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Richard L Proia

Publications and source records attributed to Richard L Proia.

12 recordsLinked to original sources
PubMed2023

SARS-CoV-2 ORF3a expression in brain disrupts the autophagy-lysosomal pathway, impairs sphingolipid homeostasis, and drives neuropathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes injury to multiple organ systems, including the brain. SARS-CoV-2's neuropathological mechanisms may include systemic inflammation and hypoxia, as well as direct cell damage resulting from viral infections of neurons and glia. How the virus directly causes injury to brain cells, acutely and over the long term, is not well understood. In order to gain insight into this process, we studied the neuropathological effects of open reading frame 3a (ORF3a), a SARS-CoV-2 accessory protein that is a key pathological factor of the virus. Forced ORF3a brain expression in mice caused the rapid onset of neurological impairment, neurodegeneration, and neuroinflammation-key neuropathological features found in coronavirus disease (COVID-19, which is caused by SARS-CoV-2 infection). Furthermore, ORF3a expression blocked autophagy progression in the brain and caused the neuronal accumulation of α-synuclein and glycosphingolipids, all of which are linked to neurodegenerative disease. Studies with ORF3-expressing HeLa cells confirmed that ORF3a disrupted the autophagy-lysosomal pathway and blocked glycosphingolipid degradation, resulting in their accumulation. These findings indicate that, in the event of neuroinvasion by SARS-CoV-2, ORF3a expression in brain cells may drive neuropathogenesis and be an important mediator of both short- and long-term neurological manifestations of COVID-19.

Animals↗
PubMed2003

G-protein-coupled receptor S1P1 acts within endothelial cells to regulate vascular maturation.

Sphingosine-1-phosphate (S1P) stimulates signaling pathways via G-protein-coupled receptors and triggers diverse cellular processes, including growth, survival, and migration. In S1P1 receptor-deficient embryos, blood vessels were incompletely covered by vascular smooth muscle cells (VSMCs), indicating the S1P1 receptor regulates vascular maturation. Because S1P1 receptor expression is not restricted to a particular cell type, it was not known whether the S1P1 receptor controlled VSMC coverage of vessels in a cell-autonomous fashion by functioning directly in VSMCs or indirectly through its activity in endothelial cells (ECs). By using the Cre/loxP system, we disrupted the S1P1 gene solely in ECs. The phenotype of the conditional mutant embryos mimicked the one obtained in the embryos globally deficient in S1P1. Thus, vessel coverage by VSMCs is directed by the activity of the S1P1 receptor in ECs.

Animals↗
PubMed2003

Glycosphingolipid functions: insights from engineered mouse models.

Glycosphingolipids are remarkable for their structural complexity and their distinctive patterns that mark different tissues and stages of development. The physiological functions of glycosphingolipids are likely to be profound and are only beginning to emerge. Much of this new information is due to the study of mutant mice lacking specific sets of glycosphingolipids, the topic of this review.

Animals↗
PubMed2003

Enhanced insulin sensitivity in mice lacking ganglioside GM3.

Gangliosides are sialic acid-containing glycosphingolipids that are present on all mammalian plasma membranes where they participate in recognition and signaling activities. We have established mutant mice that lack GM3 synthase (CMP-NeuAc:lactosylceramide alpha2,3-sialyltransferase; EC 2.4.99.-). These mutant mice were unable to synthesize GM3 ganglioside, a simple and widely distributed glycosphingolipid. The mutant mice were viable and appeared without major abnormalities but showed a heightened sensitivity to insulin. A basis for the increased insulin sensitivity in the mutant mice was found to be enhanced insulin receptor phosphorylation in skeletal muscle. Importantly, the mutant mice were protected from high-fat diet-induced insulin resistance. Our results show that GM3 ganglioside is a negative regulator of insulin signaling, making it a potential therapeutic target in type 2 diabetes.

Animals↗
PubMed2002

Early developmental expression of the gene encoding glucosylceramide synthase, the enzyme controlling the first committed step of glycosphingolipid synthesis.

Glycosphingolipids (GSLs) are ubiquitous plasma membrane components composed of a ceramide lipid anchor attached to one of a diverse complement of oligosaccharide structures. Fundamentally important activities have been attributed to GSLs including formation of plasma membrane structures involved in membrane trafficking, signal transduction and cell-cell interactions. Glucosylceramide synthase converts ceramide to glucosylceramide, a core structure of the vast majority of GSLs. Disruption of the gene encoding glucosylceramide synthase (Ugcg) caused embryonic lethality in mice during gastrulation. To further investigate the role of GSL synthesis during embryogenesis, we produced mice with a Lacz reporter gene inserted into the glucosylceramide synthase locus. These mice allowed the visualization of glucosylceramide synthase expression during early embryonic development.

Animals↗
PubMed2002

Sphingosine-1-phosphate receptors and the development of the vascular system.

Extracellular sphingolipid signaling has been implicated as an essential event in vascular development. Sphingosine-1-phosphate (S1P), through interactions with G protein-coupled receptors, regulates functions of endothelial and smooth muscle cells (SMCs)-the major cell types of the vasculature. The knockout of the gene encoding the S1P1 receptor (formally known as Edg-1) in mice blocks vascular maturation, the process where SMCs and pericytes envelop nascent endothelial tubes. The question that remains is how stimulation of S1P receptors controls this critical event in the developmental sequence leading to the formation of functional blood vessels.

Animals↗
PubMed2002

Molecular pathophysiology in Tay-Sachs and Sandhoff diseases as revealed by gene expression profiling.

Tay-Sachs and Sandhoff diseases are lysosomal storage disorders characterized by the absence of beta-hexosaminidase activity and the accumulation of GM2 ganglioside in neurons. In each disorder, a virtually identical course of neurodegeneration begins in infancy and leads to demise generally by 4-6 years of age. Through serial analysis of gene expression (SAGE), we determined gene expression profiles in cerebral cortex from a Tay-Sachs patient, a Sandhoff disease patient and a pediatric control. Examination of genes that showed altered expression in both patients revealed molecular details of the pathophysiology of the disorders relating to neuronal dysfunction and loss. A large fraction of the elevated genes in the patients could be attributed to activated macrophages/microglia and astrocytes, and included class II histocompatability antigens, the pro-inflammatory cytokine osteopontin, complement components, proteinases and inhibitors, galectins, osteonectin/SPARC, and prostaglandin D2 synthase. The results are consistent with a model of neurodegeneration that includes inflammation as a factor leading to the precipitous loss of neurons in individuals with these disorders.

Cerebral Cortex↗
PubMed2002

Kidney sulfatides in mouse models of inherited glycosphingolipid disorders: determination by nano-electrospray ionization tandem mass spectrometry.

Sulfatides show structural, and possibly physiological similarities to gangliosides. Kidney dysfunction might be correlated with changes in sulfatides, the major acidic glycosphingolipids in this organ. To elucidate their in vivo metabolic pathway these compounds were analyzed in mice afflicted with inherited glycosphingolipid disorders. The mice under study lacked the genes encoding either beta-hexosaminidase alpha-subunit (Hexa-/-), the beta-hexosaminidase beta-subunit (Hexb-/-), both beta-hexosaminidase alpha and beta-subunits (Hexa-/- and Hexb-/-), GD3 synthase (GD3S-/-), GD3 synthase and GalNAc transferase (GD3S-/- and GalNAcT-/-), GM2 activator protein (Gm2a-/-), or arylsulfatase A (ASA-/-). Quantification of the sulfatides, I(3)SO(3)(-)-GalCer (SM4s), II(3)SO(3)(-)-LacCer (SM3), II(3)SO(3)(-)-Gg(3)Cer (SM2a), and IV(3,) II(3)-(SO(3)(-))(2)-Gg(4)Cer (SB1a), was performed by nano-electrospray tandem mass spectrometry. We conclude for the in vivo situation in mouse kidneys that: 1) a single enzyme (GalNAc transferase) is responsible for the synthesis of SM2a and GM2 from SM3 and GM3, respectively. 2) In analogy to GD1a, SB1a is degraded via SM2a. 3) SM2a is hydrolyzed to SM3 by beta-hexosaminidase S (Hex S) and Hex A, but not Hex B. Both enzymes are supported by GM2-activator protein. 4) Arylsulfatase A is required to degrade SB1a. It is probably the sole sphingolipid-sulfatase cleaving the galactosyl-3-sulfate bond. In addition, a human Tay-Sachs patient's liver was investigated, which showed accumulation of SM2a along with GM2 storage. The different ceramide compositions of both compounds indicated they were probably derived from different cell types. These data demonstrate that in vivo the sulfatides of the ganglio-series follow the same metabolic pathways as the gangliosides with the replacement of sulfotransferases and sulfatases by sialyltransferases and sialidases. Furthermore, a novel neutral GSL, IV(6)GlcNAcbeta-Gb(4)Cer, was found to accumulate only in Hexa-/- and Hexb-/- mouse kidneys. From this we conclude that Hex S also efficiently cleaves terminal beta1-6-linked HexNAc residues from neutral GSLs in vivo.

Animals↗
PubMed2002

Lubricating cell signaling pathways with gangliosides.

Gangliosides--glycosphingolipids that contain sialic acid--are concentrated in plasma membrane lipid domains that are specialized for cell signaling. Recent evidence indicates that gangliosides have two different roles in cell signaling. They can act in cis to modulate tyrosine kinase receptor function and in trans as ligands for receptors that facilitate communication between cells. These signaling functions of gangliosides may be potential therapeutic targets in cancer, diabetes and nerve regeneration.

Animals↗
PubMed2002

Systemic inflammation in glucocerebrosidase-deficient mice with minimal glucosylceramide storage.

Gaucher disease, the most common lysosomal storage disease, is caused by a deficiency of glucocerebrosidase resulting in the impairment of glucosylceramide degradation. The hallmark of the disease is the presence of the Gaucher cell, a macrophage containing much of the stored glucosylceramide found in tissues, which is believed to cause many of the clinical manifestations of the disease. We have developed adult mice carrying the Gaucher disease L444P point mutation in the glucocerebrosidase (Gba) gene and exhibiting a partial enzyme deficiency. The mutant mice demonstrate multisystem inflammation, including evidence of B cell hyperproliferation, an aspect of the disease found in some patients. However, the mutant mice do not accumulate large amounts of glucosylceramide or exhibit classic Gaucher cells in tissues.

Animals↗
PubMed2001

Physiological substrates for human lysosomal beta -hexosaminidase S.

Human lysosomal beta-hexosaminidases remove terminal beta-glycosidically bound N-acetylhexosamine residues from a number of glycoconjugates. Three different isozymes composed of two noncovalently linked subunits alpha and beta exist: Hex A (alphabeta), Hex B (betabeta), and Hex S (alphaalpha). While the role of Hex A and B for the degradation of several anionic and neutral glycoconjugates has been well established, the physiological significance of labile Hex S has remained unclear. However, the striking accumulation of anionic oligosaccharides in double knockout mice totally deficient in hexosaminidase activity but not in mice expressing Hex S (Sango, K., McDonald, M. P., Crawley, J. N., Mack, M. L., Tifft, C.J., Skop, E., Starr, C. M., Hoffmann, A., Sandhoff, K., Suzuki, K., and Proia, R. L., (1996) Nat. Genet. 14, 348-352) prompted us to reinvestigate the substrate specificity of Hex S. To identify physiological substrates of Hex S, anionic and neutral oligosaccharides excreted in the urine of the double knockout mice were isolated and analyzed. Using ESI-MS/MS and glycosidase digestion the anionic glycans were identified as products of incomplete dermatan sulfate degradation whereas the neutral storage oligosaccharides were found to be fragments of N-glycan degradation. In vitro, recombinant Hex S was highly active on water-soluble and amphiphilic glycoconjugates including artificial substrates, sulfated GAG fragments, and the sulfated glycosphingolipid SM2. Hydrolysis of membrane-bound SM2 by the recombinant Hex S was synergistically stimulated by the GM2 activator protein and the lysosomal anionic phospholipid bis(monoacylglycero)phosphate.

Animals↗