Targeting de novo lipid synthesis induces lipotoxicity and impairs DNA damage repair in glioblastoma mouse models.

Deregulated de novo lipid synthesis (DNLS) is a potential druggable vulnerability in glioblastoma (GBM), a highly lethal and incurable cancer. Yet the molecular mechanisms that determine susceptibility to DNLS-targeted therapies remain unknown, and the lack of brain-penetrant inhibitors of DNLS has prevented their clinical evaluation as GBM therapeutics. Here, we report that YTX-7739, a clinical-stage inhibitor of stearoyl CoA desaturase (SCD), triggers lipotoxicity in patient-derived GBM stem-like cells (GSCs) and inhibits fatty acid desaturation in GSCs orthotopically implanted in mice. When administered as a single agent, or in combination with temozolomide (TMZ), YTX-7739 showed therapeutic efficacy in orthotopic GSC mouse models owing to its lipotoxicity and ability to impair DNA damage repair. Leveraging genetic, pharmacological, and physiological manipulation of key signaling nodes in gliomagenesis complemented with shotgun lipidomics, we show that aberrant MEK/ERK signaling and its repression of the energy sensor AMP-activated protein kinase (AMPK) primarily drive therapeutic vulnerability to SCD and other DNLS inhibitors. Conversely, AMPK activation mitigates lipotoxicity and renders GSCs resistant to the loss of DNLS, both in culture and in vivo, by decreasing the saturation state of phospholipids and diverting toxic lipids into lipid droplets. Together, our findings reveal mechanisms of metabolic plasticity in GSCs and provide a framework for the rational integration of DNLS-targeted GBM therapies.

Automated algorithm development to assess survival of human neurons using longitudinal single-cell tracking: Application to synucleinopathy.

The development of phenotypic assays with appropriate analyses is an important step in the drug discovery process. Assays using induced pluripotent stem cell (iPSC)-derived human neurons are emerging as powerful tools for drug discovery in neurological disease. We have previously shown that longitudinal single cell tracking enabled the quantification of survival and death of neurons after overexpression of α-synuclein with a familial Parkinson's disease mutation (A53T). The reliance of this method on manual counting, however, rendered the process labor intensive, time consuming and error prone. To overcome these hurdles, we have developed automated detection algorithms for neurons using the BioStation CT live imaging system and CL-Quant software. In the current study, we use these algorithms to successfully measure the risk of neuronal death caused by overexpression of α-synuclein (A53T) with similar accuracy and improved consistency as compared to manual counting. This novel method also provides additional key readouts of neuronal fitness including total neurite length and the number of neurite nodes projecting from the cell body. Finally, the algorithm reveals the neuroprotective effects of brain-derived neurotrophic factor (BDNF) treatment in neurons overexpressing α-synuclein (A53T). These data show that an automated algorithm improves the consistency and considerably shortens the analysis time of assessing neuronal health, making this method advantageous for small molecule screening for inhibitors of synucleinopathy and other neurodegenerative diseases.

Patient-derived three-dimensional cortical neurospheres to model Parkinson's disease.

There are currently no preventive or disease-modifying therapies for Parkinson's Disease (PD). Failures in clinical trials necessitate a re-evaluation of existing pre-clinical models in order to adopt systems that better recapitulate underlying disease mechanisms and better predict clinical outcomes. In recent years, models utilizing patient-derived induced pluripotent stem cells (iPSC) have emerged as attractive models to recapitulate disease-relevant neuropathology in vitro without exogenous overexpression of disease-related pathologic proteins. Here, we utilized iPSC derived from patients with early-onset PD and dementia phenotypes that harbored either a point mutation (A53T) or multiplication at the α-synuclein/SNCA gene locus. We generated a three-dimensional (3D) cortical neurosphere culture model to better mimic the tissue microenvironment of the brain. We extensively characterized the differentiation process using quantitative PCR, Western immunoblotting and immunofluorescence staining. Differentiated and aged neurospheres revealed alterations in fatty acid profiles and elevated total and pathogenic phospho-α-synuclein levels in both A53T and the triplication lines compared to their isogenic control lines. Furthermore, treatment of the neurospheres with a small molecule inhibitor of stearoyl CoA desaturase (SCD) attenuated the protein accumulation and aberrant fatty acid profile phenotypes. Our findings suggest that the 3D cortical neurosphere model is a useful tool to interrogate targets for PD and amenable to test small molecule therapeutics.

The Parkinson's disease protein alpha-synuclein is a modulator of processing bodies and mRNA stability.

Alpha-synuclein (αS) is a conformationally plastic protein that reversibly binds to cellular membranes. It aggregates and is genetically linked to Parkinson's disease (PD). Here, we show that αS directly modulates processing bodies (P-bodies), membraneless organelles that function in mRNA turnover and storage. The N terminus of αS, but not other synucleins, dictates mutually exclusive binding either to cellular membranes or to P-bodies in the cytosol. αS associates with multiple decapping proteins in close proximity on the Edc4 scaffold. As αS pathologically accumulates, aberrant interaction with Edc4 occurs at the expense of physiologic decapping-module interactions. mRNA decay kinetics within PD-relevant pathways are correspondingly disrupted in PD patient neurons and brain. Genetic modulation of P-body components alters αS toxicity, and human genetic analysis lends support to the disease-relevance of these interactions. Beyond revealing an unexpected aspect of αS function and pathology, our data highlight the versatility of conformationally plastic proteins with high intrinsic disorder.

A Brain-Penetrant Stearoyl-CoA Desaturase Inhibitor Reverses α-Synuclein Toxicity.

Increasing evidence has shown that Parkinson's disease (PD) impairs midbrain dopaminergic, cortical and other neuronal subtypes in large part due to the build-up of lipid- and vesicle-rich α-synuclein (αSyn) cytotoxic inclusions. We previously identified stearoyl-CoA desaturase (SCD) as a potential therapeutic target for synucleinopathies. A brain-penetrant SCD inhibitor, YTX-7739, was developed and has entered Phase 1 clinical trials. Here, we report the efficacy of YTX-7739 in reversing pathological αSyn phenotypes in various in vitro and in vivo PD models. In cell-based assays, YTX-7739 decreased αSyn-mediated neuronal death, reversed the abnormal membrane interaction of amplified E46K ("3K") αSyn, and prevented pathological phenotypes in A53T and αSyn triplication patient-derived neurospheres, including dysregulated fatty acid profiles and pS129 αSyn accumulation. In 3K PD-like mice, YTX-7739 crossed the blood-brain barrier, decreased unsaturated fatty acids, and prevented progressive motor deficits. Both YTX-7739 treatment and decreasing SCD activity through deletion of one copy of the SCD1 gene (SKO) restored the physiological αSyn tetramer-to-monomer ratio, dopaminergic integrity, and neuronal survival in 3K αSyn mice. YTX-7739 efficiently reduced pS129 + and PK-resistant αSyn in both human wild-type αSyn and 3K mutant mice similar to the level of 3K-SKO. Together, these data provide further validation of SCD as a PD therapeutic target and YTX-7739 as a clinical candidate for treating human α-synucleinopathies.

Non-clinical Pharmacology of YTX-7739: a Clinical Stage Stearoyl-CoA Desaturase Inhibitor Being Developed for Parkinson's Disease.

Stearoyl-CoA desaturase (SCD) is a potential therapeutic target for Parkinson's and related neurodegenerative diseases. SCD inhibition ameliorates neuronal toxicity caused by aberrant α-synuclein, a lipid-binding protein implicated in Parkinson's disease. Its inhibition depletes monounsaturated fatty acids, which may modulate α-synuclein conformations and membrane interactions. Herein, we characterize the pharmacokinetic and pharmacodynamic properties of YTX-7739, a clinical-stage SCD inhibitor. Administration of YTX-7739 to rats and monkeys for 15 days caused a dose-dependent increase in YTX-7739 concentrations that were well-tolerated and associated with concentration-dependent reductions in the fatty acid desaturation index (FADI), the ratio of monounsaturated to saturated fatty acids. An approximate 50% maximal reduction in the carbon-16 desaturation index was observed in the brain, with comparable responses in the plasma and skin. A study with a diet supplemented in SCD products indicates that changes in brain C16 desaturation were due to local SCD inhibition, rather than to changes in systemic fatty acids that reach the brain. Assessment of pharmacodynamic response onset and reversibility kinetics indicated that approximately 7 days of dosing were required to achieve maximal responses, which persisted for at least 2 days after cessation of dosing. YTX-7739 thus achieved sufficient concentrations in the brain to inhibit SCD and produce pharmacodynamic responses that were well-tolerated in rats and monkeys. These results provide a framework for evaluating YTX-7739 pharmacology clinically as a disease-modifying therapy to treat synucleinopathies.

In Situ Peroxidase Labeling and Mass Spectrometry of Alpha-Synuclein in Rat Cortical Neurons.

In this chapter, we describe a novel ascorbate peroxidase (APEX)-based labeling method that in combination with mass spectrometry identifies proteins in the immediate vicinity of αSyn in living rat cortical neurons. To isolate these interactions, we transduced primary cortical neurons with a lentivirus encoding APEX2 tagged to the C-terminus of alpha-synuclein (αSyn) and under the control of a synapsin promoter. Neural protein lysates were then incubated with streptavidin magnetic beads, washed, eluted from the beads, and digested overnight. The desalted peptides were then labeled with iTRAQ (4-plex) reagents and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). Collected data were analyzed using Spectrum Mill software, ultimately shedding light on αSyn physiological function and abnormal behavior during pathology.

Inhibiting Stearoyl-CoA Desaturase Ameliorates α-Synuclein Cytotoxicity.

The lack of disease-modifying treatments for neurodegenerative disease stems in part from our rudimentary understanding of disease mechanisms and the paucity of targets for therapeutic intervention. Here we used an integrated discovery paradigm to identify a new therapeutic target for diseases caused by α-synuclein (α-syn), a small lipid-binding protein that misfolds and aggregates in Parkinson's disease and other disorders. Using unbiased phenotypic screening, we identified a series of compounds that were cytoprotective against α-syn-mediated toxicity by inhibiting the highly conserved enzyme stearoyl-CoA desaturase (SCD). Critically, reducing the levels of unsaturated membrane lipids by inhibiting SCD reduced α-syn toxicity in human induced pluripotent stem cell (iPSC) neuronal models. Taken together, these findings suggest that inhibition of fatty acid desaturation has potential as a therapeutic approach for the treatment of Parkinson's disease and other synucleinopathies.

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

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.

In Situ Peroxidase Labeling and Mass-Spectrometry Connects Alpha-Synuclein Directly to Endocytic Trafficking and mRNA Metabolism in Neurons.

Synucleinopathies, including Parkinson's disease (PD), are associated with the misfolding and mistrafficking of alpha-synuclein (α-syn). Here, using an ascorbate peroxidase (APEX)-based labeling method combined with mass spectrometry, we defined a network of proteins in the immediate vicinity of α-syn in living neurons to shed light on α-syn function. This approach identified 225 proteins, including synaptic proteins, proteins involved in endocytic vesicle trafficking, the retromer complex, phosphatases and mRNA binding proteins. Many were in complexes with α-syn, and some were encoded by genes known to be risk factors for PD and other neurodegenerative diseases. Endocytic trafficking and mRNA translation proteins within this spatial α-syn map overlapped with genetic modifiers of α-syn toxicity, developed in an accompanying study (Khurana et al., this issue of Cell Systems). Our data suggest that perturbation of these particular pathways is directly related to the spatial localization of α-syn within the cell. These approaches provide new avenues to systematically examine protein function and pathology in living cells.

Genome-Scale Networks Link Neurodegenerative Disease Genes to α-Synuclein through Specific Molecular Pathways.

Numerous genes and molecular pathways are implicated in neurodegenerative proteinopathies, but their inter-relationships are poorly understood. We systematically mapped molecular pathways underlying the toxicity of alpha-synuclein (α-syn), a protein central to Parkinson's disease. Genome-wide screens in yeast identified 332 genes that impact α-syn toxicity. To "humanize" this molecular network, we developed a computational method, TransposeNet. This integrates a Steiner prize-collecting approach with homology assignment through sequence, structure, and interaction topology. TransposeNet linked α-syn to multiple parkinsonism genes and druggable targets through perturbed protein trafficking and ER quality control as well as mRNA metabolism and translation. A calcium signaling hub linked these processes to perturbed mitochondrial quality control and function, metal ion transport, transcriptional regulation, and signal transduction. Parkinsonism gene interaction profiles spatially opposed in the network (ATP13A2/PARK9 and VPS35/PARK17) were highly distinct, and network relationships for specific genes (LRRK2/PARK8, ATXN2, and EIF4G1/PARK18) were confirmed in patient induced pluripotent stem cell (iPSC)-derived neurons. This cross-species platform connected diverse neurodegenerative genes to proteinopathy through specific mechanisms and may facilitate patient stratification for targeted therapy.

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 baker's 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 platform.

No disease-modifying therapies are available for synucleinopathies, including Parkinson's 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 molecule's 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.

Calcineurin determines toxic versus beneficial responses to α-synuclein.

Calcineurin (CN) is a highly conserved Ca(2+)-calmodulin (CaM)-dependent phosphatase that senses Ca(2+) concentrations and transduces that information into cellular responses. Ca(2+) 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 Ca(2+), 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 CN's spectrum of substrates toward protective pathways. Modulating CN or CN's 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.

Identification and rescue of α-synuclein toxicity in Parkinson patient-derived neurons.

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.

Yeast reveal a "druggable" Rsp5/Nedd4 network that ameliorates α-synuclein toxicity in neurons.

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, Parkinson's 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.

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 Parkinson's 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.