Calcineurin determines toxic versus beneficial responses to α-synuclein

Calcineurin (CN) is a highly conserved Ca[superscript 2+]–calmodulin (CaM)-dependent phosphatase that senses Ca[superscript 2+] concentrations and transduces that information into cellular responses. Ca[superscript 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[superscript 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.Jeffry M. and Barbara Picower FoundationJPB FoundationHoward Hughes Medical Institute (Collaborative Innovation Award)Eleanor Schwartz Charitable Foundatio

Compounds from an Unbiased Chemical Screen Reverse Both Er-to-Golgi Trafficking Defects and Mitochondrial Dysfunction in Parkinson's Disease Models

α-Synuclein (α-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because α-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson’s disease (PD). We previously created a yeast model of α-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to α-syn expression. We also uncovered a core group of proteins with diverse activities related to α-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of α-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress α-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of α-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced α-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of α-syn foci, re-established ER-to-Golgi trafficking and ameliorated α-syn-mediated damage to mitochondria. They also corrected the toxicity of α-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of α-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.MGH/MIT Morris Udall Center of Excellence in Parkinson Disease Research (NS038372)Michael J. Fox Foundation for Parkinson's ResearchHoward Hughes Medical InstituteUnited States. National Institutes of Health (NS049221)American Parkinson Disease Association, Inc

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. Keywords: alpha-synuclein; iPS cell; Parkinson’s disease; stem cell; mRNA translation; RNA-binding protein; LRRK2; VPS35; vesicle trafficking; yeas

Context Dependent Neuroprotective Properties of Prion Protein (Prp)

Although it has been known for more than twenty years that an aberrant conformation of the prion protein (PrP) is the causative agent in prion diseases, the role of PrP in normal biology is undetermined. Numerous studies have suggested a protective function for PrP, including protection from ischemic and excitotoxic lesions and several apoptotic insults. On the other hand, many observations have suggested the contrary, linking changes in PrP localization or domain structure—independent of infectious prion conformation—to severe neuronal damage. Surprisingly, a recent report suggests that PrP is a receptor for toxic oligomeric species of a-β, a pathogenic fragment of the amyloid precursor protein, and likely contributes to disease pathogenesis of Alzheimer’s disease. We sought to access the role of PrP in diverse neurological disorders. First, we confirmed that PrP confers protection against ischemic damage using an acute stroke model, a well characterized association. After ischemic insult, PrP knockouts had dramatically increased infarct volumes and decreased behavioral performance compared to controls. To examine the potential of PrP’s neuroprotective or neurotoxic properties in the context of other pathologies, we deleted PrP from several transgenic models of neurodegenerative disease. Deletion of PrP did not substantially alter the disease phenotypes of mouse models of Parkinson’s disease or tauopathy. Deletion of PrP in one of two Huntington’s disease models tested, R6/2, modestly slowed motor deterioration as measured on an accelerating rotarod but otherwise did not alter other major features of the disease. Finally, transgenic overexpression of PrP did not exacerbate the Huntington’s motor phenotype. These results suggest that PrP has a context-dependent neuroprotective function and does not broadly contribute to the disease models tested herein.Ellison Medical FoundationWhitaker Health Sciences Fund Fellowshi

Modelling Parkinson\u27s disease in Drosophila: The protective role of molecular chaperones

Parkinson\u27s disease (PD) is a neurodegenerative disorder characterized by resting tremor and postural rigidity. The progressive loss of dopaminergic neurons in the substantia nigra pars compacta is the key pathologic feature underlying these symptoms. Mutations in the synaptic protein α-synuclein are linked to autosomal-dominant PD. Moreover, α-synuclein is also a major component of Lewy bodies (LBs) found in idiopathic PD. We modelled PD in Drosophila by directing the expression of asynuclein to dopaminergic neurons. Expression of α-synuclein resulted in both the age-dependent degeneration of 50% of the dopaminergic neurons in the dorsomedial clusters of the fly brain and the formation of LB-like aggregates. As a genetic organism, Drosophila is an ideal system in which to study modifiers of α-synuclein toxicity with potential therapeutic relevance. We therefore tested whether the molecular chaperone Hsp70 could protect against α-synuclein toxicity. Transgenic expression of Hsp70 fully protected against the toxicity of α-synuclein to dopaminergic neurons. Furthermore, compromising endogenous chaperone activity accelerated α-synuclein-mediated neurodegeneration. Hsp70 and other chaperones also found to localize to LBs in postmortem PD brain tissue and to LB-like aggregates in the brains of transgenic α-synuclein flies. Thus it appears that chaperone activity may be altered in PD patients and contributes to the toxicity of α-synuclein. We next examined whether pharmacological enhancement of chaperone activity might also protect against α-synuclein toxicity. Geldanamycin (GA) is an antibiotic that inhibits the activity of Hsp90 which negatively regulates heat shock factor (HSF), the transcriptional activator of Hsp70 and other chaperones. Treatment of adult flies with GA fully suppressed the toxicity of α-synuclein. Using a temperature-sensitive null allele of HSF, we found that GA-mediated neuroprotection was fully dependent upon HSF activity; genetic elimination of HSF activity abrogated the drug\u27s cytoprotective activity. Finally, we determined that other pathways modified by Hsp90 were not responsible for neuroprotection by GA. Through these studies, we have shown that enhancement of chaperone activity, both genetically and pharmacologically, is a potent mitigator of α-synuclein toxicity in Drosophila. We propose that targeted enhancement of chaperone pathways should be further investigated as a cytoprotective treatment for PD and related neurodegenerative disorders

Modelling Parkinson\u27s disease in Drosophila: The protective role of molecular chaperones

Parkinson\u27s disease (PD) is a neurodegenerative disorder characterized by resting tremor and postural rigidity. The progressive loss of dopaminergic neurons in the substantia nigra pars compacta is the key pathologic feature underlying these symptoms. Mutations in the synaptic protein α-synuclein are linked to autosomal-dominant PD. Moreover, α-synuclein is also a major component of Lewy bodies (LBs) found in idiopathic PD. We modelled PD in Drosophila by directing the expression of asynuclein to dopaminergic neurons. Expression of α-synuclein resulted in both the age-dependent degeneration of 50% of the dopaminergic neurons in the dorsomedial clusters of the fly brain and the formation of LB-like aggregates. As a genetic organism, Drosophila is an ideal system in which to study modifiers of α-synuclein toxicity with potential therapeutic relevance. We therefore tested whether the molecular chaperone Hsp70 could protect against α-synuclein toxicity. Transgenic expression of Hsp70 fully protected against the toxicity of α-synuclein to dopaminergic neurons. Furthermore, compromising endogenous chaperone activity accelerated α-synuclein-mediated neurodegeneration. Hsp70 and other chaperones also found to localize to LBs in postmortem PD brain tissue and to LB-like aggregates in the brains of transgenic α-synuclein flies. Thus it appears that chaperone activity may be altered in PD patients and contributes to the toxicity of α-synuclein. We next examined whether pharmacological enhancement of chaperone activity might also protect against α-synuclein toxicity. Geldanamycin (GA) is an antibiotic that inhibits the activity of Hsp90 which negatively regulates heat shock factor (HSF), the transcriptional activator of Hsp70 and other chaperones. Treatment of adult flies with GA fully suppressed the toxicity of α-synuclein. Using a temperature-sensitive null allele of HSF, we found that GA-mediated neuroprotection was fully dependent upon HSF activity; genetic elimination of HSF activity abrogated the drug\u27s cytoprotective activity. Finally, we determined that other pathways modified by Hsp90 were not responsible for neuroprotection by GA. Through these studies, we have shown that enhancement of chaperone activity, both genetically and pharmacologically, is a potent mitigator of α-synuclein toxicity in Drosophila. We propose that targeted enhancement of chaperone pathways should be further investigated as a cytoprotective treatment for PD and related neurodegenerative disorders

Methylation of the dopamine transporter gene in blood is associated with striatal dopamine transporter availability in ADHD: A preliminary study

Dopamine transporters (DAT) are implicated in the pathogenesis and treatment of attention-deficit hyperactivity disorder (ADHD) and are upregulated by chronic treatment with methylphenidate, commonly prescribed for ADHD. Methylation of the DAT1 gene in brain and blood has been associated with DAT expression in rodents' brains. Here we tested the association between methylation of the DAT1 promoter derived from blood and DAT availability in the striatum of unmedicated ADHD adult participants and in that of healthy age-matched controls (HC) using Positron Emission Tomography (PET) and [11 C]cocaine. Results showed no between-group differences in DAT1 promoter methylation or striatal DAT availability. However, the degree of methylation in the promoter region of DAT1 correlated negatively with DAT availability in caudate in ADHD participants only. DAT availability in VS correlated with inattention scores in ADHD participants. We verified in a postmortem cohort with ADHD diagnosis and without, that DAT1 promoter methylation in peripheral blood correlated positively with DAT1 promoter methylation extracted from substantia nigra (SN) in both groups. In the cohort without ADHD diagnosis, DAT1 gene expression in SN further correlated positively with DAT protein expression in caudate; however, the sample size of the cohort with ADHD was insufficient to investigate DAT1 and DAT expression levels. Overall, these findings suggest that peripheral DAT1 promoter methylation may be predictive of striatal DAT availability in adults with ADHD. Due to the small sample size, more work is needed to validate whether DAT1 methylation in blood predicts DAT1 methylation in SN in ADHD and controls

Development of an aggregate-selective, human-derived α-synuclein antibody BIIB054 that ameliorates disease phenotypes in Parkinson's disease models

Aggregation of α-synuclein (α-syn) is neuropathologically and genetically linked to Parkinson's disease (PD). Since stereotypic cell-to-cell spreading of α-syn pathology is believed to contribute to disease progression, immunotherapy with antibodies directed against α-syn is considered a promising therapeutic approach for slowing disease progression. Here we report the identification, binding characteristics, and efficacy in PD mouse models of the human-derived α-syn antibody BIIB054, which is currently under investigation in a Phase 2 clinical trial for PD. BIIB054 was generated by screening human memory B-cell libraries from healthy elderly individuals. Epitope mapping studies conducted using peptide scanning, X-ray crystallography, and mutagenesis show that BIIB054 binds to α-syn residues 1-10. BIIB054 is highly selective for aggregated forms of α-syn with at least an 800-fold higher apparent affinity for fibrillar versus monomeric recombinant α-syn and a strong preference for human PD brain tissue. BIIB054 discriminates between monomers and oligomeric/fibrillar forms of α-syn based on high avidity for aggregates, driven by weak monovalent affinity and fast binding kinetics. In efficacy studies in three different mouse models with intracerebrally inoculated preformed α-syn fibrils, BIIB054 treatment attenuated the spreading of α-syn pathology, rescued motor impairments, and reduced the loss of dopamine transporter density in dopaminergic terminals in striatum. The preclinical data reported here provide a compelling rationale for clinical development of BIIB054 for the treatment and prevention of PD

Identification and Rescue of -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.Howard Hughes Medical Institute (Collaborative Innovation Award)JPB FoundationNational Institutes of Health (U.S.) (Grant 5 R01CA084198)National Science Foundation (U.S.