Store-operated Ca2+ entry regulatory factor alters murine metabolic state in an age-dependent manner via hypothalamic pathways.

Store-operated calcium entry (SOCE) is a vital process aimed at refilling cellular internal Ca2+ stores and a primary cellular signaling driver for transcription factors' entry to the nucleus. SOCE-associated regulatory factor (SARAF)/TMEM66 is an endoplasmic reticulum (ER)-resident transmembrane protein that promotes SOCE inactivation and prevents Ca2+ overfilling of the cell. Here, we demonstrate that mice deficient in SARAF develop age-dependent sarcopenic obesity with decreased energy expenditure, lean mass, and locomotion without affecting food consumption. Moreover, SARAF ablation reduces hippocampal proliferation, modulates the activity of the hypothalamus-pituitary-adrenal (HPA) axis, and mediates changes in anxiety-related behaviors. Interestingly, selective SARAF ablation in the hypothalamus's paraventricular nucleus (PVN) neurons reduces old age-induced obesity and preserves locomotor activity, lean mass, and energy expenditure, suggesting a possible central control with a site-specific role for SARAF. At the cellular level, SARAF ablation in hepatocytes leads to elevated SOCE, elevated vasopressin-induced Ca2+ oscillations, and an increased mitochondrial spare respiratory capacity (SPC), thus providing insights into the cellular mechanisms that may affect the global phenotypes. These effects may be mediated via the liver X receptor (LXR) and IL-1 signaling metabolic regulators explicitly altered in SARAF ablated cells. In short, our work supports both central and peripheral roles of SARAF in regulating metabolic, behavioral, and cellular responses.

Quinone Methide-Based Organophosphate Hydrolases Inhibitors: Trans Proximity Labelers versus Cis Labeling Activity-Based Probes.

Quinone methide (QM) chemistry is widely applied including in enzyme inhibitors. Typically, enzyme-mediated bond breaking releases a phenol product that rearranges into an electrophilic QM that in turn covalently modifies protein side chains. However, the factors that govern the reactivity of QM-based inhibitors and their mode of inhibition have not been systematically explored. Foremost, enzyme inactivation might occur in cis, whereby a QM molecule inactivates the very same enzyme molecule that released it, or by trans if the released QMs diffuse away and inactivate other enzyme molecules. We examined QM-based inhibitors for enzymes exhibiting phosphoester hydrolase activity. We tested different phenolic substituents and benzylic leaving groups, thereby modulating the rates of enzymatic hydrolysis, phenolate-to-QM rearrangement, and the electrophilicity of the resulting QM. By developing assays that distinguish between cis and trans inhibition, we have identified certain combinations of leaving groups and phenyl substituents that lead to inhibition in the cis mode, while other combinations gave trans inhibition. Our results suggest that cis-acting QM-based substrates could be used as activity-based probes to identify various phospho- and phosphono-ester hydrolases, and potentially other hydrolases.

Presenilin 1 Regulates [Ca2+]i and Mitochondria/ER Interaction in Cultured Rat Hippocampal Neurons.

Mutations in the presenilin 1 (PS1) gene are a major trigger of familial Alzheimer's disease (AD), yet the mechanisms affected by mutated PS1 causing cognitive decline are not yet elucidated. In the present study, we compared rat hippocampal neurons in culture, transfected with PS1 or with mutant (M146V) PS1 (mPS1) plasmids in several neuronal functions. Initially, we confirmed earlier observations that mPS1-expressing neurons are endowed with fewer mature "mushroom" spines and more filopodial immature protrusions. The correlation between calcium changes in the cytosol, mitochondria, and endoplasmic reticulum (ER) is mitigated in the mPS1 neurons, tested by the response to an abrupt increase in ambient [Ca2+]o; cytosolic [Ca2+]i is higher in the mPS1 neurons but mitochondrial [Ca2+] is lower than in control neurons. Strikingly, mPS1-transfected neurons express higher excitability and eventual lower survival rate when exposed to the oxidative stressor, paraquat. These results highlight an impaired calcium regulation in mPS1 neurons, resulting in a reduced ability to handle oxidative stress, which may lead to cell death and AD.

SARAF Luminal Domain Structure Reveals a Novel Domain-Swapped ß-Sandwich Fold Important for SOCE Modulation.

Store-Operated Calcium Entry (SOCE) plays key roles in cell proliferation, muscle contraction, immune responses, and memory formation. The coordinated interactions of a number of proteins from the plasma and endoplasmic reticulum membranes control SOCE to replenish internal Ca2+ stores and generate intracellular Ca2+ signals. SARAF, an endoplasmic reticulum resident component of the SOCE pathway having no homology to any characterized protein, serves as an important brake on SOCE. Here, we describe the X-ray crystal structure of the SARAF luminal domain, SARAFL. This domain forms a novel 10-stranded ß-sandwich fold that includes a set of three conserved disulfide bonds, denoted the "SARAF-fold." The structure reveals a domain-swapped dimer in which the last two ß-strands (ß9 and ß10) are exchanged forming a region denoted the "SARAF luminal switch" that is essential for dimerization. Sequence comparisons reveal that the SARAF-fold is highly conserved in vertebrates and in a variety of pathologic fungi. Förster resonance energy transfer experiments using full-length SARAF validate the formation of the domain-swapped dimer in cells and demonstrate that dimerization is reversible. A designed variant lacking the SARAF luminal switch shows that the domain swapping is essential to function and indicates that the SARAF dimer accelerates SOCE inactivation.

Importin α5 Regulates Anxiety through MeCP2 and Sphingosine Kinase 1.

Importins mediate transport from synapse to soma and from cytoplasm to nucleus, suggesting that perturbation of importin-dependent pathways should have significant neuronal consequences. A behavioral screen on five importin α knockout lines revealed that reduced expression of importin α5 (KPNA1) in hippocampal neurons specifically decreases anxiety in mice. Re-expression of importin α5 in ventral hippocampus of knockout animals increased anxiety behaviors to wild-type levels. Hippocampal neurons lacking importin α5 reveal changes in presynaptic plasticity and modified expression of MeCP2-regulated genes, including sphingosine kinase 1 (Sphk1). Knockout of importin α5, but not importin α3 or α4, reduces MeCP2 nuclear localization in hippocampal neurons. A Sphk1 blocker reverses anxiolysis in the importin α5 knockout mouse, while pharmacological activation of sphingosine signaling has robust anxiolytic effects in wild-type animals. Thus, importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons.

Altered lysosome distribution is an early neuropathological event in neurological forms of Gaucher disease.

In the lysosomal storage disorder Gaucher disease (GD), glucosylceramide (GlcCer) accumulates due to the defective activity of glucocerebrosidase. A subset of GD patients develops neuropathology. We now show mislocalization of Limp2-positive puncta and a large reduction in the number of Lamp1-positive puncta, which are associated with impaired tubulin. These changes occur at an early stage in animal models of GD, prior to development of overt symptoms and considerably earlier than neuronal loss. Altered lysosomal localization and cytoskeleton disruption precede the neuroinflammatory pathways, axonal dystrophy and neuronal loss previously characterized in neuronal forms of GD.

Delineating pathological pathways in a chemically induced mouse model of Gaucher disease.

Great interest has been shown in understanding the pathology of Gaucher disease (GD) due to the recently discovered genetic relationship with Parkinson's disease. For such studies, suitable animal models of GD are required. Chemical induction of GD by inhibition of acid ß-glucosidase (GCase) using the irreversible inhibitor conduritol B-epoxide (CBE) is particularly attractive, although few systematic studies examining the effect of CBE on the development of symptoms associated with neurological forms of GD have been performed. We now demonstrate a correlation between the amount of CBE injected into mice and levels of accumulation of the GD substrates, glucosylceramide and glucosylsphingosine, and show that disease pathology, indicated by altered levels of pathological markers, depends on both the levels of accumulated lipids and the time at which their accumulation begins. Gene array analysis shows a remarkable similarity in the gene expression profiles of CBE-treated mice and a genetic GD mouse model, the Gba(flox/flox) ;nestin-Cre mouse, with 120 of the 144 genes up-regulated in CBE-treated mice also up-regulated in Gba(flox/flox) ;nestin-Cre mice. We also demonstrate that various aspects of neuropathology and some behavioural abnormalities can be arrested upon cessation of CBE treatment during a specific time window. Together, our data demonstrate that injection of mice with CBE provides a rapid and relatively easy way to induce symptoms typical of neuronal forms of GD. This is particularly useful when examining the role of specific biochemical pathways in GD pathology, since CBE can be injected into mice defective in components of putative pathological pathways, alleviating the need for time-consuming crossing of mice. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Identification of a biomarker in cerebrospinal fluid for neuronopathic forms of Gaucher disease.

Gaucher disease, a recessive inherited metabolic disorder caused by defects in the gene encoding glucosylceramidase (GlcCerase), can be divided into three subtypes according to the appearance of symptoms associated with central nervous system involvement. We now identify a protein, glycoprotein non-metastatic B (GPNMB), that acts as an authentic marker of brain pathology in neurological forms of Gaucher disease. Using three independent techniques, including quantitative global proteomic analysis of cerebrospinal fluid (CSF) in samples from Gaucher disease patients that display neurological symptoms, we demonstrate a correlation between the severity of symptoms and GPNMB levels. Moreover, GPNMB levels in the CSF correlate with disease severity in a mouse model of Gaucher disease. GPNMB was also elevated in brain samples from patients with type 2 and 3 Gaucher disease. Our data suggest that GPNMB can be used as a marker to quantify neuropathology in Gaucher disease patients and as a marker of treatment efficacy once suitable treatments towards the neurological symptoms of Gaucher disease become available.

From sheep to mice to cells: tools for the study of the sphingolipidoses.

The sphingolipidoses are a group of inherited lysosomal storage diseases in which sphingolipids accumulate due to the defective activity of one or other enzymes involved in their degradation. For most of the sphingolipidoses, little is known about the molecular mechanisms that lead to disease, which has negatively impacted attempts to develop therapies for these devastating human diseases. Use of both genetically-modified animals, ranging from mice to larger mammals, and of novel cell culture systems, is of utmost importance in delineating the molecular mechanisms that cause pathophysiology, and in providing tools that enable testing the efficacy of new therapies. In this review, we discuss eight sphingolipidoses, namely Gaucher disease, Fabry disease, metachromatic leukodystrophy, Krabbe disease, Niemann-Pick diseases A and B, Farber disease, GM1 gangliosidoses, and GM2 gangliosidoses, and describe the tools that are currently available for their study. This article is part of a Special Issue entitled Tools to study lipid functions.

RIPK3 as a potential therapeutic target for Gaucher's disease.

Gaucher's disease (GD), an inherited metabolic disorder caused by mutations in the glucocerebrosidase gene (GBA), is the most common lysosomal storage disease. Heterozygous mutations in GBA are a major risk factor for Parkinson's disease. GD is divided into three clinical subtypes based on the absence (type 1) or presence (types 2 and 3) of neurological signs. Type 1 GD was the first lysosomal storage disease (LSD) for which enzyme therapy became available, and although infusions of recombinant glucocerebrosidase (GCase) ameliorate the systemic effects of GD, the lack of efficacy for the neurological manifestations, along with the considerable expense and inconvenience of enzyme therapy for patients, renders the search for alternative or complementary therapies paramount. Glucosylceramide and glucosylsphingosine accumulation in the brain leads to massive neuronal loss in patients with neuronopathic GD (nGD) and in nGD mouse models. However, the mode of neuronal death is not known. Here, we show that modulating the receptor-interacting protein kinase-3 (Ripk3) pathway markedly improves neurological and systemic disease in a mouse model of GD. Notably, Ripk3 deficiency substantially improved the clinical course of GD mice, with increased survival and motor coordination and salutary effects on cerebral as well as hepatic injury.