UCHL1 Expression Research Articles

Characterization and Survival of Human Infant Testicular Cells After Direct Xenotransplantation.

BackgroundCryopreservation of prepubertal testicular tissue preserves spermatogonial stem cells (SSCs) that may be used to restore fertility in men at risk of infertility due to gonadotoxic treatments for either a malignant or non-malignant disease. Spermatogonial stem cell-based transplantation is a promising fertility restoration technique. Previously, we performed xenotransplantation of propagated SSCs from prepubertal testis and found human SSCs colonies within the recipient testes six weeks post-transplantation. In order to avoid the propagation step of SSCs in vitro that may cause genetic and epigenetic changes, we performed direct injection of single cell suspension in this study, which potentially may be safer and easier to be applied in future clinical applications.MethodsTestis biopsies were obtained from 11 infant boys (median age 1.3 years, range 0.5-3.5) with cryptorchidism. Following enzymatic digestion, dissociated single-cell suspensions were prelabeled with green fluorescent dye and directly transplanted into seminiferous tubules of busulfan-treated mice. Six to nine weeks post-transplantation, the presence of gonocytes and SSCs was determined by whole-mount immunofluorescence for a number of germ cell markers (MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28), somatic cell markers (SOX9, CYP17A1).ResultsFollowing xenotransplantation human infant germ cells, consisting of gonocytes and SSCs, were shown to settle on the basal membrane of the recipient seminiferous tubules and form SSC colonies with expression of MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28. The colonization efficiency was approximately 6%. No human Sertoli cells were detected in the recipient mouse testes.ConclusionXenotransplantation, without in vitro propagation, of testicular cell suspensions from infant boys with cryptorchidism resulted in colonization of mouse seminiferous tubules six to nine weeks post-transplantation. Spermatogonial stem cell-based transplantation could be a therapeutic treatment for infertility of prepubertal boys with cryptorchidism and boys diagnosed with cancer. However, more studies are required to investigate whether the low number of the transplanted SSC is sufficient to secure the presence of sperm in the ejaculate of those patients over time.

UCHL1 promotes proliferation and metastasis in head and neck squamous cell carcinoma and could be a potential therapeutic target

The purpose of this study was to research the physiological roles of ubiquitin carboxyl-terminal esterase L1 (UCHL1) in head and neck squamous cell carcinoma (HNSCC). Ten HNSCC samples and matched normal oral mucosal tissues were collected. UCHL1 expression of these tissues was detected by the immunohistochemical staining and real-time quantitative polymerase chain reaction. The human HNSCC cell line HN6 UCHL1 knockout (UCHL1 KO) cell line was constructed using CRISPR/CAS9 gene editing and verified by western blotting. Wound healing assay, cell proliferation assay, cell invasion assay, and flow cytometric analysis of the cell cycle and apoptosis were applied to research the role of UCHL1 in HNSCC. Also, an RNAseq gene expression data set and HNSCC patient survival data from The Cancer Genome Atlas were analyzed. UCHL1 was highly expressed in HNSCC tissues compared with normal oral mucosal tissues (P=.032). A decreased proliferation (P < .0001), migration (P < .0001), and invasion (P = .0049) ability of HN6 cells was exhibited after knockout of UCHL1. However, HN6 UCHL1 KO cells showed no significant differences in the cell cycle or apoptosis. The progression, nodal metastasis status, and stage of HNSCC had a positive correlation with the expression of UCHL1. UCHL1 plays an important role in HNSCC, and we consider that targeting UCHL1 may be a feasible therapeutic strategy for HNSCC.

The Epstein–Barr virus noncoding RNA EBER2 transactivates the UCHL1 deubiquitinase to accelerate cell growth

The Epstein-Barr virus (EBV) transforms resting B cells and is involved in the development of B cell lymphomas. We report here that the viral noncoding RNA EBER2 accelerates B cell growth by potentiating expression of the UCHL1 deubiquitinase that itself increased expression of the Aurora kinases and of cyclin B1. Importantly, this effect was also visible in Burkitt's lymphoma cells that express none of the virus's known oncogenes. Mechanistically, EBER2 bound the UCHL1 messenger RNA (mRNA), thereby bringing a protein complex that includes PU.1, a UCHL1 transactivator, to the vicinity of its promoter. Although the EBV oncogene LMP1 has been suggested to induce UCHL1, we show here that EBER2 plays a much more important role to reach significant levels of the deubiquitinase in infected cells. However, some viruses that carried a polymorphic LMP1 had an increased ability to achieve full UCHL1 expression. This work identifies a direct cellular target of a viral noncoding RNA that is likely to be central to EBV's oncogenic properties.

Prominent Prognostic Factors in Aggressive Breast Cancer: A Review

Context: Breast cancer (BC) is the most common cancer in women worldwide. Hereditary susceptibility created by mutations in autosomal dominant genes is responsible for 5 to 10% of all BC cases in women. Recent studies have identified genes associated with increased risk for aggressive BC, providing the basis for better risk management. Evidence Acquisition: The latest information in National Center for Biotechnology Information (NCBI), Google Scholar, ScienceDirect, and Scopus were the main databases for finding articles. A combination of keywords of ‘metastasis’, ‘invasion’, ‘aggressive breast cancer’, ‘prognostic factor’, ‘mutation’, and ‘cancer treatment’ was searched in the databases to identify related articles. Titles and abstracts of the articles were studied to choose the right articles. Results: Mutations in breast cancer type 1 susceptibility protein (BRCA1) and breast cancer type 2 susceptibility protein (BRCA2) genes are two central players related to the high risk of BC. Mutation in tumor protein p53 (TP53) is another important mutation that leads to triple-negative BC. Although the majority of BC types are not associated with high-throughput mutant genes such as BRCA1, BRCA2, and TP53, they are associated with low-throughput genes, including DNA repair protein Rad50 (RAD50), Nijmegen breakage syndrome gene (NBS1), checkpoint kinase 2 (CHEK2), BRCA1-interacting protein 1 (BRIP1), E-cadherin gene (CDH1) and PALB2, UCHL1, aldehydedehydrogenase1A3 (ALDH1A3), androgen receptor (AR), 5-bisphosphate 3-kinase (PIK3CA), phosphatidylinositol-4, and luminal gene expression that are generally mutated in the global population. High tumor mutational burden (TMB) was associated with improved progression-free survival. Conclusions: The lymph node status, early tumor size, ER, PR, human epidermal growth factor receptor-2 (HER2), and Ki-67 are conventional prognostic factors for BC. However, these factors cannot exactly predict the aggressive behavior of BC. Hence, in this review, we discussed new prognostic factors of aggressive BCs that are useful for the treatment of patients with BC.

PROTEIN EXPRESSION OF UBIQUITIN C-TERMINAL HYDROLASE L1 (UCHL1) RISES AS OOCYTES SUCCESSFULLY TRANSITION TO LONG TERM QUIESCENCE

To delineate UCHL1’s role in the dynamic foundation of ovarian reserve by evaluating its expression over time during human fetal gestation. The prenatal dynamics of ovarian reserve establishment and understanding of what selection bias is applied to oocytes is still under investigation. The proteome of these early oocytes may offer further insight, and one of the most specifically and differentially expressed proteins in early oocytes is UCHL1. 2340 oocytes across 17 weeks of gestation were analyzed (20 weeks-36 weeks). Fetal tissues from 25 autopsy specimens were obtained under IRB approval. The preserved ovarian tissue was sectioned, and quantitative immunofluorescence performed using an anti-UCHL1 antibody. Oocytes were identified by morphology; area was measured, and fluorescence intensity was then determined. This fluorescence was later corrected for area and background absorbance to gain a Corrected Total Cell Fluorescence (CTCF). CTCF was compared across gestational ages. A linear regression model was initially used. Given concerns that the linear relationship might be an oversimplification of the dynamic biologic relationship, a locally weighted scatterplot smoothing (LOESS) was also used to describe the trend. LOESS is designed to address nonlinear relationships where linear methods do not perform well by fitting a regression line through the moving central tendency of a biological attribute along the time gradient. LOESS can be used to examine the threshold change of biological activity along a time gradient, if a threshold exists. UCHL1 exhibits minimal expression in the numerous small oogonia/oocytes at 20 weeks. As gestation continues, surviving oocytes have increased density of UCHL1 expression which is correlated with oocyte area (R=0.5530, p=2.4e-41) and, less so, distance from the cortex (R=0.2053, p=3.673e-6). A linear estimation of the relationship between CTCF and weeks of gestation has a positive slope with an R= 0.31 and a p <2.2e-16. This clearly demonstrates the increased oocyte expression of UCHL1 as the fetus approaches term gestation. Furthermore, through use of a LOESS model a clear threshold of expression is reached at 27 weeks gestation and maintained through 36 weeks. This analysis of protein expression across a difficult-to-study period of human fetal oogenesis offers a window into the successful transition of oocyte quiescence. UCHL1 shows promise as a key factor in oocyte survival and acquisition of quiescence during the massive wave of atresia that occurs prior to birth. Its dense expression in primary oocytes at near term is the culmination of a biologic process with an initial threshold reached at 27 weeks gestation.

Growth arrest and DNA damage-inducible proteins (GADD45) in psoriasis

The interplay between T cells, dendritic cells and keratinocytes is crucial for the development and maintenance of inflammation in psoriasis. GADD45 proteins mediate DNA repair in different cells including keratinocytes. In the immune system, GADD45a and GADD45b regulate the function and activation of both T lymphocytes and dendritic cells and GADD45a links DNA repair and epigenetic regulation through its demethylase activity. Here, we analyzed the expression of GADD45a and GADD45b in the skin, dendritic cells and circulating T cells in a cohort of psoriasis patients and their regulation by inflammatory signals. Thirty patients (17 male/13 female) with plaque psoriasis and 15 controls subjects (7 male/8 female), were enrolled. Psoriasis patients exhibited a lower expression of GADD45a at the epidermis but a higher expression in dermal infiltrating T cells in lesional skin. The expression of GADD45a and GADD45b was also higher in peripheral T cells from psoriasis patients, although no differences were observed in p38 activation. The expression and methylation state of the GADD45a target UCHL1 were evaluated, revealing a hypermethylation of its promoter in lesional skin compared to controls. Furthermore, reduced levels of GADD45a correlated with a lower expression UCHL1 in lesional skin. We propose that the demethylase function of GADD45a may account for its pleiotropic effects, and the complex and heterogeneous pattern of expression observed in psoriatic disease.

Impact of prostatic radiation therapy on bladder contractility and innervation.

To determine the effect of prostatic radiation therapy (RT) on bladder contractility and morphology, and axon, or neuron profiles within the detrusor and major pelvic ganglia (MPG) in male rats. Male Sprague-Dawley rats (8 weeks) received a single dose of prostatic RT (0 or 22 Gy). Bladders and MPG were collected 2- and 10-weeks post-RT. Detrusor contractile responses to carbachol and electrical field stimulation (EFS) were measured. Bladders were stained with Masson's trichrome, and antibodies for nonspecific neuronal marker, cholinergic nerve marker choline acetyltransferase (ChAT), and alpha-smooth muscle actin. MPG gene expression was assessed by quantitative polymerase chain reaction for ubiquitin carboxy-terminal hydrolase L1 (Uchl1) and Chat. At 2 weeks post-RT, bladder smooth muscle, detrusor cholinergic axon profiles, and MPG Chat gene expression were increased (p < .05), while carbachol and EFS-mediated contractions were decreased (p < .05). In contrast, at 10 weeks post-RT, nerve-mediated contractions were increased compared with control (p < .05), while bladder smooth muscle, detrusor cholinergic axon profiles, MPG Chat expression, and carbachol contractions had normalized. At both 2- and 10-weeks post-RT, there was no change in detrusor nonspecific axon profiles and MPG Uchl1 expression. In a rat model, RT of the prostate and MPG was associated with early changes in MPG Chat gene expression, and bladder cholinergic axon profiles and smooth muscle content which resolved over time. After RT recovery, bladder contractility decreased early and increased by 10 weeks. Long-term changes to the MPG and increased bladder cholinergic axons may contribute to RT-induced bladder dysfunction in prostate cancer survivors.

Effects of Michigan Cancer Foundation-7/A New Adriamycin Cell-Derived Exosomes on MCF-7 Cell Apoptosis and Drug Sensitivity Through Ubiquitin Carboxyl-Terminal Hydrolase L1

Exosomes secreted by adriamycin-resistant human breast cancer cell line MCF-7/ADR can promote the resistance of normal breast cancer cells. However, the exosomes? contents remain unclear. This article investigated the effect of UCHL1 in MCF-7/ADR-derived exosomes on MCF-7 cell apoptosis and drug sensitivity. In this experiment, control group (NC group), Exosome group, Exosome+ siRNA-UCHL1 group were set followed by analysis of exosomes morphology by electron microscope, UCHL1 and Tubulin level by Western Blot, UCHL1 level by immunofluorescence, UCHL1 mRNA level by qRT-PCR, cell apoptosis by flow cytometry, and drug sensitivity by MTT assay. The size and morphology of exosomes were normal with high expression of UCHL1 and CD63. Compared to NC group, UCHL1 level in Exosome group was significantly increased, while siRNAUCHL1 could reduce UCHL1 level; in addition, Exosome group showed significantly decreased cell apoptosis (P &lt; 0.01) which was elevated in Exosome+siRNA-UCHL1 group (P &lt; 0.05); finally, drug sensitivity in Exosome group was significantly increased (P &lt; 0.01) and decreased in the Exosome+siRNA-UCHL1 group (P &lt; 0.05). MCF-7/ADR-derived UCHL1 in exosome can increase UCHL1 level in MCF-7 cells, reduce cell apoptosis and drug sensitivity of cells.

Absence of Glia Maturation Factor Protects from Axonal Injury and Motor Behavioral Impairments after Traumatic Brain Injury.

Traumatic brain injury (TBI) causes disability and death, accelerating the progression towards Alzheimer’s disease and Parkinson’s disease (PD). TBI causes serious motor and cognitive impairments, as seen in PD that arise during the period of the initial insult. However, this has been understudied relative to TBI induced neuroinflammation, motor and cognitive decline that progress towards PD. Neuronal ubiquitin-C-terminal hydrolase-L1 (UCHL1) is a thiol protease that breaks down ubiquitinated proteins and its level represents the severity of TBI. Previously, we demonstrated the molecular action of glia maturation factor (GMF); a proinflammatory protein in mediating neuroinflammation and neuronal loss. Here, we show that the weight drop method induced TBI neuropathology using behavioral tests, western blotting, and immunofluorescence techniques on sections from wild type (WT) and GMF-deficient (GMF-KO) mice. Results reveal a significant improvement in substantia nigral tyrosine hydroxylase and dopamine transporter expression with motor behavioral performance in GMF-KO mice following TBI. In addition, a significant reduction in neuroinflammation was manifested, as shown by activation of nuclear factor-kB, reduced levels of inducible nitric oxide synthase, and cyclooxygenase-2 expressions. Likewise, neurotrophins including brain-derived neurotrophic factor and glial-derived neurotrophic factor were significantly improved in GMF-KO mice than WT 72 h post-TBI. Consistently, we found that TBI enhances GFAP and UCHL-1 expression and reduces the number of dopaminergic TH-positive neurons in WT compared to GMF-KO mice 72 h post-TBI. Interestingly, we observed a reduction of TH-positive tanycytes in the median eminence of WT than GMF-KO mice. Overall, we found that absence of GMF significantly reversed these neuropathological events and improved behavioral outcome. This study provides evidence that PD-associated pathology progression can be initiated upon induction of TBI.

Ubiquitin Carboxyl-Terminal Hydrolases (UCHs): Potential Mediators for Cancer and Neurodegeneration.

Emerging evidence suggests an inverse association between cancer and neurodegenerative diseases (NDD). Although phenotypically different, both diseases display a significant imbalance in the ubiquitination/deubiquitination processes. Therefore, we particularly investigated the expression of ubiquitin C-terminal hydrolases (UCHs: UCH-L1, UCH-L3, UCH-L5 and BAP1), a subfamily of deubiquitinating enzymes (DUBs), using publically available datasets (GTEx, TCGA) and observed altered expression of UCH-L1, UCH-L3, UCH-L5 in 17 cancer types. Interestingly, UCH-L1 (known to be enriched in neurons and interacting with the Parkinson’s disease-associated protein α-synuclein) appeared to be a prognostic indicator of unfavorable outcome in endometrial and urothelial cancer, while increased expression of UCH-L3 and UCH-L5 was associated with poor survival in liver and thyroid cancer, respectively. In normal tissues, UCH-L1 was found to be strongly expressed in the cerebral cortex and hypothalamus, while UCH-L3 expression was somewhat higher in the testis. The occurrence of mutation rates in UCHs also suggests that BAP1 and UCH-L5 may play a more dominant role in cancers than UCH-L1 and UCH-L3. We also characterized the functional context and configuration of the repeat elements in the promoter of DUBs genes and found that UCHs are highly discriminatory for catabolic function and are mainly enriched with LINE/CR1 repeats. Regarding the thesis of an inverse association between cancer and NDD, we observed that among all DUBs, UCHs are the one most involved in both entities. Considering a putative therapeutic potential based on presumed common mechanisms, it will be useful to determine whether other DUBs can compensate for the loss of UCH activity under physiological conditions. However, experimental evidence is required to substantiate this argument.

Gene UCHL1 expresses specifically in mouse uterine decidual cells in response to estrogen.

UCHL1 is expressed specifically in the brain and gonads of almost all studied model organisms including Drosophila, zebrafish, amphibians, and mammals, suggesting a high degree of evolutionary conservation in its structure and function. Although UCHL1 has been involved in spermatogenesis in mice, its specific expression in mammal placenta remains elusive. Our previous work has revealed that UCHL1 is highly expressed in oocytes, and has been involved in mouse ovarian follicular development. Here, we further examined UCHL1 expression change in endometria during early natural pregnancy, with different stages of the estrous cycle and pseudopregnancy as control. The UCHL1 gene deletion model showed that UCHL1 protein is associated with endometrial development, and its deletion leads to infertility. Notably, we demonstrate evidence showing the distinct expression pattern of UCHL1: weak expression over the uterine endometria, strong expression in decidualized stromal cells at the implantation site with a peak at pregnancy D6, and a shift with primary decidualization to secondary decidualized zones. Using the delayed implantation, the delayed implantation activation, and the artificial decidualization models, we have demonstrated that strong expression of UCHL1 occurred in response to decidualization and estrogen stimulation. These observations suggest that during the early proliferation and differentiation of mouse uterine decidua, UCHL1 expression is up-regulated, and formed an unique intracellular distribution mode. Therefore, we proposed that UCHL1 is involved in decidualization, and possibly in response to estrogen regulation.

Establishment of cell lines with porcine spermatogonial stem cell properties

BackgroundSpermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to mature functional spermatozoa, being the only adult stem cells in the males that can transmit genetic information to the next generation. Porcine SSCs hold great value in transgenic pig production and in establishment of porcine models for regenerative medicine. However, studies and applications of porcine SSCs have been greatly hampered by the low number of SSCs in the testis as well as the lack of an ideal stable long-term culture system to propagate porcine SSCs perpetually.ResultsIn the present study, by lentiviral transduction of plasmids expressing the simian virus 40 (SV40) large T antigen into porcine primary SSCs, we developed two immortalized cell lines with porcine SSC attributes. The established cell lines, with the expression of porcine SSC and germ cell markers UCHL1, PLZF, THY1, VASA and DAZL, could respond to retinoic acid (RA), and could colonize the recipient mouse testis without tumor formation after transplantation. The cell lines displayed infinite proliferation potential, and have now been cultured for more than 7 months and passaged for over 35 times without morphological abnormalities.ConclusionsWe have for the first time established porcine SSC lines that could provide abundant cell sources for mechanistic studies on porcine SSC self-renewal and differentiation, thereby facilitating development of an optimal long-term culture system for porcine primary SSCs and their application to animal husbandry and medicine.

Propagation of goat putative spermatogonial stem cells under growth factors defined serum-free culture conditions.

In the present study, we used a serum-free culture media to propagate goat putative spermatogonial stem cells (SSCs) and evaluated the effect of crucial growth factors on relative expression of some SSC markers and self-renewal related genes. The enriched SSCs were cultured on a homologous Sertoli cell feeder layer in KO-DMEM supplemented with 10% KOSR. Putative SSC colonies emerged between day 6 and 10 which were then characterized by the expression of numerous spermatogonial and pluripotency related markers. After 15 days of subculture, the relative mRNA expression study revealed that 40 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of BCL6B, ID4, PLZF, and UCHL1. Moreover, the supplementation of GDNF + bFGF up-regulated the expression of PLZF and BCL6B. UCHL1 expression was higher after addition of GDNF + LIF while, THY1 overexpressed in response to the addition of GDNF + CSF1. These results demonstrated that the goat SSCs were efficiently propagated using a KOSR based serum-free media and the growth factor supplementation markedly influences their gene expression profile.

Short communication: Evaluation of α-linolenic acid–based intramammary nanosuspension for treatment of subclinical mastitis

The current study investigates the therapeutic efficacy of an α-linolenic acid (ALA, 18:3n-3)-based intramammary nanosuspension (ALA-NS) for treatment of subclinical mastitis. After confirmation of mastitis with the help of field-based testing, a total of 9 mixed-breed cows (23 udder quarter samples) were divided into 3 groups and treated with ALA-NS and cefoperazone intramammary suspension for 10 d. Subclinical mastitis on d 1 was confirmed through field-based tests such as pH, California Mastitis Test (CMT), Whiteside test (WST), and bromothymol blue test (BBT) scores. Treatment with ALA-NS (F1 and F2) exhibited significant effects on field-based parameters, along with curtailment of total microbial count [28 ± 3.16 (mean ± standard deviation) and 25 ± 4.24 cfu/50 µL] and somatic cell count (SCC; 3.9 and 2.8 log SCC cells/mL), respectively for ALA-NS F1 and F2, after 10-d treatment. The efficacy of ALA-NS was further affirmed using more stringent markers for inflammation (nuclear factor kappa-light-chain-enhancer of activated B cells, NFκB-p65), milk quality (sterol response element-binding protein-1c, SREBP-1c), and bacterial resistance (ubiquitin carboxyl-terminal hydrolase-1, UCHL-1) in milk samples. Treatment with ALA-NS (at 2 concentrations of ALA, F1 and F2) significantly decreased expression of NFκB-p65, SREBP-1c, and UCHL-1 after d 10 of treatment. Apparently, anti-inflammatory, antibacterial, peripheral analgesic properties of ALA could account for the therapeutic efficacy of the proposed regimen.

The deubiquitinating enzyme UCHL1 promotes resistance to pemetrexed in non-small cell lung cancer by upregulating thymidylate synthase.

Rationale: Resistance to pemetrexed (PEM)-based chemotherapy is a major cause of progression in non-small cell lung cancer (NSCLC) patients. The deubiquitinating enzyme UCHL1 was recently found to play important roles in chemoresistance and tumor progression. However, the potential roles and mechanisms of UCHL1 in PEM resistance remain unclear.Methods: Bioinformatics analyses and immunohistochemistry were used to evaluate UCHL1 expression in NSCLC specimens. Kaplan-Meier analysis with the log-rank test was used for survival analyses. We established PEM-resistant NSCLC cell lines by exposing them to step-wise increases in PEM concentrations, and in vitro and in vivo assays were used to explore the roles and mechanisms of UCHL1 in PEM resistance using the NSCLC cells.Results: In chemoresistant tumors from NSCLC patients, UCHL1 was highly expressed and elevated UCHL1 expression was strongly associated with poor outcomes. Furthermore, UCHL1 expression was significantly upregulated in PEM-resistant NSCLC cells, while genetic silencing or inhibiting UCHL1 suppressed resistance to PEM and other drugs in NSCLC cells. Mechanistically, UCHL1 promoted PEM resistance in NSCLC by upregulating the expression of thymidylate synthase (TS), based on reduced TS expression after UCHL1 inhibition and re-emergence of PEM resistance upon TS restoration. Furthermore, UCHL1 upregulated TS expression, which mitigated PEM-induced DNA damage and cell cycle arrest in NSCLC cells, and also conferred resistance to PEM and other drugs.Conclusions: It appears that UCHL1 promotes PEM resistance by upregulating TS in NSCLC cells, which mitigated DNA damage and cell cycle arrest. Thus, UCHL1 may be a therapeutic target for overcoming PEM resistance in NSCLC patients.

UCH-L1-mediated Down-regulation of Estrogen Receptor α Contributes to Insensitivity to Endocrine Therapy for Breast Cancer

Purpose: To determine the role of UCH-L1 in regulating ERα expression, and to evaluate whether therapeutic targeting of UCH-L1 can enhance the efficacy of anti-estrogen therapy against breast cancer with loss or reduction of ERα.Methods: Expressions of UCH-L1 and ERα were examined in breast cancer cells and patient specimens. The associations between UCH-L1 and ERα, therapeutic response and prognosis in breast cancer patients were analyzed using multiple databases. The molecular pathways by which UCH-L1 regulates ERα were analyzed using immunoblotting, qRT-PCR, immunoprecipitation, ubiquitination, luciferase and ChIP assays. The effects of UCH-L1 inhibition on the efficacy of tamoxifen in ERα (-) breast cancer cells were tested both in vivo and in vitro.Results: UCH-L1 expression was conversely correlated with ERα status in breast cancer, and the negative regulatory effect of UCH-L1 on ERα was mediated by the deubiquitinase-mediated stability of EGFR, which suppresses ERα transcription. High expression of UCH-L1 was associated with poor therapeutic response and prognosis in patients with breast cancer. Up-regulation of ERα caused by UCH-L1 inhibition could significantly enhance the efficacy of tamoxifen and fulvestrant in ERα (-) breast cancer both in vivo and in vitro.Conclusions: Our results reveal an important role of UCH-L1 in modulating ERα status and demonstrate the involvement of UCH-L1-EGFR signaling pathway, suggesting that UCH-L1 may serve as a novel adjuvant target for treatment of hormone therapy-insensitive breast cancers. Targeting UCH-L1 to sensitize ER negative breast cancer to anti-estrogen therapy might represent a new therapeutic strategy that warrants further exploration.

Chronic waterborne exposure to benzo[a]pyrene induces locomotor dysfunction and development of neurodegenerative phenotypes in zebrafish

Benzo[a]pyrene (B[a]P), a prototype of polycyclic aromatic hydrocarbons (PAHs), is an ubiquitous and notable anthropogenic toxicant. The escalating load of anthropogenic organic pollutants on water bodies and its momentous impact on aquatic life may lead to the development of potent neurodegenerative diseases. Thus, the present study was conducted on zebrafish model to address the potential role of B[a]P in inducing neurodegenerative disease like phenotypes. Waterborne B[a]P exposure was performed for a stipulated period of 21 days at a concentration of 0.4 μg/ml. Separate groups of zebrafish were subjected to methylphenidate hydrochloride (MPH: 0.15 mg/L) bath exposure to study the effect on their behaviour before B[a]P exposure. The findings of the present study advocate that chronic exposure to B[a]P significantly impairs the locomotor activity in zebrafish, which showed reduction in total distance travelled and velocity. Histopathological observation by cresyl violet staining showed that there was significant increase in pyknotic neuronal counts in the diencephalon and telencephalic region of zebrafish brain after B[a]P exposure. Protein expression study showed that there was a significant increase in α-synuclein and decrease in UCH-L1, PSEN2, Nurr1 and NeuN expression in whole brain lysate of B[a]P-exposed zebrafish. Tyrosine hydroxylase (TH), as a marker of dopaminergic neurons, was significantly reduced in the B[a]P-exposed group. MPH co-supplementation significantly ameliorated the B[a]P induced altered expression of Parkinson’s relevant proteins in zebrafish brain. These findings advocate that the locomotor impairment following chronic B[a]P exposure is associated with development of neurodegenerative phenotypes typically affecting the dopaminergic system in zebrafish.

Lyn Kinase Contributes to the Reprogramming of Fibroblasts Promoting Chronic Lymphocytic Leukemia Progression

Growth of chronic lymphocytic leukemia cells strongly depends on a nurturing microenvironmental niche that is specifically primed by diverse, bi-directional interactions to promote leukemic homing, proliferation and progression. The Src-family kinase Lyn was previously identified by our group as a key factor for the formation of this pro-leukemic niche and for the expansion of CLL cells, using the Eµ-TCL1 mouse model. In order to attribute the pro-leukemic function of Lyn to a specific cell type, chimeric mice with lineage-specific defects of Lyn within hematopoietic or non-hematopoietic compartments were generated by irradiating BL6J-mice lethally and restoring their hematopoietic system with Lyn-WT or Lyn-KO stem cells. Consecutively mice were xenotransplanted with TCL1+-malignant cells. Lyn deficiency within the non-hematopoietic compartment decelerated leukemic expansion to a higher degree than did Lyn deficiency within the hematopoietic compartment. Completely Lyn deficient mice showed a more prominent retardation of leukemic expansion compared with both lineage specific Lyn deficient mouse strains, suggesting an additive effect of the two distinct compartments for leukemic expansion. In focusing on the non-hematopoietic fibroblastic bystander cells, primary human CLL cells were cocultured in vitro with Lyn-deficient mouse embryonic fibroblasts as well as Lyn-KO human HS5 cells, generated via the CRISPR-Cas9 system, and leukemic cell survival was assessed over time. All Lyn-deficient fibroblasts showed a significantly reduced feeding capacity for CLL cells compared to WT stroma, indicating the functional relevance of Lyn in leukemia-associated fibroblasts. Subsequently, transcriptomic, proteomic and phosphoproteomic alterations related to Lyn-KO in HS5 cells were comprehensively analyzed, revealing a surprisingly extensive change in gene and protein expression pattern that appeared to be regulated mainly at the transcriptional level. The differentially expressed genes were remarkably often extracellular matrix (ECM)-, cytoskeleton- or cytokine-associated. GO-term enrichment analysis additionally suggested a correlation with ECM processes. Therefore, we hypothesized that Lyn-deficiency might induce transcriptional changes of the cancer-associated fibroblast (CAF)-like phenotype, thus leading to a reduction in leukemic feeding capacity. The diminished expression of several CAF-makers congruent with this reduced activation status was validated in Lyn-KO fibroblasts, as well as the transcriptionally regulated differential expression of chosen target genes. Amongst those, the deubiquitinating enzyme UCHL1 was most abundantly reduced in Lyn-KO HS5 cells, showing an almost complete loss of mRNA and protein expression. Application of a specific UCHL1-inhibitor -in a dose without toxic effects on CLL cells - to CLL-stroma coculture resulted in a significantly hampered feeder effect and reduced CLL cell survival, implying a functional relevance of microenvironmental UCHL1 for stromal support in our system. Additionally, stroma cell death induced by higher drug concentrations in WT cells was completely prohibited in Lyn-KO stroma, illustrating the importance of Lyn for regulating UCHL1 expression and function. In summary, we propose that the Lyn kinase contributes to the formation of a supportive microenvironment via the transcriptional reprogramming of stroma fibroblasts into a "CAF-like" phenotype, which echances viability of CLL cells. In addition, UCHL1 might be a potentially druggable mediator of this activation process. Disclosures Hallek: Roche, Gilead Sciences, Inc., Mundipharma, Janssen, Celgene, Pharmacyclics, AbbVie: Honoraria, Research Funding, Speakers Bureau.

Stroke and Amyloid-β Downregulate TREM-2 and Uch-L1 Expression that Synergistically Promote the Inflammatory Response.

Neuroinflammation is involved in the pathogenesis of Alzheimer's disease, and the transcription factor NF-κB is a player in this event. We found here that the ischemic damage alone or in association with Aβ1-42 activates the NF-κB pathway, induces an increase of BACE1 and a parallel inhibition of Uch-L1 and TREM2, both in vitro and in vivo, in Tg 5XFAD and in human brains of sporadic AD. This mechanism creates a synergistic loop that fosters inflammation. We also demonstrated a significant protection exerted by the restoration of Uch-L1 activity. The rescue of the enzyme is able to abolish the decrease of TREM2 and the parameters of neuroinflammation.

A role for DJ-1 against oxidative stress in the mammalian retina

We have previously reported the expression of Parkinson disease-associated genes encoding α-synuclein, parkin and UCH-L1 in the retina across mammals. DJ-1, or parkinsonism-associated deglycase, is a redox-sensitive protein with putative roles in cellular protection against oxidative stress, among a variety of functions, acting through distinct pathways and mechanisms in a wide variety of tissues. Its function in counteracting oxidative stress in the retina, as it occurs in Parkinson and other human neurodegenerative diseases, is, however, poorly understood. In the present study, we address the expression of DJ-1 in the mammalian retina and its putative neuroprotective role in this tissue in a well-known model of parkinsonism, the rotenone-treated rat. As a result, we demonstrate that the DJ1 gene is expressed at both mRNA and protein levels in the neural retina and retinal pigment epithelium (RPE) of all mammalian species studied. We also present evidence that DJ-1 functions in the retina as a sensor of cellular redox homeostasis, which reacts to oxidative stress by increasing its intracellular levels and additionally becoming oxidized. Levels of α-synuclein also became upregulated, although parkin and UCH-L1 expression remained unchanged. It is inferred that DJ-1 likely exerts in the retina a potential neuroprotective role against oxidative stress, including α-synuclein oxidation and aggregation, which should be operative under both physiological and pathological conditions.