Pavan K. Auluck

α-Synuclein: Membrane Interactions and Toxicity in Parkinson's Disease

In the late 1990s, mutations in the synaptic protein α-synuclein (α-syn) were identified in families with hereditary Parkinsons disease (PD). Rapidly, α-syn became the target of numerous investigations that have transformed our understanding of the pathogenesis underlying this disorder. α-Syn is the major component of Lewy bodies (LBs), cytoplasmic protein aggregates that form in the neurons of PD patients. α-Syn interacts with lipid membranes and adopts amyloid conformations that deposit within LBs. Work in yeast and other model systems has revealed that α-syn-associated toxicity might be the consequence of abnormal membrane interactions and alterations in vesicle trafficking. Here we review evidence regarding α-syns normal interactions with membranes and regulation of synaptic vesicles as well as how overexpression of α-syn yields global cellular dysfunction. Finally, we present a model linking vesicle dynamics to toxicity with the sincere hope that understanding these disease mechanisms will lead to the development of novel, potent therapeutics.

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.

Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity

Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens. By comparing the results of these two assays across various stimuli, we found that genetic screens tend to identify response regulators, whereas mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We used this approach to reveal cellular pathways responding to the toxicity of alpha-synuclein, a protein implicated in several neurodegenerative disorders including Parkinsons disease. For this we screened an established yeast model to identify genes that when overexpressed alter alpha-synuclein toxicity. Bridging these data and data from mRNA profiling provided functional explanations for many of these genes and identified previously unknown relations between alpha-synuclein toxicity and basic cellular pathways.

Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.

SUMMARY 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.

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.

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.