Subclinical Myocardial Injury and the Phenotype of Clinical Congestion in Patients With Heart Failure and Reduced Left Ventricular Ejection Fraction.

BACKGROUND: Clinical congestion is associated with adverse outcomes in patients with heart failure. The pathophysiological mediators of this association remain uncertain. METHODS AND RESULTS: We prospectively enrolled a cohort of patients with heart failure and reduced left ventricular ejection fraction and performed a detailed clinical examination followed on the same day by an invasive right heart catheterization and blood sampling for biomarkers. High-sensitivity troponin T and N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels were measured. A clinical congestion score was calculated based on jugular venous pressure (cm H20 <10 = 0, 10-14 = 1, >14 = 2 points), bendopnea (0 vs 1), a third heart sound (0 vs 1), or peripheral edema (0-2). Congestion was categorized into tiers as absent (0 points), mild (1 point), or moderate to severe (≥ 2 points). We tested for associations of high-sensitivity troponin T, NT-proBNP, and elevated ventricular filling pressures with clinical congestion in both univariate and multivariable analyses. Of 153 participants, 65 (42%) had absent, 35 mild (23%), and 53 (35%) had moderate to severe clinical congestion. Congestion tier was associated with higher NT-proBNP and hs-troponin levels, and the right atrial pressure and pulmonary capillary wedge pressure (P < .001 for each). Increased congestion tier was also associated with the coexistent presence of elevated troponin T (≥52 ng/L), NT-proBNP (≥1000 pg/mL), and pulmonary capillary wedge pressure (≥22 mm Hg). Specifically, 78% of those with absent clinical congestion had 0 to 1 of these findings, whereas 75% of those with moderate-severe congestion had 2 or all 3 of these abnormalities (P < .001). An elevated hs-troponin was associated with mild or greater clinical congestion (odds ratio 3, 95% confidence interval 1.2-7.5, P = .02) in multivariable analysis adjusting for potential confounders including the right atrial pressure, pulmonary capillary wedge pressure, and NT-proBNP levels. CONCLUSIONS: Clinical congestion is a phenotype in which there is a high coexistent presence of elevated ventricular filling pressures, elevated natriuretic peptide levels, and subclinical myocardial injury. An elevated troponin was associated with clinical congestion in multivariable models that adjusted for ventricular filling pressures and natriuretic peptide levels. These data strengthen the evidence base for an association of elevated troponin with clinical congestion, suggesting that subclinical myocardial injury may be an important contributor to the pathophysiology of the congested state.

Global Dynamics of the Stationary M2 Mode-1 Internal Tide.

A reduced-physics model is employed at 1/25° to 1/100° global resolution to determine (a) if linear dynamics can reproduce the observed low-mode M2 internal tide, (b) internal-tide sensitivity to bathymetry, stratification, surface tides, and dissipation parameterizations, and (c) the amount of power transferred to the nonstationary internal tide. The simulations predict 200 GW of mode-1 internal-tide generation, consistent with a general circulation model and semianalytical theory. Mode-1 energy is sensitive to damping, but a simulation using parameterizations for wave drag and wave-mean interaction predicts 84% of satellite observed sea-surface height amplitude variance on a 1° × 1° grid. The simulation energy balance indicates that 16% of stationary mode-1 energy is scattered to modes 2-4 and negligible energy propagates onto the shelves. The remaining 84% of energy is lost through parameterizations for high-mode scattering over rough topography (54%) and wave-mean interactions that transfer energy to the nonstationary internal tide (29%).

Absence of Batf3 reveals a new dimension of cell state heterogeneity within conventional dendritic cells.

Conventional dendritic cells (cDCs) are traditionally subdivided into cDC1 and cDC2 lineages. Batf3 is a cDC1-required transcription factor, and we observed that Batf3-/- mice harbor a population of cDC1-like cells co-expressing cDC2-associated surface molecules. Using single-cell RNA sequencing with integrated cell surface protein expression (CITE-seq), we found that Batf3-/- mitotic immature cDC1-like cells showed reduced expression of cDC1 features and increased levels of cDC2 features. In wild type, we also observed a proportion of mature cDC1 cells expressing surface features characteristic to cDC2 and found that overall cDC cell state heterogeneity was mainly driven by developmental stage, proliferation, and maturity. We detected population diversity within Sirpa+ cDC2 cells, including a Cd33+ cell state expressing high levels of Sox4 and lineage-mixed features characteristic to cDC1, cDC2, pDCs, and monocytes. In conclusion, these data suggest that multiple cDC cell states can co-express lineage-overlapping features, revealing a level of previously unappreciated cDC plasticity.

Improving FLAIR SAR efficiency at 7T by adaptive tailoring of adiabatic pulse power through deep learning B1+ estimation.

PURPOSE: The purpose of this study is to demonstrate a method for specific absorption rate (SAR) reduction for 2D T2 -FLAIR MRI sequences at 7 T by predicting the required adiabatic radiofrequency (RF) pulse power and scaling the RF amplitude in a slice-wise fashion. METHODS: We used a time-resampled frequency-offset corrected inversion (TR-FOCI) adiabatic pulse for spin inversion in a T2 -FLAIR sequence to improve B1+ homogeneity and calculated the pulse power required for adiabaticity slice-by-slice to minimize the SAR. Drawing on the implicit B1+ inhomogeneity in a standard localizer scan, we acquired 3D AutoAlign localizers and SA2RAGE B1+ maps in 28 volunteers. Then, we trained a convolutional neural network (CNN) to estimate the B1+ profile from the localizers and calculated pulse scale factors for each slice. We assessed the predicted B1+ profiles and the effect of scaled pulse amplitudes on the FLAIR inversion efficiency in oblique transverse, sagittal, and coronal orientations. RESULTS: The predicted B1+ amplitude maps matched the measured ones with a mean difference of 9.5% across all slices and participants. The slice-by-slice scaling of the TR-FOCI inversion pulse was most effective in oblique transverse orientation and resulted in a 1 min and 30 s reduction in SAR induced delay time while delivering identical image quality. CONCLUSION: We propose a SAR reduction technique based on the estimation of B1+ profiles from standard localizer scans using a CNN and show that scaling the inversion pulse power slice-by-slice for FLAIR sequences at 7T reduces SAR and scan time without compromising image quality.

The regulation of p53, p38 MAPK, JNK and XBP-1s by sphingosine kinases in human embryonic kidney cells.

Since inhibitors of sphingosine kinases (SK1, SK2) have been shown to induce p53-mediated cell death, we have further investigated their role in regulating p53, stress activated protein kinases and XBP-1s in HEK293T cells. Treatment of these cells with the sphingosine kinase inhibitor, SKi, which fails to induce apoptosis, promoted the conversion of p53 into two proteins with molecular masses of 63 and 90 kDa, and which was enhanced by over-expression of ubiquitin. The SKi induced conversion of p53 to p63/p90 was also enhanced by siRNA knockdown of SK1, but not SK2 or dihydroceramide desaturase (Degs1), suggesting that SK1 is a negative regulator of this process. In contrast, another sphingosine kinase inhibitor, ABC294640 only very weakly stimulated formation of p63/p90 and induced apoptosis of HEK293T cells. We have previously shown that SKi promotes the polyubiquitination of Degs1, and these forms positively regulate p38 MAPK/JNK pathways to promote HEK293T cell survival/growth. siRNA knockdown of SK1 enhanced the activation of p38 MAPK/JNK pathways in response to SKi, suggesting that SK1 functions to oppose these pro-survival pathways in HEK293T cells. SKi also enhanced the stimulatory effect of the proteasome inhibitor, MG132 on the expression of the pro-survival protein XBP-1s and this was reduced by siRNA knockdown of SK2 and increased by knockdown of p53. These findings suggest that SK1 and SK2 have opposing roles in regulating p53-dependent function in HEK293T cells.

Case-finding tools for the diagnosis of OAB in women: A narrative review.

AIMS: Overactive bladder (OAB) remains underdiagnosed with many patients never seeking medical help. Methods to aid early identification and treatment of OAB will be hugely beneficial, and to this end several case-finding tools have been developed. The aim of this review is to critically analyse the literature regarding case-finding tools that have been validated specifically for the detection of OAB in women. METHODS: A literature search of the PubMed database was performed until July 2019 using the search terms screening and OAB or lower urinary tract symptoms (LUTS). Names of individual case-finding tools included in the sixth International Consultation on Incontinence were also searched on the PubMed database. Original articles on the validation of patient-reported screening questionnaires for the detection of OAB in women were included. RESULTS: A total of 22 studies met the inclusion criteria and were included in this review. The validation studies of 11 case-finding tools were assessed. All demonstrated good sensitivity and specificity for OAB or incontinence symptoms, and five tools have been most extensively validated for this condition (bladder control self-assessment questionnaire [B-SAQ], OAB-V8, OAB-V3, OAB symptom score, and questionnaire for urinary incontinence diagnosis). B-SAQ and OAB-V8 demonstrated high sensitivity whilst actionable bladder symptom screening tool was the most specific. B-SAQ was the only tool in this review to encompass screening for "red-flag" symptoms (hematuria, pain), and it has also been validated in a primary care setting. CONCLUSIONS: Several case-finding tools have been demonstrated to have high accuracy for diagnosing OAB in women. B-SAQ encompasses other LUTS as well as "red-flag" symptoms; its use should be promoted in primary care.

Association of Religious Activity with Male Suicide Deaths.

OBJECTIVE: To analyze the relationship between suicide in men and stratified measures of religiosity. METHODS: We studied 192 suicides and 81 controls (nonsuicide, sudden, or accidental death). We employed the psychological autopsy method to compile diagnoses based on DSM-IV criteria. Overall, religiosity and participation in religious associations were determined using a Likert-type scale. Given the limited participation of women in the religious associations of southern Spain, only male subjects were included. RESULTS: Religious participants had decreased risk of suicide compared to nonparticipants (OR = 0.148, 95% CI = 0.049-0.447). This lower risk was further associated with the degree of involvement in religious activity. Suicides accounted for 73.47% of subjects with no religious participation, 61.17% of those with some participation, and 56.52% of frequent participants (linear trend test Z = -2.0329, p = .042). Membership in a religious association was also associated with a lower rate of suicide compared to nonmembers (OR = 0.356, 95% CI = 0.172-0.736). This effect was similarly associated with the degree of involvement in the association. Suicides accounted for 74.67% of subjects who never participated in the activities of a religious association, 69.23% of those who sometimes participated, and 42.86% of frequent participants (linear trend test Z = -3.4082, p < .001). CONCLUSIONS: Religiosity, either as general participation or through a religious association, was associated with protection against suicide proportional to the degree of involvement in religious activities.

Control of CD1d-restricted antigen presentation and inflammation by sphingomyelin.

Invariant natural killer T (iNKT) cells recognize activating self and microbial lipids presented by CD1d. CD1d can also bind non-activating lipids, such as sphingomyelin. We hypothesized that these serve as endogenous regulators and investigated humans and mice deficient in acid sphingomyelinase (ASM), an enzyme that degrades sphingomyelin. We show that ASM absence in mice leads to diminished CD1d-restricted antigen presentation and iNKT cell selection in the thymus, resulting in decreased iNKT cell levels and resistance to iNKT cell-mediated inflammatory conditions. Defective antigen presentation and decreased iNKT cells are also observed in ASM-deficient humans with Niemann-Pick disease, and ASM activity in healthy humans correlates with iNKT cell phenotype. Pharmacological ASM administration facilitates antigen presentation and restores the levels of iNKT cells in ASM-deficient mice. Together, these results demonstrate that control of non-agonistic CD1d-associated lipids is critical for iNKT cell development and function in vivo and represents a tight link between cellular sphingolipid metabolism and immunity.

Analysis of 1-Deoxysphingoid Bases and Their N-Acyl Metabolites and Exploration of Their Occurrence in Some Food Materials.

Most common sphingolipids are comprised of "typical" sphingoid bases (sphinganine, sphingosine, and structurally related compounds) and are produced via the condensation of l-serine with a fatty acyl-CoA by serine palmitoyltransferase. Some organisms, including mammals, also produce "atypical" sphingoid bases that lack a 1-hydroxyl group as a result of the utilization of l-alanine or glycine instead of l-serine, resulting in the formation of 1-deoxy- or 1-desoxymethylsphingoid bases, respectively. Elevated production of "atypical" sphingolipids has been associated with human disease, but 1-deoxysphingoid bases have also been found to have potential as anticancer compounds, hence, the importance of knowing more about the occurrence of these compounds in food. Most of the "typical" and "atypical" sphingoid bases are found as the N-acyl metabolites (e.g., ceramides and 1-deoxyceramides) in mammals, but this has not been uniformly assessed in previous studies nor determined in consumed food. Therefore, we developed a method for the quantitative analysis of "typical" and "atypical" sphingoid bases and their N-acyl derivatives by reverse-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. On the basis of these analyses, there was considerable variability in the amounts and molecular subspecies of atypical sphingoid bases and their N-acyl metabolites found in different edible sources. These findings demonstrate that a broader assessment of the types of sphingolipids in foods is needed because some diets might contain sufficient amounts of atypical as well as typical sphingolipids that could have beneficial or possibly deleterious effects on human health.

Vitamin E alleviates non-alcoholic fatty liver disease in phosphatidylethanolamine N-methyltransferase deficient mice.

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC), mainly in the liver. Pemt-/- mice are protected from high-fat diet (HFD)-induced obesity and insulin resistance, but develop severe non-alcoholic fatty liver disease (NAFLD) when fed a HFD, mostly due to impaired VLDL secretion. Oxidative stress is thought to be an essential factor in the progression from simple steatosis to steatohepatitis. Vitamin E is an antioxidant that has been clinically used to improve NAFLD pathology. Our aim was to determine whether supplementation of the diet with vitamin E could attenuate HFD-induced hepatic steatosis and its progression to NASH in Pemt-/- mice. Treatment with vitamin E (0.5 g/kg) for 3 weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis. We also observed abnormal ceramide metabolism in Pemt-/- mice fed a HFD, with elevation of ceramides and other sphingolipids and higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (Cerk). Interestingly, vitamin E supplementation restored Asah1 and Cerk mRNA and sphingolipid levels. Together this study shows that vitamin E treatment efficiently prevented the progression from simple steatosis to steatohepatitis in mice lacking PEMT.

Native and Polyubiquitinated Forms of Dihydroceramide Desaturase Are Differentially Linked to Human Embryonic Kidney Cell Survival.

There is controversy concerning the role of dihydroceramide desaturase (Degs1) in regulating cell survival, with studies showing that it can both promote and protect against apoptosis. We have therefore investigated the molecular basis for these opposing roles of Degs1. Treatment of HEK293T cells with the sphingosine kinase inhibitor SKi [2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole] or fenretinide, but not the Degs1 inhibitor GT11 {N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octan-amide}, induced the polyubiquitination of Degs1 (Mr = 40 to 140 kDa) via a mechanism involving oxidative stress, p38 mitogen-activated protein kinase (MAPK), and Mdm2 (E3 ligase). The polyubiquitinated forms of Degs1 exhibit "gain of function" and activate prosurvival pathways, p38 MAPK, c-Jun N-terminal kinase (JNK), and X-box protein 1s (XBP-1s). In contrast, another sphingosine kinase inhibitor, ABC294640 [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide], at concentrations of 25 to 50 µM failed to induce formation of the polyubiquitinated forms of Degs1. In contrast to SKi, ABC294640 (25 µM) promotes apoptosis of HEK293T cells via a Degs1-dependent mechanism that is associated with increased de novo synthesis of ceramide. These findings are the first to demonstrate that the polyubiquitination of Degs1 appears to change its function from proapoptotic to prosurvival. Thus, polyubiquitination of Degs1 might provide an explanation for the reported opposing functions of this enzyme in cell survival/apoptosis.

Eleven residues determine the acyl chain specificity of ceramide synthases.

Lipids display large structural complexity, with ∼40,000 different lipids identified to date, ∼4000 of which are sphingolipids. A critical factor determining the biological activities of the sphingolipid, ceramide, and of more complex sphingolipids is their N-acyl chain length, which in mammals is determined by a family of six ceramide synthases (CerS). Little information is available about the CerS regions that determine specificity toward different acyl-CoA substrates. We previously demonstrated that substrate specificity resides in a region of ∼150 residues in the Tram-Lag-CLN8 domain. Using site-directed mutagenesis and biochemical analyses, we now narrow specificity down to an 11-residue sequence in a loop located between the last two putative transmembrane domains (TMDs) of the CerS. The specificity of a chimeric protein, CerS5(299-309→CerS2), based on the backbone of CerS5 (which generates C16-ceramide), but containing 11 residues from CerS2 (which generates C22-C24-ceramides), was altered such that it generated C22-C24 and other ceramides. Moreover, a chimeric protein, CerS4(291-301→CerS2), based on CerS4 (which normally generates C18-C22 ceramides) displayed significant activity toward C24:1-CoA. Additional data supported the notion that substitutions of these 11 residues alter the specificities of the CerS toward their cognate acyl-CoAs. Our findings may suggest that this short loop may restrict adjacent TMDs, leading to a more open conformation in the membrane, and that the CerS acting on shorter acyl-CoAs may have a longer, more flexible loop, permitting TMD flexibility. In summary, we have identified an 11-residue region that determines the acyl-CoA specificity of CerS.

Critical Role for Very-Long Chain Sphingolipids in Invariant Natural Killer T Cell Development and Homeostasis.

The role of sphingolipids (SLs) in the immune system has come under increasing scrutiny recently due to the emerging contributions that these important membrane components play in regulating a variety of immunological processes. The acyl chain length of SLs appears particularly critical in determining SL function. Here, we show a role for very-long acyl chain SLs (VLC-SLs) in invariant natural killer T (iNKT) cell maturation in the thymus and homeostasis in the liver. Ceramide synthase 2-null mice, which lack VLC-SLs, were susceptible to a hepatotropic strain of lymphocytic choriomeningitis virus, which is due to a reduction in the number of iNKT cells. Bone marrow chimera experiments indicated that hematopoietic-derived VLC-SLs are essential for maturation of iNKT cells in the thymus, whereas parenchymal-derived VLC-SLs are crucial for iNKT cell survival and maintenance in the liver. Our findings suggest a critical role for VLC-SL in iNKT cell physiology.

Regulation of very-long acyl chain ceramide synthesis by acyl-CoA-binding protein.

Ceramide and more complex sphingolipids constitute a diverse group of lipids that serve important roles as structural entities of biological membranes and as regulators of cellular growth, differentiation, and development. Thus, ceramides are vital players in numerous diseases including metabolic and cardiovascular diseases, as well as neurological disorders. Here we show that acyl-coenzyme A-binding protein (ACBP) potently facilitates very-long acyl chain ceramide synthesis. ACBP increases the activity of ceramide synthase 2 (CerS2) by more than 2-fold and CerS3 activity by 7-fold. ACBP binds very-long-chain acyl-CoA esters, which is required for its ability to stimulate CerS activity. We also show that high-speed liver cytosol from wild-type mice activates CerS3 activity, whereas cytosol from ACBP knock-out mice does not. Consistently, CerS2 and CerS3 activities are significantly reduced in the testes of ACBP-/- mice, concomitant with a significant reduction in long- and very-long-chain ceramide levels. Importantly, we show that ACBP interacts with CerS2 and CerS3. Our data uncover a novel mode of regulation of very-long acyl chain ceramide synthesis by ACBP, which we anticipate is of crucial importance in understanding the regulation of ceramide metabolism in pathogenesis.

Novel Interconnections in Lipid Metabolism Revealed by Overexpression of Sphingomyelin Synthase-1.

This study investigates the consequences of elevating sphingomyelin synthase 1 (SMS1) activity, which generates the main mammalian sphingolipid, sphingomyelin. HepG2 cells stably transfected with SMS1 (HepG2-SMS1) exhibit elevated enzyme activity in vitro and increased sphingomyelin content (mainly C22:0- and C24:0-sphingomyelin) but lower hexosylceramide (Hex-Cer) levels. HepG2-SMS1 cells have fewer triacylglycerols than controls but similar diacylglycerol acyltransferase activity, triacylglycerol secretion, and mitochondrial function. Treatment with 1 mm palmitate increases de novo ceramide synthesis in both cell lines to a similar degree, causing accumulation of C16:0-ceramide (and some C18:0-, C20:0-, and C22:0-ceramides) as well as C16:0- and C18:0-Hex-Cers. In these experiments, the palmitic acid is delivered as a complex with delipidated BSA (2:1, mol/mol) and does not induce significant lipotoxicity. Based on precursor labeling, the flux through SM synthase also increases, which is exacerbated in HepG2-SMS1 cells. In contrast, palmitate-induced lipid droplet formation is significantly reduced in HepG2-SMS1 cells. [14C]Choline and [3H]palmitate tracking shows that SMS1 overexpression apparently affects the partitioning of palmitate-enriched diacylglycerol between the phosphatidylcholine and triacylglycerol pathways, to the benefit of the former. Furthermore, triacylglycerols from HepG2-SMS1 cells are enriched in polyunsaturated fatty acids, which is indicative of active remodeling. Together, these results delineate novel metabolic interactions between glycerolipids and sphingolipids.

Biomarkers of NAFLD progression: a lipidomics approach to an epidemic.

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an "omics" approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain.

Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that Cers with different N-acyl chain composition differentially impact cell physiology, which may in part be due to specific alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N-acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerSs). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fluid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the significance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain Cers exert their specific biological actions.

Neuronal accumulation of glucosylceramide in a mouse model of neuronopathic Gaucher disease leads to neurodegeneration.

Gaucher disease has recently received wide attention due to the unexpected discovery that it is a genetic risk factor for Parkinson's disease. Gaucher disease is caused by the defective activity of the lysosomal enzyme, glucocerebrosidase (GCase; GBA1), resulting in intracellular accumulation of the glycosphingolipids, glucosylceramide and psychosine. The rare neuronopathic forms of GD (nGD) are characterized by profound neurological impairment and neuronal cell death. We have previously described the progression of neuropathological changes in a mouse model of nGD. We now examine the relationship between glycosphingolipid accumulation and initiation of pathology at two pre-symptomatic stages of the disease in four different brain areas which display differential degrees of susceptibility to GCase deficiency. Liquid chromatography electrospray ionization tandem mass spectrometry demonstrated glucosylceramide and psychosine accumulation in nGD brains prior to the appearance of neuroinflammation, although only glucosylceramide accumulation correlated with neuroinflammation and neuron loss. Levels of other sphingolipids, including the pro-apoptotic lipid, ceramide, were mostly unaltered. Transmission electron microscopy revealed that glucosylceramide accumulation occurs in neurons, mostly in the form of membrane-delimited pseudo-tubules located near the nucleus. Highly disrupted glucosylceramide-storing cells, which are likely degenerating neurons containing massive inclusions, numerous autophagosomes and unique ultrastructural features, were also observed. Together, our results indicate that a certain level of neuronal glucosylceramide storage is required to trigger neuropathological changes in affected brain areas, while other brain areas containing similar glucosylceramide levels are unaltered, presumably because of intrinsic differences in neuronal properties, or in the neuronal environment, between various brain regions.