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Alteration to endoplasmic reticulum (ER) proteostasis is observed in a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR target genes. In this study, we designed an ATF6f/XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has a stronger effect in reducing the abnormal aggregation of mutant huntingtin and α-synuclein when compared to XBP1s or ATF6 alone. We developed a gene transfer approach to deliver UPRplus into the brain using adeno-associated viruses (AAVs) and demonstrated potent neuroprotection in vivo in preclinical models of Parkinson’s disease and Huntington’s disease. These results support the concept in which directing UPR-mediated gene expression toward specific adaptive programs may serve as a possible strategy to optimize the beneficial effects of the pathway in different disease conditions.

Increasing evidence suggests that the process of alpha-synuclein (alpha-syn) aggregation from monomers into amyloid fibrils and Lewy bodies, via oligomeric intermediates plays an essential role in the pathogenesis of different synucleinopathies, including Parkinson's disease (PD), multiple system atrophy and dementia with Lewy bodies (DLB). However, the nature of the toxic species and the mechanisms by which they contribute to neurotoxicity and disease progression remain elusive. Over the past two decades, significant efforts and resources have been invested in studies aimed at identifying and targeting toxic species along the pathway of alpha-syn fibrillization. Although this approach has helped to advance the field and provide insights into the biological properties and toxicity of different alpha-syn species, many of the fundamental questions regarding the role of alpha-syn aggregation in PD remain unanswered, and no therapeutic compounds targeting alpha-syn aggregates have passed clinical trials. Several factors have contributed to this slow progress, including the complexity of the aggregation pathways and the heterogeneity and dynamic nature of alpha-syn aggregates. In the majority of experiment, the alpha-syn samples used contain mixtures of alpha-syn species that exist in equilibrium and their ratio changes upon modifying experimental conditions. The failure to quantitatively account for the distribution of different alpha-syn species in different studies has contributed not only to experimental irreproducibility but also to misinterpretation of results and misdirection of valuable resources. Towards addressing these challenges and improving experimental reproducibility in Parkinson's research, we describe here a simple centrifugation-based filtration protocol for the isolation, quantification and assessment of the distribution of alpha-syn monomers, oligomers and fibrils, in heterogeneous alpha-syn samples of increasing complexity. The protocol is simple, does not require any special instrumentation and can be performed rapidly on multiple samples using small volumes. Here, we present and discuss several examples that illustrate the applications of this protocol and how it could contribute to improving the reproducibility of experiments aimed at elucidating the structural basis of alpha-syn aggregation, seeding activity, toxicity and pathology spreading. This protocol is applicable, with slight modifications, to other amyloid-forming proteins.

Generation of peroxynitrite (ONOO-) as a result of altered redox balance has been shown to affect cardiac function; however, inconsistencies in the data exist, particularly for myocardial contractility. The hypothesis that the cardiac impact of ONOO- formation depends on its site of generation, intravascular or intramyocardial, was examined. Cardiac contractility was assessed by pressure-volume analysis to delineate vascular versus cardiac changes on direct infusion of ONOO- into the right atria of conscious dogs both with normal cardiac function and in heart failure. Additionally, ONOO- was administered to isolated murine cardiomyocytes to mimic in situ cardiac generation. When infused in vivo, ONOO had little impact on inotropy but led to systemic arterial dilation, likely as a result of rapid decomposition to NO2- and NO3-. In contrast, infused ONOO- was long lived enough to abolish beta-adrenergic (dobutamine)-stimulated contractility/relaxation, most likely through catecholamine oxidation to aminochrome. When administered to isolated murine cardiomyocytes, ONOO- induced a rapid reduction in sarcomere shortening and whole cell calcium transients, although neither decomposed ONOO- or NaNO2 had any effect. Thus, systemic generation of ONOO- is unlikely to have primary cardiac effects, but may modulate cardiac contractile reserve, via blunted beta-adrenergic stimulation, and vascular tone, as a result of generation of NO2- and NO3. However, myocyte generation of ONOO- may impair contractile function by directly altering Ca2+ handling. These data demonstrate that the site of generation within the cardiovascular system largely dictates the ability of ONOO- to directly or indirectly modulate cardiac pump function. (c) 2006 Elsevier Inc. All rights reserved.

The nitric oxide (NO center dot) sibling, nitroxyl or nitrosyl hydride (HNO), is emerging as a molecule whose pharmacological properties include providing functional support to failing hearts. HNO also preconditions myocardial tissue, protecting it against ischemia-reperfusion injury while exerting vascular antiproliferative actions. In this review, HNO's peculiar cardiovascular assets are discussed in light of its unique chemistry that distinguish HNO from NO center dot as well as from reactive oxygen and nitrogen species such as the hydroxyl radical and peroxynitrite. Included here is a discussion of the possible routes of HNO formation in the myocardium and its chemical targets in the heart. HNO has been shown to have positive inotropic/lusitropic effects under normal and congestive heart failure conditions in animal models. The mechanistic intricacies of the beneficial cardiac effects of HNO are examined in cellular models. In contrast to beta-receptor/cyclic adenosine monophosphate/protein kinase A-dependent enhancers of myocardial performance, HNO uses its "thiophylic'' nature as a vehicle to interact with redox switches such as cysteines, which are located in key components of the cardiac electromechanical machinery ruling myocardial function. Here, we will briefly review new features of HNO's cardiovascular effects that when combined with its positive inotropic/lusitropic action may render HNO donors an attractive addition to the current therapeutic armamentarium for treating patients with acutely decompensated congestive heart failure. Antioxid. Redox Signal. 14, 1687-1698.