Indiana University-Purdue University Indianapolis (IUPUI) / Key regulatory decisions of protein synthesis and autophagy are controlled by the lysosomal nutrient sensing complex (LYNUS). To engage protein synthesis signaling, LYNUS requires cellular availability of amino acids, adenosine triphosphate (ATP), growth factors, and docking at the lysosomal membrane. The molecular determinants of LYNUS signaling and docking are not completely elucidated and may involve regulators of the lipid membrane structure and function of the lysosome. Since ceramides are both bioactive second messengers and determinants of lipid membrane stiffness, we investigated the role of the ceramide-producing lysosomal acid sphingomyelinase (ASM) in the homeostatic function of mammalian target of rapamycin (mTOR) signaling and autophagy. Using ASM inhibition with either imipramine or siRNA against SMPD1, in primary human lung cells or Smpd1+/- mice, we demonstrated that ASM is an endogenous inhibitor of autophagy. ASM was necessary for physiological mTOR signaling and maintenance of sphingosine levels. Whereas overstimulation of ASM has been shown to trigger autophagy with impaired flux, inhibition of ASM activity during homeostatic, non-stressed conditions triggered autophagy with degradative potential, associated with enhanced transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis genes, translocation to the nucleus and decreased sphingosine levels. These findings suggest LYNUS signaling and autophagy are partially regulated by ASM.

Acid Sphingomyelinase (ASMase) activity has been proposed to mediate LPS signaling in various cell types. This study shows that in macrophages, ASMase is a negative regulator of LPS-induced TNFα secretion. ASMasedeficient (asm-/-) mice and isolated peritoneal macrophages produce several fold more TNFα than their wild-type (asm+/+) counterparts when stimulated with LPS. The mechanism for these differences however is not transcriptional but post-translational. The TNFα converting enzyme (TACE) catalyzes the maturation of the 26kD precursor (proTNFα) to the active 17kD form (sTNFα). In mouse peritoneal macrophages, the activity of TACE rather than the rate of TNFα mRNA synthesis was the rate-limiting factor regulating TNFα production. Substantial portion of the translated proTNFα was not processed to sTNFα; instead it was rapidly internalized and degraded in the lysosomes. TACE activity was 2 to 3 fold higher in asm-/- macrophages as compared to asm+/+ macrophages and was suppressed when cells were treated with exogenous ceramide and SMase. In asm-/- but not in asm+/+macrophages, indirect immunofluorescence experiments revealed distinct TNFα-positive structures in close vicinity of the plasma membrane. Asm-/- cells also had higher number of EEA1-positive early endosomes. Co-localization experiments that involved inhibitors of TACE and/or lysosomal proteolysis suggest that in asm-/-cells a significant portion of proTNFα is sequestered within the early endosomes, and instead of undergoing lysosomal proteolysis it is recycled to the plasma membrane and processed to sTNFα.

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Kyeong-Ok Chang / Caliciviruses are important pathogens of humans and animals. Noroviruses are major causes of foodborne gastroenteritis cases, but their research is hindered due to the inability to grow human noroviruses in cell culture. Detailed studies on entry events of caliciviruses are lacking and may be crucial for development of cell culture models. We examined the entry events of caliciviruses using porcine enteric calicivirus (PEC), feline calicivirus (FCV) and murine norovirus-1 (MNV-1). PEC replication in LLC-PK cells requires bile acid in the medium, but the mechanism is not well understood. Our studies showed that bile acids are required in the early stage of virus replication, and while internalization of PEC is not dependent of them, they are required for endosomal escape and successful replication. Further examination on virus entry, we demonstrated that endosomal acidification and cathepsin L activity are essential in the replication of PEC, FCV and MNV-1. The results showed that inhibition of endosomal acidification or cathepsin L activity led to retention of viruses in the endosomes. Also we demonstrated that recombinant cathepsin L cleaved structural protein of PEC, FCV or MNV-1, which suggests that the enzyme may facilitate uncoating viruses in endosomes. In addition to bile acids, we found that a cold shock treatment during virus entry supported PEC replication by facilitating the endosomal escape. While PEC alone did not induce ceramide formation, bile acids or cold shock treatment induce ceramide formation on endosomes through activation acid sphingomyelinase (ASM), and this event was crucial for virus replication because inhibition of ASM blocked ceramide formation and significantly reduced PEC replication. Incubation of FCV or MNV-1 with cells led to ceramide formation during virus entry, and inhibition of ASM also significantly reduced their replication. Inhibition of ASM led to endosomal retention of PEC, FCV or MNV-1 during virus entry, which may be the reason for the reduction of viral replication. These studies revealed the important and common events during calicivirus entry for successful replication, including virus endosomal escape, cathepsin L activity and ASM/ceramide formation. This detailed information may provide clues for understanding the replication of fastidious caliciviruses and for potential therapeutic targets.

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A doença de Niemann-Pick tipo C (NPC) é uma esfingolipidose autossômica recessiva que se caracteriza pelo acúmulo lisossômico de colesterol não-esterificado em vários tecidos, resultando em neurodegeneração progressiva, hepatoesplenomegalia e paralisia ocular vertical, entre outros sintomas. Sua manifestação ocorre geralmente entre a metade da infância e a adolescência. A morte ocorre geralmente até a terceira década de vida. Associado à sintomatologia clínica, o diagnóstico de NPC é realizado através do teste de Filipin em fibroblastos cultivados, que demonstra um intenso padrão de fluorescência perinuclear, consistente com o acúmulo de colesterol nãoesterificado. Alguns pacientes, referidos como NPC variantes, apresentam quadro clínico compatível com NPC, mas resultados inconclusivos nos testes bioquímicos, com fluorescência não característica no teste de Filipin, tornando problemático o diagnóstico. O objetivo deste trabalho foi desenvolver técnicas objetivas capazes de auxiliar na confirmação do diagnóstico de NPC, quando um fenótipo apresentar característica indefinida/variante ao teste de Filipin, tais como: quantificação do padrão de fluorescência perinuclear quanto ao número de pixels em pacientes com fenótipo clássico (NPC cl) e variante (NPC var) de NPC, comparando-os entre si e com controles saudáveis (CS); medida da quantidade de colesterol citoplasmático em fibroblastos dos dois grupos de pacientes; medida da atividade das enzimas esfingomielinase ácida (ASM), quitotriosidase (QT), beta-glicosidase ácida (GBA) e beta-galactosidase (GLB). Todos estes parâmetros foram comparados com aqueles de CS. Os resultados mostraram que a quantificação da fluorescência do colesterol no teste de Filipin nos três grupos estudados através do número de pixels da imagem, é um método prático e não subjetivo que demonstrou uma diferença significativa entre o acúmulo de colesterol intracelular em CS, NPC cl e NPC var, confirmando a eficácia do método para o esclarecimento de padrões duvidosos. A dosagem de colesterol intracelular em fibroblastos de NPC apresentou-se sete vezes maior no padrão clássico e quatro vezes maior no variante, do que aquela encontrada nos CS. Com esses dados, a dosagem intracelular de colesterol mostra-se um bom parâmetro quantitativo para auxiliar no teste de Filipin quando este apresentar padrão de fluorescência apenas moderado. A medida da atividade da ASM tanto em leucócitos quanto em SPF não apresentou diferenças estatisticamente significativas entre os grupos estudados, embora seja aparente a diminuição na atividade desta enzima no fenótipo NPC cl. Com os valores obtidos para a medida da atividade da QT em plasma e SPF, verificamos que não houve diferença significativa entre os dois grupos NPC mas sim entre estes e os CS, demostrando que tanto os indivíduos com o fenótipo clássico quanto com o variante possuem uma atividade aumentada desta enzima. A análise da atividade da GBA em leucócitos não apresentou diferenças significativas entre os três grupos, embora sua atividade em NPC cl pareça maior do em CS. Já na análise em SPF, esta diferença foi estatisticamente confirmada. A atividade da GLB em leucócitos não apresentou diferenças entre os grupos estudados, embora esta pareça maior nos grupos NPC do que em CS. / Niemann-Pick disease Type C (NPC) is an autosomal recessive sphingolipidosis which is characterized by a lysosomal accumulation of unesterified cholesterol in various tissues, resulting in progressive neurodegeneration, hepatosplenomegaly and vertical eye paralysis, among other symptoms. Its onset occurs commonly between middle childhood and adolescence. Death occurs usually until the third decade of life. Along with the clinical symptoms, the diagnosis of NPC is accomplished by Filipin test in cultured fibroblasts, showing an intense perinuclear staining pattern, which is consistent with the accumulation of unesterified cholesterol. Some patients, referred to as NPC variants, present a clinical picture of NPC, but inconclusive results in biochemical tests, with no characteristic fluorescence in Filipin test, which makes the diagnosis problematic. The objective of this study was to develop objective techniques that can assist in confirming the diagnosis of NPC, when the phenotype shows undefined/variant characteristics to Filipin test, such as: quantification of perinuclear fluorescence pattern based on pixels' luminosity pattern in patients with classical (NPC cl) and variant (NPC var) NPC phenotype, comparing them among themselves and with healthy controls (CS); the amount of cytoplasmic cholesterol in fibroblasts of both groups of patients; measurement of the activity of the enzymes acid sphingomyelinase (ASM), chitotriosidase (CT), beta-glucosidase acid (GBA), and beta-galactosidase (GLB). All of these parameters were compared with those of CS. The results showed that the fluorescence quantitation of cholesterol in the Filipin test, for the three studied groups, by counting the number of image pixels, is a practical and non-subjective method, which showed a significant difference between the accumulation of intracellular cholesterol in CS, NPC cl and NPC var, confirming its effectiveness to clarify dubious patterns. Measures of intracellular cholesterol in NPC fibroblasts showed seven times higher in classic and four times higher in variant pattern, than that found in CS. With these data, the dosage of intracellular cholesterol appears to be a good parameter to aid in the quantitative Filipin test when it presents only a moderate fluorescence pattern. The measurement of ASM activity in both leukocytes and in SPF did not show statistically significant differences between the groups, despite the remarkable decrease in the enzyme's activity in NPC cl phenotype. With the data obtained for QT's activity in plasma and SPF, we found that there was no significant difference between the two NPC groups, but a significant one between the NPC and the CS, demonstrating that both individuals with the classical phenotype and with the variant one have an increased activity of this enzyme. The analysis of GBA activity in leukocytes showed no significant differences among the three groups, although its activity in NPC cl seemed to be greater than in CS. In the SPF analysis, this difference was statistically confirmed. The activity of GLB in leukocytes did not differ between the groups, although it seems greater in the NPC group than in CS.