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1.
Sci Rep ; 12(1): 11662, 2022 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-35804072

RESUMO

Neuronal ceroid lipofuscinosis is one of many neurodegenerative storage diseases characterized by excessive accumulation of lipofuscins. CLN10 disease, an early infantile neuronal ceroid lipofuscinosis, is associated with a gene that encodes cathepsin D (CtsD), one of the major lysosomal proteases. Whole body CtsD-knockout mice show neurodegenerative phenotypes with the accumulation of lipofuscins in the brain and also show defects in other tissues including intestinal necrosis. To clarify the precise role of CtsD in the central nervous system (CNS), we generated a CNS-specific CtsD-knockout mouse (CtsD-CKO). CtsD-CKO mice were born normally but developed seizures and their growth stunted at around postnatal day 23 ± 1. CtsD-CKO did not exhibit apparent intestinal symptoms as those observed in whole body knockout. Histologically, autofluorescent materials were detected in several areas of the CtsD-CKO mouse's brain, including: thalamus, cerebral cortex, hippocampus, and cerebellum. Expression of ubiquitin and autophagy-associated proteins was also increased, suggesting that the autophagy-lysosome system was impaired. Microglia and astrocytes were activated in the CtsD-CKO thalamus, and inducible nitric oxide synthase (iNOS), an inflammation marker, was increased in the microglia. Interestingly, deposits of proteinopathy-related proteins, phosphorylated α-synuclein, and Tau protein were also increased in the thalamus of CtsD-CKO infant mice. Considering these results, we propose thatt the CtsD-CKO mouse is a useful mouse model to investigate the contribution of cathepsin D to the early phases of neurodegenerative diseases in relation to lipofuscins, proteinopathy-related proteins and activation of microglia and astrocytes.


Assuntos
Catepsina D/metabolismo , Lipofuscinoses Ceroides Neuronais , Animais , Astrócitos/metabolismo , Catepsina D/genética , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Microglia/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia
2.
Cell Death Dis ; 13(6): 552, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35715412

RESUMO

Inhibition of cathepsin D (Cat D) sensitizes cancer cells to anticancer drugs via RNF183-mediated downregulation of Bcl-xL expression. Although NF-κB activation is involved in the upregulation of RNF183 expression, the molecular mechanism of NF-κB activation by Cat D inhibition is unknown. We conducted this study to investigate the molecular mechanism underlying Cat D-mediated NF-κB activation. Interestingly, Cat D inhibition-induced IκB degradation in an autophagy-dependent manner. Knockdown of autophagy-related genes (ATG7 and Beclin1) and lysosome inhibitors (chloroquine and bafilomycin A1) blocked IκB degradation via Cat D inhibition. Itch induced K63-linked ubiquitination of IκB and then modulated the protein stability of IκB by Cat D inhibition. Inhibition of Cat D-mediated Itch activation was modulated by the JNK signaling pathway, and phosphorylated Itch could bind to IκB, resulting in polyubiquitination of IκB. Additionally, inhibition of Cat D increased autophagy flux via activation of the LKB1-AMPK-ULK1 pathway. Therefore, our results suggested that Cat D inhibition activated NF-κB signaling via degradation of autophagy-dependent IκB, which is associated with the upregulation of RNF183, an E3 ligase of Bcl-xL. Cat D inhibition enhances TRAIL-induced apoptosis through Bcl-xL degradation via upregulation of RNF183.


Assuntos
Antineoplásicos , NF-kappa B , Antineoplásicos/farmacologia , Apoptose , Autofagia , Catepsina D/genética , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
3.
J Dairy Sci ; 105(8): 6997-7010, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35688731

RESUMO

Activated autophagy-lysosomal pathway (ALP) can degrade virtually all kinds of cellular components, including intracellular lipid droplets, especially during catabolic conditions. Sustained lipolysis and increased plasma fatty acids concentrations are characteristic of dairy cows with hyperketonemia. However, the status of ALP in adipose tissue during this physiological condition is not well known. The present study aimed to ascertain whether lipolysis is associated with activation of ALP in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes. In vivo, blood and subcutaneous adipose tissue (SAT) biopsies were collected from nonhyperketonemic (nonHYK) cows [blood ß-hydroxybutyrate (BHB) concentration <1.2 mM, n = 10] and hyperketonemic (HYK) cows (blood BHB concentration 1.2-3.0 mM, n = 10) with similar days in milk (range: 3-9) and parity (range: 2-4). In vitro, calf adipocytes isolated from 5 healthy Holstein calves (1 d old, female, 30-40 kg) were differentiated and used for (1) treatment with lipolysis inducer isoproterenol (ISO, 10 µM, 3 h) or mammalian target of rapamycin inhibitor Torin1 (250 nM, 3 h), and (2) pretreatment with or without the ALP inhibitor leupeptin (10 µg/mL, 4 h) followed by ISO (10 µM, 3 h) treatment. Compared with nonHYK cows, serum concentration of free fatty acids was greater and serum glucose concentration, DMI, and milk yield were lower in HYK cows. In SAT of HYK cows, ratio of phosphorylated hormone-sensitive lipase to hormone-sensitive lipase, and protein abundance of adipose triacylglycerol lipase were greater, but protein abundance of perilipin 1 (PLIN1) and cell death-inducing DNA fragmentation factor-α-like effector c (CIDEC) was lower. In addition, mRNA abundance of autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), protein abundance of lysosome-associated membrane protein 1, and cathepsin D, and activity of ß-N-acetylglucosaminidase were greater, whereas protein abundance of sequestosome-1 (p62) was lower in SAT of HYK cows. In calf adipocytes, treatment with ISO or Torin1 decreased protein abundance of PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes, but increased glycerol content in the supernatant of calf adipocytes. Moreover, the mRNA abundance of ATG5, ATG7, and MAP1LC3B was upregulated, the protein abundance of lysosome-associated membrane protein 1, cathepsin D, and activity of ß-N-acetylglucosaminidase were increased, whereas the protein abundance of p62 was decreased in calf adipocytes treated with ISO or Torin1 compared with control group. Compared with treatment with ISO alone, the protein abundance of p62, PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes were higher, but the glycerol content in the supernatant of calf adipocytes was lower in ISO and leupeptin co-treated group. Overall, these data indicated that activated ALP is associated with increased lipolysis in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes.


Assuntos
Doenças dos Bovinos , Cetose , Ácido 3-Hidroxibutírico , Acetilglucosaminidase/metabolismo , Tecido Adiposo/metabolismo , Animais , Autofagia , Catepsina D/metabolismo , Bovinos , Doenças dos Bovinos/metabolismo , Feminino , Glicerol/metabolismo , Cetose/veterinária , Lactação , Leupeptinas/metabolismo , Lipólise , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/metabolismo , Mamíferos/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Esterol Esterase/metabolismo , Triglicerídeos/metabolismo
4.
Asian Pac J Cancer Prev ; 23(6): 2017-2025, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35763644

RESUMO

OBJECTIVE: Hepatocellular carcinoma (HCC) represents a global health concern, particularly in Southeast Asia where hepatitis B virus (HBV) infection is common. In this study, we applied tissue-based proteomics to identify novel serological proteins for HCC and validated their performance in serum specimens. METHODS: In a discovery set, liver tissue specimens of HBV-related HCC, intrahepatic cholangiocarcinoma (iCCA) and colorectal cancer with liver metastasis (CRLM) were analyzed using mass spectrometry (LTQ-Orbitrap-XL). A subset of proteins that showed highly expressed in HCC were then confirmed by Western blotting. Additionally, clinical significance of selected candidate proteins was tested in serum samples of 80 patients with HBV-related HCC, 50 patients with HBV-related liver cirrhosis and 30 healthy controls. RESULTS: Based on LTQ-Orbitrap-XL mass spectrometer, various differentially expressed proteins (DEPs) between tumor and adjacent non-tumor tissues were identified. These included 77 DEPs for HCC, 77 DEPs for iCCA and 55 DEPs for CRLM. Among selected candidate proteins, annexin A2 and cathepsin D were confirmed to be overexpressed in HCC tissue by Western blot analysis. In a validate cohort, serum cathepsin D level, but not annexin A2, was significantly higher in HCC compared with the non-HCC groups. Serum cathepsin D level was also positively correlated with tumor size and tumor stage. Additionally, the combined assay of serum cathepsin D and alpha-fetoprotein had a high sensitivity in detecting early HCC (83%) and intermediate/advanced HCC (96%). Moreover, patients with low serum cathepsin D (<305 ng/mL) displayed significantly better overall survival than those whose serum levels were high (≥305 ng/mL). CONCLUSIONS: Proteomics and subsequent validation revealed cathepsin D as a novel biomarker for HCC. Apart from its diagnostic role, serum cathepsin D might also serve as a prognostic biomarker of HCC. Additional large-scale studies are needed to verify our findings.


Assuntos
Neoplasias dos Ductos Biliares , Carcinoma Hepatocelular , Catepsina D/sangue , Colangiocarcinoma , Hepatite B Crônica , Hepatite B , Neoplasias Hepáticas , Neuroblastoma , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Biomarcadores , Carcinoma Hepatocelular/patologia , Colangiocarcinoma/patologia , Hepatite B/complicações , Hepatite B/patologia , Vírus da Hepatite B , Humanos , Cirrose Hepática , Neoplasias Hepáticas/patologia
5.
Int J Mol Sci ; 23(11)2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35682572

RESUMO

A major cause of cancer cell resistance to chemotherapeutics is the blocking of apoptosis and induction of autophagy in the context of cell adaptation and survival. Therefore, new compounds are being sought, also among drugs that are commonly used in other therapies. Due to the involvement of histamine in the regulation of processes occurring during the development of many types of cancer, antihistamines are now receiving special attention. Our study concerned the identification of new mechanisms of action of azelastine hydrochloride, used in antiallergic treatment. The study was performed on HeLa cells treated with different concentrations of azelastine (15-90 µM). Cell cycle, level of autophagy (LC3 protein activity) and apoptosis (annexin V assay), activity of caspase 3/7, anti-apoptotic protein of Bcl-2 family, ROS concentration, measurement of mitochondrial membrane potential (Δψm), and level of phosphorylated H2A.X in response to DSB were evaluated by cytometric method. Cellular changes were also demonstrated at the level of transmission electron microscopy and optical and fluorescence microscopy. Lysosomal enzyme activities-cathepsin D and L and cell viability (MTT assay) were assessed spectrophotometrically. Results: Azelastine in concentrations of 15-25 µM induced degradation processes, vacuolization, increase in cathepsin D and L activity, and LC3 protein activation. By increasing ROS, it also caused DNA damage and blocked cells in the S phase of the cell cycle. At the concentrations of 45-90 µM, azelastine clearly promoted apoptosis by activation of caspase 3/7 and inactivation of Bcl-2 protein. Fragmentation of cell nucleus was confirmed by DAPI staining. Changes were also found in the endoplasmic reticulum and mitochondria, whose damage was confirmed by staining with rhodamine 123 and in the MTT test. Azelastine decreased the mitotic index and induced mitotic catastrophe. Studies demonstrated the multidirectional effects of azelastine on HeLa cells, including anti-proliferative, cytotoxic, autophagic, and apoptotic properties, which were the predominant mechanism of death. The revealed novel properties of azelastine may be practically used in anti-cancer therapy in the future.


Assuntos
Catepsina D , Neoplasias do Colo do Útero , Apoptose , Autofagia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Feminino , Células HeLa , Humanos , Ftalazinas , Proteínas Proto-Oncogênicas c-bcl-2 , Espécies Reativas de Oxigênio/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico
6.
Int J Mol Sci ; 23(9)2022 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-35563171

RESUMO

Neuroblastoma is a malignant extracranial solid tumor arising from the sympathoadrenal lineage of the neural crest and is often associated with N-MYC amplification. Cathepsin D has been associated with chemoresistance in N-MYC-overexpressing neuroblastomas. Increased EGFR expression also has been associated with the aggressive behavior of neuroblastomas. This work aimed to understand the mechanisms linking EGFR stimulation and cathepsin D expression with neuroblastoma progression and prognosis. Gene correlation analysis in pediatric neuroblastoma patients revealed that individuals bearing a high EGFR transcript level have a good prognosis only when CTSD (the gene coding for the lysosomal protease Cathepsin D, CD) is highly expressed. Low CTSD expression was associated with poor clinical outcome. CTSD expression was negatively correlated with CCNB2, CCNA2, CDK1 and CDK6 genes involved in cell cycle division. We investigated the biochemical pathways downstream to EGFR stimulation in human SH-SY5Y neuroblastoma cells engineered for overexpressing or silencing of CD expression. Cathepsin D overexpression decreased the proliferative potential of neuroblastoma cells through downregulation of the pro-oncogenic MAPK signaling pathway. EGFR stimulation downregulated cathepsin D expression, thus favoring cell cycle division. Our data suggest that chemotherapeutics that inhibit the EGFR pathway, along with stimulators of cathepsin D synthesis and activity, could benefit neuroblastoma prognosis.


Assuntos
Catepsina D , Neuroblastoma , Catepsina D/genética , Catepsina D/metabolismo , Ciclo Celular/genética , Criança , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Lisossomos/metabolismo , Neuroblastoma/metabolismo , Peptídeo Hidrolases/metabolismo
7.
Chin J Physiol ; 65(2): 53-63, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35488670

RESUMO

Our previous study demonstrated that chronic intermittent hypobaric hypoxia (CIHH) protects vascular endothelium function through ameliorating autophagy in mesenteric arteries of metabolic syndrome (MS) rats. This study aimed to investigate the role of adenosine mono-phosphate-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) signaling in CIHH effect. Six-week-old male Sprague-Dawley rats were divided into control (CON), MS model, CIHH treatment (CIHH), and MS + CIHH groups. Serum pro-inflammatory cytokines were measured. The endothelium dependent relaxation (EDR), endothelial ultrastructure and autophagosomes were observed in mesenteric arteries. The expression of phosphor (p)-AMPKα, p-mTOR, autophagy-related and endoplasmic reticulum stress-related proteins, p-endothelial nitric oxide synthase, and cathepsin D were assayed. In MS rats, pro-inflammatory cytokines were increased, EDR was attenuated, and endothelial integrity was impaired. In addition, the expression level of p-AMPKα and cathepsin D was down-regulated, but the level of p-mTOR was up-regulated. While in MS + CIHH rats, all aforementioned abnormalities were ameliorated, and the beneficial effect of CIHH was cancelled by AMPKα inhibitor. In conclusion, AMPK-mTOR signaling pathway participates in the protection of CIHH on vascular endothelium of MS rats.


Assuntos
Endotélio Vascular , Síndrome Metabólica , Proteínas Quinases Ativadas por AMP , Nucleotídeos de Adenina , Adenosina , Animais , Catepsina D , Citocinas , Hipóxia , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Serina-Treonina Quinases TOR
8.
J Biol Chem ; 298(5): 101888, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35367412

RESUMO

Adenosine A2A receptor (A2AR)-dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A2AR targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A2AR and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A2AR-interacting partners have in macrophages. Here, we aimed to identify A2AR-interacting partners in macrophages that may effect receptor trafficking and activity. To this end, we performed a yeast two-hybrid screen using the C-terminal tail of A2AR as the "bait" and a macrophage expression library as the "prey." We found that the lysosomal protease cathepsin D (CtsD) was a robust hit. The A2AR-CtsD interaction was validated in vitro and in cellular models, including RAW 264.7 and mouse peritoneal macrophage (IPMΦ) cells. We also demonstrated that the A2AR is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A2AR in macrophages. Conversely, we demonstrate that A2AR activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A2AR-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes.


Assuntos
Adenosina , Catepsina D/metabolismo , Receptor A2A de Adenosina , Adenosina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Catepsina D/genética , Macrófagos/metabolismo , Camundongos , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/metabolismo , Transdução de Sinais
9.
Diabetes Care ; 45(6): 1416-1427, 2022 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-35377940

RESUMO

OBJECTIVE: Understanding mechanisms underlying rapid estimated glomerular filtration rate (eGFR) decline is important to predict and treat kidney disease in type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS: We performed a case-control study nested within four T1D cohorts to identify urinary proteins associated with rapid eGFR decline. Case and control subjects were categorized based on eGFR decline ≥3 and <1 mL/min/1.73 m2/year, respectively. We used targeted liquid chromatography-tandem mass spectrometry to measure 38 peptides from 20 proteins implicated in diabetic kidney disease. Significant proteins were investigated in complementary human cohorts and in mouse proximal tubular epithelial cell cultures. RESULTS: The cohort study included 1,270 participants followed a median 8 years. In the discovery set, only cathepsin D peptide and protein were significant on full adjustment for clinical and laboratory variables. In the validation set, associations of cathepsin D with eGFR decline were replicated in minimally adjusted models but lost significance with adjustment for albuminuria. In a meta-analysis with combination of discovery and validation sets, the odds ratio for the association of cathepsin D with rapid eGFR decline was 1.29 per SD (95% CI 1.07-1.55). In complementary human cohorts, urine cathepsin D was associated with tubulointerstitial injury and tubulointerstitial cathepsin D expression was associated with increased cortical interstitial fractional volume. In mouse proximal tubular epithelial cell cultures, advanced glycation end product-BSA increased cathepsin D activity and inflammatory and tubular injury markers, which were further increased with cathepsin D siRNA. CONCLUSIONS: Urine cathepsin D is associated with rapid eGFR decline in T1D and reflects kidney tubulointerstitial injury.


Assuntos
Diabetes Mellitus Tipo 1 , Nefropatias Diabéticas , Albuminúria , Animais , Biomarcadores/metabolismo , Estudos de Casos e Controles , Catepsina D , Estudos de Coortes , Progressão da Doença , Taxa de Filtração Glomerular , Humanos , Camundongos , Proteômica/métodos
10.
Fish Physiol Biochem ; 48(3): 749-765, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35482165

RESUMO

Yolk processing pathways vary in the oocytes of benthophil and pelagophil teleosts. The present study investigated the yolk processing pattern in the oocytes of the fresh water catfish Clarias gariepinus at vitellogenic, maturation, and ovulated stages. This study concludes that during maturation stage, an electrophoretic shift in the major peptide band on Polyacrylamide gel electrophoresis (PAGE) occurs due to a decrease in the size of the yolk protein. The PMF spectrum of corresponding peptides from vitellogenic and ovulated oocytes revealed a difference in the minor ions. A minor difference in the molecular weight of the corresponding peptides occurs due to a difference in their amino acid composition. Maximal activity of the proteases cathepsin D and cathepsin B was observed in the vitellogenic oocytes, thus confirming their role in the processing of yolk. A significant transient increase in the activity of cathepsin B in the mature oocytes also suggests its role in oocyte maturation.


Assuntos
Peixes-Gato , Catepsina B , Catepsina D/metabolismo , Animais , Peixes-Gato/metabolismo , Catepsina B/metabolismo , Proteínas do Ovo/metabolismo , Oócitos/metabolismo , Proteólise
11.
Cells ; 11(7)2022 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-35406699

RESUMO

INTRODUCTION: Both increased activity of the complement system (CS) and the role of the pituitary hormone prolactin (PRL) are implicated in osteoarthritis (OA) pathogenesis. Besides, Cathepsin D (CatD) activity is increased in the context of OA and can exert not only proteolytic but also non-proteolytic effects on cells. For the first time, possible crosstalk between two separate humoral systems: the CS and the PRL hormone systems in chondrocytes are examined together. METHODS: Primary human articular chondrocytes (hAC) were stimulated with complement protein C5 (10 µg /mL), PRL (25 ng/mL), CatD (100 ng/mL), or anaphylatoxin C5a (25 ng/mL) for 24 h or 72 h, while unstimulated cells served as controls. In addition, co-stimulations of C5 or PRL with CatD were carried out under the same conditions. The influence of the stimulants on cell viability, cell proliferation, and metabolic activity of hAC, the chondrosarcoma cell line OUMS-27, and endothelial cells of the human umbilical cord vein (HUVEC) was investigated. Gene expression analysis of C5a receptor (C5aR1), C5, complement regulatory protein CD59, PRL, PRL receptor (PRLR), CatD, and matrix metal-loproteinases (MMP)-13 were performed using real-time PCR. Also, collagen type (Col) I, Col II, C5aR1, CD59, and PRL were detected on protein level using immunofluorescence labeling. RESULTS: The stimulation of the hAC showed no significant impairment of the cell viability. C5, C5a, and PRL induced cell growth in OUMS-27 and HUVEC, but not in chondrocytes. CatD, as well as C5, significantly reduced the gene expression of CatD, C5aR1, C5, and CD59. PRLR gene expression was likewise impaired by C5, C5a, and PRL+CatD stimulation. On the protein level, CatD, as well as C5a, decreased Col II as well as C5aR1 synthesis. CONCLUSIONS: The significant suppression of the C5 gene expression under the influence of PRL+CatD and that of CD59 via PRL+/-CatD and conversely a suppression of the PRLR gene expression via C5 alone or C5a stimulation indicates an interrelation between the two mentioned systems. In addition, CatD and C5, in contrast to PRL, directly mediate possible negative feedback of their own gene expression.


Assuntos
Condrócitos , Osteoartrite , Catepsina D/metabolismo , Condrócitos/metabolismo , Complemento C5/metabolismo , Complemento C5/farmacologia , Complemento C5a/farmacologia , Proteínas do Sistema Complemento/metabolismo , Células Endoteliais/metabolismo , Humanos , Osteoartrite/metabolismo , Prolactina/metabolismo , Prolactina/farmacologia
12.
Eur J Neurol ; 29(7): 2084-2096, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35318785

RESUMO

BACKGROUND AND PURPOSE: The therapeutic landscape of spinal muscular atrophy (SMA) has changed dramatically during the past 4 years, but treatment responses differ remarkably between individuals, and therapeutic decision-making remains challenging, underlining the persistent need for validated biomarkers. METHODS: We applied untargeted proteomic analyses to determine biomarkers in cerebrospinal fluid (CSF) samples of SMA patients under treatment with nusinersen. Identified candidate proteins were validated in CSF samples of SMA patients by Western blot and enzyme-linked immunosorbent assay. Furthermore, levels of peripheral neurofilament heavy and light chain were determined. RESULTS: Untargeted proteomic analysis of CSF samples of three SMA type 1 patients revealed the lysosomal protease cathepsin D as a candidate biomarker. Subsequent validation analysis in a larger cohort of 31 pediatric SMA patients (type 1, n = 12; type 2, n = 9; type 3, n = 6; presymptomatically treated, n = 4; age = 0-16 years) revealed a significant decline of cathepsin D levels in SMA patients aged ≥2 months at the start of treatment. Although evident in all older age categories, this decline was only significant in the group of patients who showed a positive motor response. Moreover, downregulation of cathepsin D was evident in muscle biopsies of SMA patients. CONCLUSIONS: We identified a decline of cathepsin D levels in CSF samples of SMA patients under nusinersen treatment that was more pronounced in the group of "treatment responders" than in "nonresponders." We believe that our results indicate a suitability of cathepsin D levels as a possible biomarker in SMA also in older patients, in combination with analysis of peripheral neurofilament light chain in adolescents or alone in adult patients.


Assuntos
Atrofia Muscular Espinal , Proteômica , Adolescente , Adulto , Idoso , Biomarcadores/líquido cefalorraquidiano , Catepsina D/uso terapêutico , Criança , Humanos , Oligonucleotídeos , Proteômica/métodos
13.
Autophagy ; 18(5): 1127-1151, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35287553

RESUMO

Parkinson disease (PD) is a neurodegenerative disorder characterized by the abnormal intracellular accumulation of SNCA/α-synuclein. While the exact mechanisms underlying SNCA pathology are not fully understood, increasing evidence suggests the involvement of autophagy as well as lysosomal deficiencies. Because CTSD (cathepsin D) has been proposed to be the major lysosomal protease involved in SNCA degradation, its deficiency has been linked to the presence of insoluble SNCA conformers in the brain of mice and humans as well as to the transcellular transmission of SNCA aggregates. We here postulate that SNCA degradation can be enhanced by the application of the recombinant human proform of CTSD (rHsCTSD). Our results reveal that rHsCTSD is efficiently endocytosed by neuronal cells, correctly targeted to lysosomes and matured to an enzymatically active protease. In dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the A53T mutation within the SNCA gene, we confirm the reduction of insoluble SNCA after treatment with rHsCTSD. Moreover, we demonstrate a decrease of pathological SNCA conformers in the brain and within primary neurons of a ctsd-deficient mouse model after dosing with rHsCTSD. Boosting lysosomal CTSD activity not only enhanced SNCA clearance in human and murine neurons as well as tissue, but also restored endo-lysosome and autophagy function. Our findings indicate that CTSD is critical for SNCA clearance and function. Thus, enzyme replacement strategies utilizing CTSD may also be of therapeutic interest for the treatment of PD and other synucleinopathies aiming to decrease the SNCA burden.Abbreviations: aa: amino acid; SNCA/α-synuclein: synuclein alpha; APP: amyloid beta precursor protein; BBB: blood brain barrier; BF: basal forebrain; CBB: Coomassie Brilliant Blue; CLN: neuronal ceroid lipofuscinosis; CNL10: neuronal ceroid lipofuscinosis type 10; Corr.: corrected; CTSD: cathepsin D; CTSB: cathepsin B; DA: dopaminergic; DA-iPSn: induced pluripotent stem cell-derived dopaminergic neurons; dox: doxycycline; ERT: enzyme replacement therapy; Fx: fornix, GBA/ß-glucocerebrosidase: glucosylceramidase beta; h: hour; HC: hippocampus; HT: hypothalamus; i.c.: intracranially; IF: immunofluorescence; iPSC: induced pluripotent stem cell; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LSDs: lysosomal storage disorders; MAPT: microtubule associated protein tau; M6P: mannose-6-phosphate; M6PR: mannose-6-phosphate receptor; MB: midbrain; mCTSD: mature form of CTSD; neurofil.: neurofilament; PD: Parkinson disease; proCTSD: proform of CTSD; PRNP: prion protein; RFU: relative fluorescence units; rHsCTSD: recombinant human proCTSD; SAPC: Saposin C; SIM: structured illumination microscopy; T-insol: Triton-insoluble; T-sol: Triton-soluble; TEM: transmission electron microscopy, TH: tyrosine hydroxylase; Thal: thalamus.


Assuntos
Lipofuscinoses Ceroides Neuronais , Doença de Parkinson , Sinucleinopatias , Peptídeos beta-Amiloides/metabolismo , Animais , Autofagia/fisiologia , Catepsina D/deficiência , Catepsina D/metabolismo , Neurônios Dopaminérgicos/metabolismo , Humanos , Lisossomos/metabolismo , Camundongos , Lipofuscinoses Ceroides Neuronais/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo
14.
Mol Neurobiol ; 59(5): 3091-3109, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35262870

RESUMO

We aim to investigate the mechanisms underlying the beneficial effects of exercise rehabilitation (ER) and/or astragaloside (AST) in counteracting amyloid-beta (Aß) pathology. Aß oligomers were microinjected into the bilateral ventricles to induce Aß neuropathology in rats. Neurobehavioral functions were evaluated. Cortical and hippocampal expressions of both BDNF/TrkB and cathepsin D were determined by the western blotting method. The rat primary cultured cortical neurons were incubated with BDNF and/or AST and ANA12 followed by exposure to aggregated Aß for 24 h. In vivo results showed that ER and/or AST reversed neurobehavioral disorders, downregulation of cortical and hippocampal expression of both BDNF/TrkB and cathepsin D, neural pathology, Aß accumulation, and altered microglial polarization caused by Aß. In vitro studies also confirmed that topical application of BDNF and/or AST reversed the Aß-induced cytotoxicity, apoptosis, mitochondrial distress, and synaptotoxicity and decreased expression of p-TrkB, p-Akt, p-GSK3ß, and ß-catenin in rat cortical neurons. The beneficial effects of combined ER (or BDNF) and AST therapy in vivo and in vitro were superior to ER (or BDNF) or AST alone. Furthermore, we observed that any gains from ER (or BDNF) and/or AST could be significantly eliminated by ANA-12, a potent BDNF/TrkB antagonist. These results indicate that whereas ER (or BDNF) and/or AST attenuate Aß pathology by reversing BDNF/TrkB signaling deficits and mitochondrial dysfunction, combining these two potentiates each other's therapeutic effects. In particular, AST can be an alternative therapy to replace ER.


Assuntos
Fator Neurotrófico Derivado do Encéfalo , Catepsina D , Peptídeos beta-Amiloides/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Catepsina D/metabolismo , Catepsina D/farmacologia , Hipocampo/metabolismo , Mitocôndrias/metabolismo , Ratos , Receptor trkB/metabolismo , Transdução de Sinais
15.
Cells ; 11(5)2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35269509

RESUMO

SLC17A9 (solute carrier family 17 member 9) functions as an ATP transporter in lysosomes as well as other secretory vesicles. SLC17A9 inhibition or silence leads to cell death. However, the molecular mechanisms causing cell death are unclear. In this study, we report that cell death induced by SLC17A9 deficiency is rescued by the transcription factor EB (TFEB), a master gene for lysosomal protein expression, suggesting that SLC17A9 deficiency may be the main cause of lysosome dysfunction, subsequently leading to cell death. Interestingly, Cathepsin D, a lysosomal aspartic protease, is inhibited by SLC17A9 deficiency. Heterologous expression of Cathepsin D successfully rescues lysosomal dysfunction and cell death induced by SLC17A9 deficiency. On the other hand, the activity of Cathepsin B, a lysosomal cysteine protease, is not altered by SLC17A9 deficiency, and Cathepsin B overexpression does not rescue lysosomal dysfunction and cell death induced by SLC17A9 deficiency. Our data suggest that lysosomal ATP and SLC17A9 play critical roles in lysosomal function and cell viability by regulating Cathepsin D activity.


Assuntos
Proteínas de Transporte de Nucleotídeos , Trifosfato de Adenosina/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Catepsina D/genética , Catepsina D/metabolismo , Sobrevivência Celular , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Nucleotídeos/genética
16.
Cell Death Dis ; 13(2): 115, 2022 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-35121737

RESUMO

Cathepsin D (Cat D) is well known for its roles in metastasis, angiogenesis, proliferation, and carcinogenesis in cancer. Despite Cat D being a promising target in cancer cells, effects and underlying mechanism of its inhibition remain unclear. Here, we investigated the plausibility of using Cat D inhibition as an adjuvant or sensitizer for enhancing anticancer drug-induced apoptosis. Inhibition of Cat D markedly enhanced anticancer drug-induced apoptosis in human carcinoma cell lines and xenograft models. The inhibition destabilized Bcl-xL through upregulation of the expression of RNF183, an E3 ligase of Bcl-xL, via NF-κB activation. Furthermore, Cat D inhibition increased the proteasome activity, which is another important factor in the degradation of proteins. Cat D inhibition resulted in p62-dependent activation of Nrf2, which increased the expression of proteasome subunits (PSMA5 and PSMB5), and thereby, the proteasome activity. Overall, Cat D inhibition sensitized cancer cells to anticancer drugs through the destabilization of Bcl-xL. Furthermore, human renal clear carcinoma (RCC) tissues revealed a positive correlation between Cat D and Bcl-xL expression, whereas RNF183 and Bcl-xL expression indicated inverse correlation. Our results suggest that inhibition of Cat D is promising as an adjuvant or sensitizer for enhancing anticancer drug-induced apoptosis in cancer cells.


Assuntos
Antineoplásicos , Carcinoma de Células Renais , Catepsina D , Neoplasias Renais , Ubiquitina-Proteína Ligases , Antineoplásicos/farmacologia , Apoptose , Carcinoma de Células Renais/tratamento farmacológico , Catepsina D/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Neoplasias Renais/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína bcl-X/metabolismo
17.
Biochem Biophys Res Commun ; 600: 22-28, 2022 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-35182971

RESUMO

OBJECTIVE: To investigate the role of high-sugar high-fat treatment in inducing autophagy of rat retinal microvascular endothelial cells. METHODS: The optimal concentrations and time points of glucose and oxidized low-density lipoprotein (ox-LDL) in inducing rat retinal microvascular endothelial cells were determined by examining the proliferate rate by CCK-8 assay. They were divided into control group (blank control), model group (treatment of 50 mM glucose and 10 µg/ml ox-LDL for 24 h), chloroquine group (treatment of 20 µM chloroquine, 50 mM glucose and 10 µg/ml ox-LDL for 24 h), resveratrol group (treatment of 50 µM resveratrol, 50 mM glucose and 10 µg/ml ox-LDL for 24 h) and MITO-Tempol group (treatment of 20 µM MITO-Tempol, 50 mM glucose and 10 µg/ml ox-LDL for 24 h). Reactive oxygen species (ROS) level in rat retinal microvascular endothelial cells induced with high sugar high-fat treatment was measured by flow cytometry. In addition, protein levels of cathepsin B and cathepsin D in rat retinal microvascular endothelial cells induced with high sugar high-fat treatment were examined by immunofluorescence, and protein levels of LC3 A/B and the autophagy substrate P62 were detected by Western blot. RESULTS: Primary retinal microvascular endothelial cells were isolated from neonatal Sprague-Dawley (SD) rats. ROS level was significantly higher in model group than that of control group (P < 0.05). Compared with that of model group, ROS level was significantly reduced in chloroquine group and MITO-Tempol group, which was significantly elevated in resveratrol group (P < 0.05). Positive expressions of cathepsin B and cathepsin D were significantly reduced in model group than those of control group (P < 0.05). They were significantly elevated in chloroquine group and MITO-Tempol group, and reduced in resveratrol group than those of model group (P < 0.05). LC3 A/B and P62 were significantly upregulated in model group than those of control group (P < 0.05). Compared with those of model group, LC3 A/B and P62 were significantly downregulated in chloroquine group and MITO-Tempol group, and upregulated in resveratrol group (P < 0.05). CONCLUSION: High-sugar high-fat treatment induces autophagy of rat retinal microvascular endothelial cells, which can be intervened to a certain extent by chloroquine and MITO-Tempol.


Assuntos
Catepsina D , Células Endoteliais , Animais , Autofagia , Catepsina B/metabolismo , Catepsina D/metabolismo , Cloroquina/farmacologia , Células Endoteliais/metabolismo , Glucose/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Açúcares/metabolismo
18.
Hum Mol Genet ; 31(14): 2396-2405, 2022 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-35179198

RESUMO

Cognitive impairment is a common non-motor complication of Parkinson's disease (PD). Glucocerebrosidase gene (GBA1) variants are found in 10-15% of PD cases and are numerically the most important risk factor for PD and dementia with Lewy bodies. Accumulation of α-synuclein and tau pathology is thought to underlie cognitive impairment in PD and likely involves cholinergic as well as dopaminergic neurons. Neural crest stem cells were isolated from both PD patients with the common heterozygous N370S GBA1 mutation and normal subjects without GBA1 mutations. The stem cells were used to generate a cholinergic neuronal cell model. The effects of the GBA1 variant on glucocerebrosidase (GCase) protein and activity, and cathepsin D, tau and α-synuclein protein levels in cholinergic neurons were examined. Ambroxol, a GCase chaperone, was used to investigate whether GCase enhancement was able to reverse the effects of the GBA1 variant on cholinergic neurons. Significant reductions in GCase protein and activity, as well as in cathepsin D levels, were found in GBA1 mutant (N370S/WT) cholinergic neurons. Both tau and α-synuclein levels were significantly increased in GBA1 mutant (N370S/WT) cholinergic neurons. Ambroxol significantly enhanced GCase activity and decreased both tau and α-synuclein levels in cholinergic neurons. GBA1 mutations interfere with the metabolism of α-synuclein and tau proteins and induce higher levels of α-synuclein and tau proteins in cholinergic neurons. The GCase pathway provides a potential therapeutic target for neurodegenerative disorders related to pathological α-synuclein or tau accumulation.


Assuntos
Ambroxol , Glucosilceramidase , Doença de Parkinson , Ambroxol/farmacologia , Catepsina D/genética , Células Cultivadas , Colinérgicos/farmacologia , Glucosilceramidase/genética , Humanos , Mutação , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo
19.
Bioorg Med Chem ; 57: 116646, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35121401

RESUMO

Cathepsin D (Cath D) has been evidenced as a potential target for cancer therapy. Our previous studies revealed that TB-9, a tasiamide B derivative, exhibited highly potent inhibition against Cath D with satisfactory selectivity over Cath E and BACE1. But this compound was inactive on cell level possibly due to poor membrane permeability. Herein, we report the design, synthesis, and evaluation of two novel Cath D inhibitors (2 and 3) which combining tasiamide B scaffold with a cell penetrating peptide (CPP) specifically targeting the endolysosomal compartment. The results revealed that 2 and 3 not only retained highly potent inhibition against Cath D, but also were active against MDA-MB-231 cell lines.


Assuntos
Catepsina D/antagonistas & inibidores , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Catepsina D/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Relação Estrutura-Atividade
20.
Neurobiol Dis ; 164: 105628, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35033660

RESUMO

Loss of vision due to progressive retinal degeneration is a hallmark of neuronal ceroid lipofuscinoses (NCL), a group of fatal neurodegenerative lysosomal storage diseases. Enzyme substitution therapies represent promising treatment options for NCLs caused by dysfunctions of soluble lysosomal enzymes. Here, we compared the efficacy of a cell-based enzyme substitution strategy and a gene therapy approach to attenuate the retinal pathology in cathepsin D- (CTSD) deficient mice, an animal model of CLN10 disease. Levels of enzymatically active CTSD in mutant retinas were significantly higher after an adeno-associated virus vector-mediated CTSD transfer to retinal glial cells and retinal pigment epithelial cells than after intravitreal transplantations of a CTSD overexpressing clonal neural stem cell line. In line with this finding, the gene therapy treatment restored the disrupted autophagy-lysosomal pathway more effectively than the cell-based approach, as indicated by a complete clearance of storage, significant attenuation of lysosomal hypertrophy, and normalized levels of the autophagy marker sequestosome 1/p62 and microtubule-associated protein 1 light chain 3-II. While the cell-based treatment did not prevent the rapidly progressing loss of various retinal cell types, the gene therapy approach markedly attenuated retinal degeneration as demonstrated by a pronounced rescue of photoreceptor cells and rod bipolar cells.


Assuntos
Autofagia/fisiologia , Catepsina D/genética , Terapia Genética , Lisossomos/fisiologia , Degeneração Retiniana/terapia , Animais , Catepsina D/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Degeneração Retiniana/genética
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