Correction: The mechanism of sirtuin 2-mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease.

[This corrects the article DOI: 10.1371/journal.pbio.2000374.].

The mechanism of sirtuin 2-mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease.

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies.

Analysis of Protein Oligomeric Species by Sucrose Gradients.

Protein misfolding, aggregation, and accumulation are a common hallmark in various neurodegenerative diseases. Invariably, the process of protein aggregation is associated with both a loss of the normal biological function of the protein and a gain of toxic function that ultimately leads to cell death. The precise origin of protein cytotoxicity is presently unclear but the predominant theory posits that smaller oligomeric species are more toxic than larger aggregated forms. While there is still no consensus on this subject, this is a central question that needs to be addressed in order to enable the design of novel and more effective therapeutic strategies. Accordingly, the development and utilization of approaches that allow the biochemical characterization of the formed oligomeric species in a given cellular or animal model will enable the correlation with cytotoxicity and other parameters of interest.Here, we provide a detailed description of a low-cost protocol for the analysis of protein oligomeric species from both yeast and mammalian cell lines models, based on their separation according to sedimentation velocity using high-speed centrifugation in sucrose gradients. This approach is an adaptation of existing protocols that enabled us to overcome existing technical issues and obtain reliable results that are instrumental for the characterization of the types of protein aggregates formed by different proteins of interest in the context of neurodegenerative disorders.

The small GTPase Rab11 co-localizes with α-synuclein in intracellular inclusions and modulates its aggregation, secretion and toxicity.

Alpha-synuclein (aSyn) misfolding and aggregation are pathological features common to several neurodegenerative diseases, including Parkinson's disease (PD). Mounting evidence suggests that aSyn can be secreted and transferred from cell to cell, participating in the propagation and spreading of pathological events. Rab11, a small GTPase, is an important regulator in both endocytic and secretory pathways. Here, we show that Rab11 is involved in regulating aSyn secretion. Rab11 knockdown or overexpression of either Rab11a wild-type (Rab11a WT) or Rab11a GDP-bound mutant (Rab11a S25N) increased secretion of aSyn. Furthermore, we demonstrate that Rab11 interacts with aSyn and is present in intracellular inclusions together with aSyn. Moreover, Rab11 reduces aSyn aggregation and toxicity. Our results suggest that Rab11 is involved in modulating the processes of aSyn secretion and aggregation, both of which are important mechanisms in the progression of aSyn pathology in PD and other synucleinopathies.

PLK2 modulates α-synuclein aggregation in yeast and mammalian cells.

Phosphorylation of α-synuclein (aSyn) on serine 129 is one of the major post-translation modifications found in Lewy bodies, the typical pathological hallmark of Parkinson's disease. Here, we found that both PLK2 and PLK3 phosphorylate aSyn on serine 129 in yeast. However, only PLK2 increased aSyn cytotoxicity and the percentage of cells presenting cytoplasmic foci. Consistently, in mammalian cells, PLK2 induced aSyn phosphorylation on serine 129 and induced an increase in the size of the inclusions. Our study supports a role for PLK2 in the generation of aSyn inclusions by a mechanism that does not depend directly on serine 129 phosphorylation.

4-hydroxytamoxifen leads to PrPSc clearance by conveying both PrPC and PrPSc to lysosomes independently of autophagy.

Prion diseases are fatal neurodegenerative disorders involving the abnormal folding of a native cellular protein, named PrP(C), to a malconformed aggregation-prone state, enriched in beta sheet secondary structure, denoted PrP(Sc). Recently, autophagy has garnered considerable attention as a cellular process with the potential to counteract neurodegenerative diseases of protein aggregation such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Stimulation of autophagy by chemical compounds has also been shown to reduce PrP(Sc) in infected neuronal cells and prolong survival times in mouse models. Consistent with previous reports, we demonstrate that autophagic flux is increased in chronically infected cells. However, in contrast to recent findings we show that autophagy does not cause a reduction in scrapie burden. We report that in infected neuronal cells different compounds known to stimulate autophagy are ineffective in increasing autophagic flux and in reducing PrP(Sc). We further demonstrate that tamoxifen and its metabolite 4-hydroxytamoxifen lead to prion degradation in an autophagy-independent manner by diverting the trafficking of both PrP and cholesterol to lysosomes. Our data indicate that tamoxifen, a well-characterized, widely available pharmaceutical, may have applications in the therapy of prion diseases.

Impaired proteostasis contributes to renal tubular dysgenesis.

Protein conformational disorders are associated with the appearance, persistence, accumulation, and misprocessing of aberrant proteins in the cell. The etiology of renal tubular dysgenesis (RTD) is linked to mutations in the angiotensin-converting enzyme (ACE). Here, we report the identification of a novel ACE mutation (Q1069R) in an RTD patient. ACE Q1069R is found sequestered in the endoplasmic reticulum and is also subject to increased proteasomal degradation, preventing its transport to the cell surface and extracellular fluids. Modulation of cellular proteostasis by temperature shift causes an extension in the processing time and trafficking of ACE Q1069R resulting in partial rescue of the protein processing defect and an increase in plasma membrane levels. In addition, we found that temperature shifting causes the ACE Q1069R protein to be secreted in an active state, suggesting that the mutation does not affect the enzyme's catalytic properties.

Identification of an intracellular site of prion conversion.

Prion diseases are fatal, neurodegenerative disorders in humans and animals and are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrP(C)), denoted PrP(Sc), which represents the major component of infectious scrapie prions. Characterization of the mechanism of conversion of PrP(C) into PrP(Sc) and identification of the intracellular site where it occurs are among the most important questions in prion biology. Despite numerous efforts, both of these questions remain unsolved. We have quantitatively analyzed the distribution of PrP(C) and PrP(Sc) and measured PrP(Sc) levels in different infected neuronal cell lines in which protein trafficking has been selectively impaired. Our data exclude roles for both early and late endosomes and identify the endosomal recycling compartment as the likely site of prion conversion. These findings represent a fundamental step towards understanding the cellular mechanism of prion conversion and will allow the development of new therapeutic approaches for prion diseases.

Prions hijack tunnelling nanotubes for intercellular spread.

In variant Creutzfeldt-Jakob disease, prions (PrP(Sc)) enter the body with contaminated foodstuffs and can spread from the intestinal entry site to the central nervous system (CNS) by intercellular transfer from the lymphoid system to the peripheral nervous system (PNS). Although several means and different cell types have been proposed to have a role, the mechanism of cell-to-cell spreading remains elusive. Tunnelling nanotubes (TNTs) have been identified between cells, both in vitro and in vivo, and may represent a conserved means of cell-to-cell communication. Here we show that TNTs allow transfer of exogenous and endogenous PrP(Sc) between infected and naive neuronal CAD cells. Significantly, transfer of endogenous PrP(Sc) aggregates was detected exclusively when cells chronically infected with the 139A mouse prion strain were connected to mouse CAD cells by means of TNTs, identifying TNTs as an efficient route for PrP(Sc) spreading in neuronal cells. In addition, we detected the transfer of labelled PrP(Sc) from bone marrow-derived dendritic cells to primary neurons connected through TNTs. Because dendritic cells can interact with peripheral neurons in lymphoid organs, TNT-mediated intercellular transfer would allow neurons to transport prions retrogradely to the CNS. We therefore propose that TNTs are involved in the spreading of PrP(Sc) within neurons in the CNS and from the peripheral site of entry to the PNS by neuroimmune interactions with dendritic cells.

Ligand-independent pathway that controls stability of interferon alpha receptor.

Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination, and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance.

Comparable potency of IFNalpha2 and IFNbeta on immediate JAK/STAT activation but differential down-regulation of IFNAR2.

Type I IFNs (interferons) (IFNalpha/beta) form a family of related cytokines that control a variety of cellular functions through binding to a receptor composed of IFNAR (IFNalpha receptor subunit) 1 and 2. Among type I IFNs, the alpha2 and beta subtypes exhibit a large difference in their binding affinities to IFNAR1, and it was suggested that high concentrations of IFNAR1 may compensate for its low intrinsic binding affinity for IFNalpha2. We tested whether receptor-proximal signalling events are sensitive to IFNAR1 surface concentration by investigating the relationship between relative IFNAR1/IFNAR2 surface levels and IFNalpha2 and IFNbeta signalling potencies in several cell lines. For this, we monitored the activation profile of JAK (Janus kinase)/STAT (signal transducer and activator of transcription) proteins, measured basal and ligand-induced surface decay of each receptor subunit and tested the effect of variable IFNAR1 levels on IFNalpha2 signalling potency. Our data show that the cell-surface IFNAR1 level is indeed a limiting factor for assembly of the functional complex, but an increased concentration of it does not translate into an IFNalpha/beta differential JAK/STAT signalling nor does it change the dynamics of the engaged receptor. Importantly, however, our data highlight a differential effect upon routing of IFNAR2. Following binding of IFNalpha2, IFNAR2 is internalized, but, instead of being routed towards degradation as it is when complexed to IFNbeta, it recycles back to the cell surface. These observations suggest strongly that the stability and the intracellular lifetime of the ternary complex account for the differential control of IFNAR2. Moreover, the present study opens up the attractive possibility that endosomal-initiated signalling may contribute to IFNalpha/beta differential bioactivities.

TYK2 activity promotes ligand-induced IFNAR1 proteolysis.

The type I IFNR (interferon receptor) is a heterodimer composed of two transmembrane chains, IFNAR1 (interferon-alpha receptor 1 subunit) and IFNAR2, which are associated with the tyrosine kinases Tyk2 and Jak1 (Janus kinase 1) respectively. Ligand-induced down-regulation of the type I IFNR is a major mechanism of negative regulation of cellular signalling and involves the internalization and lysosomal degradation of IFNAR1. IFNalpha promotes the phosphorylation of IFNAR1 on Ser535, followed by recruitment of the E3 ubiquitin ligase, beta-TrCP2 (beta-transducin repeats-containing protein 2), ubiquitination of IFNAR1 and proteolysis. The non-catalytic role of Tyk2 in sustaining the steady-state IFNAR1 level at the plasma membrane is well documented; however, little is known about the function of Tyk2 in the steps that precede and succeed serine phosphorylation and ubiquitination of IFNAR1 in response to ligand binding. In the present study, we show that catalytic activation of Tyk2 is not essential for IFNAR1 internalization, but is required for ligand-induced IFNAR1 serine phosphorylation, ubiquitination and efficient lysosomal proteolysis.

Closing the gap: identification of human 3-ketosteroid reductase, the last unknown enzyme of mammalian cholesterol biosynthesis.

The protein encoded by the HSD17B7 gene was originally described as a prolactin receptor-associated protein and as 17beta-hydroxysteroid dehydrogenase (HSD) type 7. Its ability to synthesize 17beta-estradiol in vitro has been reported previously. However, we demonstrate that HSD17B7 is the ortholog of the yeast 3-ketosteroid reductase Erg27p and converts zymosterone to zymosterol in vitro, using reduced nicotinamide adenine dinucleotide phosphate as cofactor. Expression of human and murine HSD17B7 in an Erg27p-deficient yeast strain complements the 3-ketosteroid reductase deficiency of the cells and restores growth on sterol-deficient medium. A fusion of HSD17B7 with green fluorescent protein is located in the endoplasmic reticulum, the site of postsqualene cholesterogenesis. Further critical evidence for a role of HSD17B7 in cholesterol metabolism is provided by the observation that its murine ortholog is a member of the same highly distinct embryonic synexpression group as hydroxymethyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme of sterol biogenesis, and is specifically expressed in tissues that are involved in the pathogenesis of congenital cholesterol-deficiency disorders. We conclude that HSD17B7 participates in postsqualene cholesterol biosynthesis, thus completing the molecular cloning of all genes of this central metabolic pathway. In its function as the 3-ketosteroid reductase of cholesterol biosynthesis, HSD17B7 is a novel candidate for inborn errors of cholesterol metabolism.