The Innate Risk: An Overview of the Genetic Rodent Models for Studying
Parkinson’s Disease
Abstract
The monogenic forms of Parkinson’s disease have opened up gates for molecular pathway
studies of the disease. From the known Parkinson-related genes, SNCA, LRRK2, PRKN,
DJ-1, PINKDJ-1, VPS35, and GBA are the most commonly identified in PD patients within the clinical
background. Understanding how their pathological mutations contribute to the disease allows
for an apprehension of the disease at its prodromal state. It helps develop therapeutic strategies
to stop the progression of neurodegeneration and restore the damaged neurons.
A large number of animal models based on those genes have been developed. These models
possess a higher construct validity when compared to traditional neurotoxin models. This thesis
will provide an overview of the available rodent models based on those genes. The behavioral
and neurobiological phenotype induced by the models will be discussed to investigate their
similarity to the features of the disease as revealed in human studies.
Introduction
Parkinson’s disease (PD) is one of the most prevalent neurodegenerative diseases affecting older people. With an increasing prevalence with age among mid-age to senior people (Pringsheim et al. 2014), it strongly impacts the quality of life of affected individuals and poses a significant economic burden on the healthcare systems worldwide (Boland & Stacy., 2012). Traditional PD diagnosis in clinical backgrounds is based on the presence of medical conditions within the motor system known as Parkinsonism, focusing on 4-6 Hz resting tremor, combined with bradykinesia, rigidity, or posture instability (Hughs et al., 1992, Gelb et al., 1999). Apart from the cardinal symptoms, PD patients suffer from an extensive range of abnormalities covering both the motor and non-motor symptoms, involving gait deficiency, speech problems, olfactory disturbances, and cognitive impairment (Moustafa et al. 2016, Pfeiffer. 2015). Depression is also a common occurrence in PD patients. It is reported that around 40% to 50% of the individuals are suffering from depressive-related disease, jeopardizing their mental state. (Reijnders et al. 2008). The introduction of non-motor symptoms in PD research redefined the possibility of reviewing the disease as a multisystem complex.
The exact mechanism of PD remains unknown to this day. Apart from the well-known dysfunctional dopaminergic system, which results from a selective loss of neurons in the substantia nigra (SN), the presence of abnormal aggregations of the alpha synuclein protein within the neurons, known as the Lewy bodies (LB), is also a major neurobiological hallmark for the disease. Researchers have conducted a large variety of animal studies in pursuit of further understanding the disease. Literature within this field has provided invaluable insights that cannot be otherwise achieved in clinical background. The most popular animal models are based on neurotoxin injection, mainly including the 6-hydroxydopamine (6-OHDA) and 1-methyl-4-pheny1-1,2,3,6-tetrahydropyridine (MPTP). Those models are able to replicate the cardinal symptoms of PD in human by selectively destroy the dopaminergic neurons in the nigrostriatal pathway (Kin et al., 2019). The major drawback of the toxin-induced animal models, however, comes from their low construct validity. According to current research, the occurrence of PD characterizing motor symptoms is considered the aftermath of a significant loss of dopaminergic neurons. The estimation of the exact number of lost neurons covers from 31% to 70%, which poses the onset of the symptoms at a relatively late stage of clinical progression (Cheng et al. 2010). PD therapies targeting the motor symptoms, from physiotherapies to dopamine-targeted medications, are usually inadequate and limited in protecting and restoring the damaged neurons
(Jankovic, J., & Aguilar, L. G.). Both conditions emphasize the necessity of valid animal models that can be used to monitor the prodromal stage of the disease and develop restorative therapies.
Early diagnostic criteria for PD excluded the role of inheritance in the disease (Hughs et al. 1992). With the technical innovations in molecular biology and the newly identified genetic loci associated with PD, researchers nowadays tend to perceive the cause of the disease as a complex of both pre- and post-natal factors. A large number of animal models have been created by targeting the hereditary component of PD. These models have been accomplished via DNA engineering that allows for insertion, ablation, silencing of genetic sequences, as well as overexpression of human transgenes of the wild type genes and their pathogenic mutations. Compared to the neurotoxin models, genetic models are based on Parkinson-related genes identified in human, and therefore possess a sounder theoretical base, primarily when used to study the cause of familial PD. Rodents are commonly used for developing genetic models providing the controllable cost of research and short breeding cycle. They bare strong similarities to human regarding genetic sequence and neuroanatomical structures, which adds up its relevance within clinical background. With this review, the writer will provide a summary of available genetic/ transgenic models that have been developed on rodents, investigating the phenotypic themes phenotypic themes induced by the models. The models based on in vivo gene-editing techniques, e.g., the adeno-associated viral vectors, will not be discussed as they do not recapitulate the hereditary features the disease. The major findings of the studies, as well as usage for future research will be discussed.
Assessment of Available Genetic Models: From the Parkinson-related Genes
Throughout decades, rare mutations in over twenty genes have been reported as potential causes of PD. So far, seven genes have been convincingly associated with the Parkinson Disease, namely SNCA, PRKN, LRRK2, VPS35, PINK1, GBA, and DJ-1 (Bandres-Ciga et al. 2020), which have also made them common targets for manipulation in animal studies. These genes have been identified via genetic linkage studies conducted on the families with a PD inheritance (Karimi-Moghadam et al., 2018). An overall of around 13% of PD patients are reported to have a positive family history (Muñoz et al. 2001). In earlier attempts, researchers sought to distinguish the familial PD (fPD) from the sporadic form of the disease (sPD) by studying the clinical features and major symptoms in patients with or without diagnosed relatives. Those few studies manage to include subjects with different genomic profiles and ethnic backgrounds. The results of these studies have manifested an overall consistency, as no major differences have been identified regarding the core features of the disease, including the presence of asymmetric movement, the leading symptom, comorbidity, and the doses of medication applied. But contradictory results exist, with some indicating an early onset and slower progression of the disease in familial PD patients (See Table 1).
Within clinical background, the centrality of PD diagnosis is usually put on the motor symptoms. According to the latest published Movement Disorder Society Clinical Diagnostic Criteria for Parkinson's disease (MDS-PD criteria, Postuma et al., 2015), a valid diagnosis is based on the presence of bradykinesia (slow movement accompanied by speed or amplitude decline during the movement), combined with at least one symptom from 4-6Hz resting tremor and rigidity. For rodent models, akinesia and rigidity are usually studied as the characterizing motor symptoms, while resting tremor is not commonly targeted as an observable measurement due to the complications for recording (Asakawa et al. 2016). A wide range of behavioral tests have been developed to measure the motor abnormalities in rodents quantitatively. They measure different aspects of the motor system, including asymmetry, somatosensory neglect, reaching abilities, and ultrasonic vocalizations, which are sensible to nigrostriatal dysfunction (Flemning et al., 2013). Among those tests, the most commonly applied are the Rotarod (Rozas et al. 1997) and Open Field Test (Hall & Ballachey. 1932). The first records the time length of the tested animals being put on a rotating cylinder, which can be set at different speeds. The second monitors the length and walking speed of rodents in a free-moving state. A combination of the behavioral tests is used to determine the presence of a pathological motor phenotype.
Apart from the motor symptoms, the MDS-PD criteria have also outlined a series of non-motor symptoms (NMS) as guidelines for prodromal PD, which facilitate the early diagnosis of the disease within a research background. The eight NMS described in the criteria are: idiopathic disturbance of the Rapid Eye Movement (REM) during sleep, excessive daytime somnolence, olfactory dysfunction, depression, hypotension, constipation, dysregulated erectile system and urinary dysfunction (Berg et al. 2015). An increasing number of PD rodent studies investigate the NMS (See Table 2). The Morris Water Maze (Morris. 1984) measuring spatial memory and learning, has been used by many for the assessment of cognitive impairment. Neuropsychological tests, like Forced Swim Test (Porsolt et al.1977) and Light / Dark Test (Crawly & Goodwin., 1980), are commonly performed to assess the neuropsychological states of the rodents (Asakawa et al., 2016). For measuring the Enteric Nervous System (ENS) function, stool weight defecated within a defined period is used. With regards to olfactory function, different experimental designs have been developed. The state of hyposmia was determined by the time spent by rodents on sniffing provided odors or before they eventually found the source of the odor (Fleming et al. 2008, Kuo et al., 2009). REM disturbance in human is a powerful marker for prodromal PD, affecting 60% - 70% PD patients. Nevertheless, it is less frequent in rodent studies, mainly due to the challenging procedure to apply a polygraph recording in small animals (Oishi et al. 2016). Behaviorally, sleeping patterns in animal studies can be restored by its duration, quality, and continuity, combined with daytime records of food consumption, water intake and energy expenditure (Toth & Bhargava., 2013).
Neurobiological similarities matching the model to the disease make up for a large part of the face validity of the model. The two hallmarks, neurodegeneration of DA cells within the SN, and the aggregates of alpha-synuclein proteins in the brain, are closely associated with PD symptoms. The six-stage Braak theory (Braak et al., 2003) proposed a propagation of toxic alpha-synuclein assembly from the lower brain stem and olfactory system to pontine tegmentum before the formation of LB in the SN, and then further projects into the neocortex. The denervation of the nigrostriatal pathway and reduced dopamine (DA) level in the striatum contributes to the motor symptoms in PD patients (Dickson., 2012). The staging theory places the NMS onset before the emergence of motor symptoms and has been supported by epidemiological research (Taguchi et al., 2020). Critics of the theory instead, argue a complimentary position between these two features, rather than ascribing the DA degeneration to the exacerbation of Lewy pathology. This alternative position is backed up by evidence of PD patients who have LB formed in the neocortex, while their lower brain structures are spared (Rietdijk et al., 2017).
Extra evidence comes from the patients as carriers of LRRK2, PRKN and Pink1 pathological mutations, who may or may not develop Lewy body pathology in their brain (Schneider & Alcalay., 2017). Therefore, when evaluating animal models based on these genes, the presence of alpha-synuclein aggregations should be kept as a secondary assessing criterion.
Table 1: Studies that compare different aspects of clinical features of familial PD (fPD) vs sporadic PD (sPD). --: no major differences have been identified on the aspects examined in the study.
SNCA
Located on chromosome 4, SNCA is the first identified Parkinson-related gene, encoding
alpha-Literature Research Design Viewed Aspects Results
Carr et al., 2003 26 fPD vs 54 sPD, Caucasian, without SNCA or Parkin mutation
Sex; Age at onset; Duration; UPDRS Motor subsection; Asymmetry; dose of Levodopa; Tremor; Dyskinesia;
--Baba et al., 2006
40 fPD (from two families with SNCA or LRRK2 mutation) vs 1277 sPD, German and Greek origin
Distribution by sex; Initial Motor Symptom; Location of Initial Motor Symptom; Frequency of Asymmetric Motor Symptoms --Papapetrop oulos et al., 2007 50 fPD (without SNCA, LRRK2 and Parkin mutations) vs 50 sPD (age and sex
matched), Greek Origin
Presenting Symptoms; UPDRS score; Cognition; Medication (dose of L-DOPA, dopamine agonists, COMT ihibitor)
--Vibha et al. 2010
30 fPD vs 104 sPD, Indian origin
Distribution by sex; Age at onset; Duration; Initial Motor Symptom; Location of Initial Motor Symptom; Clinical subtype; UPDRS score; Cognition
In fPD patients: higher rate of female patients, younger age of onset, focal dystonia present at onset, longer latency between the occurrence of dyskinesia and start of levodopa therapy
Jukkarwala A. 2017
20 fPD vs 20 sPD, Indian origin
Age of onset; Duration; Clinical subtype; Comorbid Illness (diabetes, hypertension); Medication (L-DOPA equivalent dose, other medications)
--Inzelberg et
al. 2003 29 fPD vs 211 sPD
Age of onset; the duration of disease until stage III of Hoehn and Yahr (YST3), until
dementia (YDEM)
Early Onset and slower progression in fPD patients
synuclein, the main structural protein of Lewy bodies. In PD patients, pathological SNCA mutations fall into two main categories: missenses of nucleotides (point mutations), which lead to replacements of amino acids in the protein, and the duplication or triplication of the whole gene (Nuyetemans et al. 2010). Considered as a causative factor of early-onset PD, SNCA mutations have been found in both familial and sporadic PD patients. A30P, A53T, and E46K are the most studied point mutations of the SNCA gene (Konno et al. 2016). Functional SNCA proteins are present in the brain as monomeric. The pathogenic mechanism of SNCA mutations is ascribed to the presence of higher-order structures, including fibrils, protofibrils, and oligomers (Koprich et al., 2017). It is also proposed that pathological SNCA mutations abolish the gene's neuroprotective effect and promote apoptosis by disinhibiting the p53 tumor suppressor (Siddiqui et al., 2016). SNCA mutations are rare among the general population, with an estimated prevalence of less than 0.1% in the UK population (Blauwendraat et al. 2020b). Evidence indicates a minimal incidence of known pathological SNCA mutations in PD patients within the Chinese Han population (Deng et al. 2015). Recent genome-wide associations studies have identified additional risk loci located on the SNCA gene, with an unequal distribution among different populations (Zhang et al. 2018). The pathological phenotype of SNCA mutations is not fully penetrant. Some carriers, even as relatives of diagnosed patients, remain asymptomatic as measured at a very late stage of life (Blauwendraat et al. 2020b, Nishioka et al. 2009).
Homozygous SNCA KO mice (SNCA-/-) are among the earliest genetic models created for studying the
disease (Abeliovich et al. 2000, Cabin et al. 2002). SNCA-/- mice do not manifest any motor-related
abnormalities, nor do they perform differently in the Morris Water Maze Test or Light/Dark Test when compared to wild type mice (SNCA+/+). Reduced dopamine level is observed in the striatum, while
reports of amphetamine-induced locomotor responses are inconsistent. The SNCA-/- mice are widely
used in later studies as control groups or targets for bacterial artificial chromosome (BAC) transgenic models.
In addition, two lines of mice with conditional deactivation of the SNCA gene have been recently accomplished (Ninkina et al., 2015, Roman et al., 2017). These models are based on Cre-LoxP recombination, which allows selective turnoff of the gene after birth, as to rule out the possibility of recompensating mechanisms in prenatal KO models. Conditional SNCA-/- do not produce locomotor
abnormalities or DA reduction in adult mice, although a significant reduction of major dopamine metabolites was found in mice with late-onset (12 months) SNCA knockout (Ninkina et al. 2020).
Transgenic SNCA models over express human wild type (hWT) SNCA or its pathological mutations (hA53T, hA30P and hE46K). A summary of the main findings is provided in the work of Koprich and colleagues (2017). Different promoters, including the complementary DNAs and BAC implemented endogenous promoters, were used for transgene, as to turn on human SNCA in the pan-neuronal system or directly target the neurons within a small region, usually the midbrain area.
In general, pan-neuronal transgenic mice models are able to reproduce the progression of age-related motor impairment, but they fail to simulate the DA degeneration within the substantia nigra (See Table 2). However, despite the maintained number of DA neurons, some do reveal DA dysfunction in other forms within the nigrostriatal system, as characterized by reduced presynaptic dopamine release in response to electric stimuli, depletion of dopaminergic terminals, and lower number of DA receptors. Aggregates of alpha synucleins are reported in most of the studies. Some also report inclusions in the peripheral system as the olfactory bulb (Fleming et al., 2008). The NMS, including cognitive impairment (Freichel et al., 2007), olfactory dysfunction (Fleming et al., 2008), and disturbance in the enteric nervous system (Kuo et al., 2010), have also been identified. The onset of the NMS can occur before or after the emergence of motor symptoms.
Meanwhile, selective over-expression of SNCA hWT within dopaminergic neurons have discouraging results regarding both motor deficits (Richfield et al., 2002) and DA neurodegeneration (Matsuoka et al., 2001), indicating that this effect alone cannot account for the formation of PD. Several other studies manage to produce motor impairments and a pathological phenotype in the striatal system with selective over-expressing of a truncated SNCA gene (Wakamatsu et al., 2008) or double point mutations (Kilpeläinen et al., 2019). Mice with truncated SNCA have an early sign of DA neuron loss dating back to 2-month-old. The truncation of the gene accelerates the assembly of alpha-synuclein monomeric proteins into filaments (Tofaris et al. 2006). This early-onset DA degeneration in the truncated mice model suggests that the absence of DA neuron loss in SNCA transgenic mice models may be caused by the limited life span of the tested animals.
Additionally, there are several available SNCA rat models for research, although the number of studies based on these models is limited. Study of a BAC transgenic hWT rat model managed to generate a relevant PD-alike behavioral phenotype (Nuber et al., 2013). It is presented with olfactory deficit dating back to as early as 3-months-old and age-dependent impairment in locomotor activities aggravating from 12-month-old. By the time, there is around 30% of nigral DA neurons lost and the striatum DA
content is reduced with comparable amount. BAC transgenic hE46K rats (Cannon et al., 2013), instead, have no signs of reduced DA level but synuclein aggregates within the nigrostriatal pathway. Motor assessments are not included in the latter study.
To sum, transgenic overexpression of the human SNCA wild type or its pathological mutations is able to produce a PD-alike behavioral phenotype in rodents. However, this effect cannot be observed in animals with SNCA depletion or overexpressing the hWT SNCA restrictedly within midbrain DA neurons, which suggest that human SNCA mutations are probably working through a gain-of-function mechanism.
Table 2: Pan-neuronal SNCA transgenic mice models
Key Literature Motor Dysfunctions
DA Loss Reports of Non-Motor
Symptoms hWT Fleming et al., 2004 Amschl et al., 2013 Masliah et al., 2000 Janezic et al., 2013 Progressive motor impairment and deficit responding to sensory stimuli
No significant DA cell loss Reduced DA terminals as SN input to striatum (Amschl et al., 2013) Lower levels of electric evoked dopamine in dorsal striatum (Janezic et al., 2013)
Olfactory Dysfunction (Fleming et al., 2004)
Cognitive Impairment (Mandler et al., 2013)
hA53T Rothman et al., 2013 Lee et al., 2002 Kuo et al., 2010 Progressive motor impairment. Reports of severe dysfunction in the Prnp promoted phenotype leading to death (Lee et al., 2002). No reports of DA neurodegeneration Olfactory Dysfunction
Cognitive Impairment (Kuo et al., 2010)
Neuropsychological modifications,
Potential sleep disturbances (Rothman et al., 2013)
hA30P Freichel et al., 2006; Neumann el al, 2002; Yavich et al., 2005, Gomez-Isla et al., 2003; Progressive motor impairment
No DA cell loss in the SN or reduced DA content and its metabolites in the striatum
Lower level of stimulus-evoked dopamine release (Yavich et al. 2005)
Cognitive Impairment, Neuropsychological
modifications (Freichel et al., 2006)
hE46K Emmer et al., 2011 Progressive motor impairment
Not reported Not reported
LRRK2
Like SNCA, Leucine-rich repeat kinase 2 (LRRK2) is also linked to autosomal dominant PD. It is a widely distributed genetic factor contributing to both familial and sporadic PD (Li et al., 2014). The most common LRRK2 variant is Gly2019Ser, accounting for around 29% and 36% of PD patients in Ashkenazic Jews and North African Berbers (Tolosa et al. 2020). In east Asia, Gly2385Arg instead is known as the most menacing mutation (Bonifati. 2007). Other mutations that raise the risk of PD include A419V, R1441C/G/H, R1628P, as has been reported among various populations (Shu et al., 2019). The LRRK2 gene encodes a large protein containing four interacting regions with proteins and two disparate enzymatic domains (Berwick et al., 2019), which made it viable for an expansive range of genetic manipulations. A systematic review of studies conducted with LRRK2 rodent models is provided in the work of Seegobin and colleagues (2020). In short, knocking out the LRRK2 gene do not inflict PD-like locomotor disturbances in rodents. They have normal to better performances in tests measuring coordination, balance and gait. The DA neurons, dendrites and receptors are intact compared to their WT control groups at both early (Daher et al., 2014) or late (Hinkle et al., 2012) stage of life span. LRRK2 knockin models, which consist in the replacement of hereditary information at its primary locus on the genetic sequence, are used to introduce the human missense point mutations into rodents. These models produce normal to mild motor symptoms, with no signs of DA neuron loss and limited evidence supporting nigrostriatal dysfunction (Seegobin et al. 2020). Hyposmia, and reduced level of depression-like behavior in the Forced Swim have been reported in a knock-in model of the R1441C point mutation (Giesert et al. 2017).
For LRRK2 transgenic models, animals overexpressing PD-related human LRRK2 mutations (hG2019S, hR1441C, hI2020T) are able to generate a PD-alike pathological phenotype (See Table 3). Some develop age-dependent, L-dopa responsive motor symptoms, usually combined with mild to severe dysfunction within the nigrostriatal system, characterized by DA cell loss and/or metabolic dysfunction. The nigrostriatal DA degeneration can also be present without observable motor symptoms (Liu et al., 2015), which suggests that a compensating mechanism might be playing the role.
Nevertheless, the DA pathway are largely intact in transgenic hWT mice and rats. They also have shown no observable PD motor symptoms. Phenotypes produced by transgenic LRRK2 models are also under the influence of transgenic procedures applied. A hR1441C model using murine ROSA26 promoter fails to produce PD-alike symptoms behaviorally and neurobiologically (Tsika et al., 2014), although the same mutation leads to observable motor impairment in other studies using a different promoter (Ramonet et al. 2011., Weng et al., 2016).
Many LRRK2 transgenic models include experimental design evaluating alpha synuclein aggregation, most of them has failed to identify the presence of alpha synuclein aggregates (Chen et al. 2012., Maekawa et al., 2012, Melrose et al., 2010, Tsika et al., 2014). Xiong et al (2018) reported the presence of molecular weight species of alpha synuclein proteins in mice overexpressing hG2019S mutation within the catecholaminergic neurons. Only two of the models with a PD - alike phenotype have inspected the NMS: Lim et al. (2018) have reported depression / anxiety behavior in hG2019S mice at around 8-12 months of age, prior to the onset of motor symptoms; Maekawa and colleagues (2012) examined the olfactory function in hI2020T whole body transgenic mice, they have found no differences when compared with their wild type controls.
In short, rodent studies suggest that human pathogenic LRRK2 mutations are linked to PD via a gain of function mechanism. Compared to transgenic models, the knock in models produce mild motor symptoms, less evidence of neurodegeneration and early signs of non-motor symptoms, which made them a possible target for studying the prodromal state of PD. In addition, the absence of alpha-synuclein-related pathology, despite the presence of locomotor dysfunction and DA degeneration in LRRK2 animal models, adds up to the evidence supporting a synergistic role between the two hallmarks of the disease.
Table 3: Summary of LRRK2 Transgenic Models.
Literature DA Degeneration Motor Symptoms
hR1441C Mice (pan-neuronal expression)
Ramonet et al., 2011
No DA cell loss due to lack of expression in the SN, significant reduction of DA and its metabolites
in the cortex (Ramonet et al., 2011) Age-dependent, L-dopa responsive impairment of locomotor activity Weng et al.,
2016
Progressive DA cell loss in the SN, reduced DA terminals and impaired dopamine release in the striatum (Weng et al., 2016)
hR1441C Rats (BAC)
Sloan et al., 2016
No DA cell loss, normal level of DA content, metabolites and
transporters
Progressive impairment in evoked DA release within dorsal striatum
Age-dependent, L-dopa responsive motor impairment hG2019S Mice (pan-neuronal expression) Chen et al., 2012
Significant reduction of DA neurons within the SN.
Normal locomotor activity (Ramonet et al., 2011) Ramonet et al., 2011 Lim et al., 2018 Age-dependent, L-dopa responsive impairment of locomotor activity (Chen et al., 2012) hG2019S Mice (mid-brain DA targeted) Lin et al., 2009 Not reported Age-dependent impairment in locomotor activities Xiong et al., 2018
Loss of DA neurons within the SN, reduced DA content and metabolites in the striatum Age-dependent impairment in locomotor activities Liu et al.,2015
No DA cell loss, age-dependent depletion of DA terminals and dopamine release within the striatum
Normal motor function
hG2019S Mice (BAC)
Melrose et al., 2010
No DA cell loss or, normal level of DA and its metabolites, lower
extracellular DA level in the striatum but normal DA reverse transport induced by amphetamine Reduced explotary behaviour hG2019S Rats (BAC) Sloan et al., 2016
No DA cell loss; normal level of DA metabolites in the striatum;
Progressive impairment in evoked DA release within dorsal striatum
Age-dependent, L-dopa responsive motor impairment hI2020T Mice (whole body expression) Maekawa et al., 2012
No DA cell loss, DA neurons with severely fragmented Golgi apparatus
Impaired locomotor ability hWT Mice (pan-neuronal expression) Liu et al., 2015 Chen et al., 2012
No DA cell loss or signs of degeneration No evidence of motor impairment hWT Mice (BAC) Volta et al., 2015 Melrose et al., 2010
No DA cell loss or reduced level of DA metabolites
No detectable locomotor modifications; long term mememory preserved
VPS35
VPS35, the vacuolar protein sorting ortholog 35, is a part of retromer complex, which participates in the degradation of dysfunctional components within the cell. The link between VPS35 and Parkinson’s Disease is first identified in a Swiss family with late-onset, autosomal dominant PD, and has been then reported in different ethnic groups worldwide. All known mutations of VPS35 are missense point mutations, with D620N by far the only confirmed pathogenic mutation. It is a rare mutation with an estimated prevalence between 0.1% to 1% among patients with fPD, which is barely reported in East Asian populations except for in Japan (Williams et al., 2017).
Homozygous VPS35 knockout mice do not survive the embryonic stage. The effect of VPS35 depletion is examined by a heterozygous model VPS35+/- (Tang et al., 2015). Those VPS35 deficit mice develop
a reduction in locomotor activity around 12-month-age as examined by the Open Field Test. No differences have been found in rotarod test or gaits. By the time of 6-month-old, these mice have significantly reduced DA content within the striatum and further lose around 20% of the DA neurons within the SN 6 months later. In addition, VPS35+/- mice have accumulations of oligomeric alpha
synuclein proteins within the ventral midbrain. Other behavioral tests were not included, but there is no evidence of anxiety-related behavior as measured by the Open Field Test.
Surprisingly, neither the over expression (Vanan et al., 2020) nor the knock in (Cataldi et al., 2018) model of the D620N mutation managed to produce a PD-like phenotype in mice. They have normal motor function as measured by the Rotarod and Open Field Test. No significant differences have been identified regarding the number of DA neurons or the alpha-synuclein expression. In both models, the integrity of the VPS35 protein is maintained, indicating that the D620N mutation can still be functional to a certain degree.
PRKN, Pink1 and DJ-1
Among the known PD-related genes, PRKN (Parkin), Pink1, and DJ-1 are inherited with an autosomal recessive form. Mechanisms underlying their association with PD are less understood compared to other the Park genes identified around the same period. An expansive amount of potential pathogenic mutations has been reported, including missense or nonsense point mutations, frameshifts and truncations (Lev et al. 2006, Kawajiri et al., 2011, Marder et al., 2012). PD patients carrying these mutations usually manifest an early onset, L-dopa responsive phenotype without atypic features
(Bonifati., 2012). The PRKN GENE encodes a E3 ubiquitin ligase which participates the selective degradation of marked proteins. Collaborating with Pink1, these two genes form a protective pathway of the cell by regulating the process of mitophagy, the guided removal of damaged mitochondria, which is essential for maintaining normal cellular homeostasis (Kawajiri et al., 2011). The DJ-1 encoded protein instead, is a cysteine proteinase. It plays an important role in regulating oxidative stress and participates in the upregulation of the tyrosine hydrolase (TH), the catalyzing enzyme for dopamine synthesis (Sarge et al., 2011). Reports of PRKN mutations are the most common compared to the other two, while DJ-1 mutations are the least prevalent. These genes are responsible for around 8.64% (PRKN), 3.73% (Pink1) and 0.44% (DJ-1) of the early onset PD cases. with Pink1 more frequently reported in Asia.
Models based on autosomal recessive genes are mostly loss-of-function models (See Table 4). PRKN
-/-mice (Goldberg et al., 2003) manifest mild motor symptoms emerging around 2-4 month and comparable exploratory behavior to their wild type controls as measured by the Open Field Test. They have no obvious signs of DA cell degeneration across the life span, but increased DA content in the extracellular fluid resulting from a higher DA release. The phenotype is likely to be caused by reduced striatal synaptic plasticity (Kitada et al., 2009). The expression of monoamine oxidase, a major degradative enzyme for catecholamines, is inhibited by the knocking out the PRKN gene (Jiang et al., 2006). In addition, PRKN-/- mice show impaired neurogenesis process, accompanied by the
downregulation of neurotrophin and neuron cell differentiation (Park et al., 2017). PRKN-/- rats have
results largely consistent with findings in the mouse model, with no observable motor signs and limited dysfunction within the nigrostriatal system.
Both Pink1 knockout mice and rats have reports of early-onset motor symptoms. But only in Pink1
-/-rats, researchers have identified a significant loss of around 50% of the DA neurons within the SN (Dave et al., 2014). Pink1-/- mice instead, have impaired synaptic plasticity compromising the Long-Term
Potentiation and Depression within the corticostriatal pathway (Kitada et al., 2007). PINK1 G309D mice, which is a knock in model of a human nonsense mutation leading to 97% reduction of the PINK1 gene expression, manifest a late-onset motor phenotype observed at 16-month-old (Gispert et al., 2009). They have reduced striatal DA level reported at 24-month-old, but the DA neurons within the SN seems to be intact. DJ-1-/- mice do not manifest motor dysfunctions as measured by the Open Field Test, they
levels of mitochondrial H2O2. DJ-1-/- rats, instead, develop a PD-alike phenotype similar to Pink1-/- rats
reported in the same article (Dave et al., 2014).
The only exception is a BAC transgenic model overexpressing the human PRKN Q311X point mutation developed by Lu et al. (2009). The Q311X is a nonsense mutation which results in the truncation of the PRKN gene. BAC hQ311X mice have a late-onset PD-alike phenotype, with progressive motor impairment present at 16 months, along with a dramatic loss of around 40% of the DA neurons within the SN. Disrupted nigrostriatal DA metabolism was found in mice at 12-14 months age, characterized by significant loss of DA terminals in the striatum. In addition, accumulation of alpha-synuclein proteins were found in the substantia nigra.
The knockout models of the autosomal recessive genes provide contradictory results. Homozygous PRKN-/- rodents manifest limited evidence with regards to the motor symptoms and DA
neurodegeneration. And the late-onset, PD-alike phenotype observed in PRKN hQ311X transgenic mice is different from the early-onset, Lewy-Body-absent phenotype as reported in PD patients with PRKN mutation, which suggests that the truncated PRKN protein may contribute to the disease via a gain-of-function mechanism different than other loss-of-function mutations. The gene ablation of DJ1 and Pink1 were able to replicate the phenotype only in rat models. This absence of DA degeneration within the SN may be attributed to an incomplete ablation of the genes in the mouse models. Reduced synaptic plasticity within the corticostriatal pathway is a common effect observed in PRKN, DJ-1 and PINK1 depletion models, which can be used as a direction for future studies.
Rodent Core Literature Motor Symptoms DA degeneration Other Important notes
PRKN-/- Mice Goldberg et al., 2003
Early-onset (2-4 month of age) mild locomotor impairment compared to WT, the differences disappear at 18 months
No signs of DA degeneration untill 24 months.
Normal levels of DA neurons, content and metabolites; unchanged binding affinities of DA neurons. Higher level of extracelluar DA, probably lead by higher DA releasee (Goldberg et al., 2003),
Expresison of monoamine oxidase is ihibited (Jiang et al., 2006)
Reduced synaptic plasticity, reduced capacity for Long Term Potentiaon and Depression (Kitada et al., 2009), Impaired neurogenesis and neuron cell differentiation, with lower level of BDNF(Park et al., 2017)
Rats Dave et al., 2014, Gemechu et al., 2016
Normal Motor function up to 8 months, Decreased locomotor response to methamphetamine (2 month of age) No signs of DA degeneration within the SN up to 8 months, normal level of DA content and metabolites, lower level of postsynaptic D2 receptor and reduced activity of monoamine oxidase within the striatum (Gemechu et al., 2016) BAC PRKN
hQ311X Mice Lu et al., 2009
Late-onset (16 -19 month of age) motor impairment
Significant loss of DA neurons within the SN, reduced DA level and its metabolites in the striatum
Higher level of protinase k resistent alpha-synuclein within the substantia nigra (Lu et al., 2009) Deficit of mitocondrial quality control(Siddiqui et al., 2015) PINK1 -/- Mice Kitada et al., 2009
Motor impairment and reduced locomotor activities (3-6 months)
Normal levels of DA neurons, content and metabolites. Reduced evoked DA release in the striatum.
Impaired synaptic plasticity within the corticostriatal pathway Rats Dave et al., 2014
Impaired Motor Function (starting at 4 month age)
loss of 50% DA neurons in the SN, increase of striatal DA content (measured at 8 month) PINK1 G309D Mice Gispert et al., 2009
Reduction of locomotor activity starting from 16 month
No reduction of DA neurons or DA terminals in the nigrostriatal pathway but deficient of strital DA content
Absence of Lewy bodies
DJ-1 -/- Mice Andres Mateos., 2007
No observed differences in spontaneous locomotion and Open Field Test compared to WT mice.
The number of DA neurons is maintained, no observable differences of DA and its metabolites compared to WT as measured in 2-3 month and 18-24 month
Rats Dave et al., 2014
Impaired Motor Function (staring at 4 month age)
loss of 50% DA neurons in the SN, increase of striatal DA content (measured at 8 month
GBA
The β-Glucocerebrosidase, known as GBA gene, has first been identified as a risk factor for Gaucher’s Disease, which is an autosomal recessive genetic disorder caused by degradation failure of glycolipid substrate. The link between GBA and Parkinson’s was built based on the high prevalence of PD in heterozygous relatives of patients diagnosed with Gaucher’s disease (Regan et al., 2017). To this day, GBA mutations have been recognized as the most common genetic risk factor for PD. Most GBA-related PD patients are carriers of point mutations N370S and L444P. The former is commonly found in Ashkenazic Jews, while the latter has more reports in Chinese and Japanese patients (Riboldi & Di Fonzo. 2019).
Studies for Gaucher’s disease have unveiled the interactive effective between the GBA and alpha-synuclein. Pharmacological inhibition of GBA expression can lead to higher level of alpha-synuclein protein within the substantia nigra (Manning-Boğ et al., 2009). This effect was later verified in genetic studies. Homozygous GBA-/- mice have accumulation of oligomeric alpha-synuclein proteins in the
midbrain as caused by impaired mitochondria quality control (Osellame et al., 2013), which is similar to the BAC transgenic PRKN hQ311X line. It is to notice that the heterozygous type mice (GBA+/-) do
not develop extra synuclein deposit in the brain. No tests targeting the motor symptoms have been conducted in these two models. A double mutation model was created by Fishbein et al (2014) by over-expressing the SNCA hA53T gene in a mouse heterozygous knock in model of the GBA L444P. The SNCA degradation rate was lower in this mouse line compared to hA53T, GBA wild type mouse. In addition, this double mutated mouse line manifests late-onset severer motor symptoms and disturbances in the ENS at round 14 month of age.
Discussion
Since the identification of the SNCA gene in 1997 (Polymeropoulos et al., 1997), inheritance, both as a causal and risk factor for Parkinson’s disease, has raised the increasing amount of attention within the research background. The genes that have been investigated via family-based linkage study and confirmed as Parkinson-related work as cornerstones for pathway and functional research. Among them, SNCA, LRRK2, and VPS35 are inherited with an autosomal dominant form, PINK1, PRKN, and DJ-1 are associated with autosomal recessive PD, and GBA is recognized as a widely distributed risk factor. Human epidemiological studies have identified several commonalities of these genes despite the differences underlying their neurobiological mechanisms. Pathogenic mutations of PD-linked genes are marked by their incomplete penetrance. Carriers of a pathogenic genotype (mutation) do not always develop a pathological phenotype. Non-affected carriers are found within the general population, as well as the members of the mutant-carrying families. In addition, the prevalence of the mutations can vary between different ethnic groups; these mutations can lead to various structural modifications in the encoded protein, further adding up to the uncertainly.
Furthermore, despite the intensive research, the majority of PD patients have not been reported as carrying mutations of any known Parkinson-related genes (Kumar et al., 2011). The complex nature of those genes, along with new risk loci identified via Genome-Wide Association Studies, has built up a genetic architecture for perceiving the disease as a complex trait. It follows a non - Mendelian hereditary pattern and is influenced by a combination of extensive genetic information. This polygenicity of the disease poses the first challenge for interpreting the results of animal models targeting PD-related genes, as the construct validity may always be limited.
However, when these models are used to study the familial form of PD, especially in patients with a monogenic subtype, their construct validity is strong. Therefore, more attention should be paid to the phenotype reproduced by the genetic modification. With this review, we summarized available rodent genetic/transgenic models based on the genes that are mostly reported within the clinical background. Genes related to juvenile PD with atypical features, for example, ATP13A2, DNAJC6, FOXO7, are not under discussion. A large of the models are able to induce a behavioral phenotype with motor deficits in rodents, while the loss of DA neurons is less reported. Our literature research revealed the similarities between the core features of familial PD and sporadic PD, as have been reported by previous comparative studies. The results are coherent with the presence of the pathogenic mutations among sPD
patients. Among the seven genes, LRRK2-related mutations produce a phenotype with the highest resemblance to idiopathic PD; PRKN, Pink1, and DJ-1 carriers have an early onset and relatively mild symptoms, while the SNCA gene is usually present with atypical features, highlighted with dementia and psychotic hallucinations (Weissbach et al., 2019). The phenotypic variance in humans is also reflected in animal studies. For example, the inclusion of alpha-synuclein formed in the midbrain is mostly absent in models not based on the SNCA gene; SNCA transgenic models also produce the most severe motor phenotype despite the absence of DA degeneration. As a result, the face validity of a model should also be supported by the relevance for clinical usage. In table 5, we selected the models providing a high resemblance to human PD, which can be used for drug development in the pre-clinical background. These models are able to reproduce the progressive nature of the disease, as characterized by age-dependent motor impairment and the nigrostriatal denervation caused by a significant amount of DA neuron loss within the SN. In addition, all these models manage to induce early signs of alpha-synuclein aggregation within the mid-brain area of the tested animals, which allows for pre-clinical tests of therapies targeting alpha-synuclein aggregations.
Table 5
Authors and Year Model
Kuo et al., 2010 BAC SNCA hA53T mice
Xiong et al., 2018 hG2019S LRRK2 transgenic mice (TH promoted)
Lu et al., 2009 BAC PRKN hQ311X mice
Nuber et al., 2013 BAC SNCA hWT rat
Moreover, several LRRK2 transgenic models (Weng et al., 2016, Chen et al., 2012), and the DJ-1 and Pink-1 knockout rats (Dave et al., 2014) manage to produce a PD-alike phenotype without the presence of Lewy Body pathology. These models may provide valuable insights in research of the monogenic subtypes of the disease and their underlying components, e.g., the roles of mitochondrial dysfunction, the inhibition of neurogenesis, and the reduced synaptic plasticity.
All these models have their drawbacks as limited by the DNA engineering techniques. The pre-natal ablation or genetic knock-in models may induce the compensatory mechanism of the central nervous system, which can mask the effects of genetic manipulation. Traditional transgenic approach based on complementary DNAs are likely to cause positional effects, since the transgene expression is under the
influence of the integration site (Yang et al., 2005). In most cases, the unmodified gene of the animals is still under expression except for the transgene, which lead to a mixed effect. BAC models enable predictable expression of the transgene but are instead subjective to a lower efficiency (Beil et al., 2012). A study examining the expression patterns of the LRRK2 gene have uncovered a higher expression level of the transgene expressed within the SN in mice when compared to rats. In addition, human BAC constructs can lead to the expression of hG2019S in an extra subset of neurons in addition to the endogenous mice LRRK2 distribution (West et al., 2014). When the animal models are used to study the pathogenic mechanism of the disease in human, this divergence between primates and lower mammalian species should be examined with caution.
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