Specialized cells of the innate immune system, such as macrophages, employ lysosomal enzymes, together with cationic peptides and reactive oxygen intermediates, to eliminate invading microorganisms ensnared within phagosomes. The effectiveness of this impressive armamentarium is potentiated by the acid pH generated by the vacuolar-type ATPase (V-ATPase). The determinants of the luminal pH of phagosomes and of the lysosomes they fuse with are not completely understood, but the V-ATPase is known to be electrogenic and net accumulation of protons requires charge compensation. For this reason, counter-ion pathways are thought to serve a central role in the control of acidification. It has generally been assumed that a parallel anion influx accompanies proton pumping to dissipate the voltage that tends to build up. In fact, impaired chloride channel activity in cystic fibrosis has been proposed to underlie the defective phagolysosome acidification and microbial killing reported in lung macrophages. In the first part of this thesis, I devised methods to dialyze the lumen of lysosomes in intact cells, while monitoring lysosomal pH, in order to assess the individual contribution of counter-ions to acidification. Surprisingly, anions were found to be completely dispensable for proton pumping, whereas the presence of permeant cations in the lysosomal lumen was essential. Accordingly, defects in lysosomal anion permeability cannot explain the impaired microbicidal capacity of phagocytes in cystic fibrosis. Even though counter-ion permeation pathways exist, dissipation of the electrical contribution of the V-ATPase may not be complete. If present, a transmembrane potential would alter the rate and extent of proton accumulation in phagosomes and lysosomes. However, no estimates of the voltage across the phagosomes were available. To overcome this deficiency, in the second part of this thesis, I describe a noninvasive procedure to estimate the voltage across the phagosome using fluorescence resonance energy transfer. This novel approach, in combination with organellar pH measurements, demonstrated that proton pumping is not limited by counter-ion permeability.

Phagocytosis is an innate immune response that is paramount in the clearance of pathogenic particles. Recognition of target particles by phagocytic receptors expressed on phagocytes induces modifications in the underlying actin cytoskeleton to form pseudopods that encircle and internalize the target particle into a membrane bound organelle called the phagosome. The nascent phagosome undergoes a maturation sequence that is characterized by substantial remodeling of the membrane and its luminal contents through interactions with components of the endocytic pathway, culminating in an acidic and hydrolytic organelle capable of digesting and elminating pathogens. Phagosome maturation is a complicated pathway that involves many protein and lipid signaling molecules. Several factors that influence phagosome maturation particularly the participation of pH, lysosome-associated membrane proteins-1 and –2, cholesterol, in addition to the survival and escape mechanisms used by, Burkholderica cenocepacia were explored. All three tenets are essential for phagosome maturation, although each factor has different mechanistic consequences. Acidification alters Rab5 activation, while ablation of LAMPs and accumulation of cholesterol interferes with various aspects of Rab 7 turnover in phagosomes and/or endosome membranes. Moreover, Burkholderia cenocepacia, an intracellular pathogen, inactivates Rab7 on phagosome membranes from within the vacuole lumen. Herein, mechanisms that govern phagosome maturation are explored and several molecules are added to the long list of essential players in this complicated pathway.

Specialized cells of the innate immune system, such as macrophages, employ lysosomal enzymes, together with cationic peptides and reactive oxygen intermediates, to eliminate invading microorganisms ensnared within phagosomes. The effectiveness of this impressive armamentarium is potentiated by the acid pH generated by the vacuolar-type ATPase (V-ATPase). The determinants of the luminal pH of phagosomes and of the lysosomes they fuse with are not completely understood, but the V-ATPase is known to be electrogenic and net accumulation of protons requires charge compensation. For this reason, counter-ion pathways are thought to serve a central role in the control of acidification. It has generally been assumed that a parallel anion influx accompanies proton pumping to dissipate the voltage that tends to build up. In fact, impaired chloride channel activity in cystic fibrosis has been proposed to underlie the defective phagolysosome acidification and microbial killing reported in lung macrophages. In the first part of this thesis, I devised methods to dialyze the lumen of lysosomes in intact cells, while monitoring lysosomal pH, in order to assess the individual contribution of counter-ions to acidification. Surprisingly, anions were found to be completely dispensable for proton pumping, whereas the presence of permeant cations in the lysosomal lumen was essential. Accordingly, defects in lysosomal anion permeability cannot explain the impaired microbicidal capacity of phagocytes in cystic fibrosis. Even though counter-ion permeation pathways exist, dissipation of the electrical contribution of the V-ATPase may not be complete. If present, a transmembrane potential would alter the rate and extent of proton accumulation in phagosomes and lysosomes. However, no estimates of the voltage across the phagosomes were available. To overcome this deficiency, in the second part of this thesis, I describe a noninvasive procedure to estimate the voltage across the phagosome using fluorescence resonance energy transfer. This novel approach, in combination with organellar pH measurements, demonstrated that proton pumping is not limited by counter-ion permeability.

Phagocytosis is an innate immune response that is paramount in the clearance of pathogenic particles. Recognition of target particles by phagocytic receptors expressed on phagocytes induces modifications in the underlying actin cytoskeleton to form pseudopods that encircle and internalize the target particle into a membrane bound organelle called the phagosome. The nascent phagosome undergoes a maturation sequence that is characterized by substantial remodeling of the membrane and its luminal contents through interactions with components of the endocytic pathway, culminating in an acidic and hydrolytic organelle capable of digesting and elminating pathogens. Phagosome maturation is a complicated pathway that involves many protein and lipid signaling molecules. Several factors that influence phagosome maturation particularly the participation of pH, lysosome-associated membrane proteins-1 and –2, cholesterol, in addition to the survival and escape mechanisms used by, Burkholderica cenocepacia were explored. All three tenets are essential for phagosome maturation, although each factor has different mechanistic consequences. Acidification alters Rab5 activation, while ablation of LAMPs and accumulation of cholesterol interferes with various aspects of Rab 7 turnover in phagosomes and/or endosome membranes. Moreover, Burkholderia cenocepacia, an intracellular pathogen, inactivates Rab7 on phagosome membranes from within the vacuole lumen. Herein, mechanisms that govern phagosome maturation are explored and several molecules are added to the long list of essential players in this complicated pathway.

No description available.

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.

No description available.

Tay-Sachs disease and its related disorders (GM2 gangliosidoses) are neurodegenerative diseases caused by the excessive accumulation of ganglioside GM2, an otherwise non-toxic plasma membrane component, in the lysosomes of cells of the central nervous system. The accumulation of ganglioside GM2 is the result of a defect in the gene encoding the α-subunit of β-hexosaminidase A (Hex A), an acid hydrolase responsible for the metabolism of gangloside GM2 in the lysosome. Though a debilitating disease in humans, Tay-Sachs model mice (𝘏𝘦𝘹𝘢-/-) escape symptoms by the action of lysosomal sialidase, which is expressed in mice at levels sufficient to metabolize ganglioside GM2 and effectively "bypass" Hex A deficiency. In an attempt to understand why a lysosomal sialidase-mediated bypass of Hex A deficiency is not observed in humans, we cloned ~ 2.9 kb of the human lysosomal sialdiase promoter and began characterization of the regulatory machinery that determines its activity. The transcription factor CDP (CCAAT -Displacement Protein) and truncations thereof were found to have a clear and consistent effect on promoter activity 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰, with the truncation CDP⁸³¹⁻¹⁵⁰⁵ resulting in a near 50-fold increase in activity. Adenovirus-mediated gene transfer of CDP⁸³¹⁻¹⁵⁰⁵ into CRB/TSD cells, a human Tay-Sachs neuroglia cell line, resulted in elevated lysosomal sialidase activity and a decrease in ganglioside GM2 stores. These results suggest that the regulatory machinery responsible for lysosomal sialidase expression may be manipulated in such a way as to "activate" a sialidase-mediated bypass of Hex A deficiency in human Tay-Sachs cells. Thus, induction of lysosomal sialidase may have a potential therapeutic benefit in human Tay-Sachs disease and other Hex A-deficient GM2 gangliosidoses. / Thesis / Master of Science (MS)

No description available.

Chronic exposure to solar UVA plays a causative role in skin photoaging and photocarcinogenesis. Guided by exploratory difference-in-gel-electrophoresis (DIGE)-proteomics, we identified the thiol-dependent cysteine-proteases cathepsin B and cathepsin L as novel UVA-targets undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction. In human skin fibroblasts, exposure to noncytotoxic doses of chronic UVA (9.9 J/cm ², twice a week, 3 weeks) caused pronounced photooxidative impairment of cathepsin B and L enzymatic activity suppressed by antioxidant intervention. Western blot analysis revealed extensive 4-hydroxy-2-trans-nonenal (4-HNE) modification of cathepsin B in UVA-exposed fibroblasts. Consistent with lysosomal impairment, accumulation of cellular autofluorescent material colocalizing with lysosomes was observed by confocal fluorescence microscopy, and extensive deposition of lipofuscin was detectable by transmission electron microscopy (TEM). Lysosomal expansion was further evidenced by increased immunodetection of lysosomal associated membrane protein-1 (Lamp-1) and Lysotracker-based flow cytometric analysis. While lysosomal membrane integrity remained intact, autophagic blockade was suggested by accumulation of cellular protein levels of LC3-II and p62 (sequestosome 1) in UVA-exposed fibroblasts. Furthermore, UVA-exposure modulated transcriptional levels of p62 (sequestosome 1, SQSTM1), α-synuclein (SNCA), and transglutaminase-2 (TGM2). Strikingly, pharmacological cathepsin B/L inhibition using CA074Me mimicked UVA-induced accumulation of lipofuscin and autophagic-lysosomal proteins (Lamp-1, LC3-II, and p62), as well as changes at the transcriptional levels. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring protein levels of Lamp-1, LC3-II, and p62, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not reproduce the UVA-induced phenotype. Similarly, TEM revealed massive accumulation of lipofuscin-containing lysosomal vesicles in fibroblasts only after CTSB/CTSL-double knockdown. Taken together, our data indicate for the first time that UVA impairs lysosomal function causing autophagic-lysosomal alterations downstream of cathepsin B/L enzymatic inactivation. This work provides evidence for a heretofore unrecognized 'double-hit' mechanism of UVA skin photodamage where primary photo-oxidative insult occurs simultaneously with impaired clearance of damaged molecules and organelles downstream of dual inactivation of cathepsin B and L.