Chee Yeun Chung

Identification and Rescue of α-Synuclein Toxicity in Parkinson Patient-Derived Neurons

From Yeast to Therapeutic? Yeast has shown some promise as a model system to generate lead compounds that could have therapeutic potential for the cellular problems associated with neurodegenerative diseases. Along these lines, Tardiff et al. (p. 979, published online 24 October) and Chung et al. (p. 983, published online 24 October) describe the results of multiple screens in yeast that lead to the identification of a potential therapeutic compound to combat the cytotoxic affect of α-synuclein accumulation. The compound was able to reverse the pathological hallmarks of Parkinsons disease in cultured neurons derived from patients with α-synuclein–induced Parkinsons disease dementia. Screening in yeast yields an effective therapeutic for Parkinson’s patient–derived neuronal stem cells. The induced pluripotent stem (iPS) cell field holds promise for in vitro disease modeling. However, identifying innate cellular pathologies, particularly for age-related neurodegenerative diseases, has been challenging. Here, we exploited mutation correction of iPS cells and conserved proteotoxic mechanisms from yeast to humans to discover and reverse phenotypic responses to α-synuclein (αsyn), a key protein involved in Parkinson’s disease (PD). We generated cortical neurons from iPS cells of patients harboring αsyn mutations, who are at high risk of developing PD dementia. Genetic modifiers from unbiased screens in a yeast model of αsyn toxicity led to identification of early pathogenic phenotypes in patient neurons. These included nitrosative stress, accumulation of endoplasmic reticulum (ER)–associated degradation substrates, and ER stress. A small molecule identified in a yeast screen (NAB2), and the ubiquitin ligase Nedd4 it affects, reversed pathologic phenotypes in these neurons.

Functional Links Between Aβ Toxicity, Endocytic Trafficking, and Alzheimer’s Disease Risk Factors in Yeast

The use of yeast as a model organism reveals cellular factors involved in beta-amyloid toxicity. Aβ (beta-amyloid peptide) is an important contributor to Alzheimer’s disease (AD). We modeled Aβ toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Aβ was previously unknown. The factors identified in yeast modified Aβ toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast, Aβ impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. Thus, links between Aβ, endocytosis, and human AD risk factors can be ascertained with yeast as a model system.

Functional Integration of Dopaminergic Neurons Directly Converted from Mouse Fibroblasts

Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of one somatic cell type to another by defined factors. However, it is not clear to what extent this type of reprogramming is able to generate fully functional differentiated cells. In addition, the activity of the reprogrammed cells in cell transplantation assays, such as those envisaged for cell-based therapy of Parkinsons disease (PD), remains to be determined. Here we show that ectopic expression of defined transcription factors in mouse tail tip fibroblasts is sufficient to induce Pitx3+ neurons that closely resemble midbrain dopaminergic (DA) neurons. In addition, transplantation of these induced DA (iDA) neurons alleviates symptoms in a mouse model of PD. Thus, iDA neurons generated from abundant somatic fibroblasts by direct lineage reprogramming hold promise for modeling neurodegenerative disease and for cell-based therapies of PD.

Yeast reveal a “druggable” Rsp5/Nedd4 Network that Ameliorates α–Synuclein Toxicity in Neurons

From Yeast to Therapeutic? Yeast has shown some promise as a model system to generate lead compounds that could have therapeutic potential for the cellular problems associated with neurodegenerative diseases. Along these lines, Tardiff et al. (p. 979, published online 24 October) and Chung et al. (p. 983, published online 24 October) describe the results of multiple screens in yeast that lead to the identification of a potential therapeutic compound to combat the cytotoxic affect of α-synuclein accumulation. The compound was able to reverse the pathological hallmarks of Parkinsons disease in cultured neurons derived from patients with α-synuclein–induced Parkinsons disease dementia. Screening in yeast yields an effective therapeutic for Parkinson’s patient–derived neuronal stem cells. α-Synuclein (α-syn) is a small lipid-binding protein implicated in several neurodegenerative diseases, including Parkinson’s disease, whose pathobiology is conserved from yeast to man. There are no therapies targeting these underlying cellular pathologies, or indeed those of any major neurodegenerative disease. Using unbiased phenotypic screens as an alternative to target-based approaches, we discovered an N-aryl benzimidazole (NAB) that strongly and selectively protected diverse cell types from α-syn toxicity. Three chemical genetic screens in wild-type yeast cells established that NAB promoted endosomal transport events dependent on the E3 ubiquitin ligase Rsp5/Nedd4. These same steps were perturbed by α-syn itself. Thus, NAB identifies a druggable node in the biology of α-syn that can correct multiple aspects of its underlying pathology, including dysfunctional endosomal and endoplasmic reticulum–to-Golgi vesicle trafficking.

SIRT1 protects against α-synuclein aggregation by activating molecular chaperones

α-Synuclein is a key molecule in the pathogenesis of synucleinopathy including dementia with Lewy bodies, Parkinsons disease, and multiple system atrophy. Sirtuins are NAD+-dependent protein deacetylases that are highly conserved and counter aging in lower organisms. We show that the life span of a mouse model with A53T α-synuclein mutation is increased by overexpressing SIRT1 and decreased by knocking out SIRT1 in brain. Furthermore, α-synuclein aggregates are reduced in the brains of mice with SIRT1 overexpression and increased by SIRT1 deletion. We show that SIRT1 deacetylates HSF1 (heat shock factor 1) and increases HSP70 RNA and protein levels, but only in the brains of mice with A53T and SIRT1 expression. Thus, SIRT1 responds to α-synuclein aggregation-induced stress by activating molecular chaperones to protect against disease.

Calcineurin determines toxic versus beneficial responses to α-synuclein

Significance Ca2+ homeostasis is indispensable for the well being of all living organisms. Ca2+ homeostasis is disrupted by α-synuclein (α-syn), whose misfolding plays a major role in neurodegenerative diseases termed synucleinopathies, such as Parkinson disease. We report that α-syn can induce sustained and highly elevated levels of cytoplasmic Ca2+, thereby activating a calcineurin (CN) cascade that results in toxicity. CN is a highly conserved Ca2+–calmodulin (CaM)-dependent phosphatase critical for sensing Ca2+ concentrations and transducing that information into cellular responses. Limiting, but not eliminating, the availability of CaM, CN and/or CN substrates directly with genetic or pharmacological tools shifts the α-syn–induced CN cascade to a protective mode. This has mechanistic implications for CNs activity and provides a therapeutic venue for the treatment of synucleinopathies. Calcineurin (CN) is a highly conserved Ca2+–calmodulin (CaM)-dependent phosphatase that senses Ca2+ concentrations and transduces that information into cellular responses. Ca2+ homeostasis is disrupted by α-synuclein (α-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that α-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca2+, thereby activating a CaM-CN cascade that engages substrates that result in toxicity. Surprisingly, complete inhibition of CN also results in toxicity. Limiting the availability of CaM shifts CNs spectrum of substrates toward protective pathways. Modulating CN or CNs substrates with highly selective genetic and pharmacological tools (FK506) does the same. FK506 crosses the blood brain barrier, is well tolerated in humans, and is active in neurons and glia. Thus, a tunable response to CN, which has been conserved for a billion years, can be targeted to rebalance the phosphatase’s activities from toxic toward beneficial substrates. These findings have immediate therapeutic implications for synucleinopathies.

Toward stem cell-based phenotypic screens for neurodegenerative diseases

In the absence of a single preventive or disease-modifying strategy, neurodegenerative diseases are becoming increasingly prevalent in our ageing population. The mechanisms underlying neurodegeneration are poorly understood, making the target-based drug screening strategies that are employed by the pharmaceutical industry fraught with difficulty. However, phenotypic screening in neurons and glia derived from patients is now conceivable through unprecedented developments in reprogramming, transdifferentiation, and genome editing. We outline progress in this nascent field, but also consider the formidable hurdles to identifying robust, disease-relevant and screenable cellular phenotypes in patient-derived cells. We illustrate how analysis in the simple bakers yeast cell Saccharaomyces cerevisiae is driving discovery in patient-derived neurons, and how approaches in this model organism can establish a paradigm to guide the development of stem cell-based phenotypic screens.

From yeast to patient neurons and back again: Powerful new discovery platforms

No disease‐modifying therapies are available for synucleinopathies, including Parkinsons disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA). The lack of therapies has been impeded by a paucity of validated drug targets and problematic cell‐based model systems. New approaches are therefore needed to identify genes and compounds that directly target the underlying cellular pathologies elicited by the pathological protein, α−synuclein (α−syn). This small, lipid‐binding protein impinges on evolutionarily conserved processes such as vesicle trafficking and mitochondrial function. For decades, the genetically tractable, single‐cell eukaryote, budding yeast, has been used to study nearly all aspects of cell biology. More recently, yeast has revealed key insights into the underlying cellular pathologies caused by α−syn. The robust cellular toxicity caused by α−syn expression facilitates unbiased high‐throughput small‐molecule screening. Critically, one must validate the discoveries made in yeast in disease‐relevant neuronal models. Here, we describe two recent reports that together establish yeast‐to‐human discovery platforms for synucleinopathies. In this exemplar, genes and small molecules identified in yeast were validated in patient‐derived neurons that present the same cellular phenotypes initially discovered in yeast. On validation, we returned to yeast, where unparalleled genetic approaches facilitated the elucidation of a small molecules mode of action. This approach enabled the identification and neuronal validation of a previously unknown “druggable” node that interfaces with the underlying, precipitating pathologies caused by α−syn. Such platforms can provide sorely needed leads and fresh ideas for disease‐modifying therapy for these devastating diseases.

From Yeast to Patients: The Audacity and Vision of Susan Lindquist

In this issue of Cell Systems, we present two papers (Chung et al., 2017; Khurana et al., 2017) that bring together 15 years of effort from the laboratory of Susan Lindquist, a visionary biologist we were privileged to call our mentor. Susan passed away from complications of cancer on October 27, 2016, just as the final revisions of these manuscripts were being submitted.

FKBP12 contributes to α-synuclein toxicity by regulating the calcineurin-dependent phosphoproteome

Significance Calcineurin is an essential Ca2+-dependent phosphatase in all eukaryotes. Whether calcineurin can be endogenously regulated by factors other than Ca2+ and calmodulin is not known. Using a model of Parkinsons Disease (PD) as a surrogate for high pathophysiological calcineurin activity and employing a shotgun proteomic approach, we show that the isomerase FKBP12 physiological regulates calcineurin activity by facilitating dephosphorylation of proteins involved in vesicle recycling. Using a rodent model of PD, partial inhibition of the functional interaction between FKBP12 and calcineurin blocks the phosphatase activity toward critical vesicle recycling proteins at nigral presynaptic terminals conferring strong neuroprotection. Our work reassigns to FKBP12 a novel mechanism that supports toxicity in a PD model by modulating calcineurins phosphatase activity with therapeutic implications. Calcineurin is an essential Ca2+-dependent phosphatase. Increased calcineurin activity is associated with α-synuclein (α-syn) toxicity, a protein implicated in Parkinson’s Disease (PD) and other neurodegenerative diseases. Calcineurin can be inhibited with Tacrolimus through the recruitment and inhibition of the 12-kDa cis-trans proline isomerase FK506-binding protein (FKBP12). Whether calcineurin/FKBP12 represents a native physiologically relevant assembly that occurs in the absence of pharmacological perturbation has remained elusive. We leveraged α-syn as a model to interrogate whether FKBP12 plays a role in regulating calcineurin activity in the absence of Tacrolimus. We show that FKBP12 profoundly affects the calcineurin-dependent phosphoproteome, promoting the dephosphorylation of a subset of proteins that contributes to α-syn toxicity. Using a rat model of PD, partial elimination of the functional interaction between FKBP12 and calcineurin, with low doses of the Food and Drug Administration (FDA)-approved compound Tacrolimus, blocks calcineurin’s activity toward those proteins and protects against the toxic hallmarks of α-syn pathology. Thus, FKBP12 can endogenously regulate calcineurin activity with therapeutic implications for the treatment of PD.