Parkinson's disease: Mechanisms, translational models and management strategies

Delayed Dominant-Negative TNF Gene Therapy Halts Progressive Loss of Nigral Dopaminergic Neurons in a Rat Model of Parkinson's Disease

Clinical Trial for Parkinson's Disease Gets a Green Light in the US.

Parkinson's disease (PD) is considered a multifactorial disorder, which is neuropathologically characterized by age-dependent neurodegeneration of dopaminergic neurons in the midbrain. Different neurotoxins including synthetic compounds, heavy metals, and dopamine itself have been proposed to be environmental risk factors of PD. Recent genome-wide genetic and mutational studies provide information on various genetic risk factors while microglial activation in the affected regions has emerged to be involved in the disease development as a local microenvironmental factor. A wide variety of animal models of PD substantially contribute to the understanding of these issues and the development of therapeutic approaches as an alternative to humans although none of them fully recaptures the symptoms and pathology of PD. This special issue is composed of 9 excellent reviews and 3 distinguished original articles that summarize the most recent progresses and ideas obtained from animal models in the pertinent field, while reporting the putative molecular mechanisms of neurodegeneration, therapeutic challenges and limitations using PD models, and generation of new versions of PD models. The first review paper briefly outlines animal models of PD, covering toxin-induced and genetic models of vertebrate and invertebrate animals, in which characteristic features of each model are discussed. Mishandling of monoamines including dopamine has been hypothesized to damage neurons. The second review paper describes mice with impaired functions of the vesicular monoamine transporter VMAT2, in which progressive loss of catecholamine-secreting neurons is observed. Such models may be potentially useful for the development of new therapeutic strategies, which would complement current dopamine replacement. Neuropathological analysis of the postmortem PD brain tissues suggests that an adverse interaction with surrounding glia and other nonneuronal cells may be one of critical steps in neurodegeneration. The third review highlights endotoxin-induced inflammation models, in which activation of microglia and lymphocyte by a bacterial lipopolysaccharide deteriorates a healthy relationship with neurons. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified to cause autosomal-dominant late-onset PD and are also implicated in sporadic PD. The neuropathological features of PD brain tissues with the LRRK2 mutations are characterized by typical Lewy body pathology in the brainstem. The forth paper reviews a variety of LRRK2-related models. Mutations and increased expression in the α-synuclein gene cause the development of early-onset familial PD. The formation of α-synuclein fibrils and aggregates, a main component of Lewy bodies and Lewy neurites, is considered a key process in the pathogenesis of PD and other synucleinophathies. Other genetic determinants include the genes for Mendelian forms of PD and susceptible genes. The following two papers focus on the potential of Drosophila genetic models to examine α-synuclein and other responsible genes. Deep brain stimulation (DBS) by electrical pulses could be one of useful therapeutic avenues for PD. However, DBS's technique requires advancement and poor understanding of the mechanisms involved hinder application in clinical practice. The seventh review paper discusses the optimization of a rat PD model for DBS. Hydrogen has turned out to reduce oxidative damage. The eighth paper introduces the neuroprotective effects of hydrogen on experimental animal models for PD and possible application in treatment and prevention of PD. The last review explains the limitations of animal models, showing differences between humans and animals, and difficulties in interpretation of obtained results with animal models. The first research paper investigates selective degeneration of dopaminergic neurons in the substantia nigra and associated motor dysfunction induced by inhalation of mixed manganese compounds on mice. This model could be instrumental for evaluating some aspects of a progressive loss of dopaminergic neurons. The second research paper examines the possible effects of testosterone on PD using a mouse model induced by 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. The study suggests that loss of testosterone induces remodeling in the morphology of medium spiny neurons where dopaminergic neurons of the substantia nigra project although no interaction between testosterone and loss of dopaminergic neurons by MPTP administration is observed. The third research paper of this special issue addresses improvement of potential gene therapy to compensate for impaired complex I activity of the mitochondria using the yeast single-subunit NADH-ubiquinone oxidoreductase, NDI1. NDI1 is functionally able to replace complex I, activity of which is thought to be compromised in most of PD cases. A decreased sense of smell is one of early signs of PD. Although degeneration of tyrosine hydroxylase-positive neurons in the olfactory bulbs is observed, the pathogenic mechanism underlying olfactory deficits is not well understood. The forth research paper addresses this issue using a rat model bearing the pathogenic α-synuclein. Yuzuru Imai Katerina Venderova David S. Park Huaibin Cai Enrico Schmidt

Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

Parkinson’s disease (PD) is a neurodegenerative movement disorder distinguished by resting tremor, bradykinesia, rigidity, postural instability and gait disturbances. Non-motor symptoms including dysfunction of the autonomic nervous system, neuropsychiatric changes, sensory and sleep disturbances are also common. PD may be diagnosed at any age but is most common in aged populations affecting approximately 1% of individuals over 60 years of age and rising to approximately 4% in age groups above 85 years of age (de Lau and Breteler, 2006; Van Den Eeden et al., 2003). It has become apparent that differential subgroups may be categorised by age of onset, dominant symptoms and progression. Two key subsets include lateand early-onset PD. Late-onset PD is typically identified in individuals over the age of 70 and is characterised by postural imbalance and gait impairment with accompanying rigidity and akinesia. Early-onset PD is typified by a dominant tremor and slow progression in motor decline, and is primarily identified in individuals less than 50 years of age (Lewis et al., 2005; Selikhova et al., 2009). The lead pathological identifier of PD is moderate to severe dopaminergic neuronal loss within the substantia nigra pars compacta with accompanying Lewy pathology in surviving neurons (Daniel and Lees, 1993; Dickson et al., 2009; Gelb et al., 1999). It is thought that the combination of Lewy pathology and dopaminergic cell loss in PD leads to striatal dopamine depletion, and this accounts for the motor symptoms (Obeso et al., 2008). Earlier diagnosis of PD is important as motor symptoms do not become apparent until approximately 60% of dopaminergic neurons are lost (Fearnley and Lees, 1991; Pakkenberg et al., 1991). Current treatments are available to manage the symptoms of PD. Medications such as the dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) and inhibitors of dopamine metabolism are used to supplement the reduced dopamine level. Deep brain stimulation is an alternative treatment. The patient undergoes surgery to implant an electrical stimulation device into the affected region of the basal ganglia. The electrical impulses generated by the device interfere with the abnormal signals that are causing the tremor, thereby alleviating some of the symptoms of the disease (reviewed in (Hurelbrink and Lewis, 2010)). To identify treatments that address or arrest the progressive nature of PD, an understanding of the molecular mechanisms responsible for the loss of nigrostriatal dopaminergic neurons and associated Lewy pathology must be delineated.

SUMMARYα-synuclein (α-syn) aggregation and accumulation drive neurodegeneration in Parkinson’s disease (PD). The substantia nigra of patients with PD contains excess iron, yet the underlying mechanism accounting for this iron accumulation is unclear. Here, we show that misfolded α-syn activates microglia, which release interleukin 6 (IL-6). IL-6, via its trans-signaling pathway, induces changes in the neuronal iron transcriptome that promote ferrous iron uptake and decrease cellular iron export via a pathway we term the cellular iron sequestration response, or CISR. The brains of patients with PD exhibit molecular signatures of the IL-6-mediated CISR. Genetic deletion of IL-6, or treatment with the iron chelator deferiprone, reduces pathological α-syn toxicity in a mouse model of sporadic PD. These data suggest that IL-6-induced CISR leads to toxic neuronal iron accumulation, contributing to synuclein-induced neurodegeneration.

Ventral midbrain dopaminergic neurons: From neurogenesis to neurodegeneration

Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.

Progressive accumulation of α-Synuclein (αSyn) in Lewy bodies (LBs) and loss of dopaminergic (DA) neurons are the hallmark pathological features of Parkinson's disease (PD). Although currently available in vitro and in vivo models have provided crucial information about PD pathogenesis, the mechanistic link between the progressive accumulation of αSyn into LBs and the loss of DA neurons is still unclear. To address this, it is critical to model LB formation and DA neuron loss, the two key neuropathological aspects of PD, in a relevant in vitro system. In this study, we developed a human midbrain-like organoid (hMBO) model of PD. We demonstrated that hMBOs generated from induced pluripotent stem cells (hiPSCs), derived from a familial PD (fPD) patient carrying αSyn gene (SNCA) triplication accumulate pathological αSyn over time. These cytoplasmic inclusions spatially and morphologically resembled diverse stages of LB formation and were composed of key markers of LBs. Importantly, the progressive accumulation of pathological αSyn was paralleled by the loss of DA neurons and elevated apoptosis. The model developed in this study will complement the existing in vitro models of PD and will provide a unique platform to study the spatiotemporal events governing LB formation and their relation with neurodegeneration. Furthermore, this model will also be beneficial for in vitro screening and the development of therapeutic compounds.

Parkinson's disease (PD) is a common neurodegenerative disorder, which is characterized by the selective and progressive death of dopaminergic (DA) neurons in the substantia nigra. Increasing evidence suggests that inflammation is important in the degeneration of DA neurons. The purinergic receptor subtype P2X7 receptor (P2X7R) is key in the activation and proliferation of microglia. The present study aimed to examine whether inhibiting purinergic P2X7 receptors is neuroprotective in a rat model of PD, specifically via inhibiting p38 mitogen-activated protein kinase (MAPK). In an intranigral lipopolysaccharide (LPS) rat model of PD, immunohistochemical analysis revealed enhanced expression of P2X7R was observed in microglia. The administration of the P2X7R antagonist, brilliant blue G (BBG), reduced activation of the microglia and the loss of nigral DA neurons. In addition, immunohistochemistry and western blot analysis revealed the phosphorylation level of p38 MAPK increased in the microglia of the LPS-injected rats, which was inhibited by BBG treatment. The p38 MAPK inhibitor, SB203580, reduced microglial activation and the loss of DA neurons. Thus, these findings suggested that inhibition of P2X7R by BBG attenuated microglial activation and the loss of substantia nigra DA neurons via p38 MAPK in the rat LPS model of PD.

Animal models of human pathologies remain invaluable tools for unraveling disease mechanisms and evaluating potential therapeutic strategies. For a number of diseases, the lack of a reliable animal model represents an important limiting step towards the development of efficient treatments. This holds particularly true for Parkinson's disease (PD), a major neurodegenerative disorder for which only symptomatic treatments currently exist. The difficulties encountered by researchers to reproduce PD pathology in animals stem primarily from an incomplete understanding of the disease. Indeed, the cause of the disease remains unknown in 90% of cases, referred to as sporadic or idiopathic. The discovery of familial forms of the disease, however, has led to the development of a large number of transgenic mice models based on genetic modifications that play a direct causative role in a significant proportion of human PD cases. Unfortunately, these transgenic mice fail to recapitulate the robust neurodegeneration of dopaminergic (DAergic) neurons of the substantia nigra pars compacta (SNpc) and concomitant loss of DAergic projections to the striatum, the neuropathological hallmark of the human condition. The lack of nigral pathology severely limits the usefulness of such models for pre-clinical evaluation of potential therapeutics. Viral vector gene delivery tools represent an interesting alternative to classical transgenesis as they allow for targeted and high-level transgene expression in the nigrostriatal system of adult animals. During the course of this thesis we have developed two new viral vector-based rodent models of PD. In our first model, we have used a recombinant adeno-associated virus (rAAV) vector, with a high tropism towards nigral DAergic neurons, to drive overexpression of the parkin-associated endothelin receptor-like receptor (Pael-R) in the SNpc of adult rats. Indeed, accumulation of Pael-R is implicated in the pathogenesis of autosomal-recessive juvenile parkinsonism (AR-JP), a young-onset familial form of PD. We show that insoluble accumulation of Pael-R in rats induces a rapidly progressing degeneration of nigral DAergic neurons and a loss of DAergic fibers and terminals in the striatum. Lesioned animals also displayed spontaneous behavioral abnormalities linked to depletion of striatal dopamine (DA) and persisting up to 6 months post-injection. Chronic accumulation of Pael-R in the nigrostriatal system of adult rats therefore represents a robust and highly reproducible model of PD, recapitulating key pathological and phenotypical features of the human condition. The second model developed was based on nigral delivery of the PD-associated mutant G2019S leucine-rich repeat kinase 2 (LRRK2) protein. Indeed, the G2019S mutation in the LRRK2 gene is the most important genetic determinant of PD, accounting for a significant proportion of both familial and sporadic PD cases. Due to the large size of the LRRK2 coding sequence, an adenoviral system with a high packaging capacity was used to drive expression of the protein. Recombinant adenoviral (rAd) vectors are potentially pro-inflammatory and less efficient tools as rAAV vectors for long-term gene delivery to the SNpc. Nevertheless, through retrograde axonal delivery of rAd-LRRK2 particles, we achieved robust and neuron-specific expression of full-length wild-type or mutant G2019S human LRRK2 in nigral DAergic neurons of adult rats. Expression persisted up to 6 weeks post-injection, with no visible signs of inflammation in the SNpc. We demonstrate that the wild-type form of LRRK2 does not induce neuronal loss when expressed in the SNpc. In contrast, under the same conditions and levels of expression as the wild-type form, the PD-associated G2019S mutation in LRRK2 is sufficient to cause a progressive loss of nigral dopaminergic neurons. This is the first demonstration of frank dopaminergic neuronal degeneration in rodents induced by the expression of G2019S mutant LRRK2. Our data also provide a new rodent model of LRRK2-linked PD which recapitulates one of the cardinal pathological features of the disease. In the absence of a clear understanding of human PD pathogenesis, the development of multiple transgenic models may help to identify common disease mechanisms and drug targets. A potential treatment identified in one model may be effective only in the corresponding subset of PD patients, carrying this particular genetic modification. On the other hand, the pathogenic pathway targeted may also be activated in sporadic PD. Ultimately, whether or not these models stand the test of time will depend on how effective newly-identified drugs will be, not only on AR-JP or LRRK2-linked PD patients, but above all on sporadic PD, which represents the vast majority of PD patients.

The loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) is a major pathological hallmark of Parkinson's disease (PD). Orexin B (OXB) has been reported to promote the growth of DA neurons. However, the roles of OXB in the degeneration of DA neurons still remained not fully clear. An in vivo PD model was constructed by administrating 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Pole test was performed to investigate the motor function of mice and the number of DA neurons was detected by immunofluorescence (IF). A PD cell model was established by treating SH-SY5Y cells with 1-methyl-4-phenylpyridinium (MPP+). OXB was added to the culture medium 2h after MPP + treatment. Microscopic analysis was carried out to investigate the function of OXB in the cell model of PD 24h after MPP + challenge. RNA-Seq analysis of the PD cell model was performed to explore the possible mechanisms. Western blot was used to detect the phosphorylation levels of extracellular signal-regulated kinase (ERK). OXB significantly decreased the DA neurons death caused by MPTP, alleviated MPP+-induced neurotoxicity in SH-SY5Y cells, and robustly enhanced the weight and motor ability of PD mice. Besides, RNA-Seq analysis demonstrated that the mitogen-activated protein kinase (MAPK) pathway was involved in the pathology of PD. Furthermore, MPP + led to increased levels of phosphorylation of ERK (p-ERK), OXB treatment significantly decreased the levels of p-ERK in MPP+-treated SH-SY5Y cells. This study demonstrated that OXB exerts a neuroprotective role associated with reduced ERK phosphorylation in the PD model. This suggests that OXB may have therapeutic potential for treatment of PD.

ISSN 1758-2024 10.2217/NMT.12.25 © 2012 Future Medicine Ltd Neurodegen. Dis. Manage. (2012) 2(3), 239–241 part of Parkinson’s disease (PD) is a movement disorder characterized by the degeneration of nigral dopaminergic (DA) neurons. The defining locomotor symptoms (i.e., rest tremor, rigidity, bradykinesia and postural instability) are caused by a relatively selective loss of nigral DA neurons [1]. Current pharmacological treatments focus on augmenting DA levels in the brain (e.g., levodopa, monoamine oxidase [MAO]-B inhibitors and catechol-O-methyltransferase inhibitors) or mimicking the actions of DA (e.g., DA agonists). These drugs mostly provide symptomatic relief; the unabated degeneration of nigral DA neurons and other cells invariably kills PD patients. Surgical interventions such as deep-brain stimulation are similar in that they only alleviate locomotor symptoms and have no impact on the neuro degeneration process. Thus, the central problems for PD research are to understand the cause for the degeneration of nigral DA neurons and to identify a strategy to intervene, preferably as early as possible. Mechanistic studies on PD-linked monogenic mutations have revealed significant insights into the unique vulnerabilities of nigral DA neurons and how mutations of a single gene, such as parkin, expose these vulnerabilities. A variety of mouse genetic models of PD have revealed surprisingly little information on the human disease [2]. For example, parkin knockout mice do not exhibit any robust phenotype [3], in sharp contrast with PD patients with parkin mutations [4]. It suggests that human nigral DA neurons are quite different from the murine counterpart. The landmark discovery of induced pluripotent stem cells (iPSCs) [5,6] has made it possible for us to generate patient-specific midbrain DA neurons from PD patients with parkin mutations and normal controls [7]. Our study shows that parkin mutations significantly disrupt the precision of DA transmission by increasing spontaneous DA release and decreasing DA reuptake. In addition, parkin mutations greatly elevate DA-induced oxidative stress by increasing the transcription of MAOs, which catalyze the oxidative deamination of DA – a reaction that produces a large amount of reactive oxygen species. Overexpression of wild-type parkin, but not its PD-linked mutant, significantly rescues all these phenotypes. Thus, mimicking the beneficial actions of parkin, for example, with small-molecule

BackgroundNoradrenergic neurons in the locus coeruleus (LC) are the primary source of norepinephrine (NE) in the brain and degeneration of these neurons is reported in the early stages of Parkinson’s disease (PD), even prior to dopaminergic neuron degeneration in the substantia nigra (SN), which is a hallmark of PD pathology. NE depletion is generally associated with increased PD pathology in neurotoxin-based PD models. The effect of NE depletion in other models of PD-like α-synuclein-based models is largely unexplored. In PD models and in human patients, β-adrenergic receptors’ (AR) signaling is associated with a reduction of neuroinflammation and PD pathology. However, the effect of NE depletion in the brain and the extent of NE and β-ARs signaling involvement in neuroinflammation, and dopaminergic neuron survival is poorly understood.MethodsTwo mouse models of PD, a 6OHDA neurotoxin-based model and a human α-synuclein (hα-SYN) virus-based model of PD, were used. DSP-4 was used to deplete NE levels in the brain and its effect was confirmed by HPLC with electrochemical detection. A pharmacological approach was used to mechanistically understand the impact of DSP-4 in the hα-SYN model of PD using a norepinephrine transporter (NET) and a β-AR blocker. Epifluorescence and confocal imaging were used to study changes in microglia activation and T-cell infiltration after β1-AR and β2-AR agonist treatment in the hα-SYN virus-based model of PD.ResultsConsistent with previous studies, we found that DSP-4 pretreatment increased dopaminergic neuron loss after 6OHDA injection. In contrast, DSP-4 pretreatment protected dopaminergic neurons after hα-SYN overexpression. DSP-4-mediated protection of dopaminergic neurons after hα-SYN overexpression was dependent on β-AR signaling since using a β-AR blocker prevented DSP-4-mediated dopaminergic neuron protection in this model of PD. Finally, we found that the β-2AR agonist, clenbuterol, reduced microglia activation, T-cell infiltration, and dopaminergic neuron degeneration, whereas xamoterol a β-1AR agonist showed increased neuroinflammation, blood brain barrier permeability (BBB), and dopaminergic neuron degeneration in the context of hα-SYN-mediated neurotoxicity.ConclusionsOur data demonstrate that the effects of DSP-4 on dopaminergic neuron degeneration are model specific, and suggest that in the context of α-SYN-driven neuropathology, β2-AR specific agonists may have therapeutic benefit in PD.

Cotransplantation of NSCs and ethyl stearate promotes synaptic plasticity in PD rats by Drd1/ERK/AP-1 signaling pathway