Ceramide-mediated orchestration of oxidative stress response through filopodia-derived small extracellular vesicles.

Extracellular vesicles (EVs) are shed from the plasma membrane, but the regulation and function of these EVs remain unclear. We found that oxidative stress induced by H2O2 in Hela cells stimulated filopodia formation and the secretion of EVs. EVs were small (150 nm) and labeled for CD44, indicating that they were derived from filopodia. Filopodia-derived small EVs (sEVs) were enriched with the sphingolipid ceramide, consistent with increased ceramide in the plasma membrane of filopodia. Ceramide was colocalized with neutral sphingomyelinase 2 (nSMase2) and acid sphingomyelinase (ASM), two sphingomyelinases generating ceramide at the plasma membrane. Inhibition of nSMase2 and ASM prevented oxidative stress-induced sEV shedding but only nSMase2 inhibition prevented filopodia formation. nSMase2 was S-palmitoylated and interacted with ASM in filopodia to generate ceramide for sEV shedding. sEVs contained nSMase2 and ASM and decreased the level of these two enzymes in oxidatively stressed Hela cells. A novel metabolic labeling technique for EVs showed that oxidative stress induced secretion of fluorescent sEVs labeled with NBD-ceramide. NBD-ceramide-labeled sEVs transported ceramide to mitochondria, ultimately inducing cell death in a proportion of neuronal (N2a) cells. In conclusion, using Hela cells we provide evidence that oxidative stress induces interaction of nSMase2 and ASM at filopodia, which leads to shedding of ceramide-rich sEVs that target mitochondria and propagate cell death.

Effect of hyperglycemia and rosiglitazone on renal and urinary neprilysin in db/db diabetic mice.

Alteration in renin-angiotensin system (RAS) has been implicated in the pathophysiology of diabetic kidney disease (DKD). The deleterious actions of angiotensin II (Ang II) could be antagonized by the formation of Ang-(1-7), generated by the actions of angiotensin-converting enzyme 2 (ACE2) and neprilysin (NEP). NEP degrades several peptides, including natriuretic peptides, bradykinin, amyloid beta, and Ang I. Although combination of Ang II receptor and NEP inhibitor treatment benefits patients with heart failure, the role of NEP in renal pathophysiology is a matter of active research. NEP pathway is a potent enzyme in Ang I to Ang-(1-7) conversion in the kidney of ACE2-deficient mice, suggesting a renoprotective role of NEP. The aim of the study is to test the hypothesis that chronic hyperglycemia downregulates renal NEP protein expression and activity in db/db diabetic mice and treatment with rosiglitazone normalizes hyperglycemia, renal NEP expression, and attenuates albuminuria. Mice received rosiglitazone (20 mg kg-1  day-1 ) for 10 weeks. Western blot analysis, immunohistochemistry, and enzyme activity revealed a significant decrease in renal and urinary NEP expression and activity in 16-wk db/db mice compared with lean control (p < .0001). Rosiglitazone also attenuated albuminuria and increased renal and urinary NEP expressions (p < .0001). In conclusion, data support the hypothesis that diabetes decreases intrarenal NEP, which could have a pivotal role in the pathogenesis of DKD. Urinary NEP may be used as an index of intrarenal NEP status. The renoprotective effects of rosiglitazone could be mediated by upregulation of renal NEP expression and activity in db/db diabetic mice.

Improved segmented-scan spectral stitching for stable isotope resolved metabolomics (SIRM) by ultra-high-resolution Fourier transform mass spectrometry.

We have implemented a linear ion trap (LIT)-based SIM-stitching method for ultra-high-resolution Fourier transform mass spectrometry (FTMS) that increases the S/N over a wide m/z range compared to non-segmented wide full-scan (WFS) spectra. Here we described an improved segmented spectral scan stitching method that was based on quadrupole mass filter (QMF)-SIM, which overcame previous limitations of ion signal loss in LIT. This allowed for accurate representation of isotopologue distributions, both at natural abundance and in stable isotope-resolved metabolomics (SIRM)-based experiments. We also introduced a new spectral binning method that provided more precise and resolution-independent bins for irreversibly noise-suppressed FTMS spectra. We demonstrated a substantial improvement in S/N and sensitivity (typically > 10-fold) for 13C labeled lipid extracts of human macrophages grown as three-dimensional (3D) cell culture, with detection of an increased number of 13C isotopologue ions. The method also enabled analysis of extracts from very limited biological samples.

Loss of BRUCE reduces cellular energy level and induces autophagy by driving activation of the AMPK-ULK1 autophagic initiating axis.

Autophagy is an intracellular catabolic system. It delivers cellular components to lysosomes for degradation and supplies nutrients that promote cell survival under stress conditions. Although much is known regarding starvation-induced autophagy, the regulation of autophagy by cellular energy level is less clear. BRUCE is an ubiquitin conjugase and ligase with multi-functionality. It has been reported that depletion of BRUCE inhibits starvation-induced autophagy by blockage of the fusion step. Herein we report a new function for BRUCE in the dual regulation of autophagy and cellular energy. Depletion of BRUCE alone (without starvation) in human osteosarcoma U2OS cells elevated autophagic activity as indicted by the increased LC3B-II protein and its autophagic puncta as well as further increase of both by chloroquine treatment. Such elevation results from enhanced induction of autophagy since the numbers of both autophagosomes and autolysosomes were increased, and recruitment of ATG16L onto the initiating membrane structure phagophores was increased. This concept is further supported by elevated lysosomal enzyme activities. In contrast to starvation-induced autophagy, BRUCE depletion did not block fusion of autophagosomes with lysosomes as indicated by increased lysosomal cleavage of the GFP-LC3 fusion protein. Mechanistically, BRUCE depletion lowered the cellular energy level as indicated by both a higher ratio of AMP/ATP and the subsequent activation of the cellular energy sensor AMPK (pThr-172). The lower energy status co-occurred with AMPK-specific phosphorylation and activation of the autophagy initiating kinase ULK1 (pSer-555). Interestingly, the higher autophagic activity by BRUCE depletion is coupled with enhanced cisplatin resistance in human ovarian cancer PEO4 cells. Taken together, BRUCE depletion promotes induction of autophagy by lowering cellular energy and activating the AMPK-ULK1-autophagy axis, which could contribute to ovarian cancer chemo-resistance. This study establishes a BRUCE-AMPK-ULK1 axis in the regulation of energy metabolism and autophagy, as well as provides insights into cancer chemo-resistance.

Getting the Most: Enhancing Efficacy by Promoting Erythropoiesis and Thrombopoiesis after Gene Therapy in Mice with Hurler Syndrome.

Novel strategies are needed to solve the conundrum of achieving clinical efficacy with high vector copy numbers (VCNs) in hematopoietic stem cells (HSCs) while attempting to minimize the potential risk of oncogenesis in lentiviral vector (LV)-mediated gene therapy clinical trials. We previously reported the benefits of reprogramming erythroid-megakaryocytic (EMK) cells for high-level lysosomal enzyme production with less risk of activating oncogenes in HSCs. Herein, using a murine model of mucopolysaccharidosis type I (MPS I) with a deficiency of α-L-iduronidase (IDUA), we sought to determine the transgene minimum effective doses (MEDs) in major organs, and if a transient increase of IDUA-containing red blood cells and platelets by repeated phlebotomy would provide further therapeutic benefits in diseased mice after EMK-restricted LV-mediated gene therapy. The MEDs for complete metabolic correction ranged from 0.1 to 2 VCNs in major visceral organs, which were dramatically reduced to 0.005-0.1 VCN by one cycle of stress induction and were associated with a further reduction of pathological deficits in mice with 0.005 VCN. This work provides a proof of concept that transiently stimulating erythropoiesis and thrombopoiesis can further improve therapeutic benefits in HSC-mediated gene therapy for MPS I, a repeatable and reversible approach to enhance clinical efficacy in the treatment of lysosomal storage diseases.

Stable Isotope-Resolved Metabolomics Shows Metabolic Resistance to Anti-Cancer Selenite in 3D Spheroids versus 2D Cell Cultures.

Conventional two-dimensional (2D) cell cultures are grown on rigid plastic substrates with unrealistic concentration gradients of O2, nutrients, and treatment agents. More importantly, 2D cultures lack cell⁻cell and cell⁻extracellular matrix (ECM) interactions, which are critical for regulating cell behavior and functions. There are several three-dimensional (3D) cell culture systems such as Matrigel, hydrogels, micropatterned plates, and hanging drop that overcome these drawbacks but they suffer from technical challenges including long spheroid formation times, difficult handling for high throughput assays, and/or matrix contamination for metabolic studies. Magnetic 3D bioprinting (M3DB) can circumvent these issues by utilizing nanoparticles that enable spheroid formation and growth via magnetizing cells. M3DB spheroids have been shown to emulate tissue and tumor microenvironments while exhibiting higher resistance to toxic agents than their 2D counterparts. It is, however, unclear if and how such 3D systems impact cellular metabolic networks, which may determine altered toxic responses in cells. We employed a Stable Isotope-Resolved Metabolomics (SIRM) approach with 13C6-glucose as tracer to map central metabolic networks both in 2D cells and M3DB spheroids formed from lung (A549) and pancreatic (PANC1) adenocarcinoma cells without or with an anti-cancer agent (sodium selenite). We found that the extent of 13C-label incorporation into metabolites of glycolysis, the Krebs cycle, the pentose phosphate pathway, and purine/pyrimidine nucleotide synthesis was largely comparable between 2D and M3DB culture systems for both cell lines. The exceptions were the reduced capacity for de novo synthesis of pyrimidine and sugar nucleotides in M3DB than 2D cultures of A549 and PANC1 cells as well as the presence of gluconeogenic activity in M3DB spheroids of PANC1 cells but not in the 2D counterpart. More strikingly, selenite induced much less perturbation of these pathways in the spheroids relative to the 2D counterparts in both cell lines, which is consistent with the corresponding lesser effects on morphology and growth. Thus, the increased resistance of cancer cell spheroids to selenite may be linked to the reduced capacity of selenite to perturb these metabolic pathways necessary for growth and survival.

Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition.

Primary brain microvessels (BrMV) maintain the cellular characters and molecular signatures as displayed in vivo, and serve as a vital tool for biomedical research of the blood-brain barrier (BBB) and the development/optimization of brain drug delivery. The variations of relative purities or cellular composition among different BrMV samples may have significant consequences in data interpretation and research outcome, especially for experiments with high-throughput genomics and proteomics technologies. In this study, we aimed to identify suitable reference gene (RG) for accurate normalization of real-time RT-qPCR analysis, and determine the proper marker genes (MG) for relative purity assessment in BrMV samples. Out of five housekeeping genes, ß-actin was selected as the most suitable RG that was validated by quantifying mRNA levels of alpha-L-iduronidase in BrMV isolated from mice with one or two expressing alleles. Four marker genes highly/selectively expressed in BBB-forming capillary endothelial cells were evaluated by RT-qPCR for purity assessment, resulting in Cldn5 and Pecam1 as most suitable MGs that were further confirmed by immunofluorescent analysis of cellular components. Plvap proved to be an indicator gene for the presence of fenestrated vessels in BrMV samples. This study may contribute to the building blocks toward overarching research needs on the blood-brain barrier.

Normalization and improvement of CNS deficits in mice with Hurler syndrome after long-term peripheral delivery of BBB-targeted iduronidase.

Mucopolysaccharidosis type I (MPS I) is a progressive lysosomal storage disorder with systemic and central nervous system (CNS) involvement due to deficiency of α-L-iduronidase (IDUA). We previously identified a receptor-binding peptide from apolipoprotein E (e) that facilitated a widespread delivery of IDUAe fusion protein into CNS. In this study, we evaluated the long-term CNS biodistribution, dose-correlation, and therapeutic benefits of IDUAe after systemic, sustained delivery via hematopoietic stem cell (HSC)-mediated gene therapy with expression restricted to erythroid/megakaryocyte lineages. Compared to the highest dosage group treated by nontargeted control IDUAc (165 U/ml), physiological levels of IDUAe in the circulation (12 U/ml) led to better CNS benefits in MPS I mice as demonstrated in glycosaminoglycan accumulation, histopathology analysis, and neurological behavior. Long-term brain metabolic correction and normalization of exploratory behavior deficits in MPS I mice were observed by peripheral enzyme therapy with physiological levels of IDUAe derived from clinically attainable levels of HSC transduction efficiency (0.1). Importantly, these levels of IDUAe proved to be more beneficial on correction of cerebrum pathology and behavioral deficits in MPS I mice than wild-type HSCs fully engrafted in MPS I chimeras. These results provide compelling evidence for CNS efficacy of IDUAe and its prospective translation to clinical application.

Platelets are efficient and protective depots for storage, distribution, and delivery of lysosomal enzyme in mice with Hurler syndrome.

Use of megakaryocytes/platelets for transgene expression may take advantage of their rapid turnover and protective storage in platelets and reduce the risk of activating oncogenes in hematopoietic stem and progenitor cells (HSCs). Here, we show that human megakaryocytic cells could overexpress the lysosomal enzyme, α-l-iduronidase (IDUA), which is deficient in patients with mucopolysaccharidosis type I (MPS I). Upon megakaryocytic differentiation, the amount of released enzyme increased rapidly and steadily by 30-fold. Using a murine MPS I model, we demonstrated that megakaryocyte/platelets were capable of producing, packaging, and storing large amounts of IDUA with proper catalytic activity, lysosomal trafficking, and receptor-mediated uptake. IDUA can be released directly into extracellular space or within microparticles during megakaryocyte maturation or platelet activation, while retaining the capacity for cross-correction in patient's cells. Gene transfer into 1.7% of HSCs led to long-term normalization of plasma IDUA and preferential distribution of enzyme in liver and spleen with complete metabolic correction in MPS I mice. Detection of GFP (coexpressed with IDUA) in Kupffer cells and hepatocytes suggested liver delivery of platelet-derived IDUA possibly via the clearance pathway for senile platelets. These findings provide proof of concept that cells from megakaryocytic lineage and platelets are capable of generating and storing fully functional lysosomal enzymes and can also lead to efficient delivery of both the enzymes released into the circulation and those protected within platelets/microparticles. This study opens a door for use of the megakaryocytes/platelets as a depot for efficient production, delivery, and effective tissue distribution of lysosomal enzymes.

Engineering a lysosomal enzyme with a derivative of receptor-binding domain of apoE enables delivery across the blood-brain barrier.

To realize the potential of large molecular weight substances to treat neurological disorders, novel approaches are required to surmount the blood-brain barrier (BBB). We investigated whether fusion of a receptor-binding peptide from apolipoprotein E (apoE) with a potentially therapeutic protein can bind to LDL receptors on the BBB and be transcytosed into the CNS. A lysosomal enzyme, α-L-iduronidase (IDUA), was used for biological and therapeutic evaluation in a mouse model of mucopolysaccharidosis (MPS) type I, one of the most common lysosomal storage disorders with CNS deficits. We identified two fusion candidates, IDUAe1 and IDUAe2, by in vitro screening, that exhibited desirable receptor-mediated binding, endocytosis, and transendothelial transport as well as appropriate lysosomal enzyme trafficking and biological function. Robust peripheral IDUAe1 or IDUAe2 generated by transient hepatic expression led to elevated enzyme levels in capillary-depleted, enzyme-deficient brain tissues and protein delivery into nonendothelium perivascular cells, neurons, and astrocytes within 2 d of treatment. Moreover, 5 mo after long-term delivery of moderate levels of IDUAe1 derived from maturing red blood cells, 2% to 3% of normal brain IDUA activities were obtained in MPS I mice, and IDUAe1 protein was detected in neurons and astrocytes throughout the brain. The therapeutic potential was demonstrated by normalization of brain glycosaminoglycan and ß-hexosaminidase in MPS I mice 5 mo after moderate yet sustained delivery of IDUAe1. These findings provide a noninvasive and BBB-targeted procedure for the delivery of large-molecule therapeutic agents to treat neurological lysosomal storage disorders and potentially other diseases that involve the brain.

Secreted luciferase for in vivo evaluation of systemic protein delivery in mice.

A naturally secreted Gaussia luciferase (Gluc) has been utilized as a reporter for bioluminescence imaging (BLI) evaluation. However, the potential application of Gluc for in vivo monitoring of systemic protein delivery, as well as its natural biodistribution, has not been studied. To examine Gluc secretion and uptake profile, we injected Gluc-encoding plasmids into mice by hydrodynamic tail-vein injection. Whole-body BLI showed that imaging quantification obtained at pawpad was directly correlated to blood Gluc activities. When gene expression was restricted to the liver by the use of a hepatic promoter, in vivo Gluc biodistribution analysis revealed the kidney/bladder, stomach/intestine, and lung as the major uptake organs. Three-dimensional BLI identified liver/stomach and lung as the main internal luminescent sources, demonstrating the feasibility of detecting major uptake organs in live animals by 3D BLI with high-background signals in circulation. Notably, Gluc levels in capillary-depleted brain samples from Gluc-injected mice were comparable to controls, suggesting that Gluc may not cross the blood-brain barrier. Gluc uptake kinetics and intracellular half-life were assessed in various types of cell lines, implicating the involvement of non-specific pinocytosis. These results suggest that Gluc-based system may provide a useful tool for in vivo evaluation of protein/agent biodistribution following systemic delivery.

Deletion of the cathepsin B gene improves memory deficits in a transgenic ALZHeimer's disease mouse model expressing AßPP containing the wild-type ß-secretase site sequence.

Therapeutic agents that improve the memory loss of Alzheimer's disease (AD) may eventually be developed if drug targets are identified that improve memory deficits in appropriate AD animal models. One such target is ß-secretase which, in most AD patients, cleaves the wild-type (WT) ß-secretase site sequence of the amyloid-ß protein precursor (AßPP) to produce neurotoxic amyloid-ß (Aß). Thus, an animal model representing most AD patients for evaluating ß-secretase effects on memory deficits is one that expresses human AßPP containing the WT ß-secretase site sequence. BACE1 and cathepsin B (CatB) proteases have ß-secretase activity, but gene knockout studies have not yet validated that the absence of these proteases improves memory deficits in such an animal model. This study assessed the effects of deleting these protease genes on memory deficits in the AD mouse model expressing human AßPP containing the WT ß-secretase site sequence and the London γ-secretase site (AßPPWT/Lon mice). Knockout of the CatB gene in the AßPPWT/Lon mice improved memory deficits and altered the pattern of Aß-related biomarkers in a manner consistent with CatB having WT ß-secretase activity. But deletion of the BACE1 gene had no effect on these parameters in the AßPPWT/Lon mice. These data are the first to show that knockout of a putative ß-secretase gene results in improved memory in an AD animal model expressing the WT ß-secretase site sequence of AßPP, present in the majority of AD patients. CatB may be an effective drug target for improving memory deficits in most AD patients.

Neprilysin: an enzyme candidate to slow the progression of Alzheimer's disease.

It is well established that the extracellular deposition of amyloid beta (Abeta) peptide plays a central role in the development of Alzheimer's disease (AD). Therefore, either preventing the accumulation of Abeta peptide in the brain or accelerating its clearance may slow the rate of AD onset. Neprilysin (NEP) is the dominant Abeta peptide-degrading enzyme in the brain; NEP becomes inactivated and down-regulated during both the early stages of AD and aging. In this study, we investigated the effect of human (h)NEP gene transfer to the brain in a mouse model of AD before the development of amyloid plaques, and assessed how this treatment modality affected the accumulation of Abeta peptide and associated pathogenetic changes (eg, inflammation, oxidative stress, and memory impairment). Overexpression of hNEP for 4 months in young APP/DeltaPS1 double-transgenic mice resulted in reduction in Abeta peptide levels, attenuation of amyloid load, oxidative stress, and inflammation, and improved spatial orientation. Moreover, the overall reduction in amyloidosis and associated pathogenetic changes in the brain resulted in decreased memory impairment by approximately 50%. These data suggest that restoring NEP levels in the brain at the early stages of AD is an effective strategy to prevent or attenuate disease progression.

Neprilysin protects neurons against Abeta peptide toxicity.

In recent years, studies have suggested that accumulation of amyloid beta (Abeta) peptide in the brain plays a key role in the development of Alzheimer's disease (AD). The steady-state level of Abeta peptide in the brain is determined by the rate of production from amyloid precursor protein (APP) via beta- and gamma-secretases and degradation by the activity of several enzymes. Neprilysin (NEP) appears to be the most potent Abeta peptide-degrading enzyme in the brain. Decreasing the activity of NEP (due to genetic mutations, age or diseases that alter the expression or activity of NEP) may lead to accumulation of the neurotoxic Abeta peptide in the brain; in turn this leads to neuronal loss. We investigated the efficacy of lentivirus-mediated over-expression of NEP to protect neuronal cells from Abeta peptide in vitro. Incubation of hippocampal neuronal cells (HT22) over-expressing NEP with the monomeric from of Abeta peptide decreases the toxicity of Abeta peptide on the neuronal cells, as measured through cell viability. We conclude that over-expression of NEP by a gene therapy approach in areas vulnerable to Abeta peptide aggregation in AD brain may protect the neurons from the toxicity effects of Abeta peptide and this promises a great potential target for altering the development of AD.