But as discussed earlier, PD is a multifactorial disease that affects both CNS and PNS along with multiple organs like the gut, heart, skin, etc., and symptoms affecting these organs called the prodromal symptoms that appear much before the visible motor symptoms. These prodromal symptoms include hyposmia, constipation, hypertension, and sleep disorders which results due to the accumulation of α-synuclein pathology in the gut, skin, heart and lower brain regions. The prodromal phase act as a golden opportunity; if we can recapitulate and model animals in a way that mimics these symptoms, we can identify novel disease targets and treatments.

After the Braak’s gut-first hypothesis [97], which states that the α-synuclein pathology first starts in the enteric nervous system decades before the motor symptoms start to appear and travels upto the brain stem and dopaminergic neurons through the vagus nerve, researchers started focusing on the gastrointestinal tract to model gut dysfunction in PD in animals [98, 99]. Other possible initiation sites have also been explored simultaneously for example targeting autonomic ganglia in mice mimics hyposmia, orthostatic hypotension and constipation without any motor symptoms [100] whereas targeting lower brainstem regions mimics RBD, depression, and anxiety [101]. Many transgenic rodent models that express α-synuclein pathology through BAC vectors show RBD-like dysfunction without atonia and hyposmia and loss of dopaminergic neurons later [102]. BAC-developed mice with A30P α-synuclein mutation show gut dysfunction before motor dysfunction. α-synuclein preformed fibrils (PFFs) injected in GI tract in mice is one of the most successful animal model since it recapitulates symptoms like gut dysfunction, anxiety, and Dopaminergic neuron loss [98]. PFFs injected in RBD-responsible region of mice also results in RBD-like behavior followed by decreased olfaction, GI dysfunction, and motor deficits [101]. VMAT-2 deficient animal models show α-synuclein aggregation and later DA neuron loss with increased anxiety and reduced olfaction [103]. These prodromal models have helped us gain insights into the cellular and molecular mechanism of PD initiation and progression, but it is not specific and limited. Thus, we see a plethora of symptoms overlapping in different animal models.

One of the key risk factor for PD is aging but majority of animal model used, are quite young which fail to relate to the cellular and molecular metabolism to this age. However certain studies compare treatment between young and old age animals, and have shown that the treatments are not so effective in old animals. The heterogenic nature of PD is seen in symptoms wherein some patients experience dementia much earlier than others. Pathological characters of PD include existence of α-synuclein Lewy bodies and loss of Lewy neurites and nigrostriatal dopaminergic pathway. But these pathologies are not restricted to only CNS but are spread outside CNS, which is extremely difficult to model in animals. Though the neurotoxin model has helped us study the dopaminergic system in PD, it is not similar to studying the complex pathology, temporal progression, and clinical expression seen in human PD. Likewise, overexpression studies of α-synuclein can explain its functions and other effects because of its overexpression in that part. However in PD cases with normal expression level of α-synuclein, the question remains unanswered through these models. Transgenic animal models are good in indicating about a particular gene or protein function, but that does not necessarily mean studying PD. Similarly, injecting α-synuclein PFF explains the seed pathology in that area and how it spreads, but the same pathology is seen is human PD is still not proved [104, 105]. Thus all these models failed to recapitulate the age of onset of disease, the spectrum of pathologies, and the temporal pattern of disorder similar to PD in humans. These models, as such cannot help us in understanding human PD’s core pathologies to treat the sporadic form of the disease. One such example is GDNF, which is used in rodents and non-human primates to recover the loss of dopaminergic neuron system but when tried in human PD patient was unsuccessful. It was also shown that in the α-synuclein PD model, this toxin protein interfere with the GDNF signaling pathway indicating the clinical efficacy of these models [106, 107]. Thus we can say that animals can be used to model only specific pathologies of PD but not the disease as it is. Since animal models and humans represent two different disease states, the therapies that work on animals do not necessarily work on humans.

Despite these limitations, animal models have not failed us. From the starting, these models have helped to develop previous and current drugs and treatments. For example, reserpine-treated rats and rabbits helped develop L-DOPA therapy, the rodent neurotoxin model helped develop dopamine-receptor antagonists, and MPTP-treated monkeys have paved the path of identifying sub-thalamic nucleus for deep brain stimulation therapy. This led to the fascinating first L-DOPA trial in human PD patients in 1961–1962 only in a window of 5 years of animal experimentation [108]. The prosecution was proposed based on three simple observations (i) a single shot of L-DOPA can reverse the sedative effect of reserpine in rats and mice [109], (ii)striatum harbors the highest amount of brain dopamine [110], and (iii) reduction of dopamine levels in caudate nucleus and putamen of Parkinsonian patients [111]. This happened much before the discovery of the neurotoxin PD animal model. It also led to the discovery of a variety of dopaminergic drugs and DBS (Deep Brain Stimulation). In 1997 α-synuclein pathogenesis was discovered, which enlisted PD in the category of protein misfolded disease. So accordingly, the disease modeling has also changed and adapted with time. Therefore, a single model no longer can serve the purpose, and even though the neurotoxin model is beneficial, it must be accompanied by models replicating the disease pathology and its progression. Appropriately using the currently available animal models can lead to new drug interventions. Though they are expensive and time-consuming, when it comes to the ethical background, only animal models can be used for preclinical trials. PD patient-derived stem cells and organoid culture are promising concepts, but they cannot replace the need for animal trials.

It is challenging to amalgamate all the complex biochemical pathways of PD in one animal model; therefore, we can use different models for different pathological aspects of PD. Thus the utility of animal models is indispensable for PD research. But there is no single model that can be used in all conditions. We need to choose the appropriate animal models according to our needs.

7. Conclusion

The early diagnosis of PD is still a major challenge as PD symptoms are very specific to human particularly in the later stage of the disease at which the PD is typically diagnosed in the patients. Diagnosis in early/prodromal stage is difficult due to the inaccessibility of patients. The use of` animal in PD research an also be debated considering the accuracy of the results in animal and human subject. Broadly we can categories the use of animal in PD research in two categories on the basis of purpose of the study i.e. either to develop new therapeutic interventions or to discover novel biomarkers for early diagnosis. Roger A. Barker and Anders Bjorklund (2020), discussed two sides of using animal model in PD. Barker discusses why the animal models are not useful and it is waste of resources on the other hand Anders Bjorklund explains the how the animal models are significantly useful and provide good insights for PD [112]. For therapeutic purpose the use of animal can be debated but for studies intended to discover early diagnostic marker the use of animal models appear to be the best choice. Despite of several failed attempt and the diversity of PD progression between animal models and human, cannot be the reason not use the animal models for further PD research particularly to identify early diagnostic markers. To discover the early diagnostic markers it necessary to have the case and control data from human subject. The major challenge in this step is that it is difficult to have early stage data from human subject because of inaccessibility of the PD patients at prodromal stage. PD is clinically diagnosed in human patients only when motor symptoms appear and has passed the prodromal stage nearly a decade before. Even if we collect the data from patients at prodromal stage it is difficult to say that they are going to show PD in future, which would again take years to develop. Also the symptoms at prodromal stage are very common to other ailments and overlap with the normal aging symptoms. For this reason using animal model has always been a best choice to study the early changes in the group of subject which are given a specific treatment to develop PD. The group of animals subjected to the PD induction can be monitored from very early stage and compared with the control group of similar age.

Acknowledgments

We acknowledge UGC-SAP to the Department of Biotechnology & Bioinformatics, School of Life Sciences, University of Hyderabad, for providing necessary support and infrastructure. We also acknowledge IoE Phase-II, University of Hyderabad (Grant no. 97020066) and DBT, India (No. BT/PR25787/GET/119/96/2017. We also acknowledge DBT, CSIR, UGC, and ICMR for provide fellowship to the NS, MZA, BK and MA respectively.

Conflict of interest

The authors declare no conflict of interest.

Acronyms and abbreviations

6-OHDA	6-Hydroxydopamine
α-synuclein	Alpha-synuclein
AD	Autosomal Dominant
AR	Autosomal Recessive
BBB	Blood-Brain Barrier
CNS	Central Nervous System
DAT	Dopamine Transporter
DBS	Deep Brain Stimulation
GBA	Glucocerebrosidase Gene A
GDNF	Glial cell line-Derived Neurotrophic Factor
LB	Lewy Body
L-DOPA	Levodopa and l-3,4-dihydroxyphenylalanine
MPTP	1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
NAT	Noradrenaline Transporter
PD	Parkinson’s disease
PFFs	Preformed Fibrils
PNS	Peripheral Nervous System
PQ	Paraquat
RBD	Rapid eye movement (REM) sleep Behavior Disorder
SNpc	Substantia Nigra pars compacta
VMAT2	Vesicular Monoamine Transporter 2