Tuning Hsp104 specificity to selectively detoxify α-synuclein.

Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, α-synuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms involving α-synuclein disaggregation or detoxification of soluble α-synuclein conformers. Importantly, both types of α-synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinson's disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.

Potentiated Hsp104 variants antagonize diverse proteotoxic misfolding events.

There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid but has no metazoan homolog and only limited activity against human neurodegenerative disease proteins. Here, we reprogram Hsp104 to rescue TDP-43, FUS, and α-synuclein proteotoxicity by mutating single residues in helix 1, 2, or 3 of the middle domain or the small domain of nucleotide-binding domain 1. Potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations reconfigure how Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Our work establishes that disease-associated aggregates and amyloid are tractable targets and that enhanced disaggregases can restore proteostasis and mitigate neurodegeneration.

Gaucher disease glucocerebrosidase and α-synuclein form a bidirectional pathogenic loop in synucleinopathies.

Parkinson's disease (PD), an adult neurodegenerative disorder, has been clinically linked to the lysosomal storage disorder Gaucher disease (GD), but the mechanistic connection is not known. Here, we show that functional loss of GD-linked glucocerebrosidase (GCase) in primary cultures or human iPS neurons compromises lysosomal protein degradation, causes accumulation of α-synuclein (α-syn), and results in neurotoxicity through aggregation-dependent mechanisms. Glucosylceramide (GlcCer), the GCase substrate, directly influenced amyloid formation of purified α-syn by stabilizing soluble oligomeric intermediates. We further demonstrate that α-syn inhibits the lysosomal activity of normal GCase in neurons and idiopathic PD brain, suggesting that GCase depletion contributes to the pathogenesis of sporadic synucleinopathies. These findings suggest that the bidirectional effect of α-syn and GCase forms a positive feedback loop that may lead to a self-propagating disease. Therefore, improved targeting of GCase to lysosomes may represent a specific therapeutic approach for PD and other synucleinopathies.

Integrated regulation of dopaminergic and epigenetic effectors of neuroprotection in Parkinson's disease models.

Whole-exome sequencing of Parkinson's disease (PD) patient DNA identified single-nucleotide polymorphisms (SNPs) in the tyrosine nonreceptor kinase-2 (TNK2) gene. Although this kinase had a previously demonstrated activity in preventing the endocytosis of the dopamine reuptake transporter (DAT), a causal role for TNK2-associated dysfunction in PD remains unresolved. We postulated the dopaminergic neurodegeneration resulting from patient-associated variants in TNK2 were a consequence of aberrant or prolonged TNK2 overactivity, the latter being a failure in TNK2 degradation by an E3 ubiquitin ligase, neuronal precursor cell-expressed developmentally down-regulated-4 (NEDD4). Interestingly, systemic RNA interference protein-3 (SID-3) is the sole TNK2 ortholog in the nematode Caenorhabditis elegans, where it is an established effector of epigenetic gene silencing mediated through the dsRNA-transporter, SID-1. We hypothesized that TNK2/SID-3 represents a node of integrated dopaminergic and epigenetic signaling essential to neuronal homeostasis. Use of a TNK2 inhibitor (AIM-100) or a NEDD4 activator [N-aryl benzimidazole 2 (NAB2)] in bioassays for either dopamine- or dsRNA-uptake into worm dopaminergic neurons revealed that sid-3 mutants displayed robust neuroprotection from 6-hydroxydopamine (6-OHDA) exposures, as did AIM-100 or NAB2-treated wild-type animals. Furthermore, NEDD4 activation by NAB2 in rat primary neurons correlated to a reduction in TNK2 levels and the attenuation of 6-OHDA neurotoxicity. CRISPR-edited nematodes engineered to endogenously express SID-3 variants analogous to TNK2 PD-associated SNPs exhibited enhanced susceptibility to dopaminergic neurodegeneration and circumvented the RNAi resistance characteristic of SID-3 dysfunction. This research exemplifies a molecular etiology for PD whereby dopaminergic and epigenetic signaling are coordinately regulated to confer susceptibility or resilience to neurodegeneration.

Systemic RNA Interference Defective (SID) genes modulate dopaminergic neurodegeneration in C. elegans.

The fine-tuning of gene expression is critical for all cellular processes; aberrations in this activity can lead to pathology, and conversely, resilience. As their role in coordinating organismal responses to both internal and external factors have increasingly come into focus, small non-coding RNAs have emerged as an essential component to disease etiology. Using Systemic RNA interference Defective (SID) mutants of the nematode Caenorhabditis elegans, deficient in gene silencing, we examined the potential consequences of dysfunctional epigenomic regulation in the context of Parkinson's disease (PD). Specifically, the loss of either the sid-1 or sid-3 genes, which encode a dsRNA transporter and an endocytic regulatory non-receptor tyrosine kinase, respectively, conferred neuroprotection to dopaminergic (DA) neurons in an established transgenic C. elegans strain wherein overexpression of human α-synuclein (α-syn) from a chromosomally integrated multicopy transgene causes neurodegeneration. We further show that knockout of a specific microRNA, mir-2, attenuates α-syn neurotoxicity; suggesting that the native targets of mir-2-dependent gene silencing represent putative neuroprotective modulators. In support of this, we demonstrated that RNAi knockdown of multiple mir-2 targets enhanced α-syn-induced DA neurodegeneration. Moreover, we demonstrate that mir-2 overexpression originating in the intestine can induce neurodegeneration of DA neurons, an effect that was reversed by pharmacological inhibition of SID-3 activity. Interestingly, sid-1 mutants retained mir-2-induced enhancement of neurodegeneration. Transcriptomic analysis of α-syn animals with and without a sid-1 mutation revealed 27 differentially expressed genes with human orthologs related to a variety of diseases, including PD. Among these was pgp-8, encoding a P-glycoprotein-related ABC transporter. Notably, sid-1; pgp-8 double mutants abolished the neurodegeneration resulting from intestinal mir-2 overexpression. This research positions known regulators of small RNA-dependent gene silencing within a framework that facilitates mechanistic evaluation of epigenetic responses to exogenous and endogenous factors influencing DA neurodegeneration, revealing a path toward new targets for therapeutic intervention of PD.

From bugs to bedside: functional annotation of human genetic variation for neurological disorders using invertebrate models.

The exponential accumulation of DNA sequencing data has opened new avenues for discovering the causative roles of single-nucleotide polymorphisms (SNPs) in neurological diseases. The opportunities emerging from this are staggering, yet only as good as our abilities to glean insights from this surplus of information. Whereas computational biology continues to improve with respect to predictions and molecular modeling, the differences between in silico and in vivo analysis remain substantial. Invertebrate in vivo model systems represent technically advanced, experimentally mature, high-throughput, efficient and cost-effective resources for investigating a disease. With a decades-long track record of enabling investigators to discern function from DNA, fly (Drosophila) and worm (Caenorhabditis elegans) models have never been better poised to serve as living engines of discovery. Both of these animals have already proven useful in the classification of genetic variants as either pathogenic or benign across a range of neurodevelopmental and neurodegenerative disorders-including autism spectrum disorders, ciliopathies, amyotrophic lateral sclerosis, Alzheimer's and Parkinson's disease. Pathogenic SNPs typically display distinctive phenotypes in functional assays when compared with null alleles and frequently lead to protein products with gain-of-function or partial loss-of-function properties that contribute to neurological disease pathogenesis. The utility of invertebrates is logically limited by overt differences in anatomical and physiological characteristics, and also the evolutionary distance in genome structure. Nevertheless, functional annotation of disease-SNPs using invertebrate models can expedite the process of assigning cellular and organismal consequences to mutations, ascertain insights into mechanisms of action, and accelerate therapeutic target discovery and drug development for neurological conditions.

A conformational switch driven by phosphorylation regulates the activity of the evolutionarily conserved SNARE Ykt6.

Ykt6 is a soluble N-ethylmaleimide sensitive factor activating protein receptor (SNARE) critically involved in diverse vesicular fusion pathways. While most SNAREs rely on transmembrane domains for their activity, Ykt6 dynamically cycles between the cytosol and membrane-bound compartments where it is active. The mechanism that regulates these transitions and allows Ykt6 to achieve specificity toward vesicular pathways is unknown. Using a Parkinson's disease (PD) model, we found that Ykt6 is phosphorylated at an evolutionarily conserved site which is regulated by Ca2+ signaling. Through a multidisciplinary approach, we show that phosphorylation triggers a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form, the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways, enhancing toxicity in PD models. Our studies reveal a mechanism by which Ykt6 conformation and activity are regulated with potential implications for PD.

Distinct functional roles of Vps41-mediated neuroprotection in Alzheimer's and Parkinson's disease models of neurodegeneration.

Commonalities and, in some cases, pathological overlap between neurodegenerative diseases have led to speculation that targeting of underlying mechanisms might be of potentially shared therapeutic benefit. Alzheimer's disease is characterized by the formation of plaques, composed primarily of the amyloid-ß 1-42 (Aß) peptide in the brain, resulting in neurodegeneration. Previously, we have shown that overexpression of the lysosomal-trafficking protein, human Vps41 (hVps41), is neuroprotective in a transgenic worm model of Parkinson's disease, wherein progressive dopaminergic neurodegeneration is induced by α-synuclein overexpression. Here, we report the results of a systematic comparison of hVps41-mediated neuroprotection between α-synuclein and Aß in transgenic nematode models of Caenorhabditis elegans. Our results indicate that an ARF-like GTPase gene product, ARL-8, mitigates endocytic Aß neurodegeneration in a VPS-41-dependent manner, rather than through RAB-7 and AP3 as with α-synuclein. Furthermore, the neuroprotective effect of ARL-8 or hVps41 appears to be dependent on their colocalization and the activity of ARL-8. Additionally, we demonstrate that the LC3 orthologue, LGG-2, plays a critical role in Aß toxicity with ARL-8. Further analysis of functional effectors of Aß protein processing via the lysosomal pathway will assist in the elucidation of the underlying mechanism involving VPS-41-mediated neuroprotection. These results reveal functional distinctions in the intracellular management of neurotoxic proteins that serve to better inform the path for development of therapeutic interventions to halt neurodegeneration.

Alpha-synuclein inhibits Snx3-retromer-mediated retrograde recycling of iron transporters in S. cerevisiae and C. elegans models of Parkinson's disease.

We probed the role of alpha-synuclein (α-syn) in modulating sorting nexin 3 (Snx3)-retromer-mediated recycling of iron transporters in Saccharomyces cerevisiae and Caenorhabditis elegans. In yeast, the membrane-bound heterodimer Fet3/Ftr1 is the high affinity iron importer. Fet3 is a membrane-bound multicopper ferroxidase, whose ferroxidase domain is orthologous to human ceruloplasmin (Cp), that oxidizes external Fe+2 to Fe+3; the Fe+3 ions then channel through the Ftr1 permease into the cell. When the concentration of external iron is low (<1 µM), Fet3/Ftr1 is maintained on the plasma membrane by retrograde endocytic-recycling; whereas, when the concentration of external iron is high (>10 µM), Fet3/Ftr1 is endocytosed and shunted to the vacuole for degradation. We discovered that α-syn expression phenocopies the high iron condition: under the low iron condition (<1 µM), α-syn inhibits Snx3-retromer-mediated recycling of Fet3/Ftr1 and instead shunts Fet3/Ftr1 into the multivesicular body pathway to the vacuole. α-Syn inhibits recycling by blocking the association of Snx3-mCherry molecules with endocytic vesicles, possibly by interfering with the binding of Snx3 to phosphatidylinositol-3-monophosphate. In C. elegans, transgenic worms expressing α-syn exhibit an age-dependent degeneration of dopaminergic neurons that is partially rescued by the iron chelator desferoxamine. This implies that α-syn-expressing dopaminergic neurons are susceptible to changes in iron neurotoxicity with age, whereby excess iron enhances α-syn-induced neurodegeneration. In vivo genetic analysis indicates that α-syn dysregulates iron homeostasis in worm dopaminergic neurons, possibly by inhibiting SNX-3-mediated recycling of a membrane-bound ortholog of Cp (F21D5.3), the iron exporter ferroportin (FPN1.1), or both.

NCEH-1 modulates cholesterol metabolism and protects against α-synuclein toxicity in a C. elegans model of Parkinson's disease.

Parkinson's disease (PD) is an aging-associated neurodegenerative disease affecting millions worldwide. Misfolding, oligomerization and accumulation of the human α-synuclein protein is a key pathological hallmark of PD and is associated with the progressive loss of dopaminergic neurons over the course of aging. Lifespan extension via the suppression of IGF-1/insulin-like signaling (IIS) offers a possibility to retard disease onset through induction of metabolic changes that provide neuroprotection. The nceh-1 gene of Caenorhabditis elegans encodes an ortholog of neutral cholesterol ester hydrolase 1 (NCEH-1), an IIS downstream protein that was identified in a screen as a modulator of α-synuclein accumulation in vivo. The mechanism whereby cholesterol metabolism functionally impacts neurodegeneration induced by α-synuclein is undefined. Here we report that NCEH-1 protects dopaminergic neurons from α-synuclein-dependent neurotoxicity in C. elegans via a mechanism that is independent of lifespan extension. We discovered that the presence of cholesterol, LDLR-mediated cholesterol endocytosis, and cholesterol efflux are all essential to NCEH-1-mediated neuroprotection. In protecting from α-synuclein neurotoxicity, NCEH-1 also stimulates cholesterol-derived neurosteroid formation and lowers cellular reactive oxygen species in mitochondria. Collectively, this study augments our understanding of how cholesterol metabolism can modulate a neuroprotective mechanism that attenuates α-synuclein neurotoxicity, thereby pointing toward regulation of neuronal cholesterol turnover as a potential therapeutic avenue for PD.

Genetic Defects in Mitochondrial Dynamics in Caenorhabditis elegans Impact Ultraviolet C Radiation- and 6-hydroxydopamine-Induced Neurodegeneration.

BACKGROUND: Parkinson's disease (PD) is one of the most common neurodegenerative disorders involving devastating loss of dopaminergic neurons in the substantia nigra. Early steps in PD pathogenesis include mitochondrial dysfunction, and mutations in mitochondrial genes have been linked to familial forms of the disease. However, low penetrance of mutations indicates a likely important role for environmental factors in PD risk through gene by environment interactions. Herein, we study how genetic deficiencies in mitochondrial dynamics processes including fission, fusion, and mitophagy interact with environmental exposures to impact neurodegeneration. METHODS: We utilized the powerful model organism Caenorhabditis elegans to study ultraviolet C radiation (UVC)- and 6-hydroxydopamine-induced degeneration of fluorescently-tagged dopaminergic neurons in the background of fusion deficiency (MFN1/2 homolog, fzo-1), fission deficiency (DMN1L homolog, drp-1), and mitochondria-specific autophagy (mitophagy) deficiency (PINK1 and PRKN homologs, pink-1 and pdr-1). RESULTS: Overall, we found that deficiency in either mitochondrial fusion or fission sensitizes nematodes to UVC exposure (used to model common environmental pollutants) but protects from 6-hydroxydopamine-induced neurodegeneration. By contrast, mitophagy deficiency makes animals more sensitive to these stressors with an interesting exception-pink-1 deficiency conferred remarkable protection from 6-hydroxydopamine. We found that this protection could not be explained by compensatory antioxidant gene expression in pink-1 mutants or by differences in mitochondrial morphology. CONCLUSIONS: Together, our results support a strong role for gene by environment interactions in driving dopaminergic neurodegeneration and suggest that genetic deficiency in mitochondrial processes can have complex effects on neurodegeneration.

Dysregulation of the Mitochondrial Unfolded Protein Response Induces Non-Apoptotic Dopaminergic Neurodegeneration in C. elegans Models of Parkinson's Disease.

Due to environmental insult or innate genetic deficiency, protein folding environments of the mitochondrial matrix are prone to dysregulation, prompting the activation of a specific organellar stress-response mechanism, the mitochondrial unfolded protein response (UPRMT). In Caenorhabditis elegans, mitochondrial damage leads to nuclear translocation of the ATFS-1 transcription factor to activate the UPRMT After short-term acute stress has been mitigated, the UPRMT is eventually suppressed to restore homeostasis to C. elegans hermaphrodites. In contrast, and reflective of the more chronic nature of progressive neurodegenerative disorders such as Parkinson's disease (PD), here, we report the consequences of prolonged, cell-autonomous activation of the UPRMT in C. elegans dopaminergic neurons. We reveal that neuronal function and integrity decline rapidly with age, culminating in activity-dependent, non-apoptotic cell death. In a PD-like context wherein transgenic nematodes express the Lewy body constituent protein α-synuclein (αS), we not only find that this protein and its PD-associated disease variants have the capacity to induce the UPRMT, but also that coexpression of αS and ATFS-1-associated dysregulation of the UPRMT synergistically potentiate dopaminergic neurotoxicity. This genetic interaction is in parallel to mitophagic pathways dependent on the C. elegans PINK1 homolog, which is necessary for cellular resistance to chronic malfunction of the UPRMT Given the increasingly recognized role of mitochondrial quality control in neurodegenerative diseases, these studies illustrate, for the first time, an insidious aspect of mitochondrial signaling in which the UPRMT pathway, under disease-associated, context-specific dysregulation, exacerbates disruption of dopaminergic neurons in vivo, resulting in the neurodegeneration characteristic of PD.SIGNIFICANCE STATEMENT Disruptions or alterations in the activation of pathways that regulate mitochondrial quality control have been linked to neurodegenerative diseases due in part to the central role of mitochondria in metabolism, ROS regulation, and proteostasis. The extent to which these pathways, including the mitochondrial unfolded protein response (UPRMT) and mitophagy, are active may predict severity and progression of these disorders, as well as sensitivity to compounding stressors. Furthermore, therapeutic strategies that aim to induce these pathways may benefit from increased study into cellular responses that arise from long-term or ectopic stimulation, especially in neuronal compartments. By demonstrating the detrimental consequences of prolonged cellular activation of the UPRMT, we provide evidence that this pathway is not a universally beneficial mechanism because dysregulation has neurotoxic consequences.

Glutaredoxin deficiency exacerbates neurodegeneration in C. elegans models of Parkinson's disease.

Parkinson's disease (PD) is characterized by selective degeneration of dopaminergic neurons. Although the etiology of PD remains incompletely understood, oxidative stress has been implicated as an important contributor in the development of PD. Oxidative stress can lead to oxidation and functional perturbation of proteins critical to neuronal survival. Glutaredoxin 1 (Grx1) is an evolutionally conserved antioxidant enzyme that repairs protein oxidation by reversing the oxidative modification of cysteine known as S-glutathionylation. We aimed to explore the regulatory role of Grx1 in PD. We first examined the levels of Grx1 in postmortem midbrain samples from PD patients, and observed that Grx1 content is decreased in PD, specifically within the dopaminergic neurons. We subsequently investigated the potential role of Grx1 deficiency in PD pathogenesis by examining the consequences of loss of the Caenorhabditis elegans Grx1 homolog in well-established worm models of familial PD caused by overexpression of pathogenic human LRRK2 mutants G2019S or R1441C. We found that loss of the Grx1 homolog led to significant exacerbation of the neurodegenerative phenotype in C. elegans overexpressing the human LRRK2 mutants. Re-expression in the dopaminergic neurons of the active, but not a catalytically inactive form of the Grx1 homolog rescued the exacerbated phenotype. Loss of the Grx1 homolog also exacerbated the neurodegenerative phenotype in other C. elegans models, including overexpression of human α-synuclein and overexpression of tyrosine hydroxylase (a model of sporadic PD). Therefore, our results reveal a novel neuroprotective role of glutaredoxin against dopaminergic neurodegeneration in models of familial and sporadic PD.

Clioquinol promotes the degradation of metal-dependent amyloid-ß (Aß) oligomers to restore endocytosis and ameliorate Aß toxicity.

Alzheimer's disease (AD) is a common, progressive neurodegenerative disorder without effective disease-modifying therapies. The accumulation of amyloid-ß peptide (Aß) is associated with AD. However, identifying new compounds that antagonize the underlying cellular pathologies caused by Aß has been hindered by a lack of cellular models amenable to high-throughput chemical screening. To address this gap, we use a robust and scalable yeast model of Aß toxicity where the Aß peptide transits through the secretory and endocytic compartments as it does in neurons. The pathogenic Aß 1-42 peptide forms more oligomers and is more toxic than Aß 1-40 and genome-wide genetic screens identified genes that are known risk factors for AD. Here, we report an unbiased screen of ∼140,000 compounds for rescue of Aß toxicity. Of ∼30 hits, several were 8-hydroxyquinolines (8-OHQs). Clioquinol (CQ), an 8-OHQ previously reported to reduce Aß burden, restore metal homeostasis, and improve cognition in mouse AD models, was also effective and rescued the toxicity of Aß secreted from glutamatergic neurons in Caenorhabditis elegans. In yeast, CQ dramatically reduced Aß peptide levels in a copper-dependent manner by increasing degradation, ultimately restoring endocytic function. This mirrored its effects on copper-dependent oligomer formation in vitro, which was also reversed by CQ. This unbiased screen indicates that copper-dependent Aß oligomer formation contributes to Aß toxicity within the secretory/endosomal pathways where it can be targeted with selective metal binding compounds. Establishing the ability of the Aß yeast model to identify disease-relevant compounds supports its further exploitation as a validated early discovery platform.

Phosphatidylethanolamine deficiency disrupts α-synuclein homeostasis in yeast and worm models of Parkinson disease.

Phosphatidylserine decarboxylase, which is embedded in the inner mitochondrial membrane, synthesizes phosphatidylethanolamine (PE) and, in some cells, synthesizes the majority of this important phospholipid. Normal levels of PE can decline with age in the brain. Here we used yeast and worms to test the hypothesis that low levels of PE alter the homeostasis of the Parkinson disease-associated protein α-synuclein (α-syn). In yeast, low levels of PE in the phosphatidylserine decarboxylase deletion mutant (psd1Δ) cause decreased respiration, endoplasmic reticulum (ER) stress, a defect in the trafficking of the uracil permease, α-syn accumulation and foci, and a slow growth phenotype. Supplemental ethanolamine (ETA), which can be converted to PE via the Kennedy pathway enzymes in the ER, had no effect on respiration, whereas, in contrast, this metabolite partially eliminated ER stress, decreased α-syn foci formation, and restored growth close to that of wild-type cells. In Caenorhabditis elegans, RNAi depletion of phosphatidylserine decarboxylase in dopaminergic neurons expressing α-syn accelerates neurodegeneration, which supplemental ETA rescues. ETA fails to rescue this degeneration in worms that undergo double RNAi depletion of phosphatidylserine decarboxylase (psd-1) and choline/ETA phosphotransferase (cept-1), which encodes the last enzyme in the CDP-ETA Kennedy pathway. This finding suggests that ETA exerts its protective effect by boosting PE through the Kennedy pathway. Overall, a low level of PE causes ER stress, disrupts vesicle trafficking, and causes α-syn to accumulate; such cells likely die from a combination of ER stress and excessive accumulation of α-syn.

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.

RTCB-1 mediates neuroprotection via XBP-1 mRNA splicing in the unfolded protein response pathway.

Parkinson's disease (PD), the second most prevalent neurodegenerative disorder, is characterized by the degeneration of dopamine (DA) neurons and age-dependent formation of protein inclusions that contain the α-synuclein (α-syn) protein. RNA interference (RNAi) screening using Caenorhabditis elegans identified RTCB-1, an uncharacterized gene product, as one of several significant modifiers of α-syn protein misfolding. RTCB-1 is the worm ortholog of the human HSPC117 protein, a component of RNA trafficking granules in mammalian neurons. Here we show that RTCB-1 protects C. elegans DA neurons from age-dependent degeneration induced by human α-syn. Moreover, neuronal-specific RNAi depletion of rtcb-1 enhanced α-syn-induced degeneration. Similar results were obtained when worms were exposed to the DA neurotoxin 6-hydroxydopamine. HSPC117 has been characterized recently as an essential subunit of the human tRNA splicing ligase complex. tRNA ligases have alternative functions in RNA repair and nonconventional mRNA splicing events. For example, in yeast, unconventional splicing of HAC1, a transcription factor that controls the unfolded protein response (UPR), is mediated by a tRNA ligase. In C. elegans, we demonstrate that RTCB-1 is necessary for xbp-1 (worm homolog of HAC1) mRNA splicing. Moreover, using a RNA ligase-dead mutant, we determine that the ligase activity of worm RTCB-1 is required for its neuroprotective role, which, in turn, is mediated through XBP-1 in the UPR pathway. Collectively, these studies highlight the mechanistic intersection of RNA processing and proteostasis in mediating neuroprotection.

Identification of novel ATP13A2 interactors and their role in α-synuclein misfolding and toxicity.

Lysosomes are responsible for degradation and recycling of bulky cell material, including accumulated misfolded proteins and dysfunctional organelles. Increasing evidence implicates lysosomal dysfunction in several neurodegenerative disorders, including Parkinson's disease and related synucleinopathies, which are characterized by the accumulation of α-synuclein (α-syn) in Lewy bodies. Studies of lysosomal proteins linked to neurodegenerative disorders present an opportunity to uncover specific molecular mechanisms and pathways that contribute to neurodegeneration. Loss-of-function mutations in a lysosomal protein, ATP13A2 (PARK9), cause Kufor-Rakeb syndrome that is characterized by early-onset parkinsonism, pyramidal degeneration and dementia. While loss of ATP13A2 function plays a role in α-syn misfolding and toxicity, the normal function of ATP13A2 in the brain remains largely unknown. Here, we performed a screen to identify ATP13A2 interacting partners, as a first step toward elucidating its function. Utilizing a split-ubiquitin membrane yeast two-hybrid system that was developed to identify interacting partners of full-length integral membrane proteins, we identified 43 novel interactors that primarily implicate ATP13A2 in cellular processes such as endoplasmic reticulum (ER) translocation, ER-to-Golgi trafficking and vesicular transport and fusion. We showed that a subset of these interactors modified α-syn aggregation and α-syn-mediated degeneration of dopaminergic neurons in Caenorhabditis elegans, further suggesting that ATP13A2 and α-syn are functionally linked in neurodegeneration. These results implicate ATP13A2 in vesicular trafficking and provide a platform for further studies of ATP13A2 in neurodegeneration.

Functional analysis of VPS41-mediated neuroprotection in Caenorhabditis elegans and mammalian models of Parkinson's disease.

Disruption of the lysosomal system has emerged as a key cellular pathway in the neurotoxicity of α-synuclein (α-syn) and the progression of Parkinson's disease (PD). A large-scale RNA interference (RNAi) screen using Caenorhabditis elegans identified VPS-41, a multidomain protein involved in lysosomal protein trafficking, as a modifier of α-syn accumulation and dopaminergic neuron degeneration (Hamamichi et al., 2008). Previous studies have shown a conserved neuroprotective function of human VPS41 (hVPS41) against PD-relevant toxins in mammalian cells and C. elegans neurons (Ruan et al., 2010). Here, we report that both the AP-3 (heterotetrameric adaptor protein complex) interaction domain and clathrin heavy-chain repeat domain are required for protecting C. elegans dopaminergic neurons from α-syn-induced neurodegeneration, as well as to prevent α-syn inclusion formation in an H4 human neuroglioma cell model. Using mutant C. elegans and neuron-specific RNAi, we revealed that hVPS41 requires both a functional AP-3 (heterotetrameric adaptor protein complex) and HOPS (homotypic fusion and vacuole protein sorting)-tethering complex to elicit neuroprotection. Interestingly, two nonsynonymous single-nucleotide polymorphisms found within the AP-3 interacting domain of hVPS41 attenuated the neuroprotective property, suggestive of putative susceptibility factors for PD. Furthermore, we observed a decrease in α-syn protein level when hVPS41 was overexpressed in human neuroglioma cells. Thus, the neuroprotective capacity of hVPS41 may be a consequence of enhanced clearance of misfolded and aggregated proteins, including toxic α-syn species. These data reveal the importance of lysosomal trafficking in maintaining cellular homeostasis in the presence of enhanced α-syn expression and toxicity. Our results support hVPS41 as a potential novel therapeutic target for the treatment of synucleinopathies like PD.

Different 8-hydroxyquinolines protect models of TDP-43 protein, α-synuclein, and polyglutamine proteotoxicity through distinct mechanisms.

No current therapies target the underlying cellular pathologies of age-related neurodegenerative diseases. Model organisms provide a platform for discovering compounds that protect against the toxic, misfolded proteins that initiate these diseases. One such protein, TDP-43, is implicated in multiple neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In yeast, TDP-43 expression is toxic, and genetic modifiers first discovered in yeast have proven to modulate TDP-43 toxicity in both neurons and humans. Here, we describe a phenotypic screen for small molecules that reverse TDP-43 toxicity in yeast. One group of hit compounds was 8-hydroxyquinolines (8-OHQ), a class of clinically relevant bioactive metal chelators related to clioquinol. Surprisingly, in otherwise wild-type yeast cells, different 8-OHQs had selectivity for rescuing the distinct toxicities caused by the expression of TDP-43, α-synuclein, or polyglutamine proteins. In fact, each 8-OHQ synergized with the other, clearly establishing that they function in different ways. Comparative growth and molecular analyses also revealed that 8-OHQs have distinct metal chelation and ionophore activities. The diverse bioactivity of 8-OHQs indicates that altering different aspects of metal homeostasis and/or metalloprotein activity elicits distinct protective mechanisms against several neurotoxic proteins. Indeed, phase II clinical trials of an 8-OHQ has produced encouraging results in modifying Alzheimer disease. Our unbiased identification of 8-OHQs in a yeast TDP-43 toxicity model suggests that tailoring 8-OHQ activity to a particular neurodegenerative disease may be a viable therapeutic strategy.