Arriba por la Vida Estudio: a randomized controlled trial promoting standing behavior to reduce sitting time among postmenopausal Latinas.

Postmenopausal Hispanic/Latina (N = 254) women with a body mass index (BMI) ≥ 25 kg/m2 were randomized to an intervention to reduce sitting time or a comparison condition for 12 weeks. The standing intervention group received three in-person health-counseling sessions, one home visit, and up to eight motivational interviewing calls. The heart healthy lifestyle comparison group (C) received an equal number of contact hours to discuss healthy aging. The primary outcome was 12-week change in sitting time measured via thigh-worn activPAL. Group differences in outcomes were analyzed using linear mixed-effects models. Participants had a mean age of 65 (6.5) years, preferred Spanish language (89%), BMI of 32.4 (4.8) kg/m2, and sat for an average of 540 (86) minutes/day. Significant between-group differences were observed in reductions of sitting time across the 12-week period [Mdifference (SE): C - 7.5 (9.1), SI - 71.0 (9.8), p < 0.01]. Results demonstrate that coaching models to reduce sitting are feasible and effective.

Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein.

Alpha-synuclein (aSyn) is a central player in Parkinson's disease (PD) but the precise molecular mechanisms underlying its pathogenicity remain unclear. It has recently been suggested that nuclear aSyn may modulate gene expression, possibly via interactions with DNA. However, the biological behavior of aSyn in the nucleus and the factors affecting its transcriptional role are not known. Here, we investigated the mechanisms underlying aSyn-mediated transcription deregulation by assessing its effects in the nucleus and the impact of phosphorylation in these dynamics. We found that aSyn induced severe transcriptional deregulation, including the downregulation of important cell cycle-related genes. Importantly, transcriptional deregulation was concomitant with reduced binding of aSyn to DNA. By forcing the nuclear presence of aSyn in the nucleus (aSyn-NLS), we found the accumulation of high molecular weight aSyn species altered gene expression and reduced toxicity when compared with the wild-type or exclusively cytosolic protein. Interestingly, nuclear localization of aSyn, and the effect on gene expression and cytotoxicity, was also modulated by phosphorylation on serine 129. Thus, we hypothesize that the role of aSyn on gene expression and, ultimately, toxicity, may be modulated by the phosphorylation status and nuclear presence of different aSyn species. Our findings shed new light onto the subcellular dynamics of aSyn and unveil an intricate interplay between subcellular location, phosphorylation and toxicity, opening novel avenues for the design of future strategies for therapeutic intervention in PD and other synucleinopathies.

E46K-like α-synuclein mutants increase lipid interactions and disrupt membrane selectivity.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders, and both genetic and histopathological evidence have implicated the ubiquitous presynaptic protein α-synuclein (αSyn) in its pathogenesis. Recent work has investigated how disrupting αSyn's interaction with membranes triggers trafficking defects, cellular stress, and apoptosis. Special interest has been devoted to a series of mutants exacerbating the effects of the E46K mutation (associated with autosomal dominant PD) through homologous Glu-to-Lys substitutions in αSyn's N-terminal region (i.e. E35K and E61K). Such E46K-like mutants have been shown to cause dopaminergic neuron loss and severe but L-DOPA-responsive motor defects in mouse overexpression models, presenting enormous translational potential for PD and other "synucleinopathies." In this work, using a variety of biophysical techniques, we characterize the molecular pathology of E46K-like αSyn mutants by studying their structure and membrane-binding and remodeling abilities. We find that, although a slight increase in the mutants' avidity for synaptic vesicle-like membranes can be detected, most of their deleterious effects are connected to their complete disruption of αSyn's curvature selectivity. Indiscriminate binding can shift αSyn's subcellular localization away from its physiological interactants at the synaptic bouton toward trafficking vesicles and organelles, as observed in E46K-like cellular and murine models, as well as in human pathology. In conclusion, our findings suggest that a loss of curvature selectivity, rather than increased membrane affinity, could be the critical dyshomeostasis in synucleinopathies.

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.

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.].

Environmental and genetic factors support the dissociation between α-synuclein aggregation and toxicity.

Synucleinopathies are a group of progressive disorders characterized by the abnormal aggregation and accumulation of α-synuclein (aSyn), an abundant neuronal protein that can adopt different conformations and biological properties. Recently, aSyn pathology was shown to spread between neurons in a prion-like manner. Proteins like aSyn that exhibit self-propagating capacity appear to be able to adopt different stable conformational states, known as protein strains, which can be modulated both by environmental and by protein-intrinsic factors. Here, we analyzed these factors and found that the unique combination of the neurodegeneration-related metal copper and the pathological H50Q aSyn mutation induces a significant alteration in the aggregation properties of aSyn. We compared the aggregation of WT and H50Q aSyn with and without copper, and assessed the effects of the resultant protein species when applied to primary neuronal cultures. The presence of copper induces the formation of structurally different and less-damaging aSyn aggregates. Interestingly, these aggregates exhibit a stronger capacity to induce aSyn inclusion formation in recipient cells, which demonstrates that the structural features of aSyn species determine their effect in neuronal cells and supports a lack of correlation between toxicity and inclusion formation. In total, our study provides strong support in favor of the hypothesis that protein aggregation is not a primary cause of cytotoxicity.

Small-Molecule-Induced Soluble Oligomers of α-Synuclein with Helical Structure.

Accumulation of α-synuclein (αSyn) aggregates constitutes the hallmark of synucleinopathies including Parkinson's disease. However, many steps from the innocuous, monomeric αSyn toward misfolded oligomers and fibrillar species remain unclear. Here, we show that αSyn can form in solution α-helical oligomers, which are off-pathway to fibrillization, through interaction with the tetrapyrrole phthalocyanine tetrasulfonate. Chemical cross-linking combined with mass spectrometry reveals a large number of intermolecular cross-links along the entire αSyn sequence in the phthalocyanine tetrasulfonate-stabilized αSyn oligomers. Our study suggests that stabilization of structured oligomers by small molecules provides a viable strategy to interfere with αSyn fibrillization.

S100A4 protects the myocardium against ischemic stress.

BACKGROUND: Myocardial infarction is followed by cardiac dysfunction, cellular death, and ventricular remodeling, including tissue fibrosis. S100A4 protein plays multiple roles in cellular survival, and tissue fibrosis, but the relative role of the S100A4 in the myocardium after myocardial infarction is unknown. This study aims to investigate the role of S100A4 in myocardial remodeling and cardiac function following infarct damage. METHODS AND RESULTS: S100A4 expression is low in the adult myocardium, but significantly increased following myocardial infarction. Deletion of S100A4 increased cardiac damage after myocardial infarction, whereas cardiac myocyte-specific overexpression of S100A4 protected the infarcted myocardium. Decreased cardiac function in S100A4 Knockout mice was accompanied with increased cardiac remodeling, fibrosis, and diminished capillary density in the remote myocardium. Loss of S100A4 caused increased apoptotic cell death both in vitro and in vivo in part mediated by decreased VEGF expression. Conversely, S100A4 overexpression protected cells against apoptosis in vitro and in vivo. Increased pro-survival AKT-signaling explained reduced apoptosis in S100A4 overexpressing cells. CONCLUSION: S100A4 expression protects cardiac myocytes against myocardial ischemia and is required for stabilization of cardiac function after MI.

Pathological hypertrophy amelioration by PRAS40-mediated inhibition of mTORC1.

Mechanistic target of rapamycin complex 1 (mTORC1), necessary for cellular growth, is regulated by intracellular signaling mediating inhibition of mTORC1 activation. Among mTORC1 regulatory binding partners, the role of Proline Rich AKT Substrate of 40 kDa (PRAS40) in controlling mTORC1 activity and cellular growth in response to pathological and physiological stress in the heart has never been addressed. This report shows PRAS40 is regulated by AKT in cardiomyocytes and that AKT-driven phosphorylation relieves the inhibitory function of PRAS40. PRAS40 overexpression in vitro blocks mTORC1 in cardiomyocytes and decreases pathological growth. Cardiomyocyte-specific overexpression in vivo blunts pathological remodeling after pressure overload and preserves cardiac function. Inhibition of mTORC1 by PRAS40 preferentially promotes protective mTORC2 signaling in chronic diseased myocardium. In contrast, strong PRAS40 phosphorylation by AKT allows for physiological hypertrophy both in vitro and in vivo, whereas cardiomyocyte-specific overexpression of a PRAS40 mutant lacking capacity for AKT-phosphorylation inhibits physiological growth in vivo, demonstrating that AKT-mediated PRAS40 phosphorylation is necessary for induction of physiological hypertrophy. Therefore, PRAS40 phosphorylation acts as a molecular switch allowing mTORC1 activation during physiological growth, opening up unique possibilities for therapeutic regulation of the mTORC1 complex to mitigate pathologic myocardial hypertrophy by PRAS40.

Burial of the polymorphic residue 129 in amyloid fibrils of prion stop mutants.

Misfolding of the natively α-helical prion protein into a ß-sheet rich isoform is related to various human diseases such as Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. In humans, the disease phenotype is modified by a methionine/valine polymorphism at codon 129 of the prion protein gene. Using a combination of hydrogen/deuterium exchange coupled to NMR spectroscopy, hydroxyl radical probing detected by mass spectrometry, and site-directed mutagenesis, we demonstrate that stop mutants of the human prion protein have a conserved amyloid core. The 129 residue is deeply buried in the amyloid core structure, and its mutation strongly impacts aggregation. Taken together the data support a critical role of the polymorphic residue 129 of the human prion protein in aggregation and disease.

Mechanistic target of rapamycin complex 2 protects the heart from ischemic damage.

BACKGROUND: The mechanistic target of rapamycin (mTOR) comprises 2 structurally distinct multiprotein complexes, mTOR complexes 1 and 2 (mTORC1 and mTORC2). Deregulation of mTOR signaling occurs during and contributes to the severity of myocardial damage from ischemic heart disease. However, the relative roles of mTORC1 versus mTORC2 in the pathogenesis of ischemic damage are unknown. METHODS AND RESULTS: Combined pharmacological and molecular approaches were used to alter the balance of mTORC1 and mTORC2 signaling in cultured cardiac myocytes and in mouse hearts subjected to conditions that mimic ischemic heart disease. The importance of mTOR signaling in cardiac protection was demonstrated by pharmacological inhibition of both mTORC1 and mTORC2 with Torin1, which led to increased cardiomyocyte apoptosis and tissue damage after myocardial infarction. Predominant mTORC1 signaling mediated by suppression of mTORC2 with Rictor similarly increased cardiomyocyte apoptosis and tissue damage after myocardial infarction. In comparison, preferentially shifting toward mTORC2 signaling by inhibition of mTORC1 with PRAS40 led to decreased cardiomyocyte apoptosis and tissue damage after myocardial infarction. CONCLUSIONS: These results suggest that selectively increasing mTORC2 while concurrently inhibiting mTORC1 signaling is a novel therapeutic approach for the treatment of ischemic heart disease.

Dynamic physiological α-synuclein S129 phosphorylation is driven by neuronal activity.

In Parkinson's disease and other synucleinopathies, the elevation of α-synuclein phosphorylated at Serine129 (pS129) is a widely cited marker of pathology. However, the physiological role for pS129 has remained undefined. Here we use multiple approaches to show for the first time that pS129 functions as a physiological regulator of neuronal activity. Neuronal activity triggers a sustained increase of pS129 in cultured neurons (200% within 4 h). In accord, brain pS129 is elevated in environmentally enriched mice exhibiting enhanced long-term potentiation. Activity-dependent α-synuclein phosphorylation is S129-specific, reversible, confers no cytotoxicity, and accumulates at synapsin-containing presynaptic boutons. Mechanistically, our findings are consistent with a model in which neuronal stimulation enhances Plk2 kinase activity via a calcium/calcineurin pathway to counteract PP2A phosphatase activity for efficient phosphorylation of membrane-bound α-synuclein. Patch clamping of rat SNCA-/- neurons expressing exogenous wild-type or phospho-incompetent (S129A) α-synuclein suggests that pS129 fine-tunes the balance between excitatory and inhibitory neuronal currents. Consistently, our novel S129A knock-in (S129AKI) mice exhibit impaired hippocampal plasticity. The discovery of a key physiological function for pS129 has implications for understanding the role of α-synuclein in neurotransmission and adds nuance to the interpretation of pS129 as a synucleinopathy biomarker.

Parkinson-causing mutations in LRRK2 impair the physiological tetramerization of endogenous α-synuclein in human neurons.

α-Synuclein (αSyn) aggregation in Lewy bodies and neurites defines both familial and 'sporadic' Parkinson's disease. We previously identified α-helically folded αSyn tetramers, in addition to the long-known unfolded monomers, in normal cells. PD-causing αSyn mutations decrease the tetramer:monomer (T:M) ratio, associated with αSyn hyperphosphorylation and cytotoxicity in neurons and a motor syndrome of tremor and gait deficits in transgenic mice that responds in part to L-DOPA. Here, we asked whether LRRK2 mutations, the most common genetic cause of cases previously considered sporadic PD, also alter tetramer homeostasis. Patient neurons carrying G2019S, the most prevalent LRRK2 mutation, or R1441C each had decreased T:M ratios and pSer129 hyperphosphorylation of their endogenous αSyn along with increased phosphorylation of Rab10, a widely reported substrate of LRRK2 kinase activity. Two LRRK2 kinase inhibitors normalized the T:M ratio and the hyperphosphorylation in the G2019S and R1441C patient neurons. An inhibitor of stearoyl-CoA desaturase, the rate-limiting enzyme for monounsaturated fatty acid synthesis, also restored the αSyn T:M ratio and reversed pSer129 hyperphosphorylation in both mutants. Coupled with the recent discovery that PD-causing mutations of glucocerebrosidase in Gaucher's neurons also decrease T:M ratios, our findings indicate that three dominant genetic forms of PD involve life-long destabilization of αSyn physiological tetramers as a common pathogenic mechanism that can occur upstream of progressive neuronal synucleinopathy. Based on αSyn's finely-tuned interaction with certain vesicles, we hypothesize that the fatty acid composition and fluidity of membranes regulate αSyn's correct binding to highly curved membranes and subsequent assembly into metastable tetramers.

Synphilin suppresses α-synuclein neurotoxicity in a Parkinson's disease Drosophila model.

Parkinson's disease (PD) is the second most common neurodegenerative disorder in humans. It affects 1% of the population over 65-years old. Its causes are environmental and genetic. As the world population ages, there is an urgent need for better and more detailed animal models for this kind of disease. In this work we show that the use of transgenic Drosophila is comparable to more complicated and costly animal models such as mice. The Drosophila model behaves very similar to the equivalent transgenic mice model. We show that both Synphilin-1 and α-synuclein are toxic by themselves, but when co-expressed, they suppress their toxicity reciprocally. Importantly, the symptoms induced in the fly can be treated and partially reverted using standard PD pharmacological treatments. This work showcases Drosophila as a detailed and multifaceted model for Parkinson's disease, providing a convenient platform in which to study and find new genetic modifiers of PD. genesis 49:392-402, 2011.

Altered conformation of α-synuclein drives dysfunction of synaptic vesicles in a synaptosomal model of Parkinson's disease.

While misfolding of alpha-synuclein (αSyn) is central to the pathogenesis of Parkinson's disease (PD), fundamental questions about its structure and function at the synapse remain unanswered. We examine synaptosomes from non-transgenic and transgenic mice expressing wild-type human αSyn, the E46K fPD-causing mutation, or an amplified form of E46K ("3K"). Synaptosomes from mice expressing the 3K mutant show reduced Ca2+-dependent vesicle exocytosis, altered synaptic vesicle ultrastructure, decreased SNARE complexes, and abnormal levels of certain synaptic proteins. With our intra-synaptosomal nuclear magnetic resonance (NMR) method, we reveal that WT αSyn participates in heterogeneous interactions with synaptic components dependent on endogenous αSyn and synaptosomal integrity. The 3K mutation markedly alters these interactions. The synaptic microenvironment is necessary for αSyn to reach its native conformations and establish a physiological interaction network. Its inability to populate diverse conformational ensembles likely represents an early step in αSyn dysfunction that contributes to the synaptotoxicity observed in synucleinopathies.

Refolding of helical soluble α-synuclein through transient interaction with lipid interfaces.

α-Synuclein (αSyn) is a key player in the pathogenesis of Parkinson's disease and other synucleinopathies. Here, we report the existence of a novel soluble α-helical conformer of αSyn, obtained through transient interaction with lipid interfaces, and propose dynamic oligomerization as the mechanism underlying its stability. The conformational space of αSyn appears to be highly context-dependent, and lipid bilayers might thus play crucial roles as molecular chaperones in a cellular environment.

The protonation state of histidine 111 regulates the aggregation of the evolutionary most conserved region of the human prion protein.

In a group of neurodegenerative diseases, collectively termed transmissible spongiform encephalopathies, the prion protein aggregates into ß-sheet rich amyloid-like deposits. Because amyloid structure has been connected to different prion strains and cellular toxicity, it is important to obtain insight into the structural properties of prion fibrils. Using a combination of solution NMR spectroscopy, thioflavin-T fluorescence and electron microscopy we here show that within amyloid fibrils of a peptide containing residues 108-143 of the human prion protein [humPrP (108-143)]-the evolutionary most conserved part of the prion protein - residue H111 and S135 are in close spatial proximity and their interaction is critical for fibrillization. We further show that residues H111 and H140 share the same microenvironment in the unfolded, monomeric state of the peptide, but not in the fibrillar form. While protonation of H140 has little influence on fibrillization of humPrP (108-143), a positive charge at position 111 blocks the conformational change, which is necessary for amyloid formation of humPrP (108-143). Our study thus highlights the importance of protonation of histidine residues for protein aggregation and suggests point mutations to probe the structure of infectious prion particles.

Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, yet disease-modifying treatments do not currently exist. Rho-associated protein kinase (ROCK) was recently described as a novel neuroprotective target in PD. Since alpha-synuclein (α-Syn) aggregation is a major hallmark in the pathogenesis of PD, we aimed to evaluate the anti-aggregative potential of pharmacological ROCK inhibition using the isoquinoline derivative Fasudil, a small molecule inhibitor already approved for clinical use in humans. Fasudil treatment significantly reduced α-Syn aggregation in vitro in a H4 cell culture model as well as in a cell-free assay. Nuclear magnetic resonance spectroscopy analysis revealed a direct binding of Fasudil to tyrosine residues Y133 and Y136 in the C-terminal region of α-Syn. Importantly, this binding was shown to be biologically relevant using site-directed mutagenesis of these residues in the cell culture model. Furthermore, we evaluated the impact of long-term Fasudil treatment on α-Syn pathology in vivo in a transgenic mouse model overexpressing human α-Syn bearing the A53T mutation (α-Syn(A53T) mice). Fasudil treatment improved motor and cognitive functions in α-Syn(A53T) mice as determined by Catwalk(TM) gait analysis and novel object recognition (NOR), without apparent side effects. Finally, immunohistochemical analysis revealed a significant reduction of α-Syn pathology in the midbrain of α-Syn(A53T) mice after Fasudil treatment. Our results demonstrate that Fasudil, next to its effects mediated by ROCK-inhibition, directly interacts with α-Syn and attenuates α-Syn pathology. This underscores the translational potential of Fasudil as a disease-modifying drug for the treatment of PD and other synucleinopathies.

PRAS40 prevents development of diabetic cardiomyopathy and improves hepatic insulin sensitivity in obesity.

Diabetes is a multi-organ disease and diabetic cardiomyopathy can result in heart failure, which is a leading cause of morbidity and mortality in diabetic patients. In the liver, insulin resistance contributes to hyperglycaemia and hyperlipidaemia, which further worsens the metabolic profile. Defects in mTOR signalling are believed to contribute to metabolic dysfunctions in diabetic liver and hearts, but evidence is missing that mTOR activation is causal to the development of diabetic cardiomyopathy. This study shows that specific mTORC1 inhibition by PRAS40 prevents the development of diabetic cardiomyopathy. This phenotype was associated with improved metabolic function, blunted hypertrophic growth and preserved cardiac function. In addition PRAS40 treatment improves hepatic insulin sensitivity and reduces systemic hyperglycaemia in obese mice. Thus, unlike rapamycin, mTORC1 inhibition with PRAS40 improves metabolic profile in diabetic mice. These findings may open novel avenues for therapeutic strategies using PRAS40 directed against diabetic-related diseases.

Small molecule-mediated stabilization of vesicle-associated helical α-synuclein inhibits pathogenic misfolding and aggregation.

α-synuclein is an abundant presynaptic protein that is important for regulation of synaptic vesicle trafficking, and whose misfolding plays a key role in Parkinson's disease. While α-synuclein is disordered in solution, it folds into a helical conformation when bound to synaptic vesicles. Stabilization of helical, folded α-synuclein might therefore interfere with α-synuclein-induced neurotoxicity. Here we show that several small molecules, which delay aggregation of α-synuclein in solution, including the Parkinson's disease drug selegiline, fail to interfere with misfolding of vesicle-bound α-synuclein. In contrast, the porphyrin phtalocyanine tetrasulfonate directly binds to vesicle-bound α-synuclein, stabilizes its helical conformation and thereby delays pathogenic misfolding and aggregation. Our study suggests that small-molecule-mediated stabilization of helical vesicle-bound α-synuclein opens new possibilities to target Parkinson's disease and related synucleinopathies.