Model organisms as in vivo screens for promising therapeutic compoundsPhenotypic screens for compounds that target the cellular pathologies underlying Parkinson's disease
Graphical abstract
Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting ∼2% of the population over 65 years of age. Patients suffer from progressive loss of muscle control, including tremor, rigidity, bradykinesia, and postural instability. These movement deficits are largely caused by the selective degeneration of dopaminergic (DA) neurons in a brain region called the substantia nigra pars compacta. The histopathological hallmark of PD is the presence of large intracytoplasmic spherical structures (called Lewy Bodies, LBs) that contain the α-synuclein (α-syn) protein [1]. The misfolding and accumulation of a disease-specific protein that causes proteotoxic stress is a common theme among many neurodegenerative diseases, including Alzheimer's and Huntington's diseases. Although most PD arises sporadically, α-syn pathology is almost always detected. Importantly, a causal role for α-syn was strengthened with the identification of rare mutations in the α-syn gene (SNCA) [2]. Extra genomic copies of SNCA also cause familial PD, establishing that simply increasing α-syn dose can cause disease [3]. Intriguingly, aging, which is the most significant risk factor for PD, is accompanied by an increase in α-syn protein levels, possibly sensitizing cells to α-syn misfolding and toxicity [4, 5].
The genetic complexity of PD is exceptionally rich as several other monogenic mutations cause related parkinsonisms. Some mutations, such as those in Parkin, PARK3, LRRK2, PLA2G6, and GBA (Gaucher's locus), accumulate α-syn-positive Lewy Bodies. Others, including mutations in UCHL1, DJ-1, ATP13A2, PARK10, GIGYF2, PARK12, HTRA2, FBX07, PARK16, have less defined α-syn involvement [6]. The diverse functions of these disease-associated genes creates a complexity where different pathological mechanisms, including ubiquitin–proteasome system, oxidative stress, vesicle trafficking, and mitochondrial dysfunction, manifest in related parkinsonisms. Also, several environmental substances, including the mitochondrial toxins MPTP/MPP+ and rotenone, as well as manganese, can cause related parkinsonisms.
Model organism studies of ‘PD’ have provided key insights into some basic, underlying toxicities of PD. They do not, however, truly model ‘disease’, which integrates complex interactions between cellular pathology, neuronal networks, multiple systems, and patient heterogeneity. This distinction is crucial in establishing dialogs between basic researchers and clinicians treating PD patients. Herein, we refer to ‘PD models’ without making claims to the actual human disease, but rather to the underlying precipitating toxic events.
Model organisms used to study PD have provided key molecular insights into the underlying cellular pathology. The accumulation of α-syn has been linked to mitochondrial dysfunction, proteasome inhibition, oxidative stress, vesicle trafficking defects, lipid droplet accumulation, calcium dysregulation, α-syn aggregation, and cellular toxicity. These phenotypes are studied in several model systems, including immortalized cell lines, primary neuronal cultures, yeast, fruit flies, nematodes, and rodents [7, 8]. Although no model organism faithfully recapitulates all cellular pathologies, the cross-validation of findings in multiple systems strengthens new connections. Indeed, the combination of diverse cellular pathologies with the aforementioned genetic complexity and environmental links necessitates innovative, unbiased phenotypic screening approaches in simple model organisms.
Despite the prevalence of PD and the substantial efforts in studying disease pathogenesis, no disease modifying agents exist. Current therapies largely manage symptoms through modulating neuronal activity, yet do not significantly modify disease progression [9]. Phenotypic screening provides an opportunity to both study toxic mechanisms and to provide new chemical entities that target the precipitating biology and may themselves modify disease. Herein, we will discuss the promise of phenotypic screens and early successes that can enrich the pipeline for potential therapeutics. We will not discuss the other significant hurdles in drug development, including compound optimization, the difficulties in testing drugs in a slowly progressing disease, or other approaches such as structure-based design.
Section snippets
Target-based versus phenotypic drug screening
The two main high-throughput (HT) screening approaches for discovering potential new PD-modifying compounds are target-based and phenotype-based. Target-based approaches rely on experimentally validated (although sometimes poorly so) protein activities to screen in vitro. Phenotypic screens, by contrast, exploit unbiased cell-based assays to identify compounds that elicit a desired response, such as protecting cells from α-syn toxicity. These two fundamentally different approaches have
Drug screening in cell culture models of PD-related toxicities
Academic screening centers provide an affordable and accessible entrée into HT phenotypic compound screening. However, few screens directed at ameliorating PD-relevant toxicities have been reported. This is probably because phenotypic screening requires robust, reproducible, and biologically meaningful assays. One must therefore examine the available model systems for their suitability to high-throughput drug screens (Fig. 1). Both genetic and pharmacological approaches are used to model
Yeast as a screening platform for chemical suppressors of α-synuclein toxicity
A yeast model of α-syn toxicity is a robust HTS platform with potential for identifying therapeutic leads. In exploiting the dose-dependence of α-syn toxicity, yeast was engineered to express α-syn at different levels to cause clear dose-dependent α-syn foci formation (Fig. 3a) and toxicity [18] (Fig. 3b). Whereas nontoxic levels of α-syn localized to the plasma membrane, expressing toxic levels of α-syn caused the accumulation of α-syn foci and stalled vesicles [18, 25]. Genetic overexpression
Small molecules from other screens with potential for suppressing PD-relevant toxicities
Screens not directly aimed at ameliorating PD-relevant toxicities may also identify small molecules that target potentially relevant proteins or pathways. For example, screens against basic protein homeostasis may provide protection in the context of PD-related toxicities. In one example, inducers of autophagy in yeast also functioned in mammalian cells to rescue polyglutamine toxicity and induce clearance of an A53T α-syn mutant protein [33, 34]. In addition, a yeast screen for compounds that
Untapped models for Parkinson's disease drug screens
Surprisingly few in vivo screens have been performed when considering the prevalence and burden of PD. Proof of principle experiments have been reported for some models; however, their true potential has yet to be realized. For example, the soil nematode C. elegans, with its well-defined development, relative ease of growth and manipulation, and recapitulation of PD-related phenotypes, may also be amenable to HT drug screens [39]. Worms expressing α-syn recapitulate neuronal toxicity and have
Conclusions
Our ever-aging population will continue to increase the number of patients suffering from age-related diseases, such as PD. Despite this trend, there are no disease-modifying agents on the market for most neurodegenerative diseases. Symptom management (e.g. with l-dopamine), and not disease modification, continues to be the focus of most therapeutics [9]. The limited number of published phenotypic high-throughput drug screens highlights the intrinsic difficulty in establishing robust,
Acknowledgements
We thank members of the Lindquist lab for helpful comments on the manuscript. DFT was funded by a Ruth L. Kirschstein National Research Service Award Fellowship (NS614192). SL is an investigator of the Howard Hughes Medical Institute.
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2020, Advances in GeneticsCitation Excerpt :Overall, these data point to the role of endocytic trafficking and stress response in the amelioration of αSyn toxicity, as well as to the differences in the specific pathogenic mechanisms involved in heritable and sporadic forms of PD. Yeast system was successfully used for identifying several potential therapeutic candidates, that rescue αSyn aggregation and/or toxicity (Tardiff & Lindquist, 2013). These include some flavonoids (e.g., quercetin and epigallocatechin gallate) (Griffioen et al., 2006), small molecule stimulators of the Rab GTPase, associated with PD (Fleming, Outeiro, Slack, Lindquist, & Bulawa, 2008), 1,2,3,4-tetrahydroquinolinones (Su et al., 2010), cyclic peptides (Kritzer et al., 2009), mannosylglycerate, originated from marine organisms (Faria et al., 2013), red pigment which is a polymerized intermediate in the yeast adenine biosynthesis pathway (Nevzglyadova et al., 2018), ascorbic acid which is a natural antioxidant (Fernandes et al., 2014), and N-aryl benzimidazole (NAB), that promotes endosomal transport via the E3 ubiquitin ligase, Rsp5 (a yeast ortholog of mammalian Nedd4) and apparently antagonizes the vesicular traffic disruption by αSyn (Tardiff et al., 2013).
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2017, Molecular and Cellular NeuroscienceCitation Excerpt :Once a therapeutic target has been identified, it needs to be validated, both in vitro and in animal models (Hughes et al., 2011; Tardiff and Lindquist, 2013). Target-based biochemical screening depends on knowing the exact target and the MMOA associated with a well-defined pathology (Sams-Dodd, 2005), and hypothesizes that varying the target activity will also modify the disease (Tardiff and Lindquist, 2013; Khurana et al., 2015). The process of target validation (Blake, 2007) could be done using structure-activity relationships (SAR) of analogs of a lead compound, generating a drug-resistant mutant of the presumed target, or knockdown or overexpression of the presumed target and monitoring the known signaling systems downstream of this target.
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2015, Current Opinion in Genetics and DevelopmentCitation Excerpt :This protein is associated with Parkinson's disease and related neurodegenerative diseases, termed synucleinopathies [86]. Susan Lindquist et al. used drug screens to identify compounds that inhibited aggregation of the protein α-synuclein exogenously expressed in yeast [87]. They identified a class of compounds called N-Aryl Benzimidazoles (NABs) that inhibit α-synuclein aggregation in yeast cells and animal neurons [88].
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