Regulator Of Lysosomal Function Research Articles

DNA damage-regulated autophagy modulator 1 prevents glioblastoma cells proliferation by regulating lysosomal function and autophagic flux stability

Glioblastoma (GBM) is the most aggressive and life-threatening brain tumor, characterized by its highly malignant and recurrent nature. DNA damage-regulated autophagy modulator 1 (DRAM-1) is a p53 target gene encoding a lysosomal protein that induces macro-autophagy and damage-induced programmed cell death in tumor growth. However, the precise mechanisms underlying how DRAM-1 affects tumor cell proliferation through regulation of lysosomal function and autophagic flux stability remain incompletely understood. We found that DRAM-1 expressions were evidently down-regulated in high-grade glioma and recurrent GBM tissues. The upregulation of DRAM-1 could increase mortality of primary cultured GBM cells. TEM analysis revealed an augmented accumulation of aberrant lysosomes in DRAM-1-overexpressing GBM cells. The assay for lysosomal pH and stability also demonstrated decreasing lysosomal membrane permeabilization (LMP) and impaired lysosomal acidity. Further research revealed the detrimental impact of lysosomal dysfunction, which impaired the autophagic flux stability and ultimately led to GBM cell death. Moreover, downregulation of mTOR phosphorylation was observed in GBM cells following upregulation of DRAM-1. In vivo and in vitro experiments additionally illustrated that the mTOR inhibitor rapamycin increased GBM cell mortality and exhibited an enhanced antitumor effect.

Seventy-Two-Hour LRRK2 Kinase Activity Inhibition Increases Lysosomal GBA Expression in H4, a Human Neuroglioma Cell Line.

Mutations in LRRK2 and GBA1 are key contributors to genetic risk of developing Parkinson’s disease (PD). To investigate how LRRK2 kinase activity interacts with GBA and contributes to lysosomal dysfunctions associated with the pathology of PD. The activity of the lysosomal enzyme β-Glucocerebrosidase (GCase) was assessed in a human neuroglioma cell model treated with two selective inhibitors of LRKK2 kinase activity (LRRK2-in-1 and MLi-2) and a GCase irreversible inhibitor, condutirol-beta-epoxide (CBE), under 24 and 72 h experimental conditions. We observed levels of GCase activity comparable to controls in response to 24 and 72 h treatments with LRRK2-in-1 and MLi-2. However, GBA protein levels increased upon 72 h treatment with LRRK2-in-1. Moreover, LC3-II protein levels were increased after both 24 and 72 h treatments with LRRK2-in-1, suggesting an activation of the autophagic pathway. These results highlight a possible regulation of lysosomal function through the LRRK2 kinase domain and suggest an interplay between LRRK2 kinase activity and GBA. Although further investigations are needed, the enhancement of GCase activity might restore the defective protein metabolism seen in PD.

TRPML1 and TFEB, an Intimate Affair.

Ca2+ is a universal second messenger that plays a wide variety of fundamental roles in cellular physiology. Thus, to warrant selective responses and to allow rapid mobilization upon specific stimuli, Ca2+ is accumulated in organelles to keep it at very low levels in the cytoplasm during resting conditions. Major Ca2+ storage organelles include the endoplasmic reticulum (ER), the mitochondria, and as recently demonstrated, the lysosome (Xu and Ren, Annu Rev Physiol 77:57-80, 2015). The importance of Ca2+ signaling deregulation in human physiology is underscored by its involvement in several human diseases, including lysosomal storage disorders, neurodegenerative disease and cancer (Shen et al., Nat Commun 3:731, 2012; Bae et al., J Neurosci 34:11485-11503, 2014). Recent evidence strongly suggests that lysosomal Ca2+ plays a major role in the regulation of lysosomal adaptation to nutrient availability through a lysosomal signaling pathway involving the lysosomal Ca2+ channel TRPML1 and the transcription factor TFEB, a master regulator for lysosomal function and autophagy (Sardiello et al., Science 325:473-477, 2009; Settembre et al., Science 332:1429-1433, 2011; Medina et al., Nat Cell Biol 17:288-299, 2015; Di Paola et al., Cell Calcium 69:112-121, 2018). Due to the tight relationship of this lysosomal Ca2+ channel and TFEB, in this chapter, we will focus on the role of the TRPML1/TFEB pathway in the regulation of lysosomal function and autophagy.

A deep-red lysosome-targetable fluorescent probe for detection of hypochlorous acid in pure water and its imaging application in living cells and zebrafish

Hypochlorite plays a significant role in physiological processes, particularly regulation of lysosomal functions, and is involved in various diseases. Thus, it is crucial to develop highly sensitive and selective molecule tools to detect HClO in lysosomes. Herein, a novel 2H-benzo[h]chromene-pyridine derivative (1) was synthesized through condensation reaction, which exhibited a notable deep-red emission at 640 nm in pure water. This deep-red emission was specifically quenched by adding ClO−. The response of probe 1 toward ClO− was rapid (within 10 s), sensitive (detection limit of 0.012 μM), and effective over a wide range of pH (1.0–12.0). Due to the existence of morpholine as the lysosome-targeting unit, the probe was successfully utilized to monitor lysosomal ClO−. Moreover, the probe 1 was also applied to detecting ClO− in zebrafish.

LRRK2 Kinase Inhibition Rescues Deficits in Lysosome Function Due to Heterozygous GBA1 Expression in Human iPSC-Derived Neurons

A growing number of genes associated with Parkinson’s disease are implicated in the regulation of lysosome function, including LRRK2, whose missense mutations are perhaps the most common monogenic cause of this neurodegenerative disease. These mutations are collectively thought to introduce a pathologic increase in LRRK2 kinase activity, which is currently a major target for therapeutic intervention. Heterozygous carriers of many missense mutations in the GBA1 gene have dramatically increased risk of Parkinson’s disease. A critical question has recently emerged regarding the potential interplay between the proteins encoded by these two disease-linked genes. Our group has recently demonstrated that knockin mutation of a Parkinson’s-linked GBA1 variant induces severe lysosomal and cytokine abnormalities in murine astrocytes and that these deficits were normalized via inhibition of wild-type LRRK2 kinase activity in these cells. Another group independently found that LRRK2 inhibition increases glucocerebrosidase activity in wild-type human iPSC-derived neurons, as well as those whose activity is disrupted by GBA1 or LRRK2 mutation. Fundamental questions remain in terms of the lysosomal abnormalities and the effects of LRRK2 kinase inhibition in human neurons deficient in glucocerebrosidase activity. Here, we further elucidate the physiological crosstalk between LRRK2 signaling and glucocerebrosidase activity in human iPSC-derived neurons. Our studies show that the allelic loss of GBA1 manifests broad defects in lysosomal morphology and function. Furthermore, our data show an increase in both the accumulation and secretion of oligomeric α-synuclein protein in these GBA1-heterozygous-null neurons, compared to isogenic controls. Consistent with recent findings in murine astrocytes, we observed that multiple indices of lysosomal dysfunction in GBA1-deficient human neurons were normalized by LRRK2 kinase inhibition, while some defects were preserved. Our findings demonstrate a selective but functional intersection between glucocerebrosidase dysfunction and LRRK2 signaling in the cell and may have implications in the pathogenesis and treatment of Parkinson’s disease.

Podocytopathy and Nephrotic Syndrome in Mice with Podocyte-Specific Deletion of the Asah1 Gene: Role of Ceramide Accumulation in Glomeruli

Lysosomal acid ceramidase (Ac) has been shown to be critical for ceramide hydrolysis and regulation of lysosome function and cellular homeostasis. In the present study, we generated a knockout mouse strain (Asah1fl/fl/PodoCre) with a podocyte-specific deletion of the α subunit (main catalytic subunit) of Ac. Although no significant morphologic changes in glomeruli were observed in these mice under light microscope, severe proteinuria and albuminuria were found in these podocyte-specific knockout mice compared with control genotype littermates. Transmission electron microscopic analysis showed that podocytes of the knockout mice had distinctive foot process effacement and microvillus formation. These functional and morphologic changes indicate the development of nephrotic syndrome in mice bearing the Asah1 podocyte-specific gene deletion. Ceramide accumulation determined by liquid chromatography-tandem mass spectrometry was demonstrated in isolated glomeruli of Asah1fl/fl/PodoCre mice compared with their littermates. By crossbreeding Asah1fl/fl/PodoCre mice with Smpd1-/- mice, we also produced a double knockout strain, Smpd1-/-/Asah1fl/fl/PodoCre, that also lacks Smpd1, the acid sphingomyelinase that hydrolyzes sphingomyelin to ceramide. These mice exhibited significantly lower levels of glomerular ceramide with decreased podocyte injury compared with Asah1fl/fl/PodoCre mice. These results strongly suggest that lysosomal Ac in podocytes is essential for themaintenance of the structural and functional integrity of podocytes.

Characterization of lysosomal proteins Progranulin and Prosaposin and their interactions in Alzheimer\u2019s disease and aged brains: increased levels correlate with neuropathology

Progranulin (PGRN) is a protein encoded by the GRN gene with multiple identified functions including as a neurotrophic factor, tumorigenic growth factor, anti-inflammatory cytokine and regulator of lysosomal function. A single mutation in the human GRN gene resulting in reduced PGRN expression causes types of frontotemporal lobar degeneration resulting in frontotemporal dementia. Prosaposin (PSAP) is also a multifunctional neuroprotective secreted protein and regulator of lysosomal function. Interactions of PGRN and PSAP affect their functional properties. Their roles in Alzheimer’s disease (AD), the leading cause of dementia, have not been defined. In this report, we examined in detail the cellular expression of PGRN in middle temporal gyrus samples of a series of human brain cases (n = 45) staged for increasing plaque pathology. Immunohistochemistry showed PGRN expression in cortical neurons, microglia, cerebral vessels and amyloid beta (Aβ) plaques, while PSAP expression was mainly detected in neurons and Aβ plaques, and to a limited extent in astrocytes. We showed that there were increased levels of PGRN protein in AD cases and corresponding increased levels of PSAP. Levels of PGRN and PSAP protein positively correlated with amyloid beta (Aβ), with PGRN levels correlating with phosphorylated tau (serine 205) levels in these samples. Although PGRN colocalized with lysosomal-associated membrane protein-1 in neurons, most PGRN associated with Aβ plaques did not. Aβ plaques with PGRN and PSAP deposits were identified in the low plaque non-demented cases suggesting this was an early event in plaque formation. We did not observe PGRN-positive neurofibrillary tangles. Co-immunoprecipitation studies of PGRN from brain samples identified only PSAP associated with PGRN, not sortilin or other known PGRN-binding proteins, under conditions used. Most PGRN associated with Aβ plaques were immunoreactive for PSAP showing a high degree of colocalization of these proteins that did not change between disease groups. As PGRN supplementation has been considered as a therapeutic approach for AD, the possible involvement of PGRN and PSAP interactions in AD pathology needs to be further considered.

Understanding organelle biogenesis during the autophagy process in the RPE

AbstractThe retinal pigmented epithelium (RPE) is critically important to retinal homeostasis, in part due to it's very active processes of phagocytosis and autophagy. Both of these processes depend upon the normal functioning of lysosomes, organelles which must fuse with (auto)phagosomes to deliver the hydrolases that effect degradation of cargo. It has become clear that signaling through mTOR complex 1 (mTORC1), is very important in the regulation of lysosomal function. Lysosomes are cellular organelles that modulate various processes such as autophagy and heterophagy, plasma membrane repair, cholesterol homeostasis, and cell death. The number, size, and content of lysosomes vary in different cell types. The distribution of lysosomes within the cell is determined by the nutrient sensing machinery at the lysosomal membrane, and is an important factor in lysosomal catabolic function. In this course, we will focus on the effector molecules present in RPE cells that impact the lysosomal‐autophagic pathway in retinal health and disease.

A novel target and pH dual-activatable fluorescent probe for precisely detecting hypochlorite in lysosomes

Hypochlorite (HClO) is involved in various physiological and pathological processes as well as regulation of lysosomal functions. Thus, it is appreciated to develop efficient molecule tools for precisely detecting HClO in lysosomes. Although several lysosomal-targetable fluorogenic probes for HClO have been developed to date, they still suffered from the discounted sensing performance under lysosomal acidic condition. Herein, on the basis of the “AND” logic gate strategy, a novel dual-activatable fluorogenic probe CS has been rationally designed by simultaneously incorporating HClO recognition site and pH-sensitive group with lysosomal-targetable characteristic into a coumarin fluorophore. Different from the single-activated ones previously reported, CS exhibited good sensitivity, high specificity and fast response towards HClO under the acidic conditions but was out of operation in the neutral or alkaline environment. Importantly, it had been successfully applied for spatial-resolution imaging of exogenous or endogenous HClO in lysosomes.

Nutrient deprivation and lysosomal stress induce activation of TFEB in retinal pigment epithelial cells

BackgroundInduction of lysosomal function and autophagy is regarded as an adaptive mechanism in response to cellular stress. The transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function and autophagy. TFEB is a member of the microphthalmia family of bHLH-LZ transcription factors that includes other members such as micropthalmia-associated transcription factor (MITF), TFE3, and TFEC. TFEB controls lysosome biogenesis and autophagy by upregulation of a family of genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network. Here, we investigated the expression of TFEB in cells subjected to nutrient deprivation and lysosomal stress. We studied transcriptional induction of TFEB-regulated genes in response to nutrient deprivation and lysosomal stress in retinal pigment epithelial (RPE) cells. Furthermore, we also investigated the induction of autophagy and lysosomal genes upon overexpression of constitutively active form of TFEB.MethodsExpression of TFEB and MITF protein levels were evaluated in cells subjected to prolonged periods of nutrient deprivation. mRNA levels of the CLEAR network genes was measured by quantitative real time PCR (qRT-PCR) analysis in cells deprived of nutrients, treated with ammonium chloride and upon overexpression of constitutively active TFEB. Immunostaining with LC3 antibody was used to measure autophagy flux. Labeling with lysoTracker dye was used to assess lysosomes.ResultsOur results show that nutrient deprivation increases protein levels of TFEB and MITF in ARPE-19 cells. Nutrient stress induces the expression of lysosomal (LAMP1, CTSD MCOLN1, SGSH) and autophagy (BECN1) genes. Lysosomal stress also increases the expression of lysosomal (ATP6V0A1 and LAMP1) and autophagy (p62 and BECN1) genes. Our results show that overexpression of constitutively active TFEB also induces the expression of CLEAR network genes.ConclusionsCollectively, these observations suggest that nutrient stress induces the protein expression of both MITF and TFEB in ARPE-19 cells. TFEB-regulated transcriptional program plays an important role in adaptive response of cells during both nutrient and lysosomal stress.

Multi-Granulin Domain Peptides Bind to Pro-Cathepsin D and Stimulate Its Enzymatic Activity More Effectively Than Progranulin in Vitro.

Progranulin (PGRN) is an evolutionarily conserved glycoprotein associated with several disease states, including neurodegeneration, cancer, and autoimmune disorders. This protein has recently been implicated in the regulation of lysosome function, whereby PGRN may bind to and promote the maturation and activity of the aspartyl protease cathepsin D (proCTSD, inactive precursor; matCTSD, mature, enzymatically active form). As the full-length PGRN protein can be cleaved into smaller peptides, called granulins, we assessed the function of these granulin peptides in binding to proCTSD and stimulating matCTSD enzyme activity in vitro. Here, we report that full-length PGRN and multi-granulin domain peptides bound to proCTSD with low to submicromolar binding affinities. This binding promoted proCTSD destabilization, the magnitude of which was greater for multi-granulin domain peptides than for full-length PGRN. Such destabilization correlated with enhanced matCTSD activity at acidic pH. The presence and function of multi-granulin domain peptides have typically been overlooked in previous studies. This work provides the first in vitro quantification of their binding and activity on proCTSD. Our study highlights the significance of multi-granulin domain peptides in the regulation of proCTSD maturation and enzymatic activity and suggests that attention to PGRN processing will be essential for the future understanding of the molecular mechanisms leading to neurodegenerative disease states with loss-of-function mutations in PGRN.

Two-photon fluorescence probe for precisely detecting endogenous H2S in lysosome by employing a dual lock system

Hydrogen sulfide (H2S) is involved in various pathological and physiological processes as well as regulation of lysosomal functions. Thus, it is essential to develop precise method for the detection of H2S in lysosome. Although a large number of fluorescent probes for H2S have been developed, the probe for lysosomal H2S assay still remains rare. Herein, a two-photon fluorescent probe, named Lyso-Nap, is designed and synthesized for detecting H2S in lysosome by employing a dual lock system (dual PET process and dual reaction sites). The fluorescence of Lyso-Nap with dual lock system is opened by H2S only when the probe is localized within acidic lososomes, which can avoid the disturbance from neutral cytosol and other organelles. Moreover, two-photon fluorescent probes with weak auto-fluorescence, large penetration depth and reduced photodamage, are particularly favored for bioimaging. Accordingly, Lyso-Nap can be successfully used for precisely tracking and detecting endogenous H2S in lysosome of living cells and tissue.

Regulation of Lysosomal Function by the DAF-16 Forkhead Transcription Factor Couples Reproduction to Aging in Caenorhabditis elegans.

Aging in eukaryotes is accompanied by widespread deterioration of the somatic tissue. Yet, abolishing germ cells delays the age-dependent somatic decline in Caenorhabditis elegans In adult worms lacking germ cells, the activation of the DAF-9/DAF-12 steroid signaling pathway in the gonad recruits DAF-16 activity in the intestine to promote longevity-associated phenotypes. However, the impact of this pathway on the fitness of normally reproducing animals is less clear. Here, we explore the link between progeny production and somatic aging and identify the loss of lysosomal acidity-a critical regulator of the proteolytic output of these organelles-as a novel biomarker of aging in C. elegans The increase in lysosomal pH in older worms is not a passive consequence of aging, but instead is timed with the cessation of reproduction, and correlates with the reduction in proteostasis in early adult life. Our results further implicate the steroid signaling pathway and DAF-16 in dynamically regulating lysosomal pH in the intestine of wild-type worms in response to the reproductive cycle. In the intestine of reproducing worms, DAF-16 promotes acidic lysosomes by upregulating the expression of v-ATPase genes. These findings support a model in which protein clearance in the soma is linked to reproduction in the gonad via the active regulation of lysosomal acidification.

70 - Tracking Intriguing Lysosomal Superoxide Formation and Distribution in Multiple Cellular Models with a Highly Selective Fluorescent Probe

Lysosome, as a major digestive compartment within the cell, are now increasingly recognized as a hub that integrates diverse signal to control multiple aspects of cell physiology. Superoxide (O2●–) is one of the most important and upstream reactive oxygen species (ROS), which participates in many physiological and pathological process. It is generally considered that mitochondria is the major source for cellular ROS. However, the role of ROS in regulation of lysosomal function is largely unknown. We have successfully developed a selective and sensitive lysosome-targeting O2●– fluorescent probe – HKSOX-2L. This probe has achieved robust response in detection of lysosomal O2●– in multiple cellular models such as PMA induced inflammation, amino acid starvation and anticancer drug Dp44mT-Cu complex stimulation in both confocal imaging and flow cytometry platforms. Furthermore, we have developed a specific lysosome-targeting O2●– scavenger Lyso-NAC, based on the reported O2●– scavenger N-Acetyl-L-Cysteine (NAC). With this molecule in hand, we further prove that the O2●– detected by HKSOX-2L is locally produced in lysosomes. Therefore, HKSOX-2L and Lyso-NAC serve as promising tools for monitoring lysosomal superoxide production/distribution during numerous physiological and pathological processes, and for elucidating the lysosome-related biological functions.

TFEB and TFE3: Linking Lysosomes to Cellular Adaptation to Stress

In recent years, our vision of lysosomes has drastically changed. Formerly considered to be mere degradative compartments, they are now recognized as key players in many cellular processes. The ability of lysosomes to respond to different stimuli revealed a complex and coordinated regulation of lysosomal gene expression. This review discusses the participation of the transcription factors TFEB and TFE3 in the regulation of lysosomal function and biogenesis, as well as the role of the lysosomal pathway in cellular adaptation to a variety of stress conditions, including nutrient deprivation, mitochondrial dysfunction, protein misfolding, and pathogen infection. We also describe how cancer cells make use of TFEB and TFE3 to promote their own survival and highlight the potential of these transcription factors as therapeutic targets for the treatment of neurological and lysosomal diseases.

The GATOR2 Component Wdr24 Regulates TORC1 Activity and Lysosome Function.

TORC1 is a master regulator of metabolism in eukaryotes that responds to multiple upstream signaling pathways. The GATOR complex is a newly defined upstream regulator of TORC1 that contains two sub-complexes, GATOR1, which inhibits TORC1 activity in response to amino acid starvation and GATOR2, which opposes the activity of GATOR1. While the GATOR1 complex has been implicated in a wide array of human pathologies including cancer and hereditary forms of epilepsy, the in vivo relevance of the GATOR2 complex remains poorly understood in metazoans. Here we define the in vivo role of the GATOR2 component Wdr24 in Drosophila. Using a combination of genetic, biochemical, and cell biological techniques we demonstrate that Wdr24 has both TORC1 dependent and independent functions in the regulation of cellular metabolism. Through the characterization of a null allele, we show that Wdr24 is a critical effector of the GATOR2 complex that promotes the robust activation of TORC1 and cellular growth in a broad array of Drosophila tissues. Additionally, epistasis analysis between wdr24 and genes that encode components of the GATOR1 complex revealed that Wdr24 has a second critical function, the TORC1 independent regulation of lysosome dynamics and autophagic flux. Notably, we find that two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. These findings represent a surprising and previously unrecognized function of GATOR2 complex components in the regulation of lysosomes. Consistent with our findings in Drosophila, through the characterization of a wdr24-/- knockout HeLa cell line we determined that Wdr24 promotes lysosome acidification and autophagic flux in mammalian cells. Taken together our data support the model that Wdr24 is a key effector of the GATOR2 complex, required for both TORC1 activation and the TORC1 independent regulation of lysosomes.

Abstract 3149: Targeting ERBB2-induced, lysosome-mediated invasion

Abstract ErbB2 overexpression is strongly associated with invasive cancer with high recurrence and poor prognosis. Similarly, increased expression of lysosomal proteases, cysteine cathepsins, is observed in several cancers and correlates with enhanced angiogenesis, invasion and metastasis. We recently showed that ErbB2 level correlates positively with cysteine cathepsin B and L levels in primary breast cancer and that ErbB2 regulates the expression of cathepsins B and L leading to their increased activity in several ErbB2-positive breast cancer cell lines. The increased cysteine cathepsin activity is essential for the invasion of ErbB2 overexpressing breast cancer cells in 3-dimensional (3D) Matrigel cultures and is mediated by novel signaling network that involves TGFβR, ERK2-MAPK, PAK4, PKCα and CDC42bpβ kinases and two transcription factors, MZF1 and ETS1. With a pharmacological protein kinase inhibitor screen, using the ErbB2 inhibitor Lapatinib as positive control, we have now identified three inhibitors putatively targeting this novel ErbB2-induced signaling network. Our data shows that the treatment of ErbB2-positive, highly invasive breast or ovarian cancer cells with any of these inhibitors specifically reduces cysteine cathepsin expression, activity and invasion of these cancer cells in 3D Matrigel cultures. It also reverses lysosomal distribution from the invasion supporting pericellular position to less-invasive perinuclear position, where the lysosomal exocytosis and secretion of lysosome contents to extracellular space to assist extracellular matrix proteolysis becomes less likely. In this study we have identified novel inhibitors for ErbB2-induced invasion and discovered a novel, function for clinically used ErbB2 inhibitor Lapatinib in the regulation of lysosomal function and trafficking. Citation Format: Tuula Kallunki, Ditte M. Brix, Bo Rafn, Knut Kristoffer Bundgaard Clemmensen, Sofie Hagel Andersen, Noona Ambartsumian, Marja Jäättelä. Targeting ERBB2-induced, lysosome-mediated invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3149. doi:10.1158/1538-7445.AM2014-3149

Autophagy maturation associated with CD38‐mediated regulation of lysosome function in mouse glomerular podocytes

Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38-mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3-II, a robust marker of autophagosomes (APs), suggesting a high basal level of autophagic activity. Treatment with the mTOR inhibitor, rapamycin, increased LC3-II and the content of both APs detected by Cyto-ID Green staining and autophagolysosomes (APLs) measured by acridine orange staining and colocalization of LC3 and Lamp1. Lysosome function inhibitor bafilomycin A1 increased APs, but decreased APLs content under both basal and rapamycin-induced conditions. Inhibition of CD38 activity by nicotinamide or silencing of CD38 gene produced the similar effects to that bafilomycin A1 did in podocytes. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, the fusion of APs with lysosomes in living podocytes was observed by co-transfection of GFP-LC3B and RFP-Lamp1 expression vectors. A colocalization of GFP-LC3B and RFP-Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. Moreover, blockade of the CD38-mediated regulation by PPADS completely abolished rapamycin-induced fusion of APs with lysosomes. These results indicate that CD38 importantly control lysosomal function and influence autophagy at the maturation step in podocytes.

Autophagy Maturation Controlled by CD38‐Lysosome Signaling in Glomerular Podocytes of Mice

Podocytes are well differentiated glomerular epithelial cells, and their structural and functional integrity are mainly maintained by a self‐defense or adaptive mechanism, autophagy. However, it remains unknown how podocyte autophagy is regulated to maintain the integrity of glomerular structure and function. The present study tested whether CD38‐mediated signaling, an important regulatory pathway of lysosome function, plays a critical role in the regulation of podocyte autophagy. Western blot analysis showed that rapamycin stimulation significantly increased the LC3‐II and beclin expression with or without prior treatment of nicotinamide, an inhibitor of CD38 or bafilomycin, a lysosome function inhibitor. Laser flow cytometry demonstrated that treatment of podocytes with nicotinamide enhanced the autophagosome (AP) accumulation (2.34±0.19 vs 1.63±0.21 of control), but decreased the formation of autophagolysosomes (1.12±0.18 vs 1.65±0.14 of control) under rapamycin stimulation. Using CD38 shRNA, we confirmed that CD38 gene silencing had similar effects to those observed by using pharmacological inhibition of CD38 activity or lysosome function, as analyzed both Western blot analysis and cytometry. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, we directly investigated the fusion of APs with lysosomes in living podocytes by co‐transfection of GFP‐LC3B and RFP‐Lamp1 expression vectors to express both proteins that were located to APs and lysosome, respectively. It was found that a colocalization of GFP‐LC3B and RFP‐Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. In conclusion, CD38 is a critical molecule that controls lysosome function and thereby determine autophagy maturation in podocytes (supported by NIH grants HL‐091464 and DK54927).

Role of galectin-3 in prion infections of the CNS

Galectin-3 is a multi-functional protein and participates in mediating inflammatory reactions. The pronounced overexpression of galectin-3 in prion-infected brain tissue prompted us to study the role of this protein in a murine prion model. Immunofluorescence double-labelling identified microglia as the major cell type expressing galectin-3. Ablation of galectin-3 did not affect PrPSc-deposition and development of gliosis. However, galectin-3−/−-mice showed prolonged survival times upon intracerebral and peripheral scrapie infections. Moreover, protein levels of the lysosomal activation marker LAMP-2 were markedly reduced in prion-infected galectin-3−/−-mice suggesting a role of galectin-3 in regulation of lysosomal functions. Lower mRNA levels of Beclin-1 and Atg5 in prion-infected wild-type and galectin-3−/−-mice indicated an impairment of autophagy although autophagosome formation was unchanged. The results point towards a detrimental role of galectin-3 in prion infections of the CNS and suggest that endo-/lysosomal dysfunction in combination with reduced autophagy may contribute to disease development.