Publications by authors named "Ulf Dettmer"

29 Publications

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Wild-type GBA1 increases the α-synuclein tetramer-monomer ratio, reduces lipid-rich aggregates, and attenuates motor and cognitive deficits in mice.

Proc Natl Acad Sci U S A 2021 Aug;118(31)

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115

Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson's disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient-derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer-monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD-causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)-human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.
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http://dx.doi.org/10.1073/pnas.2103425118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346893PMC
August 2021

Excess membrane binding of monomeric alpha-, beta-, and gamma-synuclein is invariably associated with inclusion formation and toxicity.

Hum Mol Genet 2021 Jul 12. Epub 2021 Jul 12.

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115 USA.

α-synuclein (αS) has been well-documented to play a role in human synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). First, the lesions found in PD/DLB brains-Lewy bodies and Lewy neurites-are rich in aggregated αS. Second, genetic evidence links missense mutations and increased αS expression to familial forms of PD/DLB. Third, toxicity and cellular stress can be caused by αS under certain experimental conditions. In contrast, the homologs β-synuclein (βS) and γ-synuclein (γS) are not typically found in Lewy bodies/neurites, have not been clearly linked to brain diseases, and have been largely non-toxic in experimental settings. In αS, the so-called non-amyloid-β component of plaques (NAC) domain, constituting amino acids 61 to 95, has been identified to be critical for aggregation in vitro. This domain is partially absent in βS and only incompletely conserved in γS, which could explain why both homologs do not cause disease. However, αS in vitro aggregation and cellular toxicity have not been firmly linked experimentally, and it has been proposed that excess αS-membrane binding is sufficient to induce neurotoxicity. Indeed, recent characterizations of Lewy bodies have highlighted the accumulation of lipids and membranous organelles, raising the possibility that βS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased βS and γS membrane affinity by strategic point mutations and demonstrate that these proteins behave like membrane-associated monomers, are cytotoxic, and form round cytoplasmic inclusions that can be prevented by inhibiting stearoyl-CoA desaturase.
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http://dx.doi.org/10.1093/hmg/ddab188DOI Listing
July 2021

Crowded organelles, lipid accumulation, and abnormal membrane tubulation in cellular models of enhanced α-synuclein membrane interaction.

Brain Res 2021 05 9;1758:147349. Epub 2021 Feb 9.

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Previous work from our group showed that certain engineered missense mutations to the α-synuclein (αS) KTKEGV repeat motifs abrogate the protein's ability to form native multimers. The resultant excess monomers accumulate in lipid-membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial Parkinson's disease mutants such as E46K. We presented an initial characterization of the lipid-rich inclusions and found similarities to the αS- and vesicle-rich inclusions that form in baker's yeast when αS is expressed. We also discussed, with some caution, a possible role of membrane-rich inclusions as precursors to filamentous Lewy bodies, the widely accepted hallmark pathology of Parkinson's disease and other synucleinopathies. In the meantime, advances in the microscopic characterization of Lewy bodies have highlighted the presence of crowded organelles and lipid membranes in addition to αS accumulation. This prompted us to revisit the αS inclusions caused by our repeat motif variants in neuroblastoma cells. In addition to our previous characterization, we found that these inclusions can often be seen by brightfield microscopy, overlap with endogenous vesicle markers in immunofluorescence experiments, stain positive for lipid dyes, and can be found to be closely associated with mitochondria. We also observed abnormal tubulation of membranes, which was subtle in inducible lines and pronounced in cells that transiently expressed high amounts of the highly disruptive KTKEGV motif mutant "KLKEGV". Membrane tubulation had been reported before as an αS activity in reductionist systems. Our in-cellulo demonstration now suggests that this mechanism could possibly be a relevant aspect of aberrant αS behavior in cells.
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http://dx.doi.org/10.1016/j.brainres.2021.147349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988302PMC
May 2021

Temperature is a key determinant of alpha- and beta-synuclein membrane interactions in neurons.

J Biol Chem 2021 Jan-Jun;296:100271. Epub 2021 Jan 9.

Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. Electronic address:

Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies. αS has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study αS under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined αS cytosol:membrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 °C or room temperature), αS was largely membrane-associated. At 37 °C, however, αS solubility was markedly increased. In contrast, the extraction of control proteins (GAPDH, cytosolic; calnexin, membrane) was not affected by temperature. When we compared the relative distribution of the synuclein homologs αS and β-synuclein (βS) under various conditions that differed in temperature and digitonin concentration (200-1200 μg/ml), we consistently found αS to be more membrane-associated than βS. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 °C and 800 μg/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% ± 9.0% for αS and 63.6% ± 6.6% for βS. PD-linked αS A30P was found to be largely cytosolic, confirming previous studies that had used different methods. Our work highlights the dynamic nature of cellular synuclein behavior and has important implications for protein-biochemical and cell-biological studies of αS proteostasis, such as testing the effects of genetic and pharmacological manipulations.
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http://dx.doi.org/10.1016/j.jbc.2021.100271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949061PMC
August 2021

Upregulation of Cellular Palmitoylation Mitigates α-Synuclein Accumulation and Neurotoxicity.

Mov Disord 2021 02 26;36(2):348-359. Epub 2020 Oct 26.

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

Background: Synucleinopathies, including Parkinson's disease (PD), are characterized by α-synuclein (αS) cytoplasmic inclusions. αS-dependent vesicle-trafficking defects are important in PD pathogenesis, but their mechanisms are not well understood. Protein palmitoylation, post-translational addition of the fatty acid palmitate to cysteines, promotes trafficking by anchoring specific proteins to the vesicle membrane. αS itself cannot be palmitoylated as it lacks cysteines, but it binds to membranes, where palmitoylation occurs, via an amphipathic helix. We hypothesized that abnormal αS membrane-binding impairs trafficking by disrupting palmitoylation. Accordingly, we investigated the therapeutic potential of increasing cellular palmitoylation.

Objectives: We asked whether upregulating palmitoylation by inhibiting the depalmitoylase acyl-protein-thioesterase-1 (APT1) ameliorates pathologic αS-mediated cellular phenotypes and sought to identify the mechanism.

Methods: Using human neuroblastoma cells, rat neurons, and iPSC-derived PD patient neurons, we examined the effects of pharmacologic and genetic downregulation of APT1 on αS-associated phenotypes.

Results: APT1 inhibition or knockdown decreased αS cytoplasmic inclusions, reduced αS serine-129 phosphorylation (a PD neuropathological marker), and protected against αS-dependent neurotoxicity. We identified the APT1 substrate microtubule-associated-protein-6 (MAP6), which binds to vesicles in a palmitoylation-dependent manner, as a key mediator of these effects. Mechanistically, we found that pathologic αS accelerated palmitate turnover on MAP6, suggesting that APT1 inhibition corrects a pathological αS-dependent palmitoylation deficit. We confirmed the disease relevance of this mechanism by demonstrating decreased MAP6 palmitoylation in neurons from αS gene triplication patients.

Conclusions: Our findings demonstrate a novel link between the fundamental process of palmitoylation and αS pathophysiology. Upregulating palmitoylation represents an unexplored therapeutic strategy for synucleinopathies. © 2020 International Parkinson and Movement Disorder Society.
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http://dx.doi.org/10.1002/mds.28346DOI Listing
February 2021

A Stearoyl-Coenzyme A Desaturase Inhibitor Prevents Multiple Parkinson Disease Phenotypes in α-Synuclein Mice.

Ann Neurol 2021 01 23;89(1):74-90. Epub 2020 Oct 23.

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

Objective: Parkinson disease (PD) has useful symptomatic treatments that do not slow the neurodegenerative process, and no significant disease-modifying treatments are approved. A key therapeutic target in PD is α-synuclein (αS), which is both genetically implicated and accumulates in Lewy bodies rich in vesicles and other lipid membranes. Reestablishing αS homeostasis is a central goal in PD. Based on previous lipidomic analyses, we conducted a mouse trial of a stearoyl-coenzyme A desaturase (SCD) inhibitor ("5b") that prevented αS-positive vesicular inclusions and cytotoxicity in cultured human neurons.

Methods: Oral dosing and brain activity of 5b were established in nontransgenic mice. 5b in drinking water was given to mice expressing wild-type human αS (WT) or an amplified familial PD αS mutation (E35K + E46K + E61K ["3K"]) beginning near the onset of nigral and cortical neurodegeneration and the robust PD-like motor syndrome in 3K. Motor phenotypes, brain cytopathology, and SCD-related lipid changes were quantified in 5b- versus placebo-treated mice. Outcomes were compared to effects of crossing 3K to SCD1 mice.

Results: 5b treatment reduced αS hyperphosphorylation in E46K-expressing human neurons, in 3K neural cultures, and in both WT and 3K αS mice. 5b prevented subtle gait deficits in WT αS mice and the PD-like resting tremor and progressive motor decline of 3K αS mice. 5b also increased αS tetramers and reduced proteinase K-resistant lipid-rich aggregates. Similar benefits accrued from genetically deleting 1 SCD allele, providing target validation.

Interpretation: Prolonged reduction of brain SCD activity prevented PD-like neuropathology in multiple PD models. Thus, an orally available SCD inhibitor potently ameliorates PD phenotypes, positioning this approach to treat human α-synucleinopathies. ANN NEUROL 2021;89:74-90.
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http://dx.doi.org/10.1002/ana.25920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756464PMC
January 2021

Rapid Alpha-Synuclein Toxicity in a Neural Cell Model and Its Rescue by a Stearoyl-CoA Desaturase Inhibitor.

Int J Mol Sci 2020 Jul 22;21(15). Epub 2020 Jul 22.

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Genetic and biochemical evidence attributes neuronal loss in Parkinson's disease (PD) and related brain diseases to dyshomeostasis of the 14 kDa protein α-synuclein (αS). There is no consensus on how αS exerts toxicity. Explanations range from disturbed vesicle biology to proteotoxicity caused by fibrillar aggregates. To probe these mechanisms further, robust cellular toxicity models are needed, but their availability is limited. We previously reported that a shift from dynamic multimers to monomers is an early event in αS dyshomeostasis, as caused by familial PD (fPD)-linked mutants such as E46K. Excess monomers accumulate in round, lipid-rich inclusions. Engineered αS '3K' (E35K+E46K+E61K) amplifies E46K, causing a PD-like, L-DOPA-responsive motor phenotype in transgenic mice. Here, we present a cellular model of αS neurotoxicity after transducing human neuroblastoma cells to express yellow fluorescent protein (YFP)-tagged αS 3K in a doxycycline-dependent manner. αS-3K::YFP induction causes pronounced growth defects that accord with cell death. We tested candidate compounds for their ability to restore growth, and stearoyl-CoA desaturase (SCD) inhibitors emerged as a molecule class with growth-restoring capacity, but the therapeutic window varied among compounds. The SCD inhibitor MF-438 fully restored growth while exerting no apparent cytotoxicity. Our αS bioassay will be useful for elucidating compound mechanisms, for pharmacokinetic studies, and for compound/genetic screens.
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http://dx.doi.org/10.3390/ijms21155193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432784PMC
July 2020

Vesicle trafficking and lipid metabolism in synucleinopathy.

Acta Neuropathol 2021 04 30;141(4):491-510. Epub 2020 Jun 30.

Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, 02115, USA.

The neuronal protein α-synuclein (αS) is central to the pathogenesis of Parkinson's disease and other progressive brain diseases such as Lewy body dementia and multiple system atrophy. These diseases, collectively referred to as 'synucleinopathies', have long been considered purely proteinopathies: diseases characterized by the misfolding of a protein into small and large aggregates mainly consisting of that protein (in this case: α-synuclein). However, recent morphological insights into Lewy bodies, the hallmark neuropathology of human synucleinopathies, suggests these lesions are also rich in vesicles and other membranous organelles. Moreover, αS physiology and pathology are both strongly associated with various aspects of intracellular vesicle trafficking and lipid biology. αS physiologically binds to synaptic and other small vesicles, and several functions of αS in regulating vesicle biology have been proposed. Familial PD-linked αS excess and missense mutations have been shown to impair vesicle trafficking and alter lipid homeostasis. On the other hand, vesicle trafficking and lipid-related genes have emerged as Parkinson's risk factors, suggesting a bidirectional relationship. The answer to the question "Does abnormal αS accumulation cause impaired vesicle trafficking and lipid dyshomeostasis or is αS aggregation the consequence of such impairments?" may be "Both". Here, we review current knowledge of the αS-lipid and αS-vesicle trafficking interplay, with a special focus on Parkinson's disease and Lewy body dementia.
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http://dx.doi.org/10.1007/s00401-020-02177-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772270PMC
April 2021

Soluble endogenous oligomeric α-synuclein species in neurodegenerative diseases: Expression, spreading, and cross-talk.

J Parkinsons Dis 2020 ;10(3):791-818

Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA.

There is growing recognition in the field of neurodegenerative diseases that mixed proteinopathies are occurring at greater frequency than originally thought. This is particularly true for three amyloid proteins defining most of these neurological disorders, amyloid-beta (Aβ), tau, and alpha-synuclein (αSyn). The co-existence and often co-localization of aggregated forms of these proteins has led to the emergence of concepts positing molecular interactions and cross-seeding between Aβ, tau, and αSyn aggregates. Amongst this trio, αSyn has received particular attention in this context during recent years due to its ability to modulate Aβ and tau aggregation in vivo, to interact at a molecular level with Aβ and tau in vivo and to cross-seed tau in mice. Here we provide a comprehensive, critical, and accessible review about the expression, role and nature of endogenous soluble αSyn oligomers because of recent developments in the understanding of αSyn multimerization, misfolding, aggregation, cross-talk, spreading and cross-seeding in neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Alzheimer's disease, and Huntington's disease. We will also discuss our current understanding about the relative toxicity of endogenous αSyn oligomers in vivo and in vitro, and introduce potential opportunities to counter their deleterious effects.
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http://dx.doi.org/10.3233/JPD-201965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458533PMC
August 2021

Parkinson's disease: proteinopathy or lipidopathy?

NPJ Parkinsons Dis 2020 3;6. Epub 2020 Jan 3.

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115 USA.

Lipids play a more significant role in Parkinson's disease and its related brain disorders than is currently recognized, supporting a "lipid cascade". The 14 kDa protein α-synuclein (αS) is strongly associated with Parkinson's disease (PD), dementia with Lewy bodies (DLB), other synucleinopathies such as multiple system atrophy, and even certain forms of Alzheimer's disease. Rigorously deciphering the biochemistry of αS in native systems is the key to developing treatments. αS is highly expressed in the brain, the second most lipid-rich organ, and has been proposed to be a lipid-binding protein that physiologically interacts with phospholipids and fatty acids (FAs). αS-rich cytoplasmic inclusions called Lewy bodies and Lewy neurites are the hallmark lesions of synucleinopathies. Excess αS-membrane interactions may trigger proteinaceous αS aggregation by stimulating its primary nucleation. However, αS may also exert its toxicity prior to or independent of its self-aggregation, e.g., via excessive membrane interactions, which may be promoted by certain lipids and FAs. A complex αS-lipid landscape exists, which comprises both physiological and pathological states of αS. As novel insights about the composition of Lewy lesions occur, new lipid-related PD drug candidates emerge, and genome-wide association studies (GWAS) increasingly validate new hits in lipid-associated pathways, it seems timely to review our current knowledge of lipids in PD and consider the roles for these pathways in synucleinopathies.Fig. 1αS ↔ lipid interplay: aspects of cellular αS homeostasis (blue oval), aspects of lipid homeostasis (green oval), and overlapping aspects.Pathological states are labeled in red. Simplified schematic of both select αS and select lipid species. Several existing publications suggest αS effects on lipids and vice versa, as indicated by arrows. DG diglyceride, ER endoplasmic reticulum, FA fatty acid, LD, lipid droplet, TG triglyceride.
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http://dx.doi.org/10.1038/s41531-019-0103-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941970PMC
January 2020

Cell models of lipid-rich α-synuclein aggregation validate known modifiers of α-synuclein biology and identify stearoyl-CoA desaturase.

Proc Natl Acad Sci U S A 2019 10 23;116(41):20760-20769. Epub 2019 Sep 23.

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115;

Microscopy of Lewy bodies in Parkinson's disease (PD) suggests they are not solely filamentous deposits of α-synuclein (αS) but also contain vesicles and other membranous material. We previously reported the existence of native αS tetramers/multimers and described engineered mutations of the αS KTKEGV repeat motifs that abrogate the multimers. The resultant excess monomers accumulate in lipid membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial PD mutants, such as E46K. Here, we use the αS "3K" (E35K+E46K+E61K) engineered mutation to probe the mechanisms of reported small-molecule modifiers of αS biochemistry and then identify compounds via a medium-throughput automated screen. αS 3K, which forms round, vesicle-rich inclusions in cultured neurons and causes a PD-like, l-DOPA-responsive motor phenotype in transgenic mice, was fused to YFP, and fluorescent inclusions were quantified. Live-cell microscopy revealed the highly dynamic nature of the αS inclusions: for example, their rapid clearance by certain known modulators of αS toxicity, including tacrolimus (FK506), isradipine, nilotinib, nortriptyline, and trifluoperazine. Our automated 3K cellular screen identified inhibitors of stearoyl-CoA desaturase (SCD) that robustly prevent the αS inclusions, reduce αS 3K neurotoxicity, and prevent abnormal phosphorylation and insolubility of αS E46K. SCD inhibition restores the E46K αS multimer:monomer ratio in human neurons, and it actually increases this ratio for overexpressed wild-type αS. In accord, conditioning 3K cells in saturated fatty acids rescued, whereas unsaturated fatty acids worsened, the αS phenotypes. Our cellular screen allows probing the mechanisms of synucleinopathy and refining drug candidates, including SCD inhibitors and other lipid modulators.
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http://dx.doi.org/10.1073/pnas.1903216116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789936PMC
October 2019

Dynamic behaviors of α-synuclein and tau in the cellular context: New mechanistic insights and therapeutic opportunities in neurodegeneration.

Neurobiol Dis 2019 12 24;132:104543. Epub 2019 Jul 24.

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address:

α-Synuclein (αS) and tau have a lot in common. Dyshomeostasis and aggregation of both proteins are central in the pathogenesis of neurodegenerative diseases: Parkinson's disease, dementia with Lewy bodies, multi-system atrophy and other 'synucleinopathies' in the case of αS; Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy and other 'tauopathies' in the case of tau. The aggregated states of αS and tau are found to be (hyper)phosphorylated, but the relevance of the phosphorylation in health or disease is not well understood. Both tau and αS are typically characterized as 'intrinsically disordered' proteins, while both engage in transient interactions with cellular components, thereby undergoing structural changes and context-specific folding. αS transiently binds to (synaptic) vesicles forming a membrane-induced amphipathic helix; tau transiently interacts with microtubules forming an 'extended structure'. The regulation and exact nature of the interactions are not fully understood. Here we review recent and previous insights into the dynamic, transient nature of αS and tau with regard to the mode of interaction with their targets, the dwell-time while bound, and the cis and trans factors underlying the frequent switching between bound and unbound states. These aspects are intimately linked to hypotheses on how subtle changes in the transient behaviors may trigger the earliest steps in the pathogenesis of the respective brain diseases. Based on a deeper understanding of transient αS and tau conformations in the cellular context, new therapeutic strategies may emerge, and it may become clearer why existing approaches have failed or how they could be optimized.
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http://dx.doi.org/10.1016/j.nbd.2019.104543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834908PMC
December 2019

Studying α-Synuclein Conformation by Intact-Cell Cross-Linking.

Methods Mol Biol 2019 ;1948:77-91

Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

β-Sheet-rich aggregates of α-synuclein (αS) are the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies, whereas the native conformations of αS in healthy cells are under debate. Cross-linking analyses in intact cells detect a large portion of endogenous αS in apparent multimeric states, most notably as putative tetramers (αS60) that run around 60 kDa on SDS-PAGE, but also point at the dynamic nature of cellular αS states. Standardization of αS cross-linking methods will facilitate efforts to study the effects of genetic, pharmacological, and environmental factors on αS conformation. Here, we present detailed protocols for cross-linking cellular αS multimers in cultured cells and brain tissues. These protocols will benefit future studies aimed at characterizing αS conformation in its cellular environment, both at steady state and upon perturbation, be it chronic or acute.
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http://dx.doi.org/10.1007/978-1-4939-9124-2_8DOI Listing
July 2019

Lipidomic Analysis of α-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment.

Mol Cell 2019 03 4;73(5):1001-1014.e8. Epub 2018 Dec 4.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, MIT, Cambridge, MA 02139, USA; HHMI, Department of Biology, MIT, Cambridge, MA 02139, USA.

In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.
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http://dx.doi.org/10.1016/j.molcel.2018.11.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408259PMC
March 2019

Rationally Designed Variants of α-Synuclein Illuminate Its Structural Properties in Health and Disease.

Authors:
Ulf Dettmer

Front Neurosci 2018 25;12:623. Epub 2018 Sep 25.

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States.

α-Synuclein (αS) is a conserved and abundant neuronal protein with unusual structural properties. It appears to partition between folded and unstructured states as well as between membrane-bound and aqueously soluble states. In addition, a switch between monomeric and tetrameric/multimeric states has been observed recently. The precise composition, localization and abundance of the multimeric species are under study and remain unsettled. Yet to interfere with disease pathogenesis, we must dissect how small changes in αS homeostasis may give rise to Parkinson's disease (PD), dementia with Lewy bodies (DLB) and other human synucleinopathies. Rationally designed αS point mutations that prevent the protein from populating all states within its normal folding repertoire have continued to be instrumental in bringing new insights into its biochemistry . This review summarizes biochemical and cell biological findings about αS homeostasis from different labs, with a special emphasis on intact-cell approaches that may preserve the complex, metastable native states of αS.
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http://dx.doi.org/10.3389/fnins.2018.00623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167557PMC
September 2018

Abrogating Native α-Synuclein Tetramers in Mice Causes a L-DOPA-Responsive Motor Syndrome Closely Resembling Parkinson's Disease.

Neuron 2018 10;100(1):75-90.e5

Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address:

α-Synuclein (αS) regulates vesicle exocytosis but forms insoluble deposits in Parkinson's disease (PD). Developing disease-modifying therapies requires animal models that reproduce cardinal features of PD. We recently described a previously unrecognized physiological form of αS, α-helical tetramers, and showed that familial PD-causing missense mutations shift tetramers to aggregation-prone monomers. Here, we generated mice expressing the fPD E46K mutation plus 2 homologous E→K mutations in adjacent KTKEGV motifs. This tetramer-abrogating mutant causes phenotypes similar to PD. αS monomers accumulate at membranes and form vesicle-rich inclusions. αS becomes insoluble, proteinase K-resistant, Ser129-phosphorylated, and C-terminally truncated, as in PD. These changes affect regions controlling motor behavior, including a decrease in nigrostriatal dopaminergic neurons. The outcome is a progressive motor syndrome including tremor and gait and limb deficits partially responsive to L-DOPA. This fully penetrant phenotype indicates that tetramers are required for normal αS homeostasis and that chronically shifting tetramers to monomers may result in PD, with attendant therapeutic implications.
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http://dx.doi.org/10.1016/j.neuron.2018.09.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211795PMC
October 2018

Loss of native α-synuclein multimerization by strategically mutating its amphipathic helix causes abnormal vesicle interactions in neuronal cells.

Hum Mol Genet 2017 09;26(18):3466-3481

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

α-Synuclein (αS) forms round cytoplasmic inclusions in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Evidence suggests a physiological function of αS in vesicle trafficking and release. In contrast to earlier tenets, recent work indicates that αS normally exists in cells in a dynamic equilibrium between monomers and tetramers/multimers. We engineered αS mutants incapable of multimerization, leading to excess monomers at vesicle membranes. By EM, such mutants induced prominent vesicle clustering, leading to round cytoplasmic inclusions. Immunogold labeling revealed abundant αS intimately associated with vesicles of varied size. Fluorescence microscopy with marker proteins showed that the αS-associated vesicles were of diverse endocytic and secretory origin. An αS '3K' mutant (E35K + E46K + E61K) that amplifies the PD/DLB-causing E46K mutation induced αS-rich vesicle clusters resembling the vesicle-rich areas of Lewy bodies, supporting pathogenic relevance. Mechanistically, E46K can increase αS vesicle binding via membrane-induced amphipathic helix formation, and '3K' further enhances this effect. Another engineered αS variant added hydrophobicity to the hydrophobic half of αS helices, thereby stabilizing αS-membrane interactions. Importantly, substituting charged for uncharged residues within the hydrophobic half of the stabilized helix not only reversed the strong membrane interaction of the multimer-abolishing αS variant but also restored multimerization and prevented the aberrant vesicle interactions. Thus, reversible αS amphipathic helix formation and dynamic multimerization regulate a normal function of αS at vesicles, and abrogating multimers has pathogenic consequences.
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http://dx.doi.org/10.1093/hmg/ddx227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5884392PMC
September 2017

Nortriptyline inhibits aggregation and neurotoxicity of alpha-synuclein by enhancing reconfiguration of the monomeric form.

Neurobiol Dis 2017 Oct 12;106:191-204. Epub 2017 Jul 12.

Department of Neurology, Washington University, Saint Louis, MO, USA.

The pathology of Parkinson's disease and other synucleinopathies is characterized by the formation of intracellular inclusions comprised primarily of misfolded, fibrillar α-synuclein (α-syn). One strategy to slow disease progression is to prevent the misfolding and aggregation of its native monomeric form. Here we present findings that support the contention that the tricyclic antidepressant compound nortriptyline (NOR) has disease-modifying potential for synucleinopathies. Findings from in vitro aggregation and kinetics assays support the view that NOR inhibits aggregation of α-syn by directly binding to the soluble, monomeric form, and by enhancing reconfiguration of the monomer, inhibits formation of toxic conformations of the protein. We go on to demonstrate that NOR inhibits the accumulation, aggregation and neurotoxicity of α-syn in multiple cell and animal models. These findings suggest that NOR, a compound with established safety and efficacy for treatment of depression, may slow progression of α-syn pathology by directly binding to soluble, native, α-syn, thereby inhibiting pathological aggregation and preserving its normal functions.
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http://dx.doi.org/10.1016/j.nbd.2017.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793922PMC
October 2017

New insights into cellular α-synuclein homeostasis in health and disease.

Curr Opin Neurobiol 2016 Feb 15;36:15-22. Epub 2015 Aug 15.

Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address:

α-Synuclein (αSyn) is a highly abundant neuronal protein whose exact structure and function are under debate. Misfolding and aggregation of this normally soluble, 140-residue polypeptide underlies a group of neurodegenerative disorders called synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). The αSyn field has focused increasing attention on the hypotheses that certain aggregates of αSyn may be directly toxic to the neurons in which they arise and/or that aggregates can be released from some neurons and diffuse by undefined mechanisms to other neurons to seed αSyn in the recipient cells, thus propagating neuropathology by a non-cell autonomous process ('pathogenic spread'). While intense interest in these hypotheses has led to new approaches and tools to model aspects of the disorders, it is important to analyze which molecular events initiate αSyn aggregation inside neurons in the first place. Here, we review new insights into how neuronal αSyn homeostasis may be maintained under physiological conditions but perturbed by pathological factors.
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http://dx.doi.org/10.1016/j.conb.2015.07.007DOI Listing
February 2016

KTKEGV repeat motifs are key mediators of normal α-synuclein tetramerization: Their mutation causes excess monomers and neurotoxicity.

Proc Natl Acad Sci U S A 2015 Aug 7;112(31):9596-601. Epub 2015 Jul 7.

Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115

α-Synuclein (αS) is a highly abundant neuronal protein that aggregates into β-sheet-rich inclusions in Parkinson's disease (PD). αS was long thought to occur as a natively unfolded monomer, but recent work suggests it also occurs normally in α-helix-rich tetramers and related multimers. To elucidate the fundamental relationship between αS multimers and monomers in living neurons, we performed systematic mutagenesis to abolish self-interactions and learn which structural determinants underlie native multimerization. Unexpectedly, tetramers/multimers still formed in cells expressing each of 14 sequential 10-residue deletions across the 140-residue polypeptide. We postulated compensatory effects among the six highly conserved and one to three additional αS repeat motifs (consensus: KTKEGV), consistent with αS and its homologs β- and γ-synuclein all forming tetramers while sharing only the repeats. Upon inserting in-register missense mutations into six or more αS repeats, certain mutations abolished tetramer formation, shown by intact-cell cross-linking and independently by fluorescent-protein complementation. For example, altered repeat motifs KLKEGV, KTKKGV, KTKEIV, or KTKEGW did not support tetramerization, indicating the importance of charged or small residues. When we expressed numerous different in-register repeat mutants in human neural cells, all multimer-abolishing but no multimer-neutral mutants caused frank neurotoxicity akin to the proapoptotic protein Bax. The multimer-abolishing variants became enriched in buffer-insoluble cell fractions and formed round cytoplasmic inclusions in primary cortical neurons. We conclude that the αS repeat motifs mediate physiological tetramerization, and perturbing them causes PD-like neurotoxicity. Moreover, the mutants we describe are valuable tools for studying normal and pathological properties of αS and screening for tetramer-stabilizing therapeutics.
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http://dx.doi.org/10.1073/pnas.1505953112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534262PMC
August 2015

Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation.

Nat Commun 2015 Jun 16;6:7314. Epub 2015 Jun 16.

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

β-Sheet-rich α-synuclein (αS) aggregates characterize Parkinson's disease (PD). αS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in α-helix-rich tetramers. Crosslinking traps principally tetrameric αS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant αS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased α-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this.
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http://dx.doi.org/10.1038/ncomms8314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490410PMC
June 2015

ExPLAining early synucleinopathies.

Brain 2015 Jun;138(Pt 6):1449-51

Ann Romney Centre for Neurologic Diseases, Harvard Medical School and Brigham and Women's Hospital, Boston, MA, USA.

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http://dx.doi.org/10.1093/brain/awv099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614127PMC
June 2015

Purification of α-synuclein from human brain reveals an instability of endogenous multimers as the protein approaches purity.

Biochemistry 2015 Jan 23;54(2):279-92. Epub 2014 Dec 23.

Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School , Boston, Massachusetts 02115, United States.

Despite two decades of research, the structure-function relationships of endogenous, physiological forms of α-synuclein (αSyn) are not well understood. Most in vitro studies of this Parkinson's disease-related protein have focused on recombinant αSyn that is unfolded and monomeric, assuming that this represents its state in the normal human brain. Recently, we have provided evidence that αSyn exists in considerable part in neurons, erythrocytes, and other cells as a metastable multimer that principally sizes as a tetramer. In contrast to recombinant αSyn, physiological tetramers purified from human erythrocytes have substantial α-helical content and resist pathological aggregation into β-sheet rich fibers. Here, we report the first method to fully purify soluble αSyn from the most relevant source, human brain. We describe protocols that purify αSyn to homogeneity from nondiseased human cortex using ammonium sulfate precipitation, gel filtration, and ion exchange, hydrophobic interaction, and affinity chromatographies. Cross-linking of the starting material and the partially purified chromatographic fractions revealed abundant αSyn multimers, including apparent tetramers, but these were destabilized in large part to monomers during the final purification step. The method also fully purified the homologue β-synuclein, with a similar outcome. Circular dichroism spectroscopy showed that purified, brain-derived αSyn can display more helical content than the recombinant protein, but this result varied. Collectively, our data suggest that purifying αSyn to homogeneity destabilizes native, α-helix-rich multimers that exist in intact and partially purified brain samples. This finding suggests existence of a stabilizing cofactor (e.g., a small lipid) present inside neurons that is lost during final purification.
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http://dx.doi.org/10.1021/bi501188aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303315PMC
January 2015

A new method for quantitative immunoblotting of endogenous α-synuclein.

PLoS One 2013 20;8(11):e81314. Epub 2013 Nov 20.

Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.

β-Sheet-rich aggregates of α-synuclein (αSyn) are the hallmark neuropathology of Parkinson's disease and related synucleinopathies, whereas the principal native structure of αSyn in healthy cells--unfolded monomer or α-helically folded oligomer--is under debate. Our recent crosslinking analysis of αSyn in intact cells showed that a large portion of endogenous αSyn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying αSyn Western blot signals among samples, as crosslinked αSyn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased αSyn immunoreactivity even after cleavage with the reducing agent β-mercaptoethanol. The effect was observed with all αSyn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous αSyn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of αSyn after DSP/β-mercaptoethanol treatment, in contrast to a marked loss of αSyn without this treatment. The treatment also enhanced immunodetection of the homologs β- and γ-synuclein and of histones, another group of small, lysine-rich proteins. We conclude that by neutralizing positive charges and increasing protein hydrophobicity, amine crosslinker treatment promotes adhesion of αSyn to blotting membranes. These data help explain the recent report of fixing αSyn blots with paraformaldehyde after transfer, which we find produces similar but weaker effects. DSP/β-mercaptoethanol treatment of Western blots should be particularly useful to quantify low-abundance αSyn forms such as extracellular and post-translationally modified αSyn and splice variants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081314PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3835431PMC
January 2015

Defining the native state of α-synuclein.

Neurodegener Dis 2014 30;13(2-3):114-7. Epub 2013 Oct 30.

Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass., USA.

Misfolding and pathogenic aggregation of α-synuclein (αSyn) is a hallmark of familial and sporadic Parkinson's disease, but the physiological state of the protein in cells remains unsettled. We have further examined our hypothesis that endogenous αSyn can occur in normal cells as a metastable, helically folded tetramer, not solely as the unfolded monomer long thought to be its native form. At this meeting, we reviewed our recent approaches for trapping αSyn in intact cells via in vivo crosslinking, a 5-step purification of αSyn from normal human brain, and the generation of new monoclonal antibodies to αSyn that enable general and oligomer-selective ELISAs. Crosslinking in intact living cells confirmed that αSyn occurs in the cytosol of neurons and non-neural cells in substantial part as metastable tetramers and related oligomers, plus varying amounts of free monomers. The non-pathogenic homolog, β-synuclein, forms closely similar oligomeric assemblies, suggesting that the oligomers we observe for αSyn are also physiological. In contrast to other normal oligomeric proteins (e.g., DJ-1), αSyn tetramers dissociate rapidly to monomers upon conventional cell lysis but are retained partially as tetramers if cells are lysed at high protein concentrations ('molecular crowding'). Thus, αSyn exists natively as helical tetramers that are in dynamic equilibrium with unfolded monomers. The tetramers appear relatively resistant to aggregation, in contrast to monomers, which may give rise to fibrillar inclusions.
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http://dx.doi.org/10.1159/000355516DOI Listing
September 2014

In vivo cross-linking reveals principally oligomeric forms of α-synuclein and β-synuclein in neurons and non-neural cells.

J Biol Chem 2013 Mar 14;288(9):6371-85. Epub 2013 Jan 14.

Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

Aggregation of α-synuclein (αSyn) in neurons produces the hallmark cytopathology of Parkinson disease and related synucleinopathies. Since its discovery, αSyn has been thought to exist normally in cells as an unfolded monomer. We recently reported that αSyn can instead exist in cells as a helically folded tetramer that resists aggregation and binds lipid vesicles more avidly than unfolded recombinant monomers (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110). However, a subsequent study again concluded that cellular αSyn is an unfolded monomer (Fauvet, B., Mbefo, M. K., Fares, M. B., Desobry, C., Michael, S., Ardah, M. T., Tsika, E., Coune, P., Prudent, M., Lion, N., Eliezer, D., Moore, D. J., Schneider, B., Aebischer, P., El-Agnaf, O. M., Masliah, E., and Lashuel, H. A. (2012) J. Biol. Chem. 287, 15345-15364). Here we describe a simple in vivo cross-linking method that reveals a major ~60-kDa form of endogenous αSyn (monomer, 14.5 kDa) in intact cells and smaller amounts of ~80- and ~100-kDa forms with the same isoelectric point as the 60-kDa species. Controls indicate that the apparent 60-kDa tetramer exists normally and does not arise from pathological aggregation. The pattern of a major 60-kDa and minor 80- and 100-kDa species plus variable amounts of free monomers occurs endogenously in primary neurons and erythroid cells as well as neuroblastoma cells overexpressing αSyn. A similar pattern occurs for the homologue, β-synuclein, which does not undergo pathogenic aggregation. Cell lysis destabilizes the apparent 60-kDa tetramer, leaving mostly free monomers and some 80-kDa oligomer. However, lysis at high protein concentrations allows partial recovery of the 60-kDa tetramer. Together with our prior findings, these data suggest that endogenous αSyn exists principally as a 60-kDa tetramer in living cells but is lysis-sensitive, making the study of natural αSyn challenging outside of intact cells.
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http://dx.doi.org/10.1074/jbc.M112.403311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585072PMC
March 2013

Transmembrane protein 147 (TMEM147) is a novel component of the Nicalin-NOMO protein complex.

J Biol Chem 2010 Aug 10;285(34):26174-81. Epub 2010 Jun 10.

German Center for Neurodegenerative Diseases (DZNE) and the Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, D-80336 Munich, Germany.

Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely gamma-secretase and the Nicalin-NOMO (Nodal modulator) complex. The gamma-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the beta-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200-220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A approximately 22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to gamma-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with gamma-secretase.
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http://dx.doi.org/10.1074/jbc.M110.132548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924024PMC
August 2010

The Nicastrin-like protein Nicalin regulates assembly and stability of the Nicalin-nodal modulator (NOMO) membrane protein complex.

J Biol Chem 2007 Apr 29;282(14):10632-8. Epub 2007 Jan 29.

Center of Integrated Protein Science and the Department of Biochemistry, Laboratory for Neurodegenerative Disease Research, Ludwig-Maximilians-University, D-80336 Munich, Germany.

The assembly of the gamma-secretase complex, an Alzheimer disease-related protease required for beta-amyloid generation, is tightly regulated and predominantly limited by the stoichiometrical availability of its components. We have identified a novel endoplasmic reticulum-located protein complex that is regulated in a similar fashion. It contains the recently identified Nodal signaling antagonists Nicalin (a distant homolog of the gamma-secretase component Nicastrin) and NOMO (Nodal modulator). Using an RNA interference approach, we found that Nicalin and NOMO became unstable in the absence of the respective binding partner, suggesting that complex formation has a stabilizing effect. Overexpression of Nicalin resulted in an increase in NOMO, whereas endogenous Nicalin was reduced below the detection limit. Both effects were shown to occur at a post-transcriptional level. Thus, NOMO is most likely produced in excess amounts and either stabilized by Nicalin or rapidly degraded. In contrast, Nicalin levels are limited independently of NOMO. We, therefore, propose that Nicalin controls the assembly and stability of the Nicalin-NOMO complex.
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http://dx.doi.org/10.1074/jbc.M611033200DOI Listing
April 2007
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